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WO2016087327A1 - Polypeptides having pullulanase activity comprising the x25, x45 and cbm41 domains - Google Patents

Polypeptides having pullulanase activity comprising the x25, x45 and cbm41 domains Download PDF

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Publication number
WO2016087327A1
WO2016087327A1 PCT/EP2015/077920 EP2015077920W WO2016087327A1 WO 2016087327 A1 WO2016087327 A1 WO 2016087327A1 EP 2015077920 W EP2015077920 W EP 2015077920W WO 2016087327 A1 WO2016087327 A1 WO 2016087327A1
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Prior art keywords
seq
pullulanase
domain
bacillus
starch
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PCT/EP2015/077920
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French (fr)
Inventor
Tomoko Matsui
Yuma KURAKATA
Keiichi Ayabe
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Novozymes A/S
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Publication of WO2016087327A1 publication Critical patent/WO2016087327A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2405—Glucanases
    • C12N9/2451—Glucanases acting on alpha-1,6-glucosidic bonds
    • C12N9/2457—Pullulanase (3.2.1.41)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/14—Preparation of compounds containing saccharide radicals produced by the action of a carbohydrase (EC 3.2.x), e.g. by alpha-amylase, e.g. by cellulase, hemicellulase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01041—Pullulanase (3.2.1.41)
    • C—CHEMISTRY; METALLURGY
    • C13—SUGAR INDUSTRY
    • C13K—SACCHARIDES OBTAINED FROM NATURAL SOURCES OR BY HYDROLYSIS OF NATURALLY OCCURRING DISACCHARIDES, OLIGOSACCHARIDES OR POLYSACCHARIDES
    • C13K1/00—Glucose; Glucose-containing syrups
    • C13K1/06—Glucose; Glucose-containing syrups obtained by saccharification of starch or raw materials containing starch
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand

Definitions

  • the present invention relates to methods for increasing pullulanase activity of a parent pullulanase towards a starch substrate. Further the invention relates to improved variant pullulanase generated by the method and to compositions comprising the variant pullulanases.
  • Starch usually consists of about 80% amylopectin and 20% amylose.
  • Amylopectin is a branched polysaccharide in which linear chains alpha-1 ,4 D-glucose residues are joined by alpha-1 ,6 glucosidic linkages.
  • Amylopectin is partially degraded by alpha-amylase, which hydrolyzes the 1 ,4-alpha-glucosidic linkages to produce branched and linear oligosaccharides.
  • Prolonged degradation of amylopectin by alpha-amylase results in the formation of so-called alpha-limit dextrins that are not susceptible to further hydrolysis by the alpha-amylase.
  • Branched oligosaccharides can be hydrolyzed into linear oligosaccharides by a debranching enzyme. The remaining branched oligosaccharides can be depolymerized to D-glucose by glucoamylase, which hydrolyzes linear oligosaccharides into D-glucose.
  • Isoamylases hydrolyses alpha-1 ,6-D-glucosidic branch linkages in amylopectin and beta-limit dextrins and can be distinguished from pullulanases by the inability of isoamylase to attack pullulan, and by their limited action on alpha-limit dextrins.
  • Pullulanase is a starch debranching enzyme having pullulan 6-glucano-hydrolase activity (EC3.2.1.41 ) that catalyzes the hydrolyses the a-1 ,6-glycosidic bonds in pullulan, releasing maltotriose with reducing carbohydrate ends.
  • pullulanase is used in combination with an alpha amylase and/or a glucoamylase.
  • Pullulanases are known in the art.
  • US 6,074,854 and US 5,817,498 disclose a pullulanase from Bacillus deramificans.
  • WO2009/075682 discloses a pullulanase derived from Bacillus acidopullulyticus.
  • the present invention provides pullulanase variants with improved properties, such as increased pullulanase activity, compared to its parent, as well as methods of increasing pullulanase activity of a parent pullulanase.
  • the present invention provides in a first aspect a method of increasing pullulanase activity of a parent pullulanase comprising the steps:
  • the invention provides a pullulanase prepared according to the method of the first aspect.
  • the invention relates to polynucleotides encoding the pullulanase of the invention, compositions and whole broth formulations comprising the pullulanases, uses of the pullulanases for starch processing, and in further aspects the invention relates to a process of producing a fermentation product from starch-containing material comprising the steps of:
  • step (c) fermenting with a fermenting organism; wherein step (a) and/or step (b) is carried out in the presence of a pullulanase of the invention or a pullulanase produced according to the method of the invention.
  • the invention relates to a process of producing a fermentation product from starch-containing material, comprising the steps of:
  • step (a) is carried out using at least a glucoamylase, and a pullulanase of the invention or a pullulanase produced according to the method the invention.
  • Pullulanase means a starch debranching enzyme having pullulan 6-glucano-hydrolase activity (EC 3.2.1 .41 ) that catalyzes the hydrolysis the a-1 ,6- glycosidic bonds in pullulan, releasing maltotriose with reducing carbohydrate ends.
  • pullulanase activity can be determined according to the procedure described in the Examples. In the context of the present invention the variant pullulanases have increased pullulanase activity. Pullulanase activity was determined as activity of the variant relative to the activity of the parent pullulanase using the PHADEBAS assay or the sweet potato starch assay as described in the examples.
  • the pullulanase variants of the invention have at least 105% relative activity when measured against the parent pullulanase activity determined as 100%, more particularly at least 1 10%, more particularly at least 120%, more particularly at least 130%, more particularly at least 140%, more particularly at least 150%.
  • the pullulanase activity is measured using the PHADEBAS assay at 65°C.
  • Pullulanase domains Naturally occurring pullulanases in particular pullulanases derived from bacterial sources, comprises a catalytic domain and in addition several or all of the following domains: CBM41 , X45, X25, and CBM48. In case an X25 domain is present it can be found within an X45 domain. The X45 domain is then split into an X45a and X45b part.
  • the following sequence elements were identified from polypeptides shown to have pullulanase activity (see Table 1 for specific examples of these elements). The elements are: a. X45a N-terminal part of X45 domain not including special composition element Linkc b. Linkc triplet of residues within X45 domain immediately prior to X25 insertion site, and with specific composition requirement for X25 insertion.
  • X45b C-terminal part of X45 domain not including special composition element Linkd.
  • X45b The complete X45 domain is comprised of elements X45a + Linkc (if present) + Linkd (if present) + X45b
  • X25 domains according to the present invention are polypeptides having a percent identity to any sequence listed in Table 2, of at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity.
  • X45 domains according to the present invention are polypeptides having a percent identity to any sequence listed in Table 3, of at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity.
  • Table 3. X45 regions in sequences showing pullulanase activity. Position of comma ',' in residue numbering and sequence shows insertion site.
  • Chimera X25-within-X45 domains are identified as polypeptides having a percent identity to any sequence listed in Table 4 (with comma symbols ',' removed), of at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity.
  • Table 4 Chimeric X25 within X45 domain.
  • PKVSNAYLDNEKTVLAKLTNPMTLSDGSSGFTVTDKTTGEQI PVVSAESAN PVTAVLVGDFQQALGASG NWSPDDDHTKLSKINSNLYQFTGTLPAGTYQYKVALDHSWSASYPNNNVNLTVPAGGTKVTFTYI PSTHQ VFDSINNPDQTFP, SSSAGTQSDLVQLTLASAPDVSHTIQVGAAGYEAVNLI PRNVLNLPRYYYS
  • PKVSNAYLDNEKTVLAKLTNPMTLSDGSSGFTVTDKTTGEQI PVTSAVSAN TVTAVLVGDLQQALGAGG NWAPTDDHTLMTKINANLYQFTGTLPAGTYQYKVALDHSWNASYPNNNVSLTVPSGGEKVTFTYI PSTHQ VYDSINNSNQTFP , ASSAGIQTDLVQLTLASAPDVSHTIQVGAAGYEAVNLI PRNVLNLPRYYYS CBM41 domain: CBM41 domains, Carbohydrate-Binding Module family 41 , are modules of approx. 100 residues found primarily in bacterial pullulanases.
  • CBM41 alias PUD (Bacterial pullulanase-associated domain) modules may be identified in a query protein sequence, by using the Pfam database 'Sequence Search' tool available at http://pfam.xfam.org/search, using Pfam version 26.0 or higher.
  • the Pfam database is a large collection of protein families, each represented by multiple sequence alignments and hidden Markov models (HMMs). Pfam is freely available under the Creative Commons Zero (“CCO") license (see http://creativecommons.0rg/publicdomain/zero/l .0/).
  • the query protein sequence in FASTA format is entered into the search field of the Pfam database Sequence Search tool available via the internet at http://pfam.xfam.org/search, and the Submit button is pressed, after which the Sequence Search results are displayed in a table showing Significant Pfam-A Matches, hereafter Table.
  • the presence of Table rows containing the Family name PUD are positive identifications of the presence of CBM41 alias PUD modules in the query protein sequence.
  • the PUD Family name may also be referred to as PF03714 without loss of ambiguity.
  • E-value refers to the statistical significance of the CBM41 alias PUD module identification.
  • Lower E-values are statistically more significant than higher E-values.
  • Significant CBM41 alias PUD module identifications are defined as those having an E-value less than 1 .0, preferably an E-value less than 1 e-2 (0.01 ), more preferably an E-value less than 1 e-4 (0.0001 ), even more preferably an E-value less than 1 e-6 (0.000001 ).
  • Catalytic domain means the region of an enzyme containing the catalytic machinery of the enzyme.
  • cDNA means a DNA molecule that can be prepared by reverse transcription from a mature, spliced, mRNA molecule obtained from a eukaryotic or prokaryotic cell. cDNA lacks intron sequences that may be present in the corresponding genomic DNA.
  • the initial, primary RNA transcript is a precursor to mRNA that is processed through a series of steps, including splicing, before appearing as mature spliced mRNA.
  • Coding sequence means a polynucleotide, which directly specifies the amino acid sequence of a polypeptide.
  • the boundaries of the coding sequence are generally determined by an open reading frame, which begins with a start codon such as ATG, GTG, or TTG and ends with a stop codon such as TAA, TAG, or TGA.
  • the coding sequence may be a genomic DNA, cDNA, synthetic DNA, or a combination thereof.
  • control sequences means nucleic acid sequences necessary for expression of a polynucleotide encoding a mature polypeptide of the present invention.
  • Each control sequence may be native (i.e., from the same gene) or foreign (i.e., from a different gene) to the polynucleotide encoding the polypeptide or native or foreign to each other.
  • control sequences include, but are not limited to, a leader, polyadenylation sequence, propeptide sequence, promoter, signal peptide sequence, and transcription terminator.
  • the control sequences include a promoter, and transcriptional and translational stop signals.
  • the control sequences may be provided with linkers for the purpose of introducing specific restriction sites facilitating ligation of the control sequences with the coding region of the polynucleotide encoding a polypeptide.
  • Isolated means a substance in a form or environment that does not occur in nature.
  • isolated substances include (1 ) any non-naturally occurring substance, (2) any substance including, but not limited to, any enzyme, variant, nucleic acid, protein, peptide or cofactor, that is at least partially removed from one or more or all of the naturally occurring constituents with which it is associated in nature; (3) any substance modified by the hand of man relative to that substance found in nature; or (4) any substance modified by increasing the amount of the substance relative to other components with which it is naturally associated (e.g., recombinant production in a host cell; multiple copies of a gene encoding the substance; and use of a stronger promoter than the promoter naturally associated with the gene encoding the substance).
  • An isolated substance may be present in a fermentation broth sample; e.g. a host cell may be genetically modified to express the polypeptide of the invention. The fermentation broth from that host cell will comprise the isolated polypeptide.
  • Mature polypeptide means a polypeptide in its final form following translation and any post-translational modifications, such as N-terminal processing, C-terminal truncation, glycosylation, phosphorylation, etc. It is known in the art that a host cell may produce a mixture of two of more different mature polypeptides (i.e., with a different C-terminal and/or N-terminal amino acid) expressed by the same polynucleotide.
  • one host cell expressing a polynucleotide may produce a different mature polypeptide (e.g., having a different C-terminal and/or N-terminal amino acid) as compared to another host cell expressing the same polynucleotide.
  • Mature polypeptide coding sequence means a polynucleotide that encodes a mature polypeptide having pullulanase activity.
  • nucleic acid construct means a nucleic acid molecule, either single- or double-stranded, which is isolated from a naturally occurring gene or is modified to contain segments of nucleic acids in a manner that would not otherwise exist in nature or which is synthetic, which comprises one or more control sequences.
  • operably linked means a configuration in which a control sequence is placed at an appropriate position relative to the coding sequence of a polynucleotide such that the control sequence directs expression of the coding sequence.
  • Sequence identity The relatedness between two amino acid sequences or between two nucleotide sequences is described by the parameter "sequence identity”.
  • the sequence identity between two amino acid sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277), preferably version 5.0.0 or later.
  • the parameters used are gap open penalty of 10, gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix.
  • the output of Needle labeled "longest identity" (obtained using the -nobrief option) is used as the percent identity and is calculated as follows:
  • the sequence identity between two deoxyribonucleotide sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, supra) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, supra), preferably version 5.0.0 or later.
  • the parameters used are gap open penalty of 10, gap extension penalty of 0.5, and the EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix.
  • the output of Needle labeled "longest identity" is used as the percent identity and is calculated as follows:
  • variant means a polypeptide having pullulanase activity comprising an alteration, i.e., a substitution, a deletion or an insertion, e.g., an insertion of a domain selected from a CBM41 or an X25 domain.
  • the present invention relates to methods for increasing pullulanase activity and to the pullulanases resulting therefrom.
  • the present invention relates to a method of increasing pullulanase activity of a parent pullulanase comprising the steps:
  • Pullulanase (EC 3.2.1 .41 ) cleaves 1 .6-alpha-glucosidic linkages in pullulan, amylopectin, and glycogen, and in alpha-and beta-amylase limit-dextrins of amylopectin and glycogen.
  • Bacillus acidopullulyticus pullulanase is used industrially in the production of high fructose corn syrup, high maltose content syrups and low calorie and "light” beers.
  • Pullulanases in addition to the catalytic domain, include several carbohydrate-binding domains (CBMs) as well as domains of unknown function (termed "X" modules).
  • X25 was identified in Bacillus acidopullulyticus pullulanase, and splits another domain of unknown function (X45). X25 is present in multiple copies in some pullulanases. It has been suggested that X25 and X45 are CBMs which target mixed alpha-1 .6/alpha-1.4 linked D-glucan polysaccharides.
  • Pullulanase (EC 3.2.1 .41 ) cleaves 1 .6-alpha-glucosidic linkages in pullulan, amylopectin, and glycogen, and in alpha-and beta-amylase limit-dextrins of amylopectin and glycogen.
  • Bacillus acidopullulyticus pullulanase is used industrially in the production of high fructose corn syrup, high maltose content syrups and low calorie and "light” beers.
  • Pullulanases in addition to the catalytic domain, include several carbohydrate-binding domains (CBMs) as well as domains of unknown function (termed "X" modules).
  • X45 was identified in Bacillus acidopullulyticus pullulanase, it is interrupted by another domain of unknown function (X25). It has been suggested that X25 and X45 are CBMs which target mixed alpha-1.6/alpha-1.4 linked D-glucan polysaccharides. Family 41 Carbohydrate-Binding Module from pullulanase-like enzymes
  • Pullulanases (EC 3.2.1 .41 ) are a group of starch-debranching enzymes, catalyzing the hydrolysis of the alpha-1 .6-glucosidic linkages of alpha-glucans, preferentially pullulan.
  • Pullulan is a polysaccharide in which alpha-1 .4 linked maltotriosyl units are combined via an alpha-1 .6 linkage. These enzymes are of importance in the starch industry, where they are used to hydrolyze amylopectin starch.
  • Pullulanases consist of multiple distinct domains, including a catalytic domain belonging to the glycoside hydrolase (GH) family 13 and carbohydrate-binding modules (CBM), including CBM41 .
  • GH glycoside hydrolase
  • CBM carbohydrate-binding modules
  • pullulanases contain all these domains at their N-terminal and some lack either one or two or all of these domains.
  • pullanase activity may be improved, in particular the improved activity is increased pullulanase activity measured as increased activity in the Phadebas assay at 65°C, by the addition of an X25 domain as defined according to the present invention.
  • This addition may be either by inserting an X25 domain within an existing X45 domain or by replacing an existing X45 domain with an X45a-X25-X45b domain. If the improved pullulanase is obtained by insertion of the X25 domain the following guidelines may be followed in order to identify the proper insertion point. i) Location of X25 insertion site within X45 domain.
  • the insertion site of X25 in identified X45 domains is identified using pair-wise sequence alignment to any sequence listed in Table 3 and designated as 'X45' domain.
  • the insertion site is identified by the location of the comma symbol counting matched positions in the alignment from the start of the sequence designated as 'X45' domain. For example, in specific instances of sequences listed in Table 3, the insertion site is between matched positions 51 and 52 of SEQ ID NO: 44, or positions 48 and 49 of SEQ ID NO: 49, or between positions 48 and 49 of SEQ ID NO: 52. ii) Composition of last three residues within X45 domain just before X25 insertion site.
  • the three residues of the X45 domain immediately prior to the X25 insertion site are of a specific composition described by sequence patterns and exemplified in Table 5.
  • the triplet residue residues immediately prior to the X25 insertion site should match the pattern [DKNS][AGLV][DKNS], or [IV][SD]D. If the three residues of the X45 domain immediately prior to the X25 insertion site do not have the described composition, for instance SEQ ID NO: 52, and SEQ ID NO: 53, then a triplet of residues matching one of the exemplified triplets in Table 5 is inserted immediately before the X25 domain.
  • the addition of three to five additional residues immediately before the X25 domain is also enabled, for example the residue triplet LGQ, designation 'Linkc2' in SEQ. NO. 30.
  • the last four or five residues within the X45 domain immediately following the X25 insertion site are of a specific composition described by sequence patterns and exemplified in Table 6.
  • the last residue of the X25 domain is most often a P or Q residue.
  • Residues immediately following the X25 insertion site should be predominantly composed of residues matching [VSTA][EDNSG][SGA] or [VSTA][EDNSG][SGA][SGA], followed often by a G residue marking the resumption of the X45 domain.
  • VES VDSS (SEQ ID NO: 57)
  • VNGS (SEQ ID NO: 58)
  • VNGSG (SEQ ID NO: 59)
  • ASSA SEQ ID NO: 62
  • ASSAG SEQ ID NO: 63
  • Another aspect of the invention relates to increasing pullulanase activity by the addition of a CBM41 domain either adjacent to the N-terminal of an X45 domain, or if a CBM41 domain is already present, adding the additional CBM41 domain adjacent to the N-terminal of the existing CBM41 or adjacent to the N-terminal of the X45 domain.
  • CBM41 domains may be determined as described in the definition section.
  • the improved pullulanase activity may in particular be increased specific activity (measured as relative specific activity).
  • the pullulanases (or parent pullulanases) that may be improved by the present method are in a particular embodiment pullulanases from a bacterium, particularly, a gram positive bacterium, more particularly a Bacillus sp.
  • the parent pullulanase is selected from a Bacillus acidopullulyticus pullulanase, Bacillus deramificans pullulanase, or a hybrid pullulanase, e.g., a hybrid comprising an N-terminal part from a Bacillus acidopullulyticus pullulanase and a C-terminal part from a Bacillus deramificans pullulanase.
  • the parent pullulanase is selected from SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 1 1 , or SEQ ID NO: 12.
  • the X25 domain as defined herein may be any X25 domain, but in a particular embodiment the X25 domain is selected from an X25 from a pullulanase, more particularly a pullulanase from a bacterium, particularly, a gram positive bacterium, more particularly a Bacillus sp., even more particularly from a Bacillus acidopullulyticus or Bacillus deramificans.
  • the X25 domain is selected from the group consisting of SEQ ID NO: 40 or an X25 domain having a sequence identity to SEQ ID NO: 40 of at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, SEQ ID NO: 41 or an X25 domain having a sequence identity to SEQ ID NO: 41 of at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, SEQ ID NO: 42 or an X25 domain having a sequence identity to SEQ ID NO: 42 of at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 9
  • the X25 domain may in one embodiment be inserted within an X45 domain already present in the parent pullulanase, or alternatively by replacing an existing X45 domain in the parent pullulanase with an X45a-X25-X45b domain.
  • a further aspect of the invention relates to increasing pullulanase activity of a parent pullulanase by adding a CBM41 domain.
  • the CBM41 domain is selected from a CBM41 domain from a pullulanase, more particularly a pullulanase from a bacterium, particularly, a gram positive bacterium, more particularly a Bacillus sp.
  • the Bacillus sp. is selected from Bacillus acidopullulyticus or Bacillus deramificans.
  • Polypeptides having increased pullulanase activity having increased pullulanase activity
  • the present invention in one embodiment relates to pullulanases prepared by the method of the present invention.
  • the invention relates to polypeptides having a sequence identity to the polypeptide of SEQ ID NO: 15 of at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, and wherein the polypeptide has at least at least 90%, at least 95%, at least 97%, at least 100% of the pullulanase activity of the polypeptide of SEQ ID NO: 15.
  • the invention relates to polypeptides having a sequence identity to the polypeptide of SEQ ID NO: 16 of at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, and wherein the polypeptide has at least at least 90%, at least 95%, at least 97%, at least 100% of the pullulanase activity of the polypeptide of SEQ ID NO: 16.
  • the invention relates to polypeptides having a sequence identity to the polypeptide of SEQ ID NO: 17 of at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, and wherein the polypeptide has at least at least 90%, at least 95%, at least 97%, at least 100% of the pullulanase activity of the polypeptide of SEQ ID NO: 17.
  • the invention relates to polypeptides having a sequence identity to the polypeptide of SEQ ID NO: 18 of at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, and wherein the polypeptide has at least at least 90%, at least 95%, at least 97%, at least 100% of the pullulanase activity of the polypeptide of SEQ ID NO: 18.
  • the invention relates to polypeptides having a sequence identity to the polypeptide of SEQ ID NO: 19 of at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, and wherein the polypeptide has at least at least 90%, at least 95%, at least 97%, at least 100% of the pullulanase activity of the polypeptide of SEQ ID NO: 19.
  • the invention relates to polypeptides having a sequence identity to the polypeptide of SEQ ID NO: 20 of at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, and wherein the polypeptide has at least at least 90%, at least 95%, at least 97%, at least 100% of the pullulanase activity of the polypeptide of SEQ ID NO: 20.
  • the invention relates to polypeptides having a sequence identity to the polypeptide of SEQ ID NO: 21 of at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, and wherein the polypeptide has at least at least 90%, at least 95%, at least 97%, at least 100% of the pullulanase activity of the polypeptide of SEQ ID NO: 21.
  • the invention relates to polypeptides having a sequence identity to the polypeptide of SEQ ID NO: 22 of at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, and wherein the polypeptide has at least at least 90%, at least 95%, at least 97%, at least 100% of the pullulanase activity of the polypeptide of SEQ ID NO: 22.
  • the invention relates to polypeptides having a sequence identity to the polypeptide of SEQ ID NO: 23 of at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, and wherein the polypeptide has at least at least 90%, at least 95%, at least 97%, at least 100% of the pullulanase activity of the polypeptide of SEQ ID NO: 23.
  • the invention relates to polypeptides having a sequence identity to the polypeptide of SEQ ID NO: 24 of at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, and wherein the polypeptide has at least at least 90%, at least 95%, at least 97%, at least 100% of the pullulanase activity of the polypeptide of SEQ ID NO: 24.
  • the invention relates to polypeptides having a sequence identity to the polypeptide of SEQ ID NO: 25 of at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, and wherein the polypeptide has at least at least 90%, at least 95%, at least 97%, at least 100% of the pullulanase activity of the polypeptide of SEQ ID NO: 25.
  • the invention relates to polypeptides having a sequence identity to the polypeptide of SEQ ID NO: 26 of at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, and wherein the polypeptide has at least at least 90%, at least 95%, at least 97%, at least 100% of the pullulanase activity of the polypeptide of SEQ ID NO: 26.
  • the invention relates to polypeptides having a sequence identity to the polypeptide of SEQ ID NO: 27 of at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, and wherein the polypeptide has at least at least 90%, at least 95%, at least 97%, at least 100% of the pullulanase activity of the polypeptide of SEQ ID NO: 27.
  • the pullulanase comprises or consists of SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ I D NO: 21 , SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27.
  • polypeptide has been isolated.
  • the present invention relates to variants of the polypeptide of SEQ ID NO: 15-27 comprising a substitution, deletion, and/or insertion at one or more (e.g., several) positions.
  • the number of amino acid substitutions, deletions and/or insertions introduced into the mature polypeptide of SEQ ID NO: 15-27 is up to 10, e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10.
  • amino acid changes may be of a minor nature, that is conservative amino acid substitutions or insertions that do not significantly affect the folding and/or activity of the protein; small deletions, typically of 1 -30 amino acids; small amino- or carboxyl-terminal extensions, such as an amino-terminal methionine residue; a small linker peptide of up to 20-25 residues; or a small extension that facilitates purification by changing net charge or another function, such as a poly-histidine tract, an antigenic epitope or a binding domain.
  • conservative substitutions are within the groups of basic amino acids (arginine, lysine and histidine), acidic amino acids (glutamic acid and aspartic acid), polar amino acids (glutamine and asparagine), hydrophobic amino acids (leucine, isoleucine and valine), aromatic amino acids (phenylalanine, tryptophan and tyrosine), and small amino acids (glycine, alanine, serine, threonine and methionine).
  • Amino acid substitutions that do not generally alter specific activity are known in the art and are described, for example, by H. Neurath and R.L. Hill, 1979, In, The Proteins, Academic Press, New York.
  • amino acid changes are of such a nature that the physico-chemical properties of the polypeptides are altered.
  • amino acid changes may improve the thermal stability of the polypeptide, alter the substrate specificity, change the pH optimum, and the like.
  • Essential amino acids in a polypeptide can be identified according to procedures known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (Cunningham and Wells, 1989, Science 244: 1081 -1085). In the latter technique, single alanine mutations are introduced at every residue in the molecule, and the resultant molecules are tested for pullulanase activity to identify amino acid residues that are critical to the activity of the molecule. See also, Hilton et al., 1996, J. Biol. Chem. 271 : 4699-4708.
  • the active site of the enzyme or other biological interaction can also be determined by physical analysis of structure, as determined by such techniques as nuclear magnetic resonance, crystallography, electron diffraction, or photo affinity labeling, in conjunction with mutation of putative contact site amino acids. See, for example, de Vos et al., 1992, Science 255: 306-312; Smith et al., 1992, J. Mol. Biol. 224: 899-904; Wlodaver et al., 1992, FEBS Lett. 309: 59-64.
  • the identity of essential amino acids can also be inferred from an alignment with a related polypeptide.
  • Single or multiple amino acid substitutions, deletions, and/or insertions can be made and tested using known methods of mutagenesis, recombination, and/or shuffling, followed by a relevant screening procedure, such as those disclosed by Reidhaar-Olson and Sauer, 1988, Science 241 : 53-57; Bowie and Sauer, 1989, Proc. Natl. Acad. Sci. USA 86: 2152-2156; WO 95/17413; or WO 95/22625.
  • Other methods that can be used include error-prone PCR, phage display (e.g., Lowman et al., 1991 , Biochemistry 30: 10832-10837; U.S. Patent No. 5,223,409; WO 92/06204), and region-directed mutagenesis (Derbyshire et al., 1986, Gene 46: 145; Ner ei a/., 1988, DNA 7: 127).
