CN111363734B

CN111363734B - Lipase mutant, composition and application thereof

Info

Publication number: CN111363734B
Application number: CN201811590902.XA
Authority: CN
Inventors: 戴小军; 周美凤; 徐正军; 牛其文
Original assignee: Wilmar Shanghai Biotechnology Research and Development Center Co Ltd
Current assignee: Wilmar Shanghai Biotechnology Research and Development Center Co Ltd
Priority date: 2018-12-25
Filing date: 2018-12-25
Publication date: 2024-08-09
Anticipated expiration: 2038-12-25
Also published as: CN111363734A

Abstract

Lipase mutants, compositions and uses thereof are provided. The lipase mutant is polypeptide which is derived from SEQ ID NO. 2, has lipase activity and is obtained by substituting, deleting and/or adding two or more amino acids in the amino acid sequence defined by SEQ ID NO. 2, wherein the two or more amino acid substitutions, deletions and/or additions comprise amino acid substitutions at 11 th and 167 th positions of the SEQ ID NO. 2. Compared with a wild type sequence, the lipase mutant has improved specific activity, methyl ester hydrolysis performance, oleic acid glycerol esterification performance and/or biodiesel application performance.

Description

Lipase mutant, composition and application thereof

Technical Field

The invention relates to lipase mutants, compositions and applications thereof.

Background

Rhizomucor miehei lipase (Rhizomucor MIEHEI LIPASE, RML) has wide industrial application. The enzyme wild type has a 24 amino acid signal peptide, a 70 amino acid leader peptide and a 269 amino acid mature peptide. Catalytic sites (Ser 144, asp203 and His 257), his143 and Thr28 with stabilizing effect on the catalytic sites; the cap region is 82-96 amino acids, and the sites related to cap chargeability are Arg86, asp91 and Arg80. The three pairs of disulfide bonds are 29-268, 40-43 and 235-244, respectively. There are references to 29-33, 39-41, 56-61, 80-97, 108-114, 42-145, 174-176, 201-215 and 245-269 in the structure of RML lipase as the contact region of the fat substrate with the enzyme (WO 1997/0074202 A1).

RML can be used for production of structural fat OPO and the like, hydrolysis of medium-length fatty acid methyl ester C8/C10, enzymatic synthesis of biodiesel, hydrolysis of acidified oil and the like. RML mutants that are more stable or have higher specific activity are of great value for industrial applications.

Disclosure of Invention

The invention adopts a random mutation mode to combine and obtain a lipase mutant, combines a solid-blue method thermal stability screening to obtain a mutant, and obtains a RML mutant with improved characteristics through sequencing, cloning, recombinant expression verification and the like.

Specifically, the invention provides an isolated polypeptide which is derived from SEQ ID NO. 2, wherein one or more amino acids are substituted, deleted and/or added in the amino acid sequence defined in SEQ ID NO. 2 and has lipase activity, and the one or more amino acid substitutions, deletions and/or additions at least comprise amino acid substitutions, deletions and/or additions at positions 11 and 167 of SEQ ID NO. 2.

In one or more embodiments, the polypeptide further comprises one or more amino acid substitutions, deletions and/or additions at any one or more of positions 19, 47, 90, 156 and 232 of SEQ ID NO. 2.

In one or more embodiments, the amino acid substitution at position 11 of SEQ ID NO.2 is an S substitution of G, H, I, L, P, Q, R or T; p is preferred.

In one or more embodiments, the amino acid substitution at position 19 of SEQ ID NO. 2 is a Y substitution of C, E, H, L, W or T; h is preferred.

In one or more embodiments, the amino acid substitution at position 47 of SEQ ID NO. 2 is an E substitution of A, C, D, G, K, R, S, T or V; g is preferred.

In one or more embodiments, the amino acid substitution occurring at position 90 of SEQ ID NO.2 is an A substitution of D, F, G, L, S, T, V or W; s is preferred.

In one or more embodiments, the amino acid substitution at position 156 of SEQ ID NO. 2 is a G substitution A, C, D, F, G, L, P or V; d is preferred.

In one or more embodiments, the amino acid substitution at position 167 of SEQ ID NO. 2 is an S substitution A, C, G or W; g is preferred.

In one or more embodiments, the amino acid substitution at position 232 of SEQ ID NO. 2 is V substitution H, I, P, R or S; preferably I.

In one or more embodiments, the isolated polypeptide is selected from the group consisting of:

(a) The amino acid sequence shown in SEQ ID NO. 4; and

(B) A polypeptide derived from SEQ ID NO. 4, which has lipase activity and is substituted, deleted and/or added with one or more amino acids in the amino acid sequence defined in SEQ ID NO. 4.

In one or more embodiments, the one or more amino acid substitutions, deletions and/or additions described in (a) occur at any one or more of positions 19, 47, 90, 156 and 232 of SEQ ID NO. 4.

In one or more embodiments, the substitution pattern of the amino acids occurring at positions 19, 47, 90, 156 and 232 of SEQ ID NO. 4 is as described in any of the previous embodiments.

In one or more embodiments, the amino acid sequence of the polypeptide is as shown in SEQ ID NO. 6, 8 or 12.

The invention also provides the coding sequences of the isolated polypeptides described herein and their complements. In certain embodiments, the coding sequence is set forth in SEQ ID NO. 3,5 or 7.

The invention also provides nucleic acid constructs comprising the coding sequences or the complements thereof. In certain embodiments, the nucleic acid construct is a vector, such as an expression vector or a cloning vector.

The invention also provides host cells comprising the coding sequences described herein.

The present invention also provides a method of producing a polypeptide described herein, comprising:

(i) Culturing a host cell described herein under conditions suitable for expression of the polypeptide; and

(Ii) Recovering the polypeptide.

The invention also provides a method of hydrolyzing a lipase substrate comprising contacting the substrate with a polypeptide as described in any of the embodiments herein.

The invention also provides a composition comprising a polypeptide as described in any of the embodiments herein.

The invention also provides a method of cleaning comprising contacting a surface or article to be cleaned with a polypeptide or composition thereof as described in any of the embodiments herein.

The invention also provides the polypeptides, coding sequences thereof, nucleic acid constructs and host cells comprising the coding sequences and the use of the compositions of any of the embodiments herein in the production of structural lipids (e.g., OPO), the hydrolysis of medium length fatty acid methyl esters (e.g., C8 fatty acids, C10 fatty acids), the enzymatic synthesis of biodiesel and the hydrolysis of acidified oils.

Drawings

Fig. 1: experimental procedures and results were obtained for mutant R9.

Fig. 2: random mutation was performed and colonies with large hydrolysis circles were selected on olive oil plates, and then these colonies were subjected to a further round of thermal stability plate screening, the results obtained. Wherein WT represents a strain containing an unmutated RML gene, R9 represents an R9 strain, and numerals 1 to 15 represent different colonies, respectively.

Detailed Description

It is understood that within the scope of the present invention, the above-described technical features of the present invention and technical features specifically described below (e.g., in the examples) may be combined with each other to constitute