  • Mutagenesis/shuffling methods can be combined with high-throughput, automated screening methods to detect activity of cloned, mutagenized polypeptides expressed by host cells (Ness et al., 1999, Nature Biotechnology 17: 893-896). Mutagenized DNA molecules that encode active polypeptides can be recovered from the host cells and rapidly sequenced using standard methods in the art. These methods allow the rapid determination of the importance of individual amino acid residues in a polypeptide.
  • the polypeptide may be a hybrid polypeptide in which a region of one polypeptide is fused at the N-terminus or the C-terminus of a region of another polypeptide.
  • the polypeptide may be a fusion polypeptide or cleavable fusion polypeptide in which another polypeptide is fused at the N-terminus or the C-terminus of the polypeptide of the present invention.
  • a fusion polypeptide is produced by fusing a polynucleotide encoding another polypeptide to a polynucleotide of the present invention.
  • Techniques for producing fusion polypeptides are known in the art, and include ligating the coding sequences encoding the polypeptides so that they are in frame and that expression of the fusion polypeptide is under control of the same promoter(s) and terminator.
  • Fusion polypeptides may also be constructed using intein technology in which fusion polypeptides are created post-translationally (Cooper et al., 1993, EMBO J. 12: 2575-2583; Dawson et ai, 1994, Science 266: 776-779).
  • a fusion polypeptide can further comprise a cleavage site between the two polypeptides. Upon secretion of the fusion protein, the site is cleaved releasing the two polypeptides.
  • cleavage sites include, but are not limited to, the sites disclosed in Martin et al., 2003, J. Ind. Microbiol. Biotechnol. 3: 568-576; Svetina et al., 2000, J. Biotechnol. 76: 245-251 ; Rasmussen-Wilson et al., 1997, Appl. Environ. Microbiol.
  • the present invention also relates to polynucleotides encoding a polypeptide of the present invention, as described herein.
  • the polynucleotide encoding the polypeptide of the present invention has been isolated.
  • the techniques used to isolate or clone a polynucleotide include isolation from genomic DNA or cDNA, or a combination thereof.
  • the cloning of the polynucleotides from genomic DNA can be effected, e.g., by using the well-known polymerase chain reaction (PCR) or antibody screening of expression libraries to detect cloned DNA fragments with shared structural features. See, e.g., Innis et al., 1990, PCR: A Guide to Methods and Application, Academic Press, New York.
  • Other nucleic acid amplification procedures such as ligase chain reaction (LCR), ligation activated transcription (LAT) and polynucleotide-based amplification (NASBA) may be used.
  • LCR ligase chain reaction
  • LAT ligation activated transcription
  • NASBA polynucleotide-based amplification
  • the present invention also relates to nucleic acid constructs comprising a polynucleotide of the present invention operably linked to one or more control sequences that direct the expression of the coding sequence in a suitable host cell under conditions compatible with the control sequences.
  • the polynucleotide may be manipulated in a variety of ways to provide for expression of the polypeptide. Manipulation of the polynucleotide prior to its insertion into a vector may be desirable or necessary depending on the expression vector. The techniques for modifying polynucleotides utilizing recombinant DNA methods are well known in the art.
  • the control sequence may be a promoter, a polynucleotide that is recognized by a host cell for expression of a polynucleotide encoding a polypeptide of the present invention.
  • the promoter contains transcriptional control sequences that mediate the expression of the polypeptide.
  • the promoter may be any polynucleotide that shows transcriptional activity in the host cell including variant, truncated, and hybrid promoters, and may be obtained from genes encoding extracellular or intracellular polypeptides either homologous or heterologous to the host cell.
  • suitable promoters for directing transcription of the nucleic acid constructs of the present invention in a bacterial host cell are the promoters obtained from the Bacillus amyloliquefaciens alpha-amylase gene (amyQ), Bacillus licheniformis alpha-amylase gene (amyL), Bacillus licheniformis penicillinase gene (penP), Bacillus stearothermophilus maltogenic amylase gene (amyM), Bacillus subtilis levansucrase gene (sacB), Bacillus subtilis xylA and xylB genes, Bacillus thuringiensis crylllA gene (Agaisse and Lereclus, 1994, Molecular Microbiology 13: 97-107), E.
  • E. coli trc promoter (Egon et al., 1988, Gene 69: 301 -315), Streptomyces coelicolor agarase gene ⁇ dagA), and prokaryotic beta- lactamase gene (Villa-Kamaroff et al., 1978, Proc. Natl. Acad. Sci. USA 75: 3727-3731 ), as well as the tac promoter (DeBoer et al., 1983, Proc. Natl. Acad. Sci. USA 80: 21 -25).
  • the control sequence may also be a transcription terminator, which is recognized by a host cell to terminate transcription.
  • the terminator is operably linked to the 3'-terminus of the polynucleotide encoding the polypeptide. Any terminator that is functional in the host cell may be used in the present invention.
  • Preferred terminators for bacterial host cells are obtained from the genes for Bacillus clausii alkaline protease ⁇ aprH), Bacillus licheniformis alpha-amylase (amyL), and Escherichia coli ribosomal RNA (rrnB).
  • control sequence may also be an mRNA stabilizer region downstream of a promoter and upstream of the coding sequence of a gene which increases expression of the gene.
  • mRNA stabilizer regions are obtained from a Bacillus thuringiensis crylllA gene (WO 94/25612) and a Bacillus subtilis SP82 gene (Hue et ai, 1995, Journal of Bacteriology 177: 3465-3471 ).
  • the control sequence may also be a signal peptide coding region that encodes a signal peptide linked to the N-terminus of a polypeptide and directs the polypeptide into the cell's secretory pathway.
  • the 5'-end of the coding sequence of the polynucleotide may inherently contain a signal peptide coding sequence naturally linked in translation reading frame with the segment of the coding sequence that encodes the polypeptide.
  • the 5'-end of the coding sequence may contain a signal peptide coding sequence that is foreign to the coding sequence.
  • a foreign signal peptide coding sequence may be required where the coding sequence does not naturally contain a signal peptide coding sequence.
  • a foreign signal peptide coding sequence may simply replace the natural signal peptide coding sequence in order to enhance secretion of the polypeptide.
  • any signal peptide coding sequence that directs the expressed polypeptide into the secretory pathway of a host cell may be used.
  • Effective signal peptide coding sequences for bacterial host cells are the signal peptide coding sequences obtained from the genes for Bacillus NCIB 1 1837 maltogenic amylase, Bacillus licheniformis subtilisin, Bacillus licheniformis beta-lactamase, Bacillus stearothermophilus alpha-amylase, Bacillus stearothermophilus neutral proteases ⁇ nprT, nprS, nprM), and Bacillus subtilis prsA. Further signal peptides are described by Simonen and Palva, 1993, Microbiological Reviews 57: 109-137.
  • the control sequence may also be a propeptide coding sequence that encodes a propeptide positioned at the N-terminus of a polypeptide.
  • the resultant polypeptide is known as a proenzyme or propolypeptide (or a zymogen in some cases).
  • a propolypeptide is generally inactive and can be converted to an active polypeptide by catalytic or autocatalytic cleavage of the propeptide from the propolypeptide.
  • the propeptide sequence is positioned next to the N-terminus of a polypeptide and the signal peptide sequence is positioned next to the N-terminus of the propeptide sequence.
  • the present invention also relates to recombinant expression vectors comprising a polynucleotide of the present invention, a promoter, and transcriptional and translational stop signals.
  • the various nucleotide and control sequences may be joined together to produce a recombinant expression vector that may include one or more convenient restriction sites to allow for insertion or substitution of the polynucleotide encoding the polypeptide at such sites.
  • the polynucleotide may be expressed by inserting the polynucleotide or a nucleic acid construct comprising the polynucleotide into an appropriate vector for expression.
  • the coding sequence is located in the vector so that the coding sequence is operably linked with the appropriate control sequences for expression.
  • the recombinant expression vector may be any vector (e.g., a plasmid or virus) that can be conveniently subjected to recombinant DNA procedures and can bring about expression of the polynucleotide.
  • the choice of the vector will typically depend on the compatibility of the vector with the host cell into which the vector is to be introduced.
  • the vector may be a linear or closed circular plasmid.
  • the vector may be an autonomously replicating vector, i.e., a vector that exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e.g., a plasmid, an extrachromosomal element, a minichromosome, or an artificial chromosome.
  • the vector may contain any means for assuring self-replication.
  • the vector may be one that, when introduced into the host cell, is integrated into the genome and replicated together with the chromosome(s) into which it has been integrated.
  • a single vector or plasmid or two or more vectors or plasmids that together contain the total DNA to be introduced into the genome of the host cell, or a transposon may be used.
  • the vector preferably contains one or more selectable markers that permit easy selection of transformed, transfected, transduced, or the like cells.
  • a selectable marker is a gene the product of which provides for biocide or viral resistance, resistance to heavy metals, prototrophy to auxotrophs, and the like.
  • bacterial selectable markers are Bacillus licheniformis or Bacillus subtilis dal genes, or markers that confer antibiotic resistance such as ampicillin, chloramphenicol, kanamycin, neomycin, spectinomycin, or tetracycline resistance.
  • the vector preferably contains an element(s) that permits integration of the vector into the host cell's genome or autonomous replication of the vector in the cell independent of the genome.
  • the vector may rely on the polynucleotide's sequence encoding the polypeptide or any other element of the vector for integration into the genome by homologous or non-homologous recombination.
  • the vector may contain additional polynucleotides for directing integration by homologous recombination into the genome of the host cell at a precise location(s) in the chromosome(s).
  • the integrational elements should contain a sufficient number of nucleic acids, such as 100 to 10,000 base pairs, 400 to 10,000 base pairs, and 800 to 10,000 base pairs, which have a high degree of sequence identity to the corresponding target sequence to enhance the probability of homologous recombination.
  • the integrational elements may be any sequence that is homologous with the target sequence in the genome of the host cell. Furthermore, the integrational elements may be non-encoding or encoding polynucleotides. On the other hand, the vector may be integrated into the genome of the host cell by non-homologous recombination.
  • the vector may further comprise an origin of replication enabling the vector to replicate autonomously in the host cell in question.
  • the origin of replication may be any plasmid replicator mediating autonomous replication that functions in a cell.
  • the term "origin of replication" or "plasmid replicator” means a polynucleotide that enables a plasmid or vector to replicate in vivo.
  • bacterial origins of replication are the origins of replication of plasmids pBR322, pUC19, pACYC177, and pACYC184 permitting replication in E. coli, and pUB1 10, pE194, pTA1060, and ⁇ permitting replication in Bacillus.
  • More than one copy of a polynucleotide of the present invention may be inserted into a host cell to increase production of a polypeptide.
  • An increase in the copy number of the polynucleotide can be obtained by integrating at least one additional copy of the sequence into the host cell genome or by including an amplifiable selectable marker gene with the polynucleotide where cells containing amplified copies of the selectable marker gene, and thereby additional copies of the polynucleotide, can be selected for by cultivating the cells in the presence of the appropriate selectable agent.
  • the present invention also relates to recombinant host cells, comprising a polynucleotide of the present invention operably linked to one or more control sequences that direct the production of a polypeptide of the present invention.
  • a construct or vector comprising a polynucleotide is introduced into a host cell so that the construct or vector is maintained as a chromosomal integrant or as a self-replicating extra-chromosomal vector as described earlier.
  • the term "host cell” encompasses any progeny of a parent cell that is not identical to the parent cell due to mutations that occur during replication. The choice of a host cell will to a large extent depend upon the gene encoding the polypeptide and its source.
  • the host cell may be any cell useful in the recombinant production of a polypeptide of the present invention, e.g., a prokaryote or a eukaryote.
  • the prokaryotic host cell may be any Gram-positive or Gram-negative bacterium.
  • Gram- positive bacteria include, but are not limited to, Bacillus, Clostridium, Enterococcus, Geobacillus, Lactobacillus, Lactococcus, Oceanobacillus, Staphylococcus, Streptococcus, and Streptomyces.
  • Gram-negative bacteria include, but are not limited to, Campylobacter, E. coli, Flavobacterium, Fusobacterium, Helicobacter, llyobacter, Neisseria, Pseudomonas, Salmonella, and Ureaplasma.
  • the bacterial host cell may be any Bacillus cell including, but not limited to, Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus firmus, Bacillus lautus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus pumilus, Bacillus stearothermophilus, Bacillus subtilis, and Bacillus thuringiensis cells.
  • the introduction of DNA into a Bacillus cell may be effected by protoplast transformation (see, e.g., Chang and Cohen, 1979, Mol. Gen. Genet. 168: 1 1 1 -1 15), competent cell transformation (see, e.g., Young and Spizizen, 1961 , J. Bacteriol. 81 : 823-829, or Dubnau and Davidoff-Abelson, 1971 , J. Mol. Biol. 56: 209-221 ), electroporation (see, e.g., Shigekawa and Dower, 1988, Biotechniques 6: 742-751 ), or conjugation (see, e.g., Koehler and Thorne, 1987, J. Bacteriol.
  • the introduction of DNA into an E. coli cell may be effected by protoplast transformation (see, e.g., Hanahan, 1983, J. Mol. Biol. 166: 557-580) or electroporation (see, e.g., Dower et al, 1988, Nucleic Acids Res. 16: 6127-6145).
  • the introduction of DNA into a Streptomyces cell may be effected by protoplast transformation, electroporation (see, e.g., Gong et al., 2004, Folia Microbiol. ⁇ Praha) 49: 399-405), conjugation (see, e.g., Mazodier et al., 1989, J. Bacteriol.
  • DNA into a Pseudomonas cell may be effected by electroporation (see, e.g., Choi et al., 2006, J. Microbiol. Methods 64: 391 -397) or conjugation (see, e.g., Pinedo and Smets, 2005, Appl. Environ. Microbiol. 71 : 51 -57).
  • the introduction of DNA into a Streptococcus cell may be effected by natural competence (see, e.g., Perry and Kuramitsu, 1981 , Infect. Immun. 32: 1295-1297), protoplast transformation (see, e.g., Catt and Jollick, 1991 , Microbios 68: 189-207), electroporation (see, e.g., Buckley et al., 1999, Appl. Environ. Microbiol. 65: 3800-3804), or conjugation (see, e.g., Clewell, 1981 , Microbiol. Rev. 45: 409-436).
  • any method known in the art for introducing DNA into a host cell can be used.
  • the host cell may be a yeast cell.
  • yeast as used herein includes ascosporogenous yeast (Endomycetales), basidiosporogenous yeast, and yeast belonging to the Fungi Imperfecti (Blastomycetes). Since the classification of yeast may change in the future, for the purposes of this invention, yeast shall be defined as described in Biology and Activities of Yeast (Skinner, Passmore, and Davenport, editors, Soc. App. Bacteriol. Symposium Series No. 9, 1980).
  • the yeast host cell may be a Candida, Hansenula, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowia cell, such as a Kluyveromyces lactis,
  • Saccharomyces carlsbergensis Saccharomyces cerevisiae, Saccharomyces diastaticus
  • Saccharomyces douglasii Saccharomyces kluyveri
  • Saccharomyces norbensis Saccharomyces norbensis
  • the present invention also relates to a fermentation broth formulation or a cell composition comprising a polypeptide of the present invention.
  • the fermentation broth product further comprises additional ingredients used in the fermentation process, such as, for example, cells (including, the host cells containing the gene encoding the polypeptide of the present invention which are used to produce the polypeptide of interest), cell debris, biomass, fermentation media and/or fermentation products.
  • the composition is a cell-killed whole broth containing organic acid(s), killed cells and/or cell debris, and culture medium.
  • fermentation broth refers to a preparation produced by cellular fermentation that undergoes no or minimal recovery and/or purification.
  • fermentation broths are produced when microbial cultures are grown to saturation, incubated under carbon-limiting conditions to allow protein synthesis (e.g., expression of enzymes by host cells) and secretion into cell culture medium.
  • the fermentation broth can contain unfractionated or fractionated contents of the fermentation materials derived at the end of the fermentation.
  • the fermentation broth is unfractionated and comprises the spent culture medium and cell debris present after the microbial cells (e.g., filamentous fungal cells) are removed, e.g., by centrifugation.
  • the fermentation broth contains spent cell culture medium, extracellular enzymes, and viable and/or nonviable microbial cells.
  • the fermentation broth formulation and cell compositions comprise a first organic acid component comprising at least one 1 -5 carbon organic acid and/or a salt thereof and a second organic acid component comprising at least one 6 or more carbon organic acid and/or a salt thereof.
  • the first organic acid component is acetic acid, formic acid, propionic acid, a salt thereof, or a mixture of two or more of the foregoing and the second organic acid component is benzoic acid, cyclohexanecarboxylic acid, 4-methylvaleric acid, phenylacetic acid, a salt thereof, or a mixture of two or more of the foregoing.
  • the composition contains an organic acid(s), and optionally further contains killed cells and/or cell debris.
  • the killed cells and/or cell debris are removed from a cell-killed whole broth to provide a composition that is free of these components.
  • the fermentation broth formulations or cell compositions may further comprise a preservative and/or anti-microbial (e.g., bacteriostatic) agent, including, but not limited to, sorbitol, sodium chloride, potassium sorbate, and others known in the art.
  • a preservative and/or anti-microbial agent including, but not limited to, sorbitol, sodium chloride, potassium sorbate, and others known in the art.
  • the cell-killed whole broth or composition may contain the unfractionated contents of the fermentation materials derived at the end of the fermentation.
  • the cell-killed whole broth or composition contains the spent culture medium and cell debris present after the microbial cells (e.g., filamentous fungal cells) are grown to saturation, incubated under carbon- limiting conditions to allow protein synthesis.
  • the cell-killed whole broth or composition contains the spent cell culture medium, extracellular enzymes, and killed filamentous fungal cells.
  • the microbial cells present in the cell-killed whole broth or composition can be permeabilized and/or lysed using methods known in the art.
  • a whole broth or cell composition as described herein is typically a liquid, but may contain insoluble components, such as killed cells, cell debris, culture media components, and/or insoluble enzyme(s). In some embodiments, insoluble components may be removed to provide a clarified liquid composition.
  • the whole broth formulations and cell compositions of the present invention may be produced by a method described in WO 90/15861 or WO 2010/096673.
  • the present invention also relates to compositions comprising a polypeptide of the present invention.
  • the compositions are enriched in such a polypeptide.
  • the term "enriched" indicates that the pullulanase activity of the composition has been increased, e.g., with an enrichment factor of at least 1 .1.
  • compositions may comprise a polypeptide of the present invention as the major enzymatic component, e.g., a mono-component composition.
  • the compositions may comprise multiple enzymatic activities, such as one or more (e.g., several) enzymes selected from the group consisting of hydrolase, isomerase, ligase, lyase, oxidoreductase, or transferase, e.g., an alpha-galactosidase, alpha-glucosidase, aminopeptidase, alpha-amylase, beta-amylase, beta-galactosidase, beta-glucosidase, beta-xylosidase, carbohydrase, carboxypeptidase, catalase, cellobiohydrolase, cellulase, chitinase, cutinase, cyclodextrin glycosyltransferase, deoxyribonuclease, end
  • additional enzyme(s) may be selected from the group consisting of an alpha amylase, glucoamylase, beta-amylase, cellulase (beta-glucosidase, cellobiohydrolase and endoglucanase), hemicellulase (e.g., xylanase), isoamylase, isomerase, lipase, phytase, protease, a further pullulanase, and/or other enzymes useful in a commercial process in conjunction with a pullulanase.
  • Such enzymes are known in the art in starch processing, sugar conversion, fermentations for alcohol and other useful end-products, commercial detergents and cleaning aids, stain removal, fabric treatment or desizing, and the like.
  • the enzyme composition of the present invention may be in any form suitable for use, such as, for example, a crude fermentation broth with or without cells removed, a cell lysate with or without cellular debris, a semi-purified or purified enzyme composition, or a host cell, e.g., Trichoderma host cell, as a source of the enzymes.
  • the enzyme composition may be a dry powder or granulate, a non-dusting granulate, a liquid, a stabilized liquid, or a stabilized protected enzyme.
  • Liquid enzyme compositions may, for instance, be stabilized by adding stabilizers such as a sugar, a sugar alcohol or another polyol, and/or lactic acid or another organic acid according to established processes.
  • composition further comprises a glucoamylase.
  • composition further comprises an alpha-amylase.
  • compositions may be prepared in accordance with methods known in the art and may be in the form of a liquid or a dry composition.
  • the compositions may be stabilized in accordance with methods known in the art.
  • compositions of the present invention are given below of preferred uses of the pullulanase and compositions of the present invention.
  • the dosage of the composition and other conditions under which the composition is used may be determined on the basis of methods known in the art.
  • the present invention is also directed to methods of using polypeptide of present invention in various industrial applications.
  • the polypeptide of the present invention may be used for starch processes, in particular starch conversion, especially liquefaction of starch (see, e.g., U.S. Patent No. 3,912,590, EP 252730 and EP 063909, WO 99/19467, and WO 96/28567, which are all hereby incorporated by reference).
  • compositions for starch conversion purposes which may beside the polypeptide of the present invention also comprise a glucoamylase (AMG), and an alpha-amylase.
  • AMG glucoamylase
  • polypeptide of the present invention is particularly useful in the production of sweeteners and ethanol (see, e.g., U.S. Patent No. 5,231 ,017, which is hereby incorporated by reference), such as fuel, drinking and industrial ethanol, from starch or whole grains.
  • the present invention relates to a use of the polypeptide according to the invention for production of a syrup and/or a fermentation product from a starch containing material.
  • the starch material may in one embodiment be gelatinized. In another embodiment the starch material is ungelatinized.
  • Native starch consists of microscopic granules, which are insoluble in water at room temperature. When an aqueous starch slurry is heated, the granules swell and eventually burst, dispersing the starch molecules into the solution. At temperatures up to about 50°C to 75°C the swelling may be reversible. However, with higher temperatures an irreversible swelling called “gelatinization” begins. During this "gelatinization” process there is a dramatic increase in viscosity.
  • Granular starch to be processed may be a highly refined starch quality, preferably at least 90%, at least 95%, at least 97% or at least 99.5% pure or it may be a more crude starch- containing materials comprising (e.g., milled) whole grains including non-starch fractions such as germ residues and fibers.
  • the raw material such as whole grains, may be reduced in particle size, e.g., by milling, in order to open up the structure and allowing for further processing.
  • whole kernels are milled and used. Wet milling gives a good separation of germ and meal (starch granules and protein) and is often applied at locations where the starch hydrolysate is used in the production of, e.g., syrups. Both dry and wet milling is well known in the art of starch processing and may be used in a process of the invention. Methods for reducing the particle size of the starch containing material are well known to those skilled in the art.
  • the starch As the solids level is 30-40% in a typical industrial process, the starch has to be thinned or "liquefied” so that it can be suitably processed. This reduction in viscosity is primarily attained by enzymatic degradation in current commercial practice.
  • Liquefaction is carried out in the presence of an alpha-amylase, preferably a bacterial alpha-amylase and/or acid fungal alpha-amylase.
  • an alpha-amylase preferably a bacterial alpha-amylase and/or acid fungal alpha-amylase.
  • a phytase is also present during liquefaction.
  • a protease is also present during liquefaction.
  • viscosity reducing enzymes such as a xylanase and/or beta-glucanase is also present during liquefaction.
  • the long-chained starch is degraded into branched and linear shorter units (maltodextrins) by an alpha-amylase.
  • Liquefaction may be carried out as a three- step hot slurry process. The slurry is heated to between 60-95°C (e.g., 70-90°C, such as 77- 86°C, 80-85°C, 83-85°C) and an alpha-amylase is added to initiate liquefaction (thinning).
  • the slurry may in an embodiment be jet-cooked at between 95-140°C, e.g., 105-125°C, for about 1 -15 minutes, e.g., about 3-10 minutes, especially around 5 minutes.
  • the slurry is then cooled to 60-95°C and more alpha-amylase is added to obtain final hydrolysis (secondary liquefaction).
  • the jet-cooking process is carried out at pH 4.5-6.5, typically at a pH between 5 and 6.
  • the alpha-amylase may be added as a single dose, e.g., before jet cooking.
  • the liquefaction process is carried out at between 70-95°C, such as 80-90°C, such as around 85°C, for about 10 minutes to 5 hours, typically for 1 -2 hours.
  • the pH is between 4 and 7, such as between 5.5 and 6.2.
  • calcium may optionally be added (to provide 1 -60 ppm free calcium ions, such as about 40 ppm free calcium ions).
  • the liquefied starch will typically have a "dextrose equivalent" (DE) of 10-15.
  • alpha-amylase examples include alpha-amylases.
  • Saccharification may be carried out using conditions well-known in the art with a carbohydrate-source generating enzyme, in particular a glucoamylase, or a beta-amylase and optionally a debranching enzyme, such as an isoamylase or a pullulanase.
  • a full saccharification step may last from about 24 to about 72 hours.
  • Saccharification is typically carried out at a temperature in the range of 20-75°C, e.g., 25-65°C and 40-70°C, typically around 60°C, and at a pH between about 4 and 5, normally at about pH 4.5.
  • saccharification and fermentation steps may be carried out either sequentially or simultaneously.
  • saccharification and fermentation are performed simultaneously (referred to as "SSF").
  • SSF simultaneous saccharification and fermentation
  • a pre-saccharification step for about 30 minutes to 2 hours (e.g., 30 to 90 minutes) at a temperature of 30 to 65°C, typically around 60°C which is followed by a complete saccharification during fermentation referred to as simultaneous saccharification and fermentation (SSF).
  • the pH is usually between 4.2-4.8, e.g., pH 4.5.
  • SSF simultaneous saccharification and fermentation
  • maltodextrins produced during liquefaction are converted into dextrose by adding a glucoamylase and a debranching enzyme, such as an isoamylase (U.S. Patent No. 4,335,208) or a pullulanase.
  • a glucoamylase U.S. Patent No. 4,335,208
  • the temperature is lowered to around 60°C, prior to the addition of the glucoamylase and debranching enzyme.
  • the saccharification process proceeds for 24-72 hours.
  • the pH Prior to addition of the saccharifying enzymes, the pH is reduced to below 4.5, while maintaining a high temperature (above 95°C), to inactivate the liquefying alpha-amylase.
  • fermentation products may be fermented at conditions and temperatures well known to persons skilled in the art, suitable for the fermenting organism in question.
  • the fermentation product may be recovered by methods well known in the art, e.g., by distillation.
  • the process of the invention further comprises, prior to the conversion of a starch-containing material to sugars/dextrins the steps of:
  • the starch-containing material is milled to reduce the particle size.
  • the particle size is reduced to between 0.05-3.0 mm, preferably 0.1 -0.5 mm, or so that at least 30%, preferably at least 50%, more preferably at least 70%, even more preferably at least 90% of the starch-containing material fits through a sieve with a 0.05-3.0 mm screen, preferably 0.1 -0.5 mm screen.
  • the aqueous slurry may contain from 10-55 wt. % dry solids (DS), preferably 25-45 wt. % dry solids (DS), more preferably 30-40 wt. % dry solids (DS) of starch-containing material.
  • the conversion process degrading starch to lower molecular weight carbohydrate components such as sugars or fat replacers includes a debranching step.
  • the starch is depolymerized.
  • a depolymerization process consists of, e.g., a pre-treatment step and two or three consecutive process steps, i.e., a liquefaction process, a saccharification process, and depending on the desired end-product, an optional isomerization process.
  • the dextrose syrup may be converted into fructose.
  • the pH is increased to a value in the range of 6-8, e.g., pH 7.5, and the calcium is removed by ion exchange.
  • the dextrose syrup is then converted into high fructose syrup using, e.g., an immobilized glucose isomerase.
  • Fermentable sugars e.g., dextrins, monosaccharides, particularly glucose
  • these fermentable sugars may be further purified and/or converted to useful sugar products.
  • the sugars may be used as a fermentation feedstock in a microbial fermentation process for producing end-products, such as alcohol (e.g., ethanol, and butanol), organic acids (e.g., succinic acid, 3-HP and lactic acid), sugar alcohols (e.g., glycerol), ascorbic acid intermediates (e.g., gluconate, 2-keto-D-gluconate, 2,5-diketo-D-gluconate, and 2-keto-L-gulonic acid), amino acids (e.g., lysine), proteins (e.g., antibodies and fragment thereof).
  • alcohol e.g., ethanol, and butanol
  • organic acids e.g., succinic acid, 3-HP and lactic acid
  • sugar alcohols e.g.,
  • the fermentable sugars obtained during the liquefaction process steps are used to produce alcohol and particularly ethanol.
  • an SSF process is commonly used wherein the saccharifying enzymes and fermenting organisms (e.g., yeast) are added together and then carried out at a temperature of 30-40°C.
  • the organism used in fermentation will depend on the desired end-product. Typically, if ethanol is the desired end product yeast will be used as the fermenting organism.
  • the ethanol-producing microorganism is a yeast and specifically Saccharomyces such as strains of S. cerevisiae (U.S. Patent No. 4,316,956).
  • S. cerevisiae are commercially available and these include but are not limited to FALI (Fleischmann's Yeast), SUPERSTART (Alltech), FERMIOL (DSM Specialties), RED STAR (Lesaffre) and Angel alcohol yeast (Angel Yeast Company, China).
  • the amount of starter yeast employed in the methods is an amount effective to produce a commercially significant amount of ethanol in a suitable amount of time, (e.g., to produce at least 10% ethanol from a substrate having between 25-40% DS in less than 72 hours).
  • Yeast cells are generally supplied in amounts of about 10 4 to about 10 12 , and preferably from about 10 7 to about 10 10 viable yeast count per ml. of fermentation broth. After yeast is added to the mash, it is typically subjected to fermentation for about 24-96 hours, e.g., 35-60 hours.
  • the temperature is between about 26- 34°C, typically at about 32°C, and the pH is from pH 3-6, e.g., around pH 4-5.
  • the fermentation may include, in addition to a fermenting microorganisms (e.g., yeast), nutrients, and additional enzymes, including phytases.
  • yeast e.g., yeast
  • additional enzymes including phytases.
  • fermentation end product including, e.g., glycerol, 1 ,3-propanediol, gluconate, 2-keto-D-gluconate, 2,5-diketo-D-gluconate, 2-keto-L-gulonic acid, succinic acid, lactic acid, amino acids, and derivatives thereof. More specifically when lactic acid is the desired end product, a Lactobacillus sp. (L case/ ' ) may be used; when glycerol or 1 ,3- propanediol are the desired end-products E.
  • Pantoea citrea may be used as the fermenting microorganism.
  • the above enumerated list are only examples and one skilled in the art will be aware of a number of fermenting microorganisms that may be used to obtain a desired end product.
  • Processes for producing fermentation products from ungelatinized starch-containing material The invention relates to processes for producing fermentation products from starch- containing material without gelatinization (i.e., without cooking) of the starch-containing material (often referred to as a "raw starch hydrolysis" process).
  • the fermentation product such as ethanol, can be produced without liquefying the aqueous slurry containing the starch-containing material and water.
  • a process of the invention includes saccharifying (e.g., milled) starch-containing material, e.g., granular starch, below the initial gelatinization temperature, preferably in the presence of alpha-amylase and/or carbohydrate-source generating enzyme(s), in particular a glucoamylase, to produce sugars that can be fermented into the fermentation product by a suitable fermenting organism.
  • the desired fermentation product e.g., ethanol
  • ungelatinized i.e., uncooked
  • milled cereal grains, such as corn.
  • the invention relates to processes for producing fermentation products from starch-containing material comprising simultaneously saccharifying and fermenting starch-containing material using a carbohydrate-source generating enzyme and a fermenting organism at a temperature below the initial gelatinization temperature of said starch- containing material. Saccharification and fermentation may also be separate.
  • the invention relates to processes of producing fermentation products, comprising the following steps: (i) saccharifying a starch-containing material at a temperature below the initial gelatinization temperature; and
  • step (i) is carried out using at least a glucoamylase, and a variant pullulanase according to the invention.
  • an alpha amylase is added in step (i). In another embodiment steps (i) and (ii) are performed simultaneously.
  • the fermenting organism is expressing the pullulanase of the invention or a pullulanase produced according to the method of the invention.
  • the fermenting organism is in particular a yeast, more particularly a Candida, Hansenula, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowia species, such as a Kluyveromyces lactis, Saccharomyces carlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomyces norbensis, Saccharomyces oviformis, or Yarrowia lipolytica.
  • the yeast is Saccharomyces cerevisiae.
  • a protease is also present.
  • the protease may be any acid fungal protease or metalloprotease.
  • the fermentation product e.g., ethanol, may optionally be recovered after fermentation, e.g., by distillation.
  • amylase(s) such as glucoamylase(s) and/or other carbohydrate-source generating enzymes, and/or alpha-amylase(s) is(are) present during fermentation.
  • glucoamylases and other carbohydrate-source generating enzymes include raw starch hydrolyzing glucoamylases.
  • alpha- amylase(s) include acid alpha-amylases such as acid fungal alpha-amylases.
  • initial gelatinization temperature means the lowest temperature at which starch gelatinization commences. In general, starch heated in water begins to gelatinize between about 50°C and 75°C; the exact temperature of gelatinization depends on the specific starch and can readily be determined by the skilled artisan. Thus, the initial gelatinization temperature may vary according to the plant species, to the particular variety of the plant species as well as with the growth conditions. In the context of this invention the initial gelatinization temperature of a given starch-containing material may be determined as the temperature at which birefringence is lost in 5% of the starch granules using the method described by Gorinstein and Lii, 1992, Starch/Starke 44(12): 461 -466.
  • a slurry of starch-containing material such as granular starch, having 10-55 w/w % dry solids (DS), preferably 25-45 w/w % dry solids, more preferably 30-40 w/w % dry solids of starch-containing material may be prepared.
  • the slurry may include water and/or process waters, such as stillage (backset), scrubber water, evaporator condensate or distillate, side-stripper water from distillation, or process water from other fermentation product plants. Because the process of the invention is carried out below the initial gelatinization temperature, and thus no significant viscosity increase takes place, high levels of stillage may be used if desired.
  • the aqueous slurry contains from about 1 to about 70 vol. %, preferably 15-60 vol. %, especially from about 30 to 50 vol. % water and/or process waters, such as stillage (backset), scrubber water, evaporator condensate or distillate, side-stripper water from distillation, or process water from other fermentation product plants, or combinations thereof, or the like.
  • the starch-containing material may be prepared by reducing the particle size, preferably by dry or wet milling, to 0.05 to 3.0 mm, preferably 0.1 -0.5 mm.
  • a process in this aspect of the invention is conducted at a temperature below the initial gelatinization temperature, which means that the temperature typically lies in the range between 30-75°C, preferably between 45-60°C.
  • the process carried at a temperature from 25°C to 40°C, such as from 28°C to 35°C, such as from 30°C to 34°C, preferably around 32°C.
  • the process is carried out so that the sugar level, such as glucose level, is kept at a low level, such as below 6 w/w %, such as below about 3 w/w %, such as below about 2 w/w %, such as below about 1 w/w %., such as below about 0.5 w/w %, or below 0.25 w/w %, such as below about 0.1 w/w %.
  • a low level can be accomplished by simply employing adjusted quantities of enzyme and fermenting organism.
  • the employed quantities of enzyme and fermenting organism may also be selected to maintain low concentrations of maltose in the fermentation broth.
  • the maltose level may be kept below about 0.5 w/w %, such as below about 0.2 w/w %.
  • the process of the invention may be carried out at a pH from about 3 and 7, preferably from pH 3.5 to 6, or more preferably from pH 4 to 5. In an embodiment fermentation is ongoing for 6 to 120 hours, in particular 24 to 96 hours.
  • the invention relates to processes for producing fermentation products, especially ethanol, from starch-containing material, which process includes a liquefaction step and sequentially or simultaneously performed saccharification and fermentation steps. Consequently, the invention relates to processes for producing fermentation products from starch-containing material comprising the steps of:
  • step (b) saccharifying the liquefied material obtained in step (a) using a glucoamylase
  • step (a) and/or step (b) is carried out in the presence of a pullulanase according to the invention.
  • saccharification and fermentation is carried out simultaneously.
  • the fermenting organism is expressing the pullulanase of the invention or a pullulanase produced according to the method of the invention.
  • the fermenting organism is in particular a yeast, more particularly a Candida, Hansenula, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowia species, such as a Kluyveromyces lactis, Saccharomyces carlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomyces norbensis, Saccharomyces oviformis, or Yarrowia lipolytica.
  • the yeast is Saccharomyces cerevisiae.
  • a protease such as an acid fungal protease or a metallo protease is added before, during and/or after liquefaction.
  • the metalloprotease is derived from a strain of Thermoascus, e.g., a strain of Thermoascus aurantiacus, especially Thermoascus aurantiacus CGMCC No. 0670.
  • the protease is a bacterial protease, particularly a protease derived from a strain of Pyrococcus, more particularly from Pyrococcus furiosus disclosed in US 6,358,726.
  • the glucoamylase derived from a strain of Aspergillus, e.g., Aspergillus niger or Aspergillus awamori, a strain of Talaromyces, especially Talaromyces emersonii; or a strain of Athelia, especially Athelia rolfsii; a strain of Trametes, e.g., Trametes cingulata; a strain of the genus Gloeophyllum, e.g., a strain of Gloeophyllum sepiarum or Gloeophyllum trabeum; or a mixture thereof. Saccharification step (b) and fermentation step (c) may be carried out either sequentially or simultaneously.
  • Saccharification step (b) and fermentation step (c) may be carried out either sequentially or simultaneously.
  • a protease may be added during saccharification and/or fermentation when the process is carried out as a sequential saccharification and fermentation process and before or during fermentation when steps (b) and (c) are carried out simultaneously (SSF process).
  • the fermentation product such as especially ethanol, may optionally be recovered after fermentation, e.g., by distillation.
  • the process of the invention further comprises, prior to step (a), the steps of:
  • the particle size is smaller than a # 7 screen, e.g., a # 6 screen.
  • a # 7 screen is usually used in conventional prior art processes.
  • the aqueous slurry may contain from 10-55, e.g., 25-45 and 30-40, w/w % dry solids (DS) of starch-containing material.
  • the slurry is heated to above the gelatinization temperature and an alpha-amylase variant may be added to initiate liquefaction (thinning).
  • the slurry may in an embodiment be jet-cooked to further gelatinize the slurry before being subjected to alpha-amylase in step (a).
  • Liquefaction may in an embodiment be carried out as a three-step hot slurry process.
  • the slurry is heated to between 60-95°C, preferably between 70-90°C, such as preferably between 80-85°C at pH 4-6, preferably 4.5-5.5, and alpha-amylase variant, optionally together with a pullulanase and/or protease, preferably metalloprotease, are added to initiate liquefaction (thinning).
  • the slurry may then be jet-cooked at a temperature between 95-140°C, preferably 100-135°C, such as 105-125°C, for about 1 -15 minutes, preferably for about 3-10 minutes, especially around about 5 minutes.
  • the slurry is cooled to 60-95°C and more alpha-amylase variant and optionally pullulanase variant and/or protease, preferably metalloprotease, is(are) added to finalize hydrolysis (secondary liquefaction).
  • the liquefaction process is usually carried out at pH 4.0-6, in particular at a pH from 4.5 to 5.5. Saccharification step (b) may be carried out using conditions well known in the art.
  • a full saccharification process may last up to from about 24 to about 72 hours, however, it is common only to do a pre-saccharification of typically 40-90 minutes at a temperature between 30-65°C, typically about 60°C, followed by complete saccharification during fermentation in a simultaneous saccharification and fermentation process (SSF process). Saccharification is typically carried out at temperatures from 20-75°C, preferably from 40-70°C, typically around 60°C, and at a pH between 4 and 5, normally at about pH 4.5.
  • SSF process simultaneous saccharification and fermentation process
  • SSF simultaneous saccharification and fermentation
  • a fermenting organism such as yeast
  • enzyme(s) may be added together.
  • SSF may typically be carried out at a temperature from 25°C to 40°C, such as from 28°C to 35°C, such as from 30°C to 34°C, preferably around about 32°C.
  • fermentation is ongoing for 6 to 120 hours, in particular 24 to 96 hours.
  • starch-containing starting material may be used in a process of the present invention.
  • the starting material is generally selected based on the desired fermentation product.
  • starch-containing starting materials suitable for use in the processes of the present invention, include barley, beans, cassava, cereals, corn, milo, peas, potatoes, rice, rye, sago, sorghum, sweet potatoes, tapioca, wheat, and whole grains, or any mixture thereof.
  • the starch-containing material may also be a waxy or non-waxy type of corn and barley.
  • the starch-containing material is corn.
  • starch-containing material is wheat. Fermentation Products
  • Fermentation product means a product produced by a method or process including fermenting using a fermenting organism. Fermentation products include alcohols (e.g., ethanol, methanol, butanol); organic acids (e.g., citric acid, acetic acid, itaconic acid, lactic acid, succinic acid, gluconic acid); ketones (e.g., acetone); amino acids (e.g., glutamic acid); gases (e.g., H 2 and C0 2 ); antibiotics (e.g., penicillin and tetracycline); enzymes; vitamins (e.g., riboflavin, B 12 , beta-carotene); and hormones.
  • alcohols e.g., ethanol, methanol, butanol
  • organic acids e.g., citric acid, acetic acid, itaconic acid, lactic acid, succinic acid, gluconic acid
  • ketones e.g., acetone
  • amino acids
  • the fermentation product is ethanol, e.g., fuel ethanol; drinking ethanol, i.e., potable neutral spirits; or industrial ethanol or products used in the consumable alcohol industry (e.g., beer and wine), dairy industry (e.g., fermented dairy products), leather industry and tobacco industry.
  • Preferred beer types comprise ales, stouts, porters, lagers, bitters, malt liquors, happoushu, high-alcohol beer, low-alcohol beer, low-calorie beer or light beer.
  • the fermentation product is ethanol.
  • Milled starch-containing material is combined with water and recycled thin-stillage resulting in an aqueous slurry.
  • the slurry can comprise between 15 to 55% ds w/w (e.g., 20 to 50%, 25 to 50%, 25 to 45%, 25 to 40%, 20 to 35% and 30-36% ds).
  • the recycled thin-stillage (backset) is in the range of about 10 to 70% v/v (e.g., 10 to 60%, 10 to 50%, 10 to 40%, 10 to 30%, 10 to 20%, 20 to 60%, 20 to 50%, 20 to 40% and also 20 to 30%).
  • the pH of the slurry will be in the range of about pH 4.5 to less than about 6.0 (e.g., pH 4.5 to 5.8, pH 4.5 to 5.6, pH 4.8 to 5.8, pH 5.0 to 5.8, pH 5.0 to 5.4, pH 5.2 to 5.5 and pH 5.2 to 5.9).
  • the pH of the slurry may be between about pH 4.5 and 5.2 depending on the amount of thin stillage added to the slurry and the type of material comprising the thin stillage.
  • the pH of the thin stillage may be between pH 3.8 and pH 4.5.
  • acids can be added to lower the pH in the beer well, to reduce the risk of microbial contamination prior to distillation.
  • a phytase is added to the slurry.
  • an alpha-amylase is added to the slurry.
  • a phytase and alpha-amylase are added to the slurry sequentially.
  • a phytase and alpha-amylase are added simultaneously.
  • the slurry comprising a phytase and optionally, an alpha-amylase are incubated (pretreated) for a period of about 5 minutes to about 8 hours (e.g., 5 minutes to 6 hours, 5 minutes to 4 hours, 5 minutes to 2 hours, and 15 minutes to 4 hours).
  • the slurry is incubated at a temperature in the range of about 40 to 1 15°C (e.g., 45 to 80°C, 50 to 70°C, 50 to 75°C, 60 to 1 10°C, 60 to 95°C, 70 to 1 10°C, 70 to 85°C and 77 to 86°C).
  • a temperature in the range of about 40 to 1 15°C (e.g., 45 to 80°C, 50 to 70°C, 50 to 75°C, 60 to 1 10°C, 60 to 95°C, 70 to 1 10°C, 70 to 85°C and 77 to 86°C).
  • the slurry is incubated at a temperature of about 0 to about 30°C (e.g., 0 to 25°C, 0 to 20°C, 0 to 15°C, 0 to 10°C and 0 to 5°C) below the starch gelatinization temperature of the starch-containing material.
  • the temperature is below about 68°C, below about 65°C, below about 62°C, below about 60°C and below about 55°C.
  • the temperature is above about 45°C, above about 50°C, above about 55°C and above about 60°C.
  • the incubation of the slurry comprising a phytase and an alpha-amylase at a temperature below the starch gelatinization temperature is referred to as a primary (1 °) liquefaction.
  • the milled starch-containing material is corn or milo.
  • the slurry comprises 25 to 40% DS, the pH is in the range of 4.8 to 5.2, and the slurry is incubated with a phytase and optionally an alpha-amylase for 5 minutes to 2 hours, at a temperature range of 60 to 75°C.
  • the incubated or pretreated starch-containing material is exposed to an increase in temperature such as about 0 to about 45°C above the starch gelatinization temperature of the starch-containing material (e.g., 70°C to 120°C, 70°C to 1 10°C, and 70°C to 90°C) for a period of time of about 2 minutes to about 6 hours (e.g., 2 minutes to 4 hours, 90 minutes, 140 minutes and 90 to 140 minutes) at a pH of about 4.0 to 5.5 more preferably between 1 hour to 2 hours.
  • the temperature can be increased by a conventional high temperature jet cooking system for a short period of time, for example, for 1 to 15 minutes.
  • the starch maybe further hydrolyzed at a temperature ranging from about 75°C to 95°C (e.g., 80°C to 90°C and 80°C to 85°C) for a period of about 15 to 150 minutes (e.g., 30 to 120 minutes).
  • the pH is not adjusted during these process steps and the pH of the liquefied mash is in the range of about pH 4.0 to pH 5.8 (e.g., pH 4.5 to 5.8, pH 4.8 to 5.4, and pH 5.0 to 5.2).
  • a second dose of thermostable alpha-amylase is added to the secondary liquefaction step, but in other embodiments there is no additional dosage of alpha-amylase.
  • the incubation and liquefaction steps may be followed by saccharification and fermentation steps well known in the art.
  • an alcohol e.g., ethanol
  • ethanol may be extracted by, for example, distillation and optionally followed by one or more process steps.
  • the yield of ethanol produced by the methods provided herein is at least 8%, at least 10%, at least 12%, at least 14%, at least 15%, at least 16%, at least 17% and at least 18% (v/v) and at least 23% v/v.
  • the ethanol obtained according to the process provided herein may be used as, for example, fuel ethanol, drinking ethanol, i.e., potable neutral spirits, or industrial ethanol.
  • the end product may include the fermentation co-products such as distiller's dried grains (DDG) and distiller's dried grain plus solubles (DDGS), which may be used, for example, as an animal feed.
  • DDG distiller's dried grains
  • DDGS distiller's dried grain plus solubles
  • the saccharification and fermentation may be carried out simultaneously or separately.
  • fermenting organism refers to any organism, including bacterial and fungal organisms, such as yeast and filamentous fungi, suitable for producing a desired fermentation product. Suitable fermenting organisms are able to ferment, i.e., convert, fermentable sugars, such as arabinose, fructose, glucose, maltose, mannose, or xylose, directly or indirectly into the desired fermentation product.
  • fermentable sugars such as arabinose, fructose, glucose, maltose, mannose, or xylose
  • fermenting organisms include fungal organisms such as yeast.
  • yeast include strains of Saccharomyces, in particular Saccharomyces cerevisiae or Saccharomyces uvarum; strains of Pichia, in particular Pichia stipitis such as Pichia stipitis CBS 5773 or Pichia pastoris; strains of Candida, in particular Candida arabinofermentans, Candida boidinii, Candida diddensii, Candida shehatae, Candida sonorensis, Candida tropicalis, or Candida utilis.
  • Other fermenting organisms include strains of Hansenula, in particular Hansenula anomala or Hansenula polymorpha; strains of Kluyveromyces, in particular Kluyveromyces fragilis or Kluyveromyces marxianus; and strains of Schizosaccharomyces, in particular Schizosaccharomyces pombe.
  • Preferred bacterial fermenting organisms include strains of Escherichia, in particular Escherichia coli, strains of Zymomonas, in particular Zymomonas mobilis, strains of Zymobacter, in particular Zymobactor palmae, strains of Klebsiella in particular Klebsiella oxytoca, strains of Leuconostoc, in particular Leuconostoc mesenteroides, strains of Clostridium, in particular Clostridium butyricum, strains of Enterobacter, in particular Enterobacter aerogenes, and strains of Thermoanaerobacter, in particular Thermoanaerobacter BG1 L1 (Appl. Microbiol.
  • Lactobacillus are also envisioned as are strains of Corynebacterium glutamicum R, Bacillus thermoglucosidaisus, and Geobacillus thermoglucosidasius.
  • the fermenting organism is a C6 sugar fermenting organism, such as a strain of, e.g., Saccharomyces cerevisiae.
  • the fermenting organism is a C5 sugar fermenting organism, such as a strain of, e.g., Saccharomyces cerevisiae.
  • the fermenting organism is added to the fermentation medium so that the viable fermenting organism, such as yeast, count per ml. of fermentation medium is in the range from 10 5 to 10 12 , preferably from 10 7 to 10 10 , especially about 5x10 7 .
  • Yeast is the preferred fermenting organism for ethanol fermentation.
  • Preferred are strains of Saccharomyces, especially strains of the species Saccharomyces cerevisiae, preferably strains which are resistant towards high levels of ethanol, i.e., up to, e.g., about 10, 12, 15 or 20 vol. % or more ethanol.
  • the C5 utilizing yeast is a Saccharomyces cerevisea strain disclosed in WO 2004/085627.
  • the fermenting organism is a C5 eukaryotic microbial cell concerned in
  • the fermenting organism is a transformed C5 eukaryotic cell capable of directly isomerize xylose to xylulose disclosed in US 2008/0014620.
  • the fermenting organism is a C5 sugar fermentating cell disclosed in WO 2009/109633.
  • yeast Commercially available yeast include LNF SA-1 , LNF BG-1 , LNF PE-2,and LNF CAT-1 (available from LNF Brazil), RED STARTM and ETHANOL REDTM yeast (available from Fermentis/Lesaffre, USA), FALI (available from Fleischmann's Yeast, USA), SUPERSTART and THERMOSACCTM fresh yeast (available from Ethanol Technology, Wl, USA), BIOFERM AFT and XR (available from NABC - North American Bioproducts Corporation, GA, USA), GERT STRAND (available from Gert Strand AB, Sweden), and FERMIOL (available from DSM Specialties).
  • the fermenting organism capable of producing a desired fermentation product from fermentable sugars is preferably grown under precise conditions at a particular growth rate.
  • the inoculated fermenting organism pass through a number of stages. Initially growth does not occur. This period is referred to as the "lag phase” and may be considered a period of adaptation.
  • the growth rate gradually increases. After a period of maximum growth the rate ceases and the fermenting organism enters "stationary phase”. After a further period of time the fermenting organism enters the "death phase" where the number of viable cells declines.
  • the fermentation conditions are determined based on, e.g., the kind of plant material, the available fermentable sugars, the fermenting organism(s) and/or the desired fermentation product.
  • One skilled in the art can easily determine suitable fermentation conditions.
  • the fermentation may be carried out at conventionally used conditions.
  • Preferred fermentation processes are anaerobic processes.
  • fermentations may be carried out at temperatures as high as 75°C, e.g., between 40-70°C, such as between 50-60°C.
  • temperatures as high as 75°C, e.g., between 40-70°C, such as between 50-60°C.
  • bacteria with a significantly lower temperature optimum down to around room temperature around 20°C are also known. Examples of suitable fermenting organisms can be found in the "Fermenting Organisms" section above.
  • the fermentation may go on for 24 to 96 hours, in particular for 35 to 60 hours.
  • the fermentation is carried out at a temperature between 20 to 40°C, preferably 26 to 34°C, in particular around 32°C.
  • the pH is from pH 3 to 6, preferably around pH 4 to 5.
  • Fermentation is typically carried out at a pH in the range between 3 and 7, preferably from pH 3.5 to 6, such as around pH 5. Fermentations are typically ongoing for 6-96 hours.
  • the processes of the invention may be performed as a batch or as a continuous process. Fermentations may be conducted in an ultrafiltration system wherein the retentate is held under recirculation in the presence of solids, water, and the fermenting organism, and wherein the permeate is the desired fermentation product containing liquid. Equally contemplated are methods/processes conducted in continuous membrane reactors with ultrafiltration membranes and where the retentate is held under recirculation in presence of solids, water, and the fermenting organism(s) and where the permeate is the fermentation product containing liquid.
  • the fermenting organism may be separated from the fermented slurry and recycled.
  • fermentation media refers to the environment in which fermentation is carried out and comprises the fermentation substrate, that is, the carbohydrate source that is metabolized by the fermenting organism(s).
  • the fermentation medium may comprise other nutrients and growth stimulator(s) for the fermenting organism(s).
  • Nutrient and growth stimulators are widely used in the art of fermentation and include nitrogen sources, such as ammonia; vitamins and minerals, or combinations thereof.
  • the fermentation product may be separated from the fermentation medium.
  • the fermentation medium may be distilled to extract the desired fermentation product or the desired fermentation product may be extracted from the fermentation medium by micro or membrane filtration techniques. Alternatively, the fermentation product may be recovered by stripping. Methods for recovery are well known in the art.
  • a method of increasing pullulanase activity of a parent pullulanase comprising the steps: a) inserting an X25 domain within an X45 domain; and /or
  • the parent pullulanase is from a bacterium, particularly, a gram positive bacterium, more particularly a Bacillus sp. [3].
  • the Bacillus sp. parent pullulanase is selected from a Bacillus acidopullulyticus pullulanase, Bacillus deramificans pullulanase, or a hybrid pullulanase, e.g., a hybrid comprising an N-terminal part from a Bacillus acidopullulyticus pullulanase and a C-terminal part from a Bacillus deramificans pullulanase. [4].
  • the X25 domain is selected from an X25 from a pullulanase, more particularly a pullulanase from a bacterium, particularly, a gram positive bacterium, more particularly a Bacillus sp.
  • Bacillus sp. is selected from Bacillus acidopullulyticus or Bacillus deramificans.
  • a polypeptide having pullulanase activity and obtained by the method according to any of the paragraphs 1 to 1 1.
  • polypeptides according to paragraph 12 selected from the group consisting of the polypeptide of SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21 , SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27 or a polypeptide having at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the mature polypeptide of SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21 , SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO:
  • polypeptide of any of paragraphs 12-13 comprising or consisting of SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21 , SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27.
  • composition comprising the polypeptide of any of paragraphs 12-14.
  • composition according to paragraph 15 further comprising a glucoamylase and/or an alpha-amylase.
  • a whole broth formulation or cell culture composition comprising a polypeptide of any of paragraphs 12-14.
  • a process of producing a fermentation product from starch-containing material comprising the steps of:
  • step (c) fermenting with a fermenting organism; wherein step (a) and/or step (b) is carried out in the presence of a pullulanase of any of paragraphs 13-14 or a pullulanase produced according to the method of paragraphs 1 -1 1.
  • a process of producing a fermentation product from starch-containing material comprising the steps of:
  • step (a) is carried out using at least a glucoamylase, and a pullulanase of any of paragraphs 13-14 or a pullulanase produced according to the method of paragraphs 1 -1 1.
  • the fermenting organism is a yeast, particularly a Candida, Hansenula, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowia species, such as a Kluyveromyces lactis, Saccharomyces carlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomyces norbensis, Saccharomyces oviformis, or Yarrowia lipolytica.
  • yeast particularly a Candida, Hansenula, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowia species, such as a Kluyveromyces lactis, Saccharomyces carlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomyces
  • EXAMPLE 1 Description of parent pullulanases used in the examples.
  • These parent pullulanases are hybrid pullulanases (P6, P8, P190, P202) having improved properties, e.g., higher thermal activity and/or higher themo-stability.
  • These hybrids were constructed by combining N-terminal fragments of a naturally occurring pullulanase from Bacillus acidopullulyticus termed GMM and disclosed as SEQ ID NO: 2 with C-terminal fragments of a naturally occurring truncated pullulanase from Bacillus deramificans termed ProD homolog and disclosed as SEQ ID NO: 4.
  • SEQ ID NO: 2 is derived from Bacillus acidopullulyticus NCI MB 1 1639 described in EP 0063909 A1 as a pullulanase producer.
  • SEQ ID NO: 2 The sequence of SEQ ID NO: 2 can be found in WO 2009/075682.
  • the pullulanase of SEQ ID NO: 4 is derived from a Bacillus deramificans strain isolated from a humus sample collected in Denmark.
  • Two hybrid pullulanases, P6 and P8, have been used as parent pullulanases in the present invention and the sequences are disclosed herein as SEQ ID NO: 9 and SEQ OID NO: 10 respectively.
  • ProD homolog, SEQ ID NO: 4 has itself been used as a parent herein.
  • Specific pullulanase variants of P8, termed P190 and P202 have also been used as parents to illustrate the improved effect according to the invention.
  • P190 is disclosed in SEQ ID NO: 1 1 .
  • P202 is disclosed in SEQ ID NO: 12.
  • NCBI1 17842 (SEQ ID NO: 6) under the control of a triple promoter system (as described in WO 99/43835) consisting of the promoters from Bacillus licheniformis alpha-amylase gene (amyl), Bacillus amyloliquefaciens alpha-amylase gene (amyQ), and the Bacillus thuringiensis crylllA promoter including stabilizing sequence, were isolated using NucleoSpin® Tissue kit [MACHEREY-NAGEL] according to its procedure. All chimera variants were constructed by multi fragments overlapping cloning using In-Fusion HD cloning kit (Clontech).
  • PCR amplifications were carried out using corresponding primer pairs which were designed to have at least 15bp overlapping with the neighboring fragments or a vector under the below conditions.
  • PCR fragments were isolated in 0.7% agarose gel and recovered by Qiagen Gel extraction kit.
  • the isolated PCR fragments with overlapping ends and a linearized E. coli/Bacillus subtilis shuttle vector (Clal Mlul digests) described in EXAMPLE1 in EP patent application no.10173848 were mixed and fused by In-Fusion HD cloning kit (Clontech) following to their manual.
  • the In-Fusion mixture was transformed into E.coli DH 12S and the derived constructs were recovered from E.coli by Qiagen plasmid extraction kit (QIAGEN). Then, the plasmids were transformed into a suitable B.
  • subtilis host and the gene coding pullulanase chimera variants were integrated into the B. subtilis genome by homologous recombination into the pectate lyase (pel) locus.
  • the gene was expressed under the control of a triple promoter system consisting of the promoters from Bacillus licheniformis alpha-amylase gene (amyl), Bacillus amyloliquefaciens alpha-amylase gene (amyQ), and the Bacillus thuringiensis crylllA described in WO 99/43835.
  • chloramphenicol acetyltransferase was used as marker as described in Diderichsen, B; Poulsen, G.B.; Joergensen, ST.; A useful cloning vector for Bacillus subtilis. Plasmid 30:312(1993). Chloramphenicol resistant clones were analyzed by DNA sequencing to verify the correct DNA sequences of the constructs. Clones having the correct sequences were selected and cultivated in 24 well micro titer plates containing TB-gly medium (13.3 g/L BactoTM Tryptone, 26.6 g/L BactoTM Yeast extract
  • Pullulanase (EC 3.2.1 .41 ) cleaves 1 ,6-alpha-glucosidic linkages in pullulan, amylopectin, and glycogen, and in alpha-and beta-amylase limit-dextrins of amylopectin and glycogen.
  • BaPul is used industrially in the production of high fructose corn syrup, high maltose content syrups and low calorie and "light” beers.
  • Pullulanases in addition to the catalytic domain, include several carbohydrate-binding domains (CBMs) as well as domains of unknown function (termed "X" modules).
  • X25 was identified in Bacillus acidopullulyticus pullulanase, and splits another domain of unknown function (X45). X25 is present in multiple copy in some pullulanases. It has been suggested that X25 and X45 are CBMs which target mixed alpha-1 ,6/alpha-1 ,4 linked D- glucan polysaccharides.
  • Pullulanase (EC 3.2.1 .41 ) cleaves 1 ,6-alpha-glucosidic linkages in pullulan, amylopectin, and glycogen, and in alpha-and beta-amylase limit-dextrins of amylopectin and glycogen.
  • BaPul is used industrially in the production of high fructose corn syrup, high maltose content syrups and low calorie and "light” beers.
  • Pullulanases in addition to the catalytic domain, include several carbohydrate-binding domains (CBMs) as well as domains of unknown function (termed "X" modules).
  • X45 was identified in Bacillus acidopullulyticus pullulanase, it is interrupted by another domain of unknown function (X25). It has been suggested that X25 and X45 are CBMs which target mixed alpha-1 ,6/alpha-1 ,4 linked D-glucan polysaccharides. Family 41 Carbohydrate-Binding Module from pullulanase-like enzymes
  • Pullulanases (EC 3.2.1 .41 ) are a group of starch-debranching enzymes, catalyzing the hydrolysis of the alpha-1 ,6-glucosidic linkages of alpha-glucans, preferentially pullulan.
  • Pullulan is a polysaccharide in which alpha-1 ,4 linked maltotriosyl units are combined via an alpha-1 ,6 linkage. These enzymes are of importance in the starch industry, where they are used to hydrolyze amylopectin starch.
  • Pullulanases consist of multiple distinct domains, including a catalytic domain belonging to the glycoside hydrolase (GH) family 13 and carbohydrate-binding modules (CBM), including CBM41 . Some pullulanases contain all these domains at their N-terminal and some lack either one or two or all of these domains.
  • GH glycoside hydrolase
  • CBM carbohydrate-binding modules
  • New pullulanase chimera variants were designed to have an additional domain from other pullulanases.
  • P8 - P153, P154, P182, P183, P184, P185, P399 (P153 without LGQ triplet at the end of X45a domain) ⁇ 190 ⁇ P195, P196, P197, P198
  • Endo-pullulanase activity in NPUN is measured relative to a Novozymes pullulanase standard, e.g., Novozym 26062 having a declared activity of 400 PUN/g.
  • One pullulanase unit is defined as the amount of enzyme that releases 1 micro mol glucose per minute under the standard conditions (0.7% red pullulan (Megazyme), pH 5, 40° C, 20 minutes). The activity is measured in NPUN/ml using red pullulan.
  • Sweet potato starch (Satsumayawaragi from NIHON STARCH CO., LTD)
  • maltodextrin pindex100 or pinedex from MATSUTANI chemical industry Co., Ltd. 25ml 50mM Na acetate buffer, pH5 (or pH4.3)
  • Substrate, DE1 1 was prepared according to the method described in EXAMPLE 1 in US 4335208;
  • the pH of the liquefied starch was adjusted to 4.5 at 95°C the reaction and the batch was then spray-dried without purification.
  • the DE of the spray-dried maltodextrin was 1 1.
  • Activity unit toward pullulan in culture supernatants of chimera pullulanase variants was determined by red-pullulan assay using commercial Pullulanase product, Novozym R 26062, as an enzyme standard (400 PUN(G)/g). The activities were adjusted to 1 PUN(G)/g and then activities toward sweet potato and phadebas were determined by PAHBAH-sweet potato starch assay and Phadebas assay.
  • Bacillus culture supernatants were concentrated by Amicon ultra 50K centrifuge filters (Millipore) and ran in SDS-PAGE. The gels were stained with Coomassie Fluor Orange (BIO- RAD) and pullulanases protein amounts were quantified by Gel Doc EZ (BIO-RAD). Enzyme solutions were adjusted to 0.1 mg/ml in 100mM sodium acetate buffer, pH5, containing 0.01 % tween20 to measure activities by Phadebas assay and PAHBAH-sweet potato starch assay.
  • B. sutilis strains were fermented on a rotary shaking table in 500 ml baffled flasks containing 100ml TB-gly with 6mg/L chloramphenicol at 220rpm, 37°C.
  • the culture was centrifuged (20000 x g, 20min) and the supernatants were carefully decanted from the precipitates.
  • the supernatants were filtered through a 0.45um filter unit to remove the rest of the Bacillus host cells.
  • pullulanases Purification of pullulanases was carried out by ⁇ -cyclodextrin affinity column and followed by anion exchange column chromatography. After purification, pullulanases were dialyzed against 20 mM sodium acetate buffer (pH 5.5) and concentrated. Enzyme concentrations were determined by A280 value and efficiency from amino acid sequence.
  • Newly constructed chimera pullulanases showed higher A405 values than their control pullulanases.
  • Sweet potato starch pH 5 50°C (P202 vs. P216)

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Abstract

The present invention relates to a method of increasing pullulanase activity of a parent pullulanase comprising the steps: a) inserting an X25 domain within an X45 domain; and /or b) adding a CBM41 domain either adjacent to the N-terminal of an X45 domain, or if a CBM41 domain is already present, adding the additional CBM41 domain adjacent to the N-terminal of the existing CBM41 or adjacent to the N-terminal of the X45 domain.

Description

POLYPEPTIDES HAVING PULLULANASE ACTIVITY COMPRISING THE X25, X45 AND CBM41
DOMAINS
Reference to a Sequence Listing
This application contains a Sequence Listing in computer readable form, which is incorporated herein by reference.
Background of the Invention
Field of the Invention
The present invention relates to methods for increasing pullulanase activity of a parent pullulanase towards a starch substrate. Further the invention relates to improved variant pullulanase generated by the method and to compositions comprising the variant pullulanases.
Description of the Related Art
Starch usually consists of about 80% amylopectin and 20% amylose. Amylopectin is a branched polysaccharide in which linear chains alpha-1 ,4 D-glucose residues are joined by alpha-1 ,6 glucosidic linkages. Amylopectin is partially degraded by alpha-amylase, which hydrolyzes the 1 ,4-alpha-glucosidic linkages to produce branched and linear oligosaccharides. Prolonged degradation of amylopectin by alpha-amylase results in the formation of so-called alpha-limit dextrins that are not susceptible to further hydrolysis by the alpha-amylase. Branched oligosaccharides can be hydrolyzed into linear oligosaccharides by a debranching enzyme. The remaining branched oligosaccharides can be depolymerized to D-glucose by glucoamylase, which hydrolyzes linear oligosaccharides into D-glucose.
Debranching enzymes which can attack amylopectin are divided into two classes: isoamylases (E.C. 3.2.1 .68) and pullulanases (E.C. 3.2.1.41 ), respectively. Isoamylase hydrolyses alpha-1 ,6-D-glucosidic branch linkages in amylopectin and beta-limit dextrins and can be distinguished from pullulanases by the inability of isoamylase to attack pullulan, and by their limited action on alpha-limit dextrins.
It is well-known in the art to add isoamylases or pullulanases in starch conversion processes. Pullulanase is a starch debranching enzyme having pullulan 6-glucano-hydrolase activity (EC3.2.1.41 ) that catalyzes the hydrolyses the a-1 ,6-glycosidic bonds in pullulan, releasing maltotriose with reducing carbohydrate ends. Usually pullulanase is used in combination with an alpha amylase and/or a glucoamylase.
Pullulanases are known in the art. US 6,074,854 and US 5,817,498 disclose a pullulanase from Bacillus deramificans. WO2009/075682 discloses a pullulanase derived from Bacillus acidopullulyticus. The present invention provides pullulanase variants with improved properties, such as increased pullulanase activity, compared to its parent, as well as methods of increasing pullulanase activity of a parent pullulanase.
Summary of the Invention
The present invention provides in a first aspect a method of increasing pullulanase activity of a parent pullulanase comprising the steps:
a) inserting an X25 domain within an X45 domain; and /or
b) adding a CBM41 domain either adjacent to the N-terminal of an X45 domain, or if a CBM41 domain is already present, adding an additional CBM41 domain adjacent to the N-terminal of the existing CBM41 or adjacent to the N-terminal of the X45 domain.
In a second aspect the invention provides a pullulanase prepared according to the method of the first aspect.
In further aspects the invention relates to polynucleotides encoding the pullulanase of the invention, compositions and whole broth formulations comprising the pullulanases, uses of the pullulanases for starch processing, and in further aspects the invention relates to a process of producing a fermentation product from starch-containing material comprising the steps of:
(a) liquefying starch-containing material in the presence of an alpha amylase;
(b) saccharifying the liquefied material in the presence of a glucoamylase; and
(c) fermenting with a fermenting organism; wherein step (a) and/or step (b) is carried out in the presence of a pullulanase of the invention or a pullulanase produced according to the method of the invention.
In a still further aspect the invention relates to a process of producing a fermentation product from starch-containing material, comprising the steps of:
(a) saccharifying starch-containing material at a temperature below the initial gelatinization temperature of said starch-containing material; and
(b) fermenting with a fermenting organism
wherein step (a) is carried out using at least a glucoamylase, and a pullulanase of the invention or a pullulanase produced according to the method the invention.
Definitions
Pullulanase: The term "pullulanase" means a starch debranching enzyme having pullulan 6-glucano-hydrolase activity (EC 3.2.1 .41 ) that catalyzes the hydrolysis the a-1 ,6- glycosidic bonds in pullulan, releasing maltotriose with reducing carbohydrate ends. For purposes of the present invention, pullulanase activity can be determined according to the procedure described in the Examples. In the context of the present invention the variant pullulanases have increased pullulanase activity. Pullulanase activity was determined as activity of the variant relative to the activity of the parent pullulanase using the PHADEBAS assay or the sweet potato starch assay as described in the examples.
In particular the pullulanase variants of the invention have at least 105% relative activity when measured against the parent pullulanase activity determined as 100%, more particularly at least 1 10%, more particularly at least 120%, more particularly at least 130%, more particularly at least 140%, more particularly at least 150%. Preferably the pullulanase activity is measured using the PHADEBAS assay at 65°C.
Pullulanase domains: Naturally occurring pullulanases in particular pullulanases derived from bacterial sources, comprises a catalytic domain and in addition several or all of the following domains: CBM41 , X45, X25, and CBM48. In case an X25 domain is present it can be found within an X45 domain. The X45 domain is then split into an X45a and X45b part. The following sequence elements were identified from polypeptides shown to have pullulanase activity (see Table 1 for specific examples of these elements). The elements are: a. X45a N-terminal part of X45 domain not including special composition element Linkc b. Linkc triplet of residues within X45 domain immediately prior to X25 insertion site, and with specific composition requirement for X25 insertion.
c. X25 X25 domain
d. Linkd special composition element consisting of four or five residues within X45 domain immediately following X25 insertion site, and with specific composition requirement for X25 insertion.
e. X45b C-terminal part of X45 domain not including special composition element Linkd. f. X45 The complete X45 domain is comprised of elements X45a + Linkc (if present) + Linkd (if present) + X45b
Table 1. Examples of identified regions in sequences showing pullulanase activity.
SEQUENCE NO. 28, position
> Signal/propeptide [ absent
> CBM41 [ 1. .101
> Linkb [ 102. .110
> X45a [ 111. .158
> Linkc [ 159. .161
> X25 [ 162. .262
> Linkd [ 263. .267
> X45b [ 268. .313
> Linke [ 314. .316
> CBM48 [ 317. .412
> Linkf [ 413. .428
> GH13 14 [ 429. .929 SEQUENCE NO. 29,
> Signal/propeptide 1. ,27
> CBM41 28. ,128
> Linkb 129. ,137 > X45a 138. .185
> Linkc 186. .188
> X25 189. .289
> Linkd 290. .294
> X45b 295. .340 > Linke 341. .343
> CBM48 344. .439
> Linkf 440. .455
> GH13_14 456. .956 SEQUENCE NO. 30,
> Signal/propeptide 1..27
> CBM41 absent
> Linkb absent
> X45a 28. 74 > Linkc 75. 77
> Linkc2 78. 80
> X25 81. 181
> Linkd 182. 186
> X45b 187. 232 > Linke 233. 235
> CBM48 236. 331
> Linkf 332. 347
> GH13_14 348. SEQUENCE NO. 31,
> Signal/propeptide 1. 36
> CBM41 37. 137
> Linkb 138. 146
> X45a 147. 194 > Linkc 195. 197
> X25 198. 298
> Linkd 299. 303
> X45b 304. 349
> Linke 350. 352 > CBM48 353. 448
> Linkf 449. 464
> GH13_14 465. 965
SEQUENCE NO. 32,
> Signal/propeptide 1. ,37
> CBM41 38. ,138
> Linkb 139. ,147
> X45a 148. .195
> Linkc 196. .198 > X25 199. .299
> Linkd 300. .304
> X45b 305. .350
> Linke 351. .353
> CBM48 354. .449 > Linkf 450. .465
> GH13 14 466. .957 SEQUENCE NO. 33,
> Signal/propeptide absent
> CBM41 absent
> Linkb absent > X45a 1. 48
> Linkc 49. 51
> X25 52. 152
> Linkd 153. 157
> X45b 158. 203 > Linke 204. 206
> CBM48 207. 302
> Linkf 303. 318
> GH13_14 319. ,809 SEQUENCE NO. 34,
> Signal/propeptide 1..16
> CBM41 17..118
> Linkb 119..123
> X45a 124..168 > Linkc 169..171
> X25 absent
> Linkd 172..175
> X45b 176..222
> Linke 223..224 > CBM48 225..321
> Linkf 322..337
> GH13_14 338..843
SEQUENCE NO. 35,
> Signal/propeptide absent
> CBM41 1..101
> Linkb 102..110
> X45a 111..158
> Linkc 159..161 > X25 absent
> Linkd 162..166
> X45b 167..212
> Linke 213..215
> CBM48 216..311 > Linkf 312..327
> GH13_14 328..829
SEQUENCE NO. 36,
> Signal/propeptide 1..25 > CBM41 26..127
> Linkb 128..132
> X45a 133..176
> Linkc 177..179
> X25 absent > Linkd 180..183
> X45b 184..230
> Linke 231..232
> CBM48 233..329
> Linkf 330..345 > GH13 14 346..849
SEQUENCE NO. 37, > Signal/propeptide 1. 52
> CBM41 53. 151
> Linkb 152. 158
> X45a 159, 206
> Linkc absent
> X25 absent
> Linkd 207..209
> X45b 210. ,255
> Linke 256. ,258
> CBM48 259, , 354
> Linkf 355. ,370
> GH13_14 371. ,865
SEQUENCE NO. 38,
> Signal/propeptide 1. 52
> CBM41 53. 151
> Linkb 152. 158
> X45a 159. 206
> Linkc absent
> X25 absent
> Linkd 207..209
> X45b 210. ,255
> Linke 256. ,258
> CBM48 259. ,354
> Linkf 355. ,370
> GH13_14 371 , ,865
SEQUENCE NO. 39,
> Signal/propeptide 1 16
> CBM41 17 118
> Linkb 119 123
> X45a 124 168
> Linkc 169 171
> X25 absent
> Linkd
> X45b 176 222
> Linke 58 59
> CBM48 225 321
> Linkf 322 337
> GH13 14 338 843
X25 domain: X25 domains according to the present invention are polypeptides having a percent identity to any sequence listed in Table 2, of at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity.
Table 2. X25 regions in sequences showing pullulanase activity.
SEQUENCE NO. 40,
> X25 [ 162..262 ]
PVTAVLVGDFQQALGASGNWSPDDDHTKLSKINSNLYQFTGTLPAGTYQYKVALDHSWSASYPNNNVNLT VPAGGTKVTFTYI PSTHQVFDSINNPDQTFP
SEQUENCE NO. 41, > X25 [ 81..181 ]
NVTAVLAGTFQHI FGGSDWAPDNHSTLLKKVNNNLYQFSGDLPEGNYQYKVALNDSWNNPSYPSNNI DLT VPTGGAHVTFSYVPSTHAVYDSINNPGADLP SEQUENCE NO. 42,
> X25 [ 198..298 ]
TVTAVLVGDLQQALGAGGNWAPTDDHTLMTKINANLYQFTGTLPAGTYQYKVALDHSWNASYPNNNVSLT VPSGGEKVTFTYI PSTHQVYDSINNSNQTFP SEQUENCE NO. 43,
> X25 [ 52..152 ]
PVTAVLVGDLQQALGAANNWSPDDDHTLLKKINPNLYQLSGTLPAGTYQYKIALDHSWNTSYPGNNVSLT VPEGGEKVTFTYI PSTNQVFDSVNHPNQAFP X45 domain: X45 domains according to the present invention are polypeptides having a percent identity to any sequence listed in Table 3, of at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity. Table 3. X45 regions in sequences showing pullulanase activity. Position of comma ',' in residue numbering and sequence shows insertion site.
SEQUENCE NO. 44,
> X45 [ 111..161, 263..313 ] 1..51 , 52..102
PKVSNAYLDNEKTVLAKLTNPMTLSDGSSGFTVTDKTTGEQI PVVSAESAN, SSSAGTQSDLVQLTLASA PDVSHTIQVGAAGYEAVNLI PRNVLNLPRYYYS
SEQUENCE NO. 45,
> X45 [ 27..77, 182..232 ] 1..51 , 52..102
AAVSNAYLDASNQVLVKLSQPFTLGEGASGFTVHDDTVNKDIPVTSVTDAS, VNGSGVKTDLVTVTLGED PDVSHTLSIQTDGYQAKQVISRNVLDSSQYYYS
SEQUENCE NO. 46,
> X45 [ 147..197, 299..349 ] 1..51 , 52..102
PWSNAYLDDEKTVLTKLNIPMTLTDGASSFTVTDTTTGQQI PVTSAVSAN, ASSAGIQTDLVQLTLASA PDVTHTFQVAADGYQAGNMLPRNVLNLPRYVYS
SEQUENCE NO. 47,
> X45 [ 148..198, 300..350 ] 1..51 , 52..102
PAVSNAYLDNEKTVLAKLSSPMTLTDGASGFTVTDETTGEQI PVVSAESAN, SSSAGTQSDLVQLTLASA PDITHDLQVVADGYKGGKILPRNVLNLSRYYYS
SEQUENCE NO. 48,
> X45 [ 1..51, 153..203 ] 1..51 , 52..102
PSVSNAYLDDEKTVLAKLSMPMTLADAASGFTVI DKTTGEKI PVTSAVSAN, TSSAGVQTNLVQLTLASA PDVTHNLDVAADGYKAHNILPRNVLNLPRYDYS
SEQUENCE NO. 49,
> X45 [ 124..171, 172..222 ] 1..48, 49..99
PRI FFAQARSNKVIEAYLTNPADTKKKGLFKVTVDGKEI PI SRVEKAD, PTDVDVTNYVRVVLSEPLKEE DLRKDVELIVEGYKPARVIMMEILDDYYYD
SEQUENCE NO.
> X45 [ 111..161,162..212 ] 1..51 , 52..102 PKVSNAYLDNEKTVLAKLTNPMTLSDGSSGFTVTDKTTGEQI PVTAATNAN, SASSSEQTDLVQLTLASA PDVSHTIQVGAAGYEAVNLI PRNVLNLPRYYYS
SEQUENCE NO. 51,
> X45 [ 133..179, 180..230 ] 1..47, 48..98
PRVLFAQARDQYTIEAYLTGQVDTTKVGAKVTVDGQPLKIARVEKAN, PTDI SRTNHVKVVLAEPIKLED VNKDVQVEIEGYKPARVIMMEILDKIYYD
SEQUENCE NO. 52,
> X45 [ 159..206,207..255 ] 1..48,49..97
PRIVSAYMDGKDEI IVSLTHQHKLTDGDNGFVIKGENKEYNVKKVEIV, GSGSSGNVLKLTLEKELDITA NYTIESPDYTGNYVIKRRVLDLPEFYYA
SEQUENCE NO. 53,
> X45 [ 159..206,207..255 ] 1..48,49..97
PRI ISAYMDGKDEI IVSLTHQHKLTDGDNGFVIKGEDKDYNVKKVEW, GSGTSGNVLKLTLEKELDITA NYTIESPDYTGNYWKRRVLDLPEFYYP
SEQUENCE NO. 54,
> X45 [ 124..171, 172..222 ] 1..48, 49..99
PRI FFAQARSNKVIEAFLTNPVDTKKKELFKVTVDGKEIPVSRVEKAD, PTDIDVTNYVRIVLSESLKEE DLRKDVELI IEGYKPARVIMMEILDDYYYD Chimera X25-within-X45 domain: Chimera X25-within-X45 domains are identified as polypeptides having a percent identity to any sequence listed in Table 4 (with comma symbols ',' removed), of at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity. Table 4. Chimeric X25 within X45 domain.
SEQUENCE NO. 55,
> chimeraX25inX45 [ 111..161 , 162..262 , 263..313 ]
1..51,52..152, 153..203
PKVSNAYLDNEKTVLAKLTNPMTLSDGSSGFTVTDKTTGEQI PVVSAESAN, PVTAVLVGDFQQALGASG NWSPDDDHTKLSKINSNLYQFTGTLPAGTYQYKVALDHSWSASYPNNNVNLTVPAGGTKVTFTYI PSTHQ VFDSINNPDQTFP, SSSAGTQSDLVQLTLASAPDVSHTIQVGAAGYEAVNLI PRNVLNLPRYYYS
SEQUENCE NO. 56,
> X45a [ 138..188, 189..289, 290..340 ]
1..51, 52..152, 153..203
PKVSNAYLDNEKTVLAKLTNPMTLSDGSSGFTVTDKTTGEQI PVTSAVSAN, TVTAVLVGDLQQALGAGG NWAPTDDHTLMTKINANLYQFTGTLPAGTYQYKVALDHSWNASYPNNNVSLTVPSGGEKVTFTYI PSTHQ VYDSINNSNQTFP , ASSAGIQTDLVQLTLASAPDVSHTIQVGAAGYEAVNLI PRNVLNLPRYYYS CBM41 domain: CBM41 domains, Carbohydrate-Binding Module family 41 , are modules of approx. 100 residues found primarily in bacterial pullulanases. CBM41 alias PUD (Bacterial pullulanase-associated domain) modules may be identified in a query protein sequence, by using the Pfam database 'Sequence Search' tool available at http://pfam.xfam.org/search, using Pfam version 26.0 or higher. The Pfam database is a large collection of protein families, each represented by multiple sequence alignments and hidden Markov models (HMMs). Pfam is freely available under the Creative Commons Zero ("CCO") license (see http://creativecommons.0rg/publicdomain/zero/l .0/).
The query protein sequence in FASTA format is entered into the search field of the Pfam database Sequence Search tool available via the internet at http://pfam.xfam.org/search, and the Submit button is pressed, after which the Sequence Search results are displayed in a table showing Significant Pfam-A Matches, hereafter Table.
The presence of Table rows containing the Family name PUD are positive identifications of the presence of CBM41 alias PUD modules in the query protein sequence. The PUD Family name may also be referred to as PF03714 without loss of ambiguity.
Additional columns in the Table show Envelope Start and End coordinates, which define respectively start and end coordinates of the CBM41 alias PUD module in the query sequence, hereafter sequence Region which encompasses sequence start to end.
An additional column in the Table shows E-value, which refers to the statistical significance of the CBM41 alias PUD module identification. Lower E-values are statistically more significant than higher E-values. Significant CBM41 alias PUD module identifications are defined as those having an E-value less than 1 .0, preferably an E-value less than 1 e-2 (0.01 ), more preferably an E-value less than 1 e-4 (0.0001 ), even more preferably an E-value less than 1 e-6 (0.000001 ).
Catalytic domain: The term "catalytic domain" means the region of an enzyme containing the catalytic machinery of the enzyme.
cDNA: The term "cDNA" means a DNA molecule that can be prepared by reverse transcription from a mature, spliced, mRNA molecule obtained from a eukaryotic or prokaryotic cell. cDNA lacks intron sequences that may be present in the corresponding genomic DNA. The initial, primary RNA transcript is a precursor to mRNA that is processed through a series of steps, including splicing, before appearing as mature spliced mRNA.
Coding sequence: The term "coding sequence" means a polynucleotide, which directly specifies the amino acid sequence of a polypeptide. The boundaries of the coding sequence are generally determined by an open reading frame, which begins with a start codon such as ATG, GTG, or TTG and ends with a stop codon such as TAA, TAG, or TGA. The coding sequence may be a genomic DNA, cDNA, synthetic DNA, or a combination thereof.
Control sequences: The term "control sequences" means nucleic acid sequences necessary for expression of a polynucleotide encoding a mature polypeptide of the present invention. Each control sequence may be native (i.e., from the same gene) or foreign (i.e., from a different gene) to the polynucleotide encoding the polypeptide or native or foreign to each other. Such control sequences include, but are not limited to, a leader, polyadenylation sequence, propeptide sequence, promoter, signal peptide sequence, and transcription terminator. At a minimum, the control sequences include a promoter, and transcriptional and translational stop signals. The control sequences may be provided with linkers for the purpose of introducing specific restriction sites facilitating ligation of the control sequences with the coding region of the polynucleotide encoding a polypeptide.
Isolated: The term "isolated" means a substance in a form or environment that does not occur in nature. Non-limiting examples of isolated substances include (1 ) any non-naturally occurring substance, (2) any substance including, but not limited to, any enzyme, variant, nucleic acid, protein, peptide or cofactor, that is at least partially removed from one or more or all of the naturally occurring constituents with which it is associated in nature; (3) any substance modified by the hand of man relative to that substance found in nature; or (4) any substance modified by increasing the amount of the substance relative to other components with which it is naturally associated (e.g., recombinant production in a host cell; multiple copies of a gene encoding the substance; and use of a stronger promoter than the promoter naturally associated with the gene encoding the substance). An isolated substance may be present in a fermentation broth sample; e.g. a host cell may be genetically modified to express the polypeptide of the invention. The fermentation broth from that host cell will comprise the isolated polypeptide.
Mature polypeptide: The term "mature polypeptide" means a polypeptide in its final form following translation and any post-translational modifications, such as N-terminal processing, C-terminal truncation, glycosylation, phosphorylation, etc. It is known in the art that a host cell may produce a mixture of two of more different mature polypeptides (i.e., with a different C-terminal and/or N-terminal amino acid) expressed by the same polynucleotide. It is also known in the art that different host cells process polypeptides differently, and thus, one host cell expressing a polynucleotide may produce a different mature polypeptide (e.g., having a different C-terminal and/or N-terminal amino acid) as compared to another host cell expressing the same polynucleotide.
Mature polypeptide coding sequence: The term "mature polypeptide coding sequence" means a polynucleotide that encodes a mature polypeptide having pullulanase activity.
Nucleic acid construct: The term "nucleic acid construct" means a nucleic acid molecule, either single- or double-stranded, which is isolated from a naturally occurring gene or is modified to contain segments of nucleic acids in a manner that would not otherwise exist in nature or which is synthetic, which comprises one or more control sequences.
Operably linked: The term "operably linked" means a configuration in which a control sequence is placed at an appropriate position relative to the coding sequence of a polynucleotide such that the control sequence directs expression of the coding sequence.
Sequence identity: The relatedness between two amino acid sequences or between two nucleotide sequences is described by the parameter "sequence identity".
For purposes of the present invention, the sequence identity between two amino acid sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277), preferably version 5.0.0 or later. The parameters used are gap open penalty of 10, gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. The output of Needle labeled "longest identity" (obtained using the -nobrief option) is used as the percent identity and is calculated as follows:
(Identical Residues x 100)/(Length of Alignment - Total Number of Gaps in Alignment) For purposes of the present invention, the sequence identity between two deoxyribonucleotide sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, supra) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, supra), preferably version 5.0.0 or later. The parameters used are gap open penalty of 10, gap extension penalty of 0.5, and the EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix. The output of Needle labeled "longest identity" (obtained using the -nobrief option) is used as the percent identity and is calculated as follows:
(Identical Deoxyribonucleotides x 100)/(Length of Alignment - Total Number of Gaps in Alignment)
Variant: The term "variant" means a polypeptide having pullulanase activity comprising an alteration, i.e., a substitution, a deletion or an insertion, e.g., an insertion of a domain selected from a CBM41 or an X25 domain.
Detailed Description of the Invention
The present invention relates to methods for increasing pullulanase activity and to the pullulanases resulting therefrom. In particular the present invention relates to a method of increasing pullulanase activity of a parent pullulanase comprising the steps:
a) inserting an X25 domain within an X45 domain; and /or
b) adding a CBM41 domain either adjacent to the N-terminal of an X45 domain, or if a CBM41 domain is already present, adding the additional CBM41 domain adjacent to the N-terminal of the existing CBM41 or adjacent to the N-terminal of the X45 domain. According to NCBI's CCD (conserved domain database, Marchler-Bauer et al., Nucleic acids research 201 1 , vol. 39, D225-229), X25, X45 and CBM41 domains in pullulanases are described as follows:
X25 domain of Bacillus acidopullulyticus pullulanase and similar proteins.
Pullulanase (EC 3.2.1 .41 ) cleaves 1 .6-alpha-glucosidic linkages in pullulan, amylopectin, and glycogen, and in alpha-and beta-amylase limit-dextrins of amylopectin and glycogen. Bacillus acidopullulyticus pullulanase is used industrially in the production of high fructose corn syrup, high maltose content syrups and low calorie and "light" beers. Pullulanases, in addition to the catalytic domain, include several carbohydrate-binding domains (CBMs) as well as domains of unknown function (termed "X" modules). X25 was identified in Bacillus acidopullulyticus pullulanase, and splits another domain of unknown function (X45). X25 is present in multiple copies in some pullulanases. It has been suggested that X25 and X45 are CBMs which target mixed alpha-1 .6/alpha-1.4 linked D-glucan polysaccharides.
X45 domain of Bacillus acidopullulyticus pullulanase and similar proteins.
Pullulanase (EC 3.2.1 .41 ) cleaves 1 .6-alpha-glucosidic linkages in pullulan, amylopectin, and glycogen, and in alpha-and beta-amylase limit-dextrins of amylopectin and glycogen. Bacillus acidopullulyticus pullulanase is used industrially in the production of high fructose corn syrup, high maltose content syrups and low calorie and "light" beers. Pullulanases, in addition to the catalytic domain, include several carbohydrate-binding domains (CBMs) as well as domains of unknown function (termed "X" modules). X45 was identified in Bacillus acidopullulyticus pullulanase, it is interrupted by another domain of unknown function (X25). It has been suggested that X25 and X45 are CBMs which target mixed alpha-1.6/alpha-1.4 linked D-glucan polysaccharides. Family 41 Carbohydrate-Binding Module from pullulanase-like enzymes
Pullulanases (EC 3.2.1 .41 ) are a group of starch-debranching enzymes, catalyzing the hydrolysis of the alpha-1 .6-glucosidic linkages of alpha-glucans, preferentially pullulan. Pullulan is a polysaccharide in which alpha-1 .4 linked maltotriosyl units are combined via an alpha-1 .6 linkage. These enzymes are of importance in the starch industry, where they are used to hydrolyze amylopectin starch. Pullulanases consist of multiple distinct domains, including a catalytic domain belonging to the glycoside hydrolase (GH) family 13 and carbohydrate-binding modules (CBM), including CBM41 .
Some pullulanases contain all these domains at their N-terminal and some lack either one or two or all of these domains.
According to the present invention it has been discovered that pullanase activity may be improved, in particular the improved activity is increased pullulanase activity measured as increased activity in the Phadebas assay at 65°C, by the addition of an X25 domain as defined according to the present invention. This addition may be either by inserting an X25 domain within an existing X45 domain or by replacing an existing X45 domain with an X45a-X25-X45b domain. If the improved pullulanase is obtained by insertion of the X25 domain the following guidelines may be followed in order to identify the proper insertion point. i) Location of X25 insertion site within X45 domain.
The insertion site of X25 in identified X45 domains is identified using pair-wise sequence alignment to any sequence listed in Table 3 and designated as 'X45' domain. The insertion site is identified by the location of the comma symbol counting matched positions in the alignment from the start of the sequence designated as 'X45' domain. For example, in specific instances of sequences listed in Table 3, the insertion site is between matched positions 51 and 52 of SEQ ID NO: 44, or positions 48 and 49 of SEQ ID NO: 49, or between positions 48 and 49 of SEQ ID NO: 52. ii) Composition of last three residues within X45 domain just before X25 insertion site.
The three residues of the X45 domain immediately prior to the X25 insertion site are of a specific composition described by sequence patterns and exemplified in Table 5. The triplet residue residues immediately prior to the X25 insertion site should match the pattern [DKNS][AGLV][DKNS], or [IV][SD]D. If the three residues of the X45 domain immediately prior to the X25 insertion site do not have the described composition, for instance SEQ ID NO: 52, and SEQ ID NO: 53, then a triplet of residues matching one of the exemplified triplets in Table 5 is inserted immediately before the X25 domain.
Table 5. Specific triplets
DAN
DAS
KAD
KVD
KLD KAN
KAK
NAN
SAN
SGN I S D
VDD
In some instances, the addition of three to five additional residues immediately before the X25 domain is also enabled, for example the residue triplet LGQ, designation 'Linkc2' in SEQ. NO. 30. iii) Composition of last four or five residues within X45 domain just after the X25 insertion site. The last four or five residues within the X45 domain immediately following the X25 insertion site are of a specific composition described by sequence patterns and exemplified in Table 6. The last residue of the X25 domain is most often a P or Q residue. Residues immediately following the X25 insertion site should be predominantly composed of residues matching [VSTA][EDNSG][SGA] or [VSTA][EDNSG][SGA][SGA], followed often by a G residue marking the resumption of the X45 domain.
If the four or five residues of the X45 domain immediately prior tofollowing the X25 insertion site do not have the described composition, for instance SEQ ID NO: 37, region 'Linkd', and SEQ ID NO: 39, region 'Linkd', then a sequence of four or five residues matching one of the exemplified triplets in Table 6 is inserted immediately after the X25 domain.
Table 6 Specific examples of residues immediately following the X25 insertion site
VES VDSS (SEQ ID NO: 57)
VNGS (SEQ ID NO: 58)
VNGSG (SEQ ID NO: 59)
SSSA (SEQ ID NO: 60)
TSSA (SEQ ID NO: 61)
ASSA (SEQ ID NO: 62)
ASSAG (SEQ ID NO: 63)
Another aspect of the invention relates to increasing pullulanase activity by the addition of a CBM41 domain either adjacent to the N-terminal of an X45 domain, or if a CBM41 domain is already present, adding the additional CBM41 domain adjacent to the N-terminal of the existing CBM41 or adjacent to the N-terminal of the X45 domain. CBM41 domains may be determined as described in the definition section.
The improved pullulanase activity may in particular be increased specific activity (measured as relative specific activity).
Thus in a particular embodiment the present invention relates to a method of increasing pullulanase activity of a parent pullulanase comprising the steps:
a) inserting an X25 domain within an X45 domain; and /or
b) adding a CBM41 domain either adjacent to the N-terminal of an X45 domain, or if a CBM41 domain is already present, adding an additional CBM41 domain adjacent to the N-terminal of the existing CBM41 or adjacent to the N-terminal of the X45 domain.
The pullulanases (or parent pullulanases) that may be improved by the present method are in a particular embodiment pullulanases from a bacterium, particularly, a gram positive bacterium, more particularly a Bacillus sp. In a specific embodiment the parent pullulanase is selected from a Bacillus acidopullulyticus pullulanase, Bacillus deramificans pullulanase, or a hybrid pullulanase, e.g., a hybrid comprising an N-terminal part from a Bacillus acidopullulyticus pullulanase and a C-terminal part from a Bacillus deramificans pullulanase. In one particular embodiment the parent pullulanase is selected from SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 1 1 , or SEQ ID NO: 12. The X25 domain as defined herein may be any X25 domain, but in a particular embodiment the X25 domain is selected from an X25 from a pullulanase, more particularly a pullulanase from a bacterium, particularly, a gram positive bacterium, more particularly a Bacillus sp., even more particularly from a Bacillus acidopullulyticus or Bacillus deramificans.
In one embodiment the X25 domain is selected from the group consisting of SEQ ID NO: 40 or an X25 domain having a sequence identity to SEQ ID NO: 40 of at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, SEQ ID NO: 41 or an X25 domain having a sequence identity to SEQ ID NO: 41 of at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, SEQ ID NO: 42 or an X25 domain having a sequence identity to SEQ ID NO: 42 of at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, SEQ ID NO: 43, or an X25 domain having a sequence identity to SEQ ID NO: 43 of at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%.
The X25 domain may in one embodiment be inserted within an X45 domain already present in the parent pullulanase, or alternatively by replacing an existing X45 domain in the parent pullulanase with an X45a-X25-X45b domain.
A further aspect of the invention relates to increasing pullulanase activity of a parent pullulanase by adding a CBM41 domain. In particular, the CBM41 domain is selected from a CBM41 domain from a pullulanase, more particularly a pullulanase from a bacterium, particularly, a gram positive bacterium, more particularly a Bacillus sp. In one embodiment the Bacillus sp. is selected from Bacillus acidopullulyticus or Bacillus deramificans.
Polypeptides having increased pullulanase activity
The present invention, in one embodiment relates to pullulanases prepared by the method of the present invention. In a particular embodiment the invention relates to polypeptides having a sequence identity to the polypeptide of SEQ ID NO: 15 of at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, and wherein the polypeptide has at least at least 90%, at least 95%, at least 97%, at least 100% of the pullulanase activity of the polypeptide of SEQ ID NO: 15.
In a particular embodiment the invention relates to polypeptides having a sequence identity to the polypeptide of SEQ ID NO: 16 of at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, and wherein the polypeptide has at least at least 90%, at least 95%, at least 97%, at least 100% of the pullulanase activity of the polypeptide of SEQ ID NO: 16.
In a particular embodiment the invention relates to polypeptides having a sequence identity to the polypeptide of SEQ ID NO: 17 of at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, and wherein the polypeptide has at least at least 90%, at least 95%, at least 97%, at least 100% of the pullulanase activity of the polypeptide of SEQ ID NO: 17.
In a particular embodiment the invention relates to polypeptides having a sequence identity to the polypeptide of SEQ ID NO: 18 of at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, and wherein the polypeptide has at least at least 90%, at least 95%, at least 97%, at least 100% of the pullulanase activity of the polypeptide of SEQ ID NO: 18.
In a particular embodiment the invention relates to polypeptides having a sequence identity to the polypeptide of SEQ ID NO: 19 of at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, and wherein the polypeptide has at least at least 90%, at least 95%, at least 97%, at least 100% of the pullulanase activity of the polypeptide of SEQ ID NO: 19.
In a particular embodiment the invention relates to polypeptides having a sequence identity to the polypeptide of SEQ ID NO: 20 of at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, and wherein the polypeptide has at least at least 90%, at least 95%, at least 97%, at least 100% of the pullulanase activity of the polypeptide of SEQ ID NO: 20.
In a particular embodiment the invention relates to polypeptides having a sequence identity to the polypeptide of SEQ ID NO: 21 of at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, and wherein the polypeptide has at least at least 90%, at least 95%, at least 97%, at least 100% of the pullulanase activity of the polypeptide of SEQ ID NO: 21.
In a particular embodiment the invention relates to polypeptides having a sequence identity to the polypeptide of SEQ ID NO: 22 of at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, and wherein the polypeptide has at least at least 90%, at least 95%, at least 97%, at least 100% of the pullulanase activity of the polypeptide of SEQ ID NO: 22. In a particular embodiment the invention relates to polypeptides having a sequence identity to the polypeptide of SEQ ID NO: 23 of at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, and wherein the polypeptide has at least at least 90%, at least 95%, at least 97%, at least 100% of the pullulanase activity of the polypeptide of SEQ ID NO: 23.
In a particular embodiment the invention relates to polypeptides having a sequence identity to the polypeptide of SEQ ID NO: 24 of at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, and wherein the polypeptide has at least at least 90%, at least 95%, at least 97%, at least 100% of the pullulanase activity of the polypeptide of SEQ ID NO: 24.
In a particular embodiment the invention relates to polypeptides having a sequence identity to the polypeptide of SEQ ID NO: 25 of at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, and wherein the polypeptide has at least at least 90%, at least 95%, at least 97%, at least 100% of the pullulanase activity of the polypeptide of SEQ ID NO: 25.
In a particular embodiment the invention relates to polypeptides having a sequence identity to the polypeptide of SEQ ID NO: 26 of at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, and wherein the polypeptide has at least at least 90%, at least 95%, at least 97%, at least 100% of the pullulanase activity of the polypeptide of SEQ ID NO: 26.
In a particular embodiment the invention relates to polypeptides having a sequence identity to the polypeptide of SEQ ID NO: 27 of at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, and wherein the polypeptide has at least at least 90%, at least 95%, at least 97%, at least 100% of the pullulanase activity of the polypeptide of SEQ ID NO: 27.
In another embodiment the pullulanase comprises or consists of SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ I D NO: 21 , SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27.
In an embodiment, the polypeptide has been isolated.
In another embodiment, the present invention relates to variants of the polypeptide of SEQ ID NO: 15-27 comprising a substitution, deletion, and/or insertion at one or more (e.g., several) positions. In an embodiment, the number of amino acid substitutions, deletions and/or insertions introduced into the mature polypeptide of SEQ ID NO: 15-27 is up to 10, e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10. The amino acid changes may be of a minor nature, that is conservative amino acid substitutions or insertions that do not significantly affect the folding and/or activity of the protein; small deletions, typically of 1 -30 amino acids; small amino- or carboxyl-terminal extensions, such as an amino-terminal methionine residue; a small linker peptide of up to 20-25 residues; or a small extension that facilitates purification by changing net charge or another function, such as a poly-histidine tract, an antigenic epitope or a binding domain.
Examples of conservative substitutions are within the groups of basic amino acids (arginine, lysine and histidine), acidic amino acids (glutamic acid and aspartic acid), polar amino acids (glutamine and asparagine), hydrophobic amino acids (leucine, isoleucine and valine), aromatic amino acids (phenylalanine, tryptophan and tyrosine), and small amino acids (glycine, alanine, serine, threonine and methionine). Amino acid substitutions that do not generally alter specific activity are known in the art and are described, for example, by H. Neurath and R.L. Hill, 1979, In, The Proteins, Academic Press, New York. Common substitutions are Ala/Ser, Val/lle, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg, Asp/Asn, Leu/lle, LeuA al, Ala/Glu, and Asp/Gly.
Alternatively, the amino acid changes are of such a nature that the physico-chemical properties of the polypeptides are altered. For example, amino acid changes may improve the thermal stability of the polypeptide, alter the substrate specificity, change the pH optimum, and the like.
Essential amino acids in a polypeptide can be identified according to procedures known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (Cunningham and Wells, 1989, Science 244: 1081 -1085). In the latter technique, single alanine mutations are introduced at every residue in the molecule, and the resultant molecules are tested for pullulanase activity to identify amino acid residues that are critical to the activity of the molecule. See also, Hilton et al., 1996, J. Biol. Chem. 271 : 4699-4708. The active site of the enzyme or other biological interaction can also be determined by physical analysis of structure, as determined by such techniques as nuclear magnetic resonance, crystallography, electron diffraction, or photo affinity labeling, in conjunction with mutation of putative contact site amino acids. See, for example, de Vos et al., 1992, Science 255: 306-312; Smith et al., 1992, J. Mol. Biol. 224: 899-904; Wlodaver et al., 1992, FEBS Lett. 309: 59-64. The identity of essential amino acids can also be inferred from an alignment with a related polypeptide.
Single or multiple amino acid substitutions, deletions, and/or insertions can be made and tested using known methods of mutagenesis, recombination, and/or shuffling, followed by a relevant screening procedure, such as those disclosed by Reidhaar-Olson and Sauer, 1988, Science 241 : 53-57; Bowie and Sauer, 1989, Proc. Natl. Acad. Sci. USA 86: 2152-2156; WO 95/17413; or WO 95/22625. Other methods that can be used include error-prone PCR, phage display (e.g., Lowman et al., 1991 , Biochemistry 30: 10832-10837; U.S. Patent No. 5,223,409; WO 92/06204), and region-directed mutagenesis (Derbyshire et al., 1986, Gene 46: 145; Ner ei a/., 1988, DNA 7: 127).
Mutagenesis/shuffling methods can be combined with high-throughput, automated screening methods to detect activity of cloned, mutagenized polypeptides expressed by host cells (Ness et al., 1999, Nature Biotechnology 17: 893-896). Mutagenized DNA molecules that encode active polypeptides can be recovered from the host cells and rapidly sequenced using standard methods in the art. These methods allow the rapid determination of the importance of individual amino acid residues in a polypeptide.
The polypeptide may be a hybrid polypeptide in which a region of one polypeptide is fused at the N-terminus or the C-terminus of a region of another polypeptide.
The polypeptide may be a fusion polypeptide or cleavable fusion polypeptide in which another polypeptide is fused at the N-terminus or the C-terminus of the polypeptide of the present invention. A fusion polypeptide is produced by fusing a polynucleotide encoding another polypeptide to a polynucleotide of the present invention. Techniques for producing fusion polypeptides are known in the art, and include ligating the coding sequences encoding the polypeptides so that they are in frame and that expression of the fusion polypeptide is under control of the same promoter(s) and terminator. Fusion polypeptides may also be constructed using intein technology in which fusion polypeptides are created post-translationally (Cooper et al., 1993, EMBO J. 12: 2575-2583; Dawson et ai, 1994, Science 266: 776-779).
A fusion polypeptide can further comprise a cleavage site between the two polypeptides. Upon secretion of the fusion protein, the site is cleaved releasing the two polypeptides. Examples of cleavage sites include, but are not limited to, the sites disclosed in Martin et al., 2003, J. Ind. Microbiol. Biotechnol. 3: 568-576; Svetina et al., 2000, J. Biotechnol. 76: 245-251 ; Rasmussen-Wilson et al., 1997, Appl. Environ. Microbiol. 63: 3488-3493; Ward et al., 1995, Biotechnology 13: 498-503; and Contreras et al., 1991 , Biotechnology 9: 378-381 ; Eaton et al., 1986, Biochemistry 25: 505-512; Collins-Racie et al., 1995, Biotechnology 13: 982- 987; Carter et al., 1989, Proteins: Structure, Function, and Genetics 6: 240-248; and Stevens, 2003, Drug Discovery World 4: 35-48.
Polynucleotides
The present invention also relates to polynucleotides encoding a polypeptide of the present invention, as described herein. In an embodiment, the polynucleotide encoding the polypeptide of the present invention has been isolated.
The techniques used to isolate or clone a polynucleotide are known in the art and include isolation from genomic DNA or cDNA, or a combination thereof. The cloning of the polynucleotides from genomic DNA can be effected, e.g., by using the well-known polymerase chain reaction (PCR) or antibody screening of expression libraries to detect cloned DNA fragments with shared structural features. See, e.g., Innis et al., 1990, PCR: A Guide to Methods and Application, Academic Press, New York. Other nucleic acid amplification procedures such as ligase chain reaction (LCR), ligation activated transcription (LAT) and polynucleotide-based amplification (NASBA) may be used. Nucleic Acid Constructs
The present invention also relates to nucleic acid constructs comprising a polynucleotide of the present invention operably linked to one or more control sequences that direct the expression of the coding sequence in a suitable host cell under conditions compatible with the control sequences.
The polynucleotide may be manipulated in a variety of ways to provide for expression of the polypeptide. Manipulation of the polynucleotide prior to its insertion into a vector may be desirable or necessary depending on the expression vector. The techniques for modifying polynucleotides utilizing recombinant DNA methods are well known in the art.
The control sequence may be a promoter, a polynucleotide that is recognized by a host cell for expression of a polynucleotide encoding a polypeptide of the present invention. The promoter contains transcriptional control sequences that mediate the expression of the polypeptide. The promoter may be any polynucleotide that shows transcriptional activity in the host cell including variant, truncated, and hybrid promoters, and may be obtained from genes encoding extracellular or intracellular polypeptides either homologous or heterologous to the host cell.
Examples of suitable promoters for directing transcription of the nucleic acid constructs of the present invention in a bacterial host cell are the promoters obtained from the Bacillus amyloliquefaciens alpha-amylase gene (amyQ), Bacillus licheniformis alpha-amylase gene (amyL), Bacillus licheniformis penicillinase gene (penP), Bacillus stearothermophilus maltogenic amylase gene (amyM), Bacillus subtilis levansucrase gene (sacB), Bacillus subtilis xylA and xylB genes, Bacillus thuringiensis crylllA gene (Agaisse and Lereclus, 1994, Molecular Microbiology 13: 97-107), E. coli lac operon, E. coli trc promoter (Egon et al., 1988, Gene 69: 301 -315), Streptomyces coelicolor agarase gene {dagA), and prokaryotic beta- lactamase gene (Villa-Kamaroff et al., 1978, Proc. Natl. Acad. Sci. USA 75: 3727-3731 ), as well as the tac promoter (DeBoer et al., 1983, Proc. Natl. Acad. Sci. USA 80: 21 -25). Further promoters are described in "Useful proteins from recombinant bacteria" in Gilbert et al., 1980, Scientific American 242: 74-94; and in Sambrook et al., 1989, supra. Examples of tandem promoters are disclosed in WO 99/43835.
The control sequence may also be a transcription terminator, which is recognized by a host cell to terminate transcription. The terminator is operably linked to the 3'-terminus of the polynucleotide encoding the polypeptide. Any terminator that is functional in the host cell may be used in the present invention. Preferred terminators for bacterial host cells are obtained from the genes for Bacillus clausii alkaline protease {aprH), Bacillus licheniformis alpha-amylase (amyL), and Escherichia coli ribosomal RNA (rrnB).
The control sequence may also be an mRNA stabilizer region downstream of a promoter and upstream of the coding sequence of a gene which increases expression of the gene.
Examples of suitable mRNA stabilizer regions are obtained from a Bacillus thuringiensis crylllA gene (WO 94/25612) and a Bacillus subtilis SP82 gene (Hue et ai, 1995, Journal of Bacteriology 177: 3465-3471 ).
The control sequence may also be a signal peptide coding region that encodes a signal peptide linked to the N-terminus of a polypeptide and directs the polypeptide into the cell's secretory pathway. The 5'-end of the coding sequence of the polynucleotide may inherently contain a signal peptide coding sequence naturally linked in translation reading frame with the segment of the coding sequence that encodes the polypeptide. Alternatively, the 5'-end of the coding sequence may contain a signal peptide coding sequence that is foreign to the coding sequence. A foreign signal peptide coding sequence may be required where the coding sequence does not naturally contain a signal peptide coding sequence. Alternatively, a foreign signal peptide coding sequence may simply replace the natural signal peptide coding sequence in order to enhance secretion of the polypeptide. However, any signal peptide coding sequence that directs the expressed polypeptide into the secretory pathway of a host cell may be used.
Effective signal peptide coding sequences for bacterial host cells are the signal peptide coding sequences obtained from the genes for Bacillus NCIB 1 1837 maltogenic amylase, Bacillus licheniformis subtilisin, Bacillus licheniformis beta-lactamase, Bacillus stearothermophilus alpha-amylase, Bacillus stearothermophilus neutral proteases {nprT, nprS, nprM), and Bacillus subtilis prsA. Further signal peptides are described by Simonen and Palva, 1993, Microbiological Reviews 57: 109-137.
The control sequence may also be a propeptide coding sequence that encodes a propeptide positioned at the N-terminus of a polypeptide. The resultant polypeptide is known as a proenzyme or propolypeptide (or a zymogen in some cases). A propolypeptide is generally inactive and can be converted to an active polypeptide by catalytic or autocatalytic cleavage of the propeptide from the propolypeptide.
Where both signal peptide and propeptide sequences are present, the propeptide sequence is positioned next to the N-terminus of a polypeptide and the signal peptide sequence is positioned next to the N-terminus of the propeptide sequence. Expression Vectors
The present invention also relates to recombinant expression vectors comprising a polynucleotide of the present invention, a promoter, and transcriptional and translational stop signals. The various nucleotide and control sequences may be joined together to produce a recombinant expression vector that may include one or more convenient restriction sites to allow for insertion or substitution of the polynucleotide encoding the polypeptide at such sites. Alternatively, the polynucleotide may be expressed by inserting the polynucleotide or a nucleic acid construct comprising the polynucleotide into an appropriate vector for expression. In creating the expression vector, the coding sequence is located in the vector so that the coding sequence is operably linked with the appropriate control sequences for expression.
The recombinant expression vector may be any vector (e.g., a plasmid or virus) that can be conveniently subjected to recombinant DNA procedures and can bring about expression of the polynucleotide. The choice of the vector will typically depend on the compatibility of the vector with the host cell into which the vector is to be introduced. The vector may be a linear or closed circular plasmid.
The vector may be an autonomously replicating vector, i.e., a vector that exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e.g., a plasmid, an extrachromosomal element, a minichromosome, or an artificial chromosome. The vector may contain any means for assuring self-replication. Alternatively, the vector may be one that, when introduced into the host cell, is integrated into the genome and replicated together with the chromosome(s) into which it has been integrated. Furthermore, a single vector or plasmid or two or more vectors or plasmids that together contain the total DNA to be introduced into the genome of the host cell, or a transposon, may be used.
The vector preferably contains one or more selectable markers that permit easy selection of transformed, transfected, transduced, or the like cells. A selectable marker is a gene the product of which provides for biocide or viral resistance, resistance to heavy metals, prototrophy to auxotrophs, and the like.
Examples of bacterial selectable markers are Bacillus licheniformis or Bacillus subtilis dal genes, or markers that confer antibiotic resistance such as ampicillin, chloramphenicol, kanamycin, neomycin, spectinomycin, or tetracycline resistance.
The vector preferably contains an element(s) that permits integration of the vector into the host cell's genome or autonomous replication of the vector in the cell independent of the genome.
For integration into the host cell genome, the vector may rely on the polynucleotide's sequence encoding the polypeptide or any other element of the vector for integration into the genome by homologous or non-homologous recombination. Alternatively, the vector may contain additional polynucleotides for directing integration by homologous recombination into the genome of the host cell at a precise location(s) in the chromosome(s). To increase the likelihood of integration at a precise location, the integrational elements should contain a sufficient number of nucleic acids, such as 100 to 10,000 base pairs, 400 to 10,000 base pairs, and 800 to 10,000 base pairs, which have a high degree of sequence identity to the corresponding target sequence to enhance the probability of homologous recombination. The integrational elements may be any sequence that is homologous with the target sequence in the genome of the host cell. Furthermore, the integrational elements may be non-encoding or encoding polynucleotides. On the other hand, the vector may be integrated into the genome of the host cell by non-homologous recombination.
For autonomous replication, the vector may further comprise an origin of replication enabling the vector to replicate autonomously in the host cell in question. The origin of replication may be any plasmid replicator mediating autonomous replication that functions in a cell. The term "origin of replication" or "plasmid replicator" means a polynucleotide that enables a plasmid or vector to replicate in vivo.
Examples of bacterial origins of replication are the origins of replication of plasmids pBR322, pUC19, pACYC177, and pACYC184 permitting replication in E. coli, and pUB1 10, pE194, pTA1060, and ρΑΜβΙ permitting replication in Bacillus.
More than one copy of a polynucleotide of the present invention may be inserted into a host cell to increase production of a polypeptide. An increase in the copy number of the polynucleotide can be obtained by integrating at least one additional copy of the sequence into the host cell genome or by including an amplifiable selectable marker gene with the polynucleotide where cells containing amplified copies of the selectable marker gene, and thereby additional copies of the polynucleotide, can be selected for by cultivating the cells in the presence of the appropriate selectable agent.
The procedures used to ligate the elements described above to construct the recombinant expression vectors of the present invention are well known to one skilled in the art (see, e.g., Sambrook et al., 1989, supra).
Host Cells
The present invention also relates to recombinant host cells, comprising a polynucleotide of the present invention operably linked to one or more control sequences that direct the production of a polypeptide of the present invention. A construct or vector comprising a polynucleotide is introduced into a host cell so that the construct or vector is maintained as a chromosomal integrant or as a self-replicating extra-chromosomal vector as described earlier. The term "host cell" encompasses any progeny of a parent cell that is not identical to the parent cell due to mutations that occur during replication. The choice of a host cell will to a large extent depend upon the gene encoding the polypeptide and its source.
The host cell may be any cell useful in the recombinant production of a polypeptide of the present invention, e.g., a prokaryote or a eukaryote.
The prokaryotic host cell may be any Gram-positive or Gram-negative bacterium. Gram- positive bacteria include, but are not limited to, Bacillus, Clostridium, Enterococcus, Geobacillus, Lactobacillus, Lactococcus, Oceanobacillus, Staphylococcus, Streptococcus, and Streptomyces. Gram-negative bacteria include, but are not limited to, Campylobacter, E. coli, Flavobacterium, Fusobacterium, Helicobacter, llyobacter, Neisseria, Pseudomonas, Salmonella, and Ureaplasma.
The bacterial host cell may be any Bacillus cell including, but not limited to, Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus firmus, Bacillus lautus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus pumilus, Bacillus stearothermophilus, Bacillus subtilis, and Bacillus thuringiensis cells.
The introduction of DNA into a Bacillus cell may be effected by protoplast transformation (see, e.g., Chang and Cohen, 1979, Mol. Gen. Genet. 168: 1 1 1 -1 15), competent cell transformation (see, e.g., Young and Spizizen, 1961 , J. Bacteriol. 81 : 823-829, or Dubnau and Davidoff-Abelson, 1971 , J. Mol. Biol. 56: 209-221 ), electroporation (see, e.g., Shigekawa and Dower, 1988, Biotechniques 6: 742-751 ), or conjugation (see, e.g., Koehler and Thorne, 1987, J. Bacteriol. 169: 5271 -5278). The introduction of DNA into an E. coli cell may be effected by protoplast transformation (see, e.g., Hanahan, 1983, J. Mol. Biol. 166: 557-580) or electroporation (see, e.g., Dower et al, 1988, Nucleic Acids Res. 16: 6127-6145). The introduction of DNA into a Streptomyces cell may be effected by protoplast transformation, electroporation (see, e.g., Gong et al., 2004, Folia Microbiol. {Praha) 49: 399-405), conjugation (see, e.g., Mazodier et al., 1989, J. Bacteriol. 171 : 3583-3585), or transduction (see, e.g., Burke et al., 2001 , Proc. Natl. Acad. Sci. USA 98: 6289-6294). The introduction of DNA into a Pseudomonas cell may be effected by electroporation (see, e.g., Choi et al., 2006, J. Microbiol. Methods 64: 391 -397) or conjugation (see, e.g., Pinedo and Smets, 2005, Appl. Environ. Microbiol. 71 : 51 -57). The introduction of DNA into a Streptococcus cell may be effected by natural competence (see, e.g., Perry and Kuramitsu, 1981 , Infect. Immun. 32: 1295-1297), protoplast transformation (see, e.g., Catt and Jollick, 1991 , Microbios 68: 189-207), electroporation (see, e.g., Buckley et al., 1999, Appl. Environ. Microbiol. 65: 3800-3804), or conjugation (see, e.g., Clewell, 1981 , Microbiol. Rev. 45: 409-436). However, any method known in the art for introducing DNA into a host cell can be used.
The host cell may be a yeast cell. "Yeast" as used herein includes ascosporogenous yeast (Endomycetales), basidiosporogenous yeast, and yeast belonging to the Fungi Imperfecti (Blastomycetes). Since the classification of yeast may change in the future, for the purposes of this invention, yeast shall be defined as described in Biology and Activities of Yeast (Skinner, Passmore, and Davenport, editors, Soc. App. Bacteriol. Symposium Series No. 9, 1980).
The yeast host cell may be a Candida, Hansenula, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowia cell, such as a Kluyveromyces lactis,
Saccharomyces carlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus,
Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomyces norbensis,
Saccharomyces oviformis, or Yarrowia lipolytica cell. Fermentation Broth Formulations or Cell Compositions
The present invention also relates to a fermentation broth formulation or a cell composition comprising a polypeptide of the present invention. The fermentation broth product further comprises additional ingredients used in the fermentation process, such as, for example, cells (including, the host cells containing the gene encoding the polypeptide of the present invention which are used to produce the polypeptide of interest), cell debris, biomass, fermentation media and/or fermentation products. In some embodiments, the composition is a cell-killed whole broth containing organic acid(s), killed cells and/or cell debris, and culture medium.
The term "fermentation broth" as used herein refers to a preparation produced by cellular fermentation that undergoes no or minimal recovery and/or purification. For example, fermentation broths are produced when microbial cultures are grown to saturation, incubated under carbon-limiting conditions to allow protein synthesis (e.g., expression of enzymes by host cells) and secretion into cell culture medium. The fermentation broth can contain unfractionated or fractionated contents of the fermentation materials derived at the end of the fermentation. Typically, the fermentation broth is unfractionated and comprises the spent culture medium and cell debris present after the microbial cells (e.g., filamentous fungal cells) are removed, e.g., by centrifugation. In some embodiments, the fermentation broth contains spent cell culture medium, extracellular enzymes, and viable and/or nonviable microbial cells.
In an embodiment, the fermentation broth formulation and cell compositions comprise a first organic acid component comprising at least one 1 -5 carbon organic acid and/or a salt thereof and a second organic acid component comprising at least one 6 or more carbon organic acid and/or a salt thereof. In a specific embodiment, the first organic acid component is acetic acid, formic acid, propionic acid, a salt thereof, or a mixture of two or more of the foregoing and the second organic acid component is benzoic acid, cyclohexanecarboxylic acid, 4-methylvaleric acid, phenylacetic acid, a salt thereof, or a mixture of two or more of the foregoing.
In one aspect, the composition contains an organic acid(s), and optionally further contains killed cells and/or cell debris. In one embodiment, the killed cells and/or cell debris are removed from a cell-killed whole broth to provide a composition that is free of these components.
The fermentation broth formulations or cell compositions may further comprise a preservative and/or anti-microbial (e.g., bacteriostatic) agent, including, but not limited to, sorbitol, sodium chloride, potassium sorbate, and others known in the art.
The cell-killed whole broth or composition may contain the unfractionated contents of the fermentation materials derived at the end of the fermentation. Typically, the cell-killed whole broth or composition contains the spent culture medium and cell debris present after the microbial cells (e.g., filamentous fungal cells) are grown to saturation, incubated under carbon- limiting conditions to allow protein synthesis. In some embodiments, the cell-killed whole broth or composition contains the spent cell culture medium, extracellular enzymes, and killed filamentous fungal cells. In some embodiments, the microbial cells present in the cell-killed whole broth or composition can be permeabilized and/or lysed using methods known in the art.
A whole broth or cell composition as described herein is typically a liquid, but may contain insoluble components, such as killed cells, cell debris, culture media components, and/or insoluble enzyme(s). In some embodiments, insoluble components may be removed to provide a clarified liquid composition.
The whole broth formulations and cell compositions of the present invention may be produced by a method described in WO 90/15861 or WO 2010/096673.
Enzyme Compositions
The present invention also relates to compositions comprising a polypeptide of the present invention. Preferably, the compositions are enriched in such a polypeptide. The term "enriched" indicates that the pullulanase activity of the composition has been increased, e.g., with an enrichment factor of at least 1 .1.
The compositions may comprise a polypeptide of the present invention as the major enzymatic component, e.g., a mono-component composition. Alternatively, the compositions may comprise multiple enzymatic activities, such as one or more (e.g., several) enzymes selected from the group consisting of hydrolase, isomerase, ligase, lyase, oxidoreductase, or transferase, e.g., an alpha-galactosidase, alpha-glucosidase, aminopeptidase, alpha-amylase, beta-amylase, beta-galactosidase, beta-glucosidase, beta-xylosidase, carbohydrase, carboxypeptidase, catalase, cellobiohydrolase, cellulase, chitinase, cutinase, cyclodextrin glycosyltransferase, deoxyribonuclease, endoglucanase, esterase, glucoamylase, invertase, laccase, lipase, mannosidase, mutanase, oxidase, pectinolytic enzyme, peroxidase, phytase, polyphenoloxidase, proteolytic enzyme, ribonuclease, transglutaminase, or xylanase. Preferably, additional enzyme(s) may be selected from the group consisting of an alpha amylase, glucoamylase, beta-amylase, cellulase (beta-glucosidase, cellobiohydrolase and endoglucanase), hemicellulase (e.g., xylanase), isoamylase, isomerase, lipase, phytase, protease, a further pullulanase, and/or other enzymes useful in a commercial process in conjunction with a pullulanase. Such enzymes are known in the art in starch processing, sugar conversion, fermentations for alcohol and other useful end-products, commercial detergents and cleaning aids, stain removal, fabric treatment or desizing, and the like.
The enzyme composition of the present invention may be in any form suitable for use, such as, for example, a crude fermentation broth with or without cells removed, a cell lysate with or without cellular debris, a semi-purified or purified enzyme composition, or a host cell, e.g., Trichoderma host cell, as a source of the enzymes. The enzyme composition may be a dry powder or granulate, a non-dusting granulate, a liquid, a stabilized liquid, or a stabilized protected enzyme. Liquid enzyme compositions may, for instance, be stabilized by adding stabilizers such as a sugar, a sugar alcohol or another polyol, and/or lactic acid or another organic acid according to established processes.
In one particular embodiment the composition further comprises a glucoamylase.
In one particular embodiment the composition further comprises an alpha-amylase.
The compositions may be prepared in accordance with methods known in the art and may be in the form of a liquid or a dry composition. The compositions may be stabilized in accordance with methods known in the art.
Examples are given below of preferred uses of the pullulanase and compositions of the present invention. The dosage of the composition and other conditions under which the composition is used may be determined on the basis of methods known in the art.
Methods of Using the Pullulanase Variants - Industrial Applications
The present invention is also directed to methods of using polypeptide of present invention in various industrial applications.
The polypeptide of the present invention may be used for starch processes, in particular starch conversion, especially liquefaction of starch (see, e.g., U.S. Patent No. 3,912,590, EP 252730 and EP 063909, WO 99/19467, and WO 96/28567, which are all hereby incorporated by reference). Also contemplated are compositions for starch conversion purposes, which may beside the polypeptide of the present invention also comprise a glucoamylase (AMG), and an alpha-amylase.
Further, the polypeptide of the present invention is particularly useful in the production of sweeteners and ethanol (see, e.g., U.S. Patent No. 5,231 ,017, which is hereby incorporated by reference), such as fuel, drinking and industrial ethanol, from starch or whole grains.
In one embodiment the present invention relates to a use of the polypeptide according to the invention for production of a syrup and/or a fermentation product from a starch containing material. The starch material may in one embodiment be gelatinized. In another embodiment the starch material is ungelatinized.
Starch Processing
Native starch consists of microscopic granules, which are insoluble in water at room temperature. When an aqueous starch slurry is heated, the granules swell and eventually burst, dispersing the starch molecules into the solution. At temperatures up to about 50°C to 75°C the swelling may be reversible. However, with higher temperatures an irreversible swelling called "gelatinization" begins. During this "gelatinization" process there is a dramatic increase in viscosity. Granular starch to be processed may be a highly refined starch quality, preferably at least 90%, at least 95%, at least 97% or at least 99.5% pure or it may be a more crude starch- containing materials comprising (e.g., milled) whole grains including non-starch fractions such as germ residues and fibers. The raw material, such as whole grains, may be reduced in particle size, e.g., by milling, in order to open up the structure and allowing for further processing. In dry milling whole kernels are milled and used. Wet milling gives a good separation of germ and meal (starch granules and protein) and is often applied at locations where the starch hydrolysate is used in the production of, e.g., syrups. Both dry and wet milling is well known in the art of starch processing and may be used in a process of the invention. Methods for reducing the particle size of the starch containing material are well known to those skilled in the art.
As the solids level is 30-40% in a typical industrial process, the starch has to be thinned or "liquefied" so that it can be suitably processed. This reduction in viscosity is primarily attained by enzymatic degradation in current commercial practice.
Liquefaction is carried out in the presence of an alpha-amylase, preferably a bacterial alpha-amylase and/or acid fungal alpha-amylase. In an embodiment, a phytase is also present during liquefaction. In an embodiment, a protease is also present during liquefaction. In an embodiment, viscosity reducing enzymes such as a xylanase and/or beta-glucanase is also present during liquefaction.
During liquefaction, the long-chained starch is degraded into branched and linear shorter units (maltodextrins) by an alpha-amylase. Liquefaction may be carried out as a three- step hot slurry process. The slurry is heated to between 60-95°C (e.g., 70-90°C, such as 77- 86°C, 80-85°C, 83-85°C) and an alpha-amylase is added to initiate liquefaction (thinning).
The slurry may in an embodiment be jet-cooked at between 95-140°C, e.g., 105-125°C, for about 1 -15 minutes, e.g., about 3-10 minutes, especially around 5 minutes. The slurry is then cooled to 60-95°C and more alpha-amylase is added to obtain final hydrolysis (secondary liquefaction). The jet-cooking process is carried out at pH 4.5-6.5, typically at a pH between 5 and 6. The alpha-amylase may be added as a single dose, e.g., before jet cooking.
The liquefaction process is carried out at between 70-95°C, such as 80-90°C, such as around 85°C, for about 10 minutes to 5 hours, typically for 1 -2 hours. The pH is between 4 and 7, such as between 5.5 and 6.2. In order to ensure optimal enzyme stability under these conditions, calcium may optionally be added (to provide 1 -60 ppm free calcium ions, such as about 40 ppm free calcium ions). After such treatment, the liquefied starch will typically have a "dextrose equivalent" (DE) of 10-15.
Generally liquefaction and liquefaction conditions are well known in the art.
Examples of alpha-amylase are disclosed in the "Alpha-Amylases" section below.
Saccharification may be carried out using conditions well-known in the art with a carbohydrate-source generating enzyme, in particular a glucoamylase, or a beta-amylase and optionally a debranching enzyme, such as an isoamylase or a pullulanase. For instance, a full saccharification step may last from about 24 to about 72 hours. However, it is common to do a pre-saccharification of typically 40-90 minutes at a temperature between 30-65°C, typically about 60°C, followed by complete saccharification during fermentation in a simultaneous saccharification and fermentation (SSF) process. Saccharification is typically carried out at a temperature in the range of 20-75°C, e.g., 25-65°C and 40-70°C, typically around 60°C, and at a pH between about 4 and 5, normally at about pH 4.5.
The saccharification and fermentation steps may be carried out either sequentially or simultaneously. In an embodiment, saccharification and fermentation are performed simultaneously (referred to as "SSF"). However, it is common to perform a pre-saccharification step for about 30 minutes to 2 hours (e.g., 30 to 90 minutes) at a temperature of 30 to 65°C, typically around 60°C which is followed by a complete saccharification during fermentation referred to as simultaneous saccharification and fermentation (SSF). The pH is usually between 4.2-4.8, e.g., pH 4.5. In a simultaneous saccharification and fermentation (SSF) process, there is no holding stage for saccharification, rather, the yeast and enzymes are added together.
In a typical saccharification process, maltodextrins produced during liquefaction are converted into dextrose by adding a glucoamylase and a debranching enzyme, such as an isoamylase (U.S. Patent No. 4,335,208) or a pullulanase. The temperature is lowered to around 60°C, prior to the addition of the glucoamylase and debranching enzyme. The saccharification process proceeds for 24-72 hours. Prior to addition of the saccharifying enzymes, the pH is reduced to below 4.5, while maintaining a high temperature (above 95°C), to inactivate the liquefying alpha-amylase. This process reduces the formation of short oligosaccharides called "panose precursors," which cannot be hydrolyzed properly by the debranching enzyme. Normally, about 0.2-0.5% of the saccharification product is the branched trisaccharide panose (Glc pa1 -6Glc pa1 -4Glc), which cannot be degraded by a pullulanase. If active amylase from the liquefaction remains present during saccharification (i.e., no denaturing), the amount of panose can be as high as 1 -2%, which is highly undesirable since it lowers the saccharification yield significantly.
Other fermentation products may be fermented at conditions and temperatures well known to persons skilled in the art, suitable for the fermenting organism in question.
The fermentation product may be recovered by methods well known in the art, e.g., by distillation.
In a particular embodiment, the process of the invention further comprises, prior to the conversion of a starch-containing material to sugars/dextrins the steps of:
(x) reducing the particle size of the starch-containing material; and
(y) forming a slurry comprising the starch-containing material and water.
In an embodiment, the starch-containing material is milled to reduce the particle size. In an embodiment the particle size is reduced to between 0.05-3.0 mm, preferably 0.1 -0.5 mm, or so that at least 30%, preferably at least 50%, more preferably at least 70%, even more preferably at least 90% of the starch-containing material fits through a sieve with a 0.05-3.0 mm screen, preferably 0.1 -0.5 mm screen. The aqueous slurry may contain from 10-55 wt. % dry solids (DS), preferably 25-45 wt. % dry solids (DS), more preferably 30-40 wt. % dry solids (DS) of starch-containing material.
Conventional starch-conversion processes, such as liquefaction and saccharification processes are described, e.g., in U.S. Patent No. 3,912,590, EP 252730 and EP 063909.
In an embodiment, the conversion process degrading starch to lower molecular weight carbohydrate components such as sugars or fat replacers includes a debranching step.
In the case of converting starch into a sugar, the starch is depolymerized. Such a depolymerization process consists of, e.g., a pre-treatment step and two or three consecutive process steps, i.e., a liquefaction process, a saccharification process, and depending on the desired end-product, an optional isomerization process.
When the desired final sugar product is, e.g., high fructose syrup the dextrose syrup may be converted into fructose. After the saccharification process, the pH is increased to a value in the range of 6-8, e.g., pH 7.5, and the calcium is removed by ion exchange. The dextrose syrup is then converted into high fructose syrup using, e.g., an immobilized glucose isomerase.
Production of Fermentation Products
Fermentable sugars (e.g., dextrins, monosaccharides, particularly glucose) are produced from enzymatic saccharification. These fermentable sugars may be further purified and/or converted to useful sugar products. In addition, the sugars may be used as a fermentation feedstock in a microbial fermentation process for producing end-products, such as alcohol (e.g., ethanol, and butanol), organic acids (e.g., succinic acid, 3-HP and lactic acid), sugar alcohols (e.g., glycerol), ascorbic acid intermediates (e.g., gluconate, 2-keto-D-gluconate, 2,5-diketo-D-gluconate, and 2-keto-L-gulonic acid), amino acids (e.g., lysine), proteins (e.g., antibodies and fragment thereof).
In an embodiment, the fermentable sugars obtained during the liquefaction process steps are used to produce alcohol and particularly ethanol. In ethanol production, an SSF process is commonly used wherein the saccharifying enzymes and fermenting organisms (e.g., yeast) are added together and then carried out at a temperature of 30-40°C.
The organism used in fermentation will depend on the desired end-product. Typically, if ethanol is the desired end product yeast will be used as the fermenting organism. In some preferred embodiments, the ethanol-producing microorganism is a yeast and specifically Saccharomyces such as strains of S. cerevisiae (U.S. Patent No. 4,316,956). A variety of S. cerevisiae are commercially available and these include but are not limited to FALI (Fleischmann's Yeast), SUPERSTART (Alltech), FERMIOL (DSM Specialties), RED STAR (Lesaffre) and Angel alcohol yeast (Angel Yeast Company, China). The amount of starter yeast employed in the methods is an amount effective to produce a commercially significant amount of ethanol in a suitable amount of time, (e.g., to produce at least 10% ethanol from a substrate having between 25-40% DS in less than 72 hours). Yeast cells are generally supplied in amounts of about 104 to about 1012, and preferably from about 107 to about 1010 viable yeast count per ml. of fermentation broth. After yeast is added to the mash, it is typically subjected to fermentation for about 24-96 hours, e.g., 35-60 hours. The temperature is between about 26- 34°C, typically at about 32°C, and the pH is from pH 3-6, e.g., around pH 4-5.
The fermentation may include, in addition to a fermenting microorganisms (e.g., yeast), nutrients, and additional enzymes, including phytases. The use of yeast in fermentation is well known in the art.
In further embodiments, use of appropriate fermenting microorganisms, as is known in the art, can result in fermentation end product including, e.g., glycerol, 1 ,3-propanediol, gluconate, 2-keto-D-gluconate, 2,5-diketo-D-gluconate, 2-keto-L-gulonic acid, succinic acid, lactic acid, amino acids, and derivatives thereof. More specifically when lactic acid is the desired end product, a Lactobacillus sp. (L case/') may be used; when glycerol or 1 ,3- propanediol are the desired end-products E. coli may be used; and when 2-keto-D-gluconate, 2,5-diketo-D-gluconate, and 2-keto-L-gulonic acid are the desired end products, Pantoea citrea may be used as the fermenting microorganism. The above enumerated list are only examples and one skilled in the art will be aware of a number of fermenting microorganisms that may be used to obtain a desired end product.
Processes for producing fermentation products from ungelatinized starch-containing material The invention relates to processes for producing fermentation products from starch- containing material without gelatinization (i.e., without cooking) of the starch-containing material (often referred to as a "raw starch hydrolysis" process). The fermentation product, such as ethanol, can be produced without liquefying the aqueous slurry containing the starch-containing material and water. In one embodiment a process of the invention includes saccharifying (e.g., milled) starch-containing material, e.g., granular starch, below the initial gelatinization temperature, preferably in the presence of alpha-amylase and/or carbohydrate-source generating enzyme(s), in particular a glucoamylase, to produce sugars that can be fermented into the fermentation product by a suitable fermenting organism. In this embodiment the desired fermentation product, e.g., ethanol, is produced from ungelatinized (i.e., uncooked), preferably milled, cereal grains, such as corn.
Accordingly, in one aspect the invention relates to processes for producing fermentation products from starch-containing material comprising simultaneously saccharifying and fermenting starch-containing material using a carbohydrate-source generating enzyme and a fermenting organism at a temperature below the initial gelatinization temperature of said starch- containing material. Saccharification and fermentation may also be separate. Thus in another aspect the invention relates to processes of producing fermentation products, comprising the following steps: (i) saccharifying a starch-containing material at a temperature below the initial gelatinization temperature; and
(ii) fermenting using a fermentation organism;
wherein step (i) is carried out using at least a glucoamylase, and a variant pullulanase according to the invention.
In one embodiment, an alpha amylase is added in step (i). In another embodiment steps (i) and (ii) are performed simultaneously. In a particular embodiment the fermenting organism is expressing the pullulanase of the invention or a pullulanase produced according to the method of the invention. The fermenting organism is in particular a yeast, more particularly a Candida, Hansenula, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowia species, such as a Kluyveromyces lactis, Saccharomyces carlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomyces norbensis, Saccharomyces oviformis, or Yarrowia lipolytica. Preferably, the yeast is Saccharomyces cerevisiae.
In one embodiment, a protease is also present. The protease may be any acid fungal protease or metalloprotease. The fermentation product, e.g., ethanol, may optionally be recovered after fermentation, e.g., by distillation. Typically amylase(s), such as glucoamylase(s) and/or other carbohydrate-source generating enzymes, and/or alpha-amylase(s), is(are) present during fermentation. Examples of glucoamylases and other carbohydrate-source generating enzymes include raw starch hydrolyzing glucoamylases. Examples of alpha- amylase(s) include acid alpha-amylases such as acid fungal alpha-amylases. The term "initial gelatinization temperature" means the lowest temperature at which starch gelatinization commences. In general, starch heated in water begins to gelatinize between about 50°C and 75°C; the exact temperature of gelatinization depends on the specific starch and can readily be determined by the skilled artisan. Thus, the initial gelatinization temperature may vary according to the plant species, to the particular variety of the plant species as well as with the growth conditions. In the context of this invention the initial gelatinization temperature of a given starch-containing material may be determined as the temperature at which birefringence is lost in 5% of the starch granules using the method described by Gorinstein and Lii, 1992, Starch/Starke 44(12): 461 -466. Before initiating the process a slurry of starch-containing material, such as granular starch, having 10-55 w/w % dry solids (DS), preferably 25-45 w/w % dry solids, more preferably 30-40 w/w % dry solids of starch-containing material may be prepared. The slurry may include water and/or process waters, such as stillage (backset), scrubber water, evaporator condensate or distillate, side-stripper water from distillation, or process water from other fermentation product plants. Because the process of the invention is carried out below the initial gelatinization temperature, and thus no significant viscosity increase takes place, high levels of stillage may be used if desired. In an embodiment the aqueous slurry contains from about 1 to about 70 vol. %, preferably 15-60 vol. %, especially from about 30 to 50 vol. % water and/or process waters, such as stillage (backset), scrubber water, evaporator condensate or distillate, side-stripper water from distillation, or process water from other fermentation product plants, or combinations thereof, or the like. The starch-containing material may be prepared by reducing the particle size, preferably by dry or wet milling, to 0.05 to 3.0 mm, preferably 0.1 -0.5 mm. After being subjected to a process of the invention at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or preferably at least 99% of the dry solids in the starch-containing material are converted into a soluble starch hydrolyzate. A process in this aspect of the invention is conducted at a temperature below the initial gelatinization temperature, which means that the temperature typically lies in the range between 30-75°C, preferably between 45-60°C. In a preferred embodiment the process carried at a temperature from 25°C to 40°C, such as from 28°C to 35°C, such as from 30°C to 34°C, preferably around 32°C. In an embodiment the process is carried out so that the sugar level, such as glucose level, is kept at a low level, such as below 6 w/w %, such as below about 3 w/w %, such as below about 2 w/w %, such as below about 1 w/w %., such as below about 0.5 w/w %, or below 0.25 w/w %, such as below about 0.1 w/w %. Such low levels of sugar can be accomplished by simply employing adjusted quantities of enzyme and fermenting organism. A skilled person in the art can easily determine which doses/quantities of enzyme and fermenting organism to use. The employed quantities of enzyme and fermenting organism may also be selected to maintain low concentrations of maltose in the fermentation broth. For instance, the maltose level may be kept below about 0.5 w/w %, such as below about 0.2 w/w %. The process of the invention may be carried out at a pH from about 3 and 7, preferably from pH 3.5 to 6, or more preferably from pH 4 to 5. In an embodiment fermentation is ongoing for 6 to 120 hours, in particular 24 to 96 hours.
Processes for producing fermentation products from gelatinized starch-containing material
In this aspect, the invention relates to processes for producing fermentation products, especially ethanol, from starch-containing material, which process includes a liquefaction step and sequentially or simultaneously performed saccharification and fermentation steps. Consequently, the invention relates to processes for producing fermentation products from starch-containing material comprising the steps of:
(a) liquefying starch-containing material in the presence of an alpha-amylase; or
(b) saccharifying the liquefied material obtained in step (a) using a glucoamylase;
(c) fermenting using a fermenting organism;
wherein step (a) and/or step (b) is carried out in the presence of a pullulanase according to the invention. In one embodiment saccharification and fermentation is carried out simultaneously.
In a particular embodiment the fermenting organism is expressing the pullulanase of the invention or a pullulanase produced according to the method of the invention. The fermenting organism is in particular a yeast, more particularly a Candida, Hansenula, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowia species, such as a Kluyveromyces lactis, Saccharomyces carlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomyces norbensis, Saccharomyces oviformis, or Yarrowia lipolytica. Preferably, the yeast is Saccharomyces cerevisiae.
In an embodiment, a protease, such as an acid fungal protease or a metallo protease is added before, during and/or after liquefaction. In an embodiment the metalloprotease is derived from a strain of Thermoascus, e.g., a strain of Thermoascus aurantiacus, especially Thermoascus aurantiacus CGMCC No. 0670. In another embodiment the protease is a bacterial protease, particularly a protease derived from a strain of Pyrococcus, more particularly from Pyrococcus furiosus disclosed in US 6,358,726.
In an embodiment the glucoamylase derived from a strain of Aspergillus, e.g., Aspergillus niger or Aspergillus awamori, a strain of Talaromyces, especially Talaromyces emersonii; or a strain of Athelia, especially Athelia rolfsii; a strain of Trametes, e.g., Trametes cingulata; a strain of the genus Gloeophyllum, e.g., a strain of Gloeophyllum sepiarum or Gloeophyllum trabeum; or a mixture thereof. Saccharification step (b) and fermentation step (c) may be carried out either sequentially or simultaneously. A protease may be added during saccharification and/or fermentation when the process is carried out as a sequential saccharification and fermentation process and before or during fermentation when steps (b) and (c) are carried out simultaneously (SSF process). The fermentation product, such as especially ethanol, may optionally be recovered after fermentation, e.g., by distillation. In a particular embodiment, the process of the invention further comprises, prior to step (a), the steps of:
x) reducing the particle size of the starch-containing material, preferably by milling
(e.g., using a hammer mill);
y) forming a slurry comprising the starch-containing material and water.
In an embodiment, the particle size is smaller than a # 7 screen, e.g., a # 6 screen. A # 7 screen is usually used in conventional prior art processes. The aqueous slurry may contain from 10-55, e.g., 25-45 and 30-40, w/w % dry solids (DS) of starch-containing material. The slurry is heated to above the gelatinization temperature and an alpha-amylase variant may be added to initiate liquefaction (thinning). The slurry may in an embodiment be jet-cooked to further gelatinize the slurry before being subjected to alpha-amylase in step (a). Liquefaction may in an embodiment be carried out as a three-step hot slurry process. The slurry is heated to between 60-95°C, preferably between 70-90°C, such as preferably between 80-85°C at pH 4-6, preferably 4.5-5.5, and alpha-amylase variant, optionally together with a pullulanase and/or protease, preferably metalloprotease, are added to initiate liquefaction (thinning). In an embodiment the slurry may then be jet-cooked at a temperature between 95-140°C, preferably 100-135°C, such as 105-125°C, for about 1 -15 minutes, preferably for about 3-10 minutes, especially around about 5 minutes. The slurry is cooled to 60-95°C and more alpha-amylase variant and optionally pullulanase variant and/or protease, preferably metalloprotease, is(are) added to finalize hydrolysis (secondary liquefaction). The liquefaction process is usually carried out at pH 4.0-6, in particular at a pH from 4.5 to 5.5. Saccharification step (b) may be carried out using conditions well known in the art. For instance, a full saccharification process may last up to from about 24 to about 72 hours, however, it is common only to do a pre-saccharification of typically 40-90 minutes at a temperature between 30-65°C, typically about 60°C, followed by complete saccharification during fermentation in a simultaneous saccharification and fermentation process (SSF process). Saccharification is typically carried out at temperatures from 20-75°C, preferably from 40-70°C, typically around 60°C, and at a pH between 4 and 5, normally at about pH 4.5. The most widely used process to produce a fermentation product, especially ethanol, is a simultaneous saccharification and fermentation (SSF) process, in which there is no holding stage for the saccharification, meaning that a fermenting organism, such as yeast, and enzyme(s), may be added together. SSF may typically be carried out at a temperature from 25°C to 40°C, such as from 28°C to 35°C, such as from 30°C to 34°C, preferably around about 32°C. In an embodiment fermentation is ongoing for 6 to 120 hours, in particular 24 to 96 hours. Starch-Containing Materials
Any suitable starch-containing starting material may be used in a process of the present invention. The starting material is generally selected based on the desired fermentation product. Examples of starch-containing starting materials, suitable for use in the processes of the present invention, include barley, beans, cassava, cereals, corn, milo, peas, potatoes, rice, rye, sago, sorghum, sweet potatoes, tapioca, wheat, and whole grains, or any mixture thereof. The starch-containing material may also be a waxy or non-waxy type of corn and barley. In a preferred embodiment the starch-containing material is corn. In a preferred embodiment the starch-containing material is wheat. Fermentation Products
The term "fermentation product" means a product produced by a method or process including fermenting using a fermenting organism. Fermentation products include alcohols (e.g., ethanol, methanol, butanol); organic acids (e.g., citric acid, acetic acid, itaconic acid, lactic acid, succinic acid, gluconic acid); ketones (e.g., acetone); amino acids (e.g., glutamic acid); gases (e.g., H2 and C02); antibiotics (e.g., penicillin and tetracycline); enzymes; vitamins (e.g., riboflavin, B12, beta-carotene); and hormones. In a preferred embodiment the fermentation product is ethanol, e.g., fuel ethanol; drinking ethanol, i.e., potable neutral spirits; or industrial ethanol or products used in the consumable alcohol industry (e.g., beer and wine), dairy industry (e.g., fermented dairy products), leather industry and tobacco industry. Preferred beer types comprise ales, stouts, porters, lagers, bitters, malt liquors, happoushu, high-alcohol beer, low-alcohol beer, low-calorie beer or light beer. In an preferred embodiment the fermentation product is ethanol.
Starch Slurry Processing with Stillage
Milled starch-containing material is combined with water and recycled thin-stillage resulting in an aqueous slurry. The slurry can comprise between 15 to 55% ds w/w (e.g., 20 to 50%, 25 to 50%, 25 to 45%, 25 to 40%, 20 to 35% and 30-36% ds). In some embodiments, the recycled thin-stillage (backset) is in the range of about 10 to 70% v/v (e.g., 10 to 60%, 10 to 50%, 10 to 40%, 10 to 30%, 10 to 20%, 20 to 60%, 20 to 50%, 20 to 40% and also 20 to 30%).
Once the milled starch-containing material is combined with water and backset, the pH is not adjusted in the slurry. Further the pH is not adjusted after the addition of a phytase and optionally an alpha-amylase to the slurry. In an embodiment, the pH of the slurry will be in the range of about pH 4.5 to less than about 6.0 (e.g., pH 4.5 to 5.8, pH 4.5 to 5.6, pH 4.8 to 5.8, pH 5.0 to 5.8, pH 5.0 to 5.4, pH 5.2 to 5.5 and pH 5.2 to 5.9). The pH of the slurry may be between about pH 4.5 and 5.2 depending on the amount of thin stillage added to the slurry and the type of material comprising the thin stillage. For example, the pH of the thin stillage may be between pH 3.8 and pH 4.5.
During ethanol production, acids can be added to lower the pH in the beer well, to reduce the risk of microbial contamination prior to distillation.
In some embodiments, a phytase is added to the slurry. In other embodiments, in addition to phytase, an alpha-amylase is added to the slurry. In some embodiments, a phytase and alpha-amylase are added to the slurry sequentially. In other embodiments, a phytase and alpha-amylase are added simultaneously. In some embodiments, the slurry comprising a phytase and optionally, an alpha-amylase, are incubated (pretreated) for a period of about 5 minutes to about 8 hours (e.g., 5 minutes to 6 hours, 5 minutes to 4 hours, 5 minutes to 2 hours, and 15 minutes to 4 hours). In other embodiments, the slurry is incubated at a temperature in the range of about 40 to 1 15°C (e.g., 45 to 80°C, 50 to 70°C, 50 to 75°C, 60 to 1 10°C, 60 to 95°C, 70 to 1 10°C, 70 to 85°C and 77 to 86°C).
In other embodiments, the slurry is incubated at a temperature of about 0 to about 30°C (e.g., 0 to 25°C, 0 to 20°C, 0 to 15°C, 0 to 10°C and 0 to 5°C) below the starch gelatinization temperature of the starch-containing material. In some embodiments, the temperature is below about 68°C, below about 65°C, below about 62°C, below about 60°C and below about 55°C. In some embodiments, the temperature is above about 45°C, above about 50°C, above about 55°C and above about 60°C. In some embodiments, the incubation of the slurry comprising a phytase and an alpha-amylase at a temperature below the starch gelatinization temperature is referred to as a primary (1 °) liquefaction. In one embodiment, the milled starch-containing material is corn or milo. The slurry comprises 25 to 40% DS, the pH is in the range of 4.8 to 5.2, and the slurry is incubated with a phytase and optionally an alpha-amylase for 5 minutes to 2 hours, at a temperature range of 60 to 75°C.
Currently, it is believed that commercially-available microbial alpha-amylases used in the liquefaction process are generally not stable enough to produce liquefied starch substrate from a dry mill process using whole ground grain at a temperature above about 80°C at a pH level that is less than pH 5.6. The stability of many commercially available alpha-amylases is reduced at a pH of less than about 4.0.
In a further liquefaction step, the incubated or pretreated starch-containing material is exposed to an increase in temperature such as about 0 to about 45°C above the starch gelatinization temperature of the starch-containing material (e.g., 70°C to 120°C, 70°C to 1 10°C, and 70°C to 90°C) for a period of time of about 2 minutes to about 6 hours (e.g., 2 minutes to 4 hours, 90 minutes, 140 minutes and 90 to 140 minutes) at a pH of about 4.0 to 5.5 more preferably between 1 hour to 2 hours. The temperature can be increased by a conventional high temperature jet cooking system for a short period of time, for example, for 1 to 15 minutes. Then the starch maybe further hydrolyzed at a temperature ranging from about 75°C to 95°C (e.g., 80°C to 90°C and 80°C to 85°C) for a period of about 15 to 150 minutes (e.g., 30 to 120 minutes). In a preferred embodiment, the pH is not adjusted during these process steps and the pH of the liquefied mash is in the range of about pH 4.0 to pH 5.8 (e.g., pH 4.5 to 5.8, pH 4.8 to 5.4, and pH 5.0 to 5.2). In some embodiments, a second dose of thermostable alpha-amylase is added to the secondary liquefaction step, but in other embodiments there is no additional dosage of alpha-amylase.
The incubation and liquefaction steps may be followed by saccharification and fermentation steps well known in the art.
Distillation
Optionally, following fermentation, an alcohol (e.g., ethanol) may be extracted by, for example, distillation and optionally followed by one or more process steps.
In some embodiments, the yield of ethanol produced by the methods provided herein is at least 8%, at least 10%, at least 12%, at least 14%, at least 15%, at least 16%, at least 17% and at least 18% (v/v) and at least 23% v/v. The ethanol obtained according to the process provided herein may be used as, for example, fuel ethanol, drinking ethanol, i.e., potable neutral spirits, or industrial ethanol.
By-Products
Left over from the fermentation is the grain, which is typically used for animal feed either in liquid or dried form. In further embodiments, the end product may include the fermentation co-products such as distiller's dried grains (DDG) and distiller's dried grain plus solubles (DDGS), which may be used, for example, as an animal feed.
Further details on how to carry out liquefaction, saccharification, fermentation, distillation, and recovery of ethanol are well known to the skilled person.
According to the process provided herein, the saccharification and fermentation may be carried out simultaneously or separately.
Fermenting Organisms
The term "fermenting organism" refers to any organism, including bacterial and fungal organisms, such as yeast and filamentous fungi, suitable for producing a desired fermentation product. Suitable fermenting organisms are able to ferment, i.e., convert, fermentable sugars, such as arabinose, fructose, glucose, maltose, mannose, or xylose, directly or indirectly into the desired fermentation product.
Examples of fermenting organisms include fungal organisms such as yeast. Preferred yeast include strains of Saccharomyces, in particular Saccharomyces cerevisiae or Saccharomyces uvarum; strains of Pichia, in particular Pichia stipitis such as Pichia stipitis CBS 5773 or Pichia pastoris; strains of Candida, in particular Candida arabinofermentans, Candida boidinii, Candida diddensii, Candida shehatae, Candida sonorensis, Candida tropicalis, or Candida utilis. Other fermenting organisms include strains of Hansenula, in particular Hansenula anomala or Hansenula polymorpha; strains of Kluyveromyces, in particular Kluyveromyces fragilis or Kluyveromyces marxianus; and strains of Schizosaccharomyces, in particular Schizosaccharomyces pombe.
Preferred bacterial fermenting organisms include strains of Escherichia, in particular Escherichia coli, strains of Zymomonas, in particular Zymomonas mobilis, strains of Zymobacter, in particular Zymobactor palmae, strains of Klebsiella in particular Klebsiella oxytoca, strains of Leuconostoc, in particular Leuconostoc mesenteroides, strains of Clostridium, in particular Clostridium butyricum, strains of Enterobacter, in particular Enterobacter aerogenes, and strains of Thermoanaerobacter, in particular Thermoanaerobacter BG1 L1 (Appl. Microbiol. Biotech. 77: 61 - 86), Thermoanarobacter ethanolicus, Thermoanaerobacter mathranii, or Thermoanaerobacter thermosaccharolyticum. Strains of Lactobacillus are also envisioned as are strains of Corynebacterium glutamicum R, Bacillus thermoglucosidaisus, and Geobacillus thermoglucosidasius.
In an embodiment, the fermenting organism is a C6 sugar fermenting organism, such as a strain of, e.g., Saccharomyces cerevisiae.
In an embodiment, the fermenting organism is a C5 sugar fermenting organism, such as a strain of, e.g., Saccharomyces cerevisiae. In one embodiment, the fermenting organism is added to the fermentation medium so that the viable fermenting organism, such as yeast, count per ml. of fermentation medium is in the range from 105 to 1012, preferably from 107 to 1010, especially about 5x107.
Yeast is the preferred fermenting organism for ethanol fermentation. Preferred are strains of Saccharomyces, especially strains of the species Saccharomyces cerevisiae, preferably strains which are resistant towards high levels of ethanol, i.e., up to, e.g., about 10, 12, 15 or 20 vol. % or more ethanol.
In an embodiment, the C5 utilizing yeast is a Saccharomyces cerevisea strain disclosed in WO 2004/085627.
In an embodiment, the fermenting organism is a C5 eukaryotic microbial cell concerned in
WO 2010/074577 (Nedalco).
In an embodiment, the fermenting organism is a transformed C5 eukaryotic cell capable of directly isomerize xylose to xylulose disclosed in US 2008/0014620.
In an embodiment, the fermenting organism is a C5 sugar fermentating cell disclosed in WO 2009/109633.
Commercially available yeast include LNF SA-1 , LNF BG-1 , LNF PE-2,and LNF CAT-1 (available from LNF Brazil), RED STAR™ and ETHANOL RED™ yeast (available from Fermentis/Lesaffre, USA), FALI (available from Fleischmann's Yeast, USA), SUPERSTART and THERMOSACC™ fresh yeast (available from Ethanol Technology, Wl, USA), BIOFERM AFT and XR (available from NABC - North American Bioproducts Corporation, GA, USA), GERT STRAND (available from Gert Strand AB, Sweden), and FERMIOL (available from DSM Specialties).
The fermenting organism capable of producing a desired fermentation product from fermentable sugars is preferably grown under precise conditions at a particular growth rate. When the fermenting organism is introduced into/added to the fermentation medium the inoculated fermenting organism pass through a number of stages. Initially growth does not occur. This period is referred to as the "lag phase" and may be considered a period of adaptation. During the next phase referred to as the "exponential phase" the growth rate gradually increases. After a period of maximum growth the rate ceases and the fermenting organism enters "stationary phase". After a further period of time the fermenting organism enters the "death phase" where the number of viable cells declines.
Fermentation
The fermentation conditions are determined based on, e.g., the kind of plant material, the available fermentable sugars, the fermenting organism(s) and/or the desired fermentation product. One skilled in the art can easily determine suitable fermentation conditions. The fermentation may be carried out at conventionally used conditions. Preferred fermentation processes are anaerobic processes.
For example, fermentations may be carried out at temperatures as high as 75°C, e.g., between 40-70°C, such as between 50-60°C. However, bacteria with a significantly lower temperature optimum down to around room temperature (around 20°C) are also known. Examples of suitable fermenting organisms can be found in the "Fermenting Organisms" section above.
For ethanol production using yeast, the fermentation may go on for 24 to 96 hours, in particular for 35 to 60 hours. In an embodiment the fermentation is carried out at a temperature between 20 to 40°C, preferably 26 to 34°C, in particular around 32°C. In an embodiment the pH is from pH 3 to 6, preferably around pH 4 to 5.
Other fermentation products may be fermented at temperatures known to the skilled person in the art to be suitable for the fermenting organism in question.
Fermentation is typically carried out at a pH in the range between 3 and 7, preferably from pH 3.5 to 6, such as around pH 5. Fermentations are typically ongoing for 6-96 hours.
The processes of the invention may be performed as a batch or as a continuous process. Fermentations may be conducted in an ultrafiltration system wherein the retentate is held under recirculation in the presence of solids, water, and the fermenting organism, and wherein the permeate is the desired fermentation product containing liquid. Equally contemplated are methods/processes conducted in continuous membrane reactors with ultrafiltration membranes and where the retentate is held under recirculation in presence of solids, water, and the fermenting organism(s) and where the permeate is the fermentation product containing liquid.
After fermentation the fermenting organism may be separated from the fermented slurry and recycled.
Fermentation Medium
The phrase "fermentation media" or "fermentation medium" refers to the environment in which fermentation is carried out and comprises the fermentation substrate, that is, the carbohydrate source that is metabolized by the fermenting organism(s).
The fermentation medium may comprise other nutrients and growth stimulator(s) for the fermenting organism(s). Nutrient and growth stimulators are widely used in the art of fermentation and include nitrogen sources, such as ammonia; vitamins and minerals, or combinations thereof.
Recovery
Subsequent to fermentation, the fermentation product may be separated from the fermentation medium. The fermentation medium may be distilled to extract the desired fermentation product or the desired fermentation product may be extracted from the fermentation medium by micro or membrane filtration techniques. Alternatively, the fermentation product may be recovered by stripping. Methods for recovery are well known in the art.
The invention is further described by the following numbered paragraphs:
[1 ]. A method of increasing pullulanase activity of a parent pullulanase comprising the steps: a) inserting an X25 domain within an X45 domain; and /or
b) adding a CBM41 domain either adjacent to the N-terminal of an X45 domain, or if a CBM41 domain is already present, adding an additional CBM41 domain adjacent to the N-terminal of the existing CBM41 or adjacent to the N-terminal of the X45 domain.
[2]. The method according to paragraph 1 , wherein the parent pullulanase is from a bacterium, particularly, a gram positive bacterium, more particularly a Bacillus sp. [3]. The method according to paragraph 2, wherein the Bacillus sp. parent pullulanase is selected from a Bacillus acidopullulyticus pullulanase, Bacillus deramificans pullulanase, or a hybrid pullulanase, e.g., a hybrid comprising an N-terminal part from a Bacillus acidopullulyticus pullulanase and a C-terminal part from a Bacillus deramificans pullulanase. [4]. The method according to any of the preceding paragraphs, wherein the X25 domain is selected from an X25 from a pullulanase, more particularly a pullulanase from a bacterium, particularly, a gram positive bacterium, more particularly a Bacillus sp.
[5]. The method according to paragraph 4, wherein the Bacillus sp. is selected from Bacillus acidopullulyticus or Bacillus deramificans.
[6]. The method according to any of paragraphs 1 -5, wherein the X25 domain is selected from the list consisting of SEQ ID NO:40, SEQ ID NO:41 , SEQ ID NO:42, SEQ ID NO:43, or an X25 domain having at least 75% identity to any of SEQ ID NO:40, SEQ ID NO:41 , SEQ ID NO:42, SEQ ID NO:43.
[7]. The method according to any of paragraphs 1 -6, wherein the X25 domain is inserted by replacing an existing X45 domain with an X45a-X25-X45b domain. [8]. The method according to any of paragraphs 1 -7, wherein the CBM41 domain is selected from a CBM41 domain from a pullulanase, more particularly a pullulanase from a bacterium, particularly, a gram positive bacterium, more particularly a Bacillus sp. [9]. The method according to paragraph 8, wherein the Bacillus sp. is selected from Bacillus acidopullulyticus or Bacillus deramificans.
[10]. The method according to any of the paragraphs 1 -9, wherein the parent pullulanase is selected from SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 1 1 , or SEQ ID NO: 12.
[1 1]. The method according to any of the preceding paragraphs, wherein the increase in pullulanase activity is measured as increased activity in the Phadebas assay at 65°C.
[12]. A polypeptide having pullulanase activity and obtained by the method according to any of the paragraphs 1 to 1 1.
[13]. The polypeptides according to paragraph 12, selected from the group consisting of the polypeptide of SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21 , SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27 or a polypeptide having at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the mature polypeptide of SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21 , SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26 or SEQ ID NO: 27, and wherein the polypeptide have pullulanase activity.
[14]. The polypeptide of any of paragraphs 12-13, comprising or consisting of SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21 , SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27.
[15]. A composition comprising the polypeptide of any of paragraphs 12-14.
[16]. The composition according to paragraph 15, further comprising a glucoamylase and/or an alpha-amylase.
[17]. A whole broth formulation or cell culture composition comprising a polypeptide of any of paragraphs 12-14.
[18]. A polynucleotide encoding the polypeptide of any of paragraphs 12-14. [19]. A use of a pullulanase of any of paragraphs 13-14 or produced according to the method of paragraphs 1 -1 1 for production of a syrup and/ or a fermentation product, e.g., ethanol, from a starch containing material. [20]. The use according to paragraph 19 wherein the starch material is gelatinized or un- gelatinized starch material.
[21]. A process of producing a fermentation product from starch-containing material comprising the steps of:
(a) liquefying starch-containing material in the presence of an alpha amylase;
(b) saccharifying the liquefied material in the presence of a glucoamylase; and
(c) fermenting with a fermenting organism; wherein step (a) and/or step (b) is carried out in the presence of a pullulanase of any of paragraphs 13-14 or a pullulanase produced according to the method of paragraphs 1 -1 1.
[22]. A process of producing a fermentation product from starch-containing material, comprising the steps of:
(a) saccharifying starch-containing material at a temperature below the initial gelatinization temperature of said starch-containing material; and
(b) fermenting with a fermenting organism
wherein step (a) is carried out using at least a glucoamylase, and a pullulanase of any of paragraphs 13-14 or a pullulanase produced according to the method of paragraphs 1 -1 1.
[23]. The process according to paragraphs 21 or 22, wherein saccharification and fermentation is carried out simultaneously.
[24]. The process according to any of the paragraphs 21 -23, wherein the fermentation product is alcohol, particularly ethanol. [25]. The process according to any of the paragraphs 21 -24, wherein the fermenting organism is expressing the pullulanase of any of paragraphs 13-14 or a pullulanase produced according to the method of paragraphs 1 -1 1 .
[26]. The process according to paragraph 25, wherein the fermenting organism is a yeast, particularly a Candida, Hansenula, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowia species, such as a Kluyveromyces lactis, Saccharomyces carlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomyces norbensis, Saccharomyces oviformis, or Yarrowia lipolytica.
[27]. The process according to paragraph 26, wherein the yeast is Saccharomyces cerevisiae.
The present invention is further described by the following examples.
Examples
EXAMPLE 1 : Description of parent pullulanases used in the examples.
The parent pullulanases used to illustrate the improvement of adding X25 or CBM41 domains according to the invention have been described in WO2015007639 and WO20151 10473.
These parent pullulanases are hybrid pullulanases (P6, P8, P190, P202) having improved properties, e.g., higher thermal activity and/or higher themo-stability. These hybrids were constructed by combining N-terminal fragments of a naturally occurring pullulanase from Bacillus acidopullulyticus termed GMM and disclosed as SEQ ID NO: 2 with C-terminal fragments of a naturally occurring truncated pullulanase from Bacillus deramificans termed ProD homolog and disclosed as SEQ ID NO: 4. SEQ ID NO: 2 is derived from Bacillus acidopullulyticus NCI MB 1 1639 described in EP 0063909 A1 as a pullulanase producer. The sequence of SEQ ID NO: 2 can be found in WO 2009/075682. The pullulanase of SEQ ID NO: 4 is derived from a Bacillus deramificans strain isolated from a humus sample collected in Denmark. Two hybrid pullulanases, P6 and P8, have been used as parent pullulanases in the present invention and the sequences are disclosed herein as SEQ ID NO: 9 and SEQ OID NO: 10 respectively. ProD homolog, SEQ ID NO: 4, has itself been used as a parent herein. Specific pullulanase variants of P8, termed P190 and P202, have also been used as parents to illustrate the improved effect according to the invention. P190 is disclosed in SEQ ID NO: 1 1 . P202 is disclosed in SEQ ID NO: 12.
EXAMPLE 2: Construction of chimera pullulanase variants
Genomic DNA from Bacillus subtilis strains harboring the gene encoding the P8 pullulanase (or P190 or P202) (SEQ ID NOs: 10, 1 1 , 12) and the gene encoding the ProD2 homolog pullulanase (SEQ ID NO: 4), and the genes encoding the pullulanase from Bacillus sp. NCBI 17841 (SEQ ID NO: 8) and Bacillus sp. NCBI1 17842 (SEQ ID NO: 6) under the control of a triple promoter system (as described in WO 99/43835) consisting of the promoters from Bacillus licheniformis alpha-amylase gene (amyl), Bacillus amyloliquefaciens alpha-amylase gene (amyQ), and the Bacillus thuringiensis crylllA promoter including stabilizing sequence, were isolated using NucleoSpin® Tissue kit [MACHEREY-NAGEL] according to its procedure. All chimera variants were constructed by multi fragments overlapping cloning using In-Fusion HD cloning kit (Clontech).
PCR amplifications were carried out using corresponding primer pairs which were designed to have at least 15bp overlapping with the neighboring fragments or a vector under the below conditions.
Phusion polymerase (thermo scientific)
Total 20μΙ
"Ι .ΟμΙ Template (I OOng/μΙ)
12.8μΙ H20
4μΙ Phusion HF Buffer
1 .6μΙ dNTP (2,5mM)
0.2μΙ Primers (20μΜ)
0.4μΙ Phusion (21Ι/μΙ)
PCR-program:
98°C/30sec
30x(98°C/10sec, 60°C/20sec, 72°C/3min)
72°C/5min
4°C/∞
PCR fragments were isolated in 0.7% agarose gel and recovered by Qiagen Gel extraction kit. The isolated PCR fragments with overlapping ends and a linearized E. coli/Bacillus subtilis shuttle vector (Clal Mlul digests) described in EXAMPLE1 in EP patent application no.10173848 were mixed and fused by In-Fusion HD cloning kit (Clontech) following to their manual. The In-Fusion mixture was transformed into E.coli DH 12S and the derived constructs were recovered from E.coli by Qiagen plasmid extraction kit (QIAGEN). Then, the plasmids were transformed into a suitable B. subtilis host and the gene coding pullulanase chimera variants were integrated into the B. subtilis genome by homologous recombination into the pectate lyase (pel) locus. The gene was expressed under the control of a triple promoter system consisting of the promoters from Bacillus licheniformis alpha-amylase gene (amyl), Bacillus amyloliquefaciens alpha-amylase gene (amyQ), and the Bacillus thuringiensis crylllA described in WO 99/43835. The gene coding for chloramphenicol acetyltransferase was used as marker as described in Diderichsen, B; Poulsen, G.B.; Joergensen, ST.; A useful cloning vector for Bacillus subtilis. Plasmid 30:312(1993). Chloramphenicol resistant clones were analyzed by DNA sequencing to verify the correct DNA sequences of the constructs. Clones having the correct sequences were selected and cultivated in 24 well micro titer plates containing TB-gly medium (13.3 g/L BactoTM Tryptone, 26.6 g/L BactoTM Yeast extract
D, 4.4 g/L Glycerol) supplemented with 6mg/L chroramphenicol.
To purify the pullulanase variants, shake flask or jar fermentations were carried out.
Primers with overlapping with the E. coli/Bacillus shuttle vector
In fusion fw Clal 48 mer
TTGCTTTTAGTTCATCGATAGCATCAGCAGATTCTACCTCGACAGAAG (SEQ ID NO:
64 )
In fusion rev Mlul 36 mer
TTATTGATTAACGCGTTTACTTTTTACCGTGGTCTG (SEQ ID NO: 65)
EXAMPLE 3: Design of chimeric pullulanase with extra domains
According to NCBI's CCD (conserved domain database, Marchler-Bauer et al, Nucleic acids research 201 1 , vol 39, D225-229), X25, X45 and CBM41 domains in pullulanases are described as follows:
X25 domain of Bacillus acidopullulyticus pullulanase and similar proteins.
Pullulanase (EC 3.2.1 .41 ) cleaves 1 ,6-alpha-glucosidic linkages in pullulan, amylopectin, and glycogen, and in alpha-and beta-amylase limit-dextrins of amylopectin and glycogen. BaPul is used industrially in the production of high fructose corn syrup, high maltose content syrups and low calorie and "light" beers. Pullulanases, in addition to the catalytic domain, include several carbohydrate-binding domains (CBMs) as well as domains of unknown function (termed "X" modules). X25 was identified in Bacillus acidopullulyticus pullulanase, and splits another domain of unknown function (X45). X25 is present in multiple copy in some pullulanases. It has been suggested that X25 and X45 are CBMs which target mixed alpha-1 ,6/alpha-1 ,4 linked D- glucan polysaccharides.
X45 domain of Bacillus acidopullulyticus pullulanase and similar proteins.
Pullulanase (EC 3.2.1 .41 ) cleaves 1 ,6-alpha-glucosidic linkages in pullulan, amylopectin, and glycogen, and in alpha-and beta-amylase limit-dextrins of amylopectin and glycogen. BaPul is used industrially in the production of high fructose corn syrup, high maltose content syrups and low calorie and "light" beers. Pullulanases, in addition to the catalytic domain, include several carbohydrate-binding domains (CBMs) as well as domains of unknown function (termed "X" modules). X45 was identified in Bacillus acidopullulyticus pullulanase, it is interrupted by another domain of unknown function (X25). It has been suggested that X25 and X45 are CBMs which target mixed alpha-1 ,6/alpha-1 ,4 linked D-glucan polysaccharides. Family 41 Carbohydrate-Binding Module from pullulanase-like enzymes
Pullulanases (EC 3.2.1 .41 ) are a group of starch-debranching enzymes, catalyzing the hydrolysis of the alpha-1 ,6-glucosidic linkages of alpha-glucans, preferentially pullulan. Pullulan is a polysaccharide in which alpha-1 ,4 linked maltotriosyl units are combined via an alpha-1 ,6 linkage. These enzymes are of importance in the starch industry, where they are used to hydrolyze amylopectin starch. Pullulanases consist of multiple distinct domains, including a catalytic domain belonging to the glycoside hydrolase (GH) family 13 and carbohydrate-binding modules (CBM), including CBM41 . Some pullulanases contain all these domains at their N-terminal and some lack either one or two or all of these domains.
New pullulanase chimera variants were designed to have an additional domain from other pullulanases.
i.e.
• Pullulanase which lacks CBM41
Original pullulanase: X45a-X25-X45b-CBM48-catalytic domain
CBM41 - X45a-X25-X45b-CBM48-catalytic domain
Example: ProD homolog -> P5
• Pullulanase which lacks X25
Original pullulanase: CBM41 -X45-CBM48-catalytic domain
CBM41 - X45a-X25-X45b-CBM48-catalytic domain
Only X25 from other pullulanases, or X25 together with X45ab, CBM41 or CBM48 were introduced.
Examples: P6^ P5
P8 -» P153, P154, P182, P183, P184, P185, P399 (P153 without LGQ triplet at the end of X45a domain) Ρ190→· P195, P196, P197, P198
P202^ P216
• Pullulanase which lacks X25
Original pullulanase: CBM41 -X45-CBM48-catalytic domain
CBM41 -CBM41 -X45-CBM48-catalytic
Example: P8^ P232 According to the above the following hybrid pullulanases were constructed.
Figure imgf000049_0001
P198 (SEQ No SEQ ID 2 SEQ ID 2 SEQ ID 6 SEQ ID 2 SEQ ID 2 SEQ ID 1 1 ID 25)
P202 (SEQ No SEQ ID 2 SEQ ID 2 No SEQ ID 2 SEQ ID 2 SEQ ID 12 ID 12)
P216 (SEQ SEQ ID 6 SEQ ID 6 SEQ ID 6 SEQ ID 6 SEQ ID 2 SEQ ID 12 ID 26)
EXAMPLE 4: Pullulanase assays
Determination of Pullulanase Activity (NPUN)
Endo-pullulanase activity in NPUN is measured relative to a Novozymes pullulanase standard, e.g., Novozym 26062 having a declared activity of 400 PUN/g. One pullulanase unit (NPUN) is defined as the amount of enzyme that releases 1 micro mol glucose per minute under the standard conditions (0.7% red pullulan (Megazyme), pH 5, 40° C, 20 minutes). The activity is measured in NPUN/ml using red pullulan.
1 ml diluted sample or standard is incubated at 40° C. for 2 minutes. 0.5 ml 2% red pullulan, 0.5 M KCI, 50 mM citric acid, pH 5 are added and mixed. The tubes are incubated at 40° C. for 20 minutes and stopped by adding 2.5 ml 80% ethanol. The tubes are left at room temperature for 10-60 minutes followed by centrifugation for 10 minutes at 4000 rpm. OD of the supernatants is then measured at 510 nm and the activity calculated using a standard curve.
Red-pullulan assay (Megazyme)
Substrate solution
0.1 g red-pullulan (megazyme S-RPUL)
0.75ml 2M sodium acetate, pH5
14.25ml H20
10 μΙ of enzyme samples were mixed with 80ul of substrate soln. and incubated at set temperatures (ex. 50, 55, 60°C) for 20min. 50 μΙ of ethanol was added to the reaction mixtures and centrifuge for 10min. at 3500rpm.
The supernatants were carefully taken out and the absorbance at A510 was read.
PAHBAH-pullulan assay
Substrate solution
0.15g BH4-pullulan
25ml 50mM Na acetate buffer, pH5
PAHBAH solution
0.0552g Bismuth (lll)-acetate 0.2g PAH BAH
0.5g Potassium sodium tartrate, tetrahydrate
10ml 500mM NaOH
Ten μΙ of enzyme samples were mixed with 1 10μΙ of substrate soln. and incubated at set temperatures (50°C, 55°C, 60°C or 65°C) for 20min. 40 μΙ of PAH BAH solution was added to the reaction mixtures, incubated for another 20min at 50°C and the absorbance at A405 was read.
PAHBAH-Sweet potato starch assay
Substrate solution
0.125g Sweet potato starch (Satsumayawaragi from NIHON STARCH CO., LTD)
25ml 50mM Na acetate buffer, pH5
PAHBAH solution
0.0552g Bismuth (lll)-acetate
0.2g PAHBAH
0.5g Potassium sodium tartrate, tetrahydrate
10ml 500mM NaOH
Ten μΙ of enzyme samples were mixed with 1 10μΙ of substrate soln. and incubated at set temperatures (50°C) for 20min. 40 μΙ of PAHBAH solution was added to the reaction mixtures, incubated for another 20min at 50°C and the absorbance at A405 was read.
PAHBAH -maltodextrin (DE3) assay
Substrate solution
0.125 g maltodextrin (pindex100 or pinedex from MATSUTANI chemical industry Co., Ltd.) 25ml 50mM Na acetate buffer, pH5 (or pH4.3)
PAHBAH solution
0.0552g Bismuth (lll)-acetate
0.2g PAHBAH
0.5g Potassium sodium tartrate, tetrahydrate
10ml 500mM NaOH
10 μΙ of enzyme samples were mixed with 1 10μΙ of substrate soln. and incubated at set temperatures (50°C, 60°C or 65°C) for 20min. 40 μΙ of PAHBAH solution was added to the reaction mixtures, incubated for another 20min at 50°C and the absorbance at A405 was read.
PHADEBAS assay
Substrate solution.
1 tablet of PHADEBAS alpha-amylase tablet (dyed amylopectin)
5 ml 50 mM Na acetate buffer, pH 5 40 sec. microwave oven up to boiling
Stop solution.
18% acetic acid
Assay method
Enzyme reaction in 96 well PCR plate
10 μ I of enzyme samples were mixed with 10ΟμΙ of substrate solution and incubated at set temperatures (ex. 55, 60, 65°C) for 20-60min. 50 μΙ of stop solution was added to the reaction mixtures and centrifuge for 10min. at 3500rpm. The supernatants were carefully taken out and the absorbance at A600 was read.
PAHBAH- NaBH4 treated-maltodextrin (DE1 1 ) assay
Substrate, DE1 1 , was prepared according to the method described in EXAMPLE 1 in US 4335208;
100 kg of corn starch (containing 100 ppm Ca 2+ and the volume adjusted to 225 liters with water). The pH was adjusted to 6.3 and 135 g of TERMAMYL (Novozymes A/S) (a thermostable alpha-amylase from Bacillus licheniformis) was added. This suspension was continuously pumped through a jet cooker (Hydro-Thermal Corp. Milwaukee) where it was heated to 105°C and maintained at 105°C. Starch suspension was flash-cooled and pumped over into a saccharification tank where it was held for 1 hour at 95°C
The pH of the liquefied starch was adjusted to 4.5 at 95°C the reaction and the batch was then spray-dried without purification. The DE of the spray-dried maltodextrin was 1 1.
DE1 1 maltodextrin was then reduced by NaBH4 as described below:
20 g of DE1 1 maltodextrin was dissolved in 270 ml Milli-Q water with heating and sodium borohydride solution was added into the solution. 6 ml of acetone was added into the solution and then neutralize by adding acetic acid to approx. pH 7. 600ml of ethanol was added and centrifuged at 8,000 rpm for 30min to precipitate the material. The supernatant was discarded and the precipitate was dried up by vacuum drying. Substrate solution
0.125g NaBH4 treated maltodextrin DE1 1
25 ml 50mM Na acetate buffer, pH 4.3
PAHBAH solution
0.0552g Bismuth (lll)-acetate
0.2g PAHBAH
0.5g Potassium sodium tartrate, tetrahydrate
10ml 500mM NaOH 10 μΙ of enzyme samples were mixed with 1 10μΙ of substrate solution and incubated at set temperatures (50°C, 60°C or 65°C) for 20min. 40 μΙ of PAH BAH solution was added to the reaction mixtures, incubated for another 20min at 50°C and the absorbance at A405 was read. EXAMPLE 4: Ratio between activity toward starch-related substrates and pullulan
Activity unit toward pullulan in culture supernatants of chimera pullulanase variants was determined by red-pullulan assay using commercial Pullulanase product, NovozymR 26062, as an enzyme standard (400 PUN(G)/g). The activities were adjusted to 1 PUN(G)/g and then activities toward sweet potato and phadebas were determined by PAHBAH-sweet potato starch assay and Phadebas assay.
Relative activity of sweet potato starch degradation/PUNG at 50°C (P8 as 100%)
P8 100%
P154 170%
P184 134%
Relative activity of sweet potato starch degradation/PUNG at 50°C (ProD homolog as 100%)
ProD homolog 100%
P5 140%
Relative activity of sweet potato starch degradation/PUNG at 65 C (P8 as 100%)
P8 100%
P152 140%
Relative activity of phadebas degradation/PUNG at 65°C (P190 as 100%)
P190 100%
P195 1 18%
P196 107%
P197 122%
P198 120%
Relative activity of phadebas degradation/PUNG at 65C (P8 as 100%)
P8 100%
P153 160%
P154 140%
P182 151 %
P183 103% P184 138%
P185 104%
Relative activity of phadebas degradation/PUNG at 60°C (ProD homolog as 100%)
ProD homolog 100%
P5 127%
These new chimeras having extra domain were confirmed to have higher activity toward starch substrates.
EXAMPLE 5: Activity toward starch-related substrates after partial-purification
Bacillus culture supernatants were concentrated by Amicon ultra 50K centrifuge filters (Millipore) and ran in SDS-PAGE. The gels were stained with Coomassie Fluor Orange (BIO- RAD) and pullulanases protein amounts were quantified by Gel Doc EZ (BIO-RAD). Enzyme solutions were adjusted to 0.1 mg/ml in 100mM sodium acetate buffer, pH5, containing 0.01 % tween20 to measure activities by Phadebas assay and PAHBAH-sweet potato starch assay.
Relative specific activity toward Phadebas and sweet potato starch at 60°C
(P190 as 100%)
Phadebas Sweet potato starch
P190 100% 100%
P195 174% 144%
P196 192% 141 %
P197 198% 135%
P198 163% 124%
Relative specific activity toward Phadebas and Sweet potato starch at 60 and 65°C
(P202 as 100%)
Sweet potato starch Phadebas
60°C 65°C 60°C 65°C
P202 100% 100% 100% 100%
P216 102% 106% 1 14% 125%
Relative specific activity toward sweet potato starch at 60 and 65C
(P8 as 100%)
Sweet potato starch
60°C 65°C
P8 100% 100% P154 128% 121 %
P232 163% 142%
Relative specific activity toward Phadebas at 60 and 65°C
(P8 as 100%)
60°C 65°C
P8 100% 100%
P153 105% 121 %
P399 128% 151 %
These new chimeras having extra domain were confirmed to have higher activity toward starch substrates.
EXAMPLE 6: Fermentation of Bacillus strains
B. sutilis strains were fermented on a rotary shaking table in 500 ml baffled flasks containing 100ml TB-gly with 6mg/L chloramphenicol at 220rpm, 37°C. The culture was centrifuged (20000 x g, 20min) and the supernatants were carefully decanted from the precipitates. The supernatants were filtered through a 0.45um filter unit to remove the rest of the Bacillus host cells.
EXAMPLE 7: Purification of pullulanases
Purification of pullulanases was carried out by β-cyclodextrin affinity column and followed by anion exchange column chromatography. After purification, pullulanases were dialyzed against 20 mM sodium acetate buffer (pH 5.5) and concentrated. Enzyme concentrations were determined by A280 value and efficiency from amino acid sequence.
EXAMPLE 8: Dose-response of chimera variants
Dose-response curves of pullulanase variants toward various starch substrates were taken using purified enzymes in PAHBAH-sweet potato starch and maltodextrins.
Newly constructed chimera pullulanases showed higher A405 values than their control pullulanases.
Sweet potato starch pH 5 50°C (P202 vs. P216)
Figure imgf000055_0001
0.2 0.205722 0.233772
0.1 0.173672 0.219022
0.05 0.125122 0.153272
0.02 0.087022 0.100922
0.01 0.051672 0.055822
Sweet potato starch pH 5 50°C (P8, P6 vs P5)
Figure imgf000056_0001
Maltodextrin (DE3) pH 5, 50°C (P8, P202 vs. P216 )
Figure imgf000056_0002
Maltodextrin (DE1 1 ) pH 4.3, 65°C (P6 vs. P5 )
Figure imgf000056_0003
Figure imgf000057_0001
Maltodextrin (DE1 1 ) pH 4.3, 65°C (P8 vs. P153, P184)
Figure imgf000057_0002
The invention described and claimed herein is not to be limited in scope by the specific aspects herein disclosed, since these aspects are intended as illustrations of several aspects of the invention. Any equivalent aspects are intended to be within the scope of this invention. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims. In the case of conflict, the present disclosure including definitions will control.

Claims

Claims
1 . A method of increasing pullulanase activity of a parent pullulanase comprising the steps: a) inserting an X25 domain within an X45 domain; and /or
b) adding a CBM41 domain either adjacent to the N-terminal of an X45 domain, or if a CBM41 domain is already present, adding an additional CBM41 domain adjacent to the N-terminal of the existing CBM41 or adjacent to the N-terminal of the X45 domain.
2. The method according to claim 1 , wherein the parent pullulanase is from a bacterium, particularly, a gram positive bacterium, more particularly a Bacillus sp.
3. The method according to claim 2, wherein the Bacillus sp. parent pullulanase is selected from a Bacillus acidopullulyticus pullulanase, Bacillus deramificans pullulanase, or a hybrid pullulanase, e.g., a hybrid comprising an N-terminal part from a Bacillus acidopullulyticus pullulanase and a C-terminal part from a Bacillus deramificans pullulanase.
4. The method according to any of the preceding claims, wherein the X25 domain is selected from an X25 from a pullulanase, more particularly a pullulanase from a bacterium, particularly, a gram positive bacterium, more particularly a Bacillus sp.
5. The method according to claim 4, wherein the Bacillus sp. is selected from Bacillus acidopullulyticus or Bacillus deramificans.
6. The method according to any of claims 1 -5, wherein the X25 domain is selected from the list consisting of SEQ ID NO:40, SEQ ID NO:41 , SEQ ID NO:42, SEQ ID NO:43, or an X25 domain having at least 75% identity to any of SEQ ID NO:40, SEQ ID NO:41 , SEQ ID NO:42, SEQ ID NO:43.
7. The method according to any of claims 1 -6, wherein the X25 domain is inserted by replacing an existing X45 domain with an X45a-X25-X45b domain.
8. The method according to any of claims 1 -7, wherein the CBM41 domain is selected from a CBM41 domain from a pullulanase, more particularly a pullulanase from a bacterium, particularly, a gram positive bacterium, more particularly a Bacillus sp.
9. The method according to claim 8, wherein the Bacillus sp. is selected from Bacillus acidopullulyticus or Bacillus deramificans.
10. The method according to any of the claims 1-9, wherein the parent pullulanase is selected from SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 1 1 , or SEQ ID NO: 12.
1 1 . The method according to any of the preceding claims, wherein the increase in pullulanase activity is measured as increased activity in the Phadebas assay at 65°C.
12. A polypeptide having pullulanase activity and obtained by the method according to any of the claims 1 to 1 1 .
13. The polypeptides according to claim 12, selected from the group consisting of the polypeptide of SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ I D NO:
19, SEQ ID NO: 20, SEQ ID NO: 21 , SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27 or a polypeptide having at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the mature polypeptide of SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21 , SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26 or SEQ ID NO: 27, and wherein the polypeptide have pullulanase activity.
14. The polypeptide of any of claims 12-13, comprising or consisting of SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21 ,
SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27.
15. A composition comprising the polypeptide of any of claims 12-14.
16. The composition according to claim 15, further comprising a glucoamylase and/or an alpha-amylase.
17. A whole broth formulation or cell culture composition comprising a polypeptide of any of claims 12-14.
18. A polynucleotide encoding the polypeptide of any of claims 12-14.
19. A use of a pullulanase of any of claims 13-14 or produced according to the method of claims 1 -1 1 for production of a syrup and/ or a fermentation product, e.g., ethanol, from a starch containing material.
20. The use according to claim 19 wherein the starch material is gelatinized or un-gelatinized starch material.
21 . A process of producing a fermentation product from starch-containing material comprising the steps of:
(a) liquefying starch-containing material in the presence of an alpha amylase;
(b) saccharifying the liquefied material in the presence of a glucoamylase; and
(c) fermenting with a fermenting organism; wherein step (a) and/or step (b) is carried out in the presence of a pullulanase of any of claims 13-14 or a pullulanase produced according to the method of claims 1 -1 1 .
22. A process of producing a fermentation product from starch-containing material, comprising the steps of:
(a) saccharifying starch-containing material at a temperature below the initial gelatinization temperature of said starch-containing material; and
(b) fermenting with a fermenting organism
wherein step (a) is carried out using at least a glucoamylase, and a pullulanase of any of claims 13-14 or a pullulanase produced according to the method of claims 1 -1 1 .
23. The process according to claims 21 or 22, wherein saccharification and fermentation is carried out simultaneously.
24. The process according to any of the claims 21 -23, wherein the fermentation product is alcohol, particularly ethanol.
25. The process according to any of the claims 21 -24, wherein the fermenting organism is expressing the pullulanase of any of claims 13-14 or a pullulanase produced according to the method of claims 1 -1 1 .
26. The process according to claim 25, wherein the fermenting organism is a yeast, particularly a Candida, Hansenula, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowia species, such as a Kluyveromyces lactis, Saccharomyces carlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomyces norbensis, Saccharomyces oviformis, or Yarrowia lipolytics.
27. The process according to claim 26, wherein the yeast is Saccharomyces cerevisiae.
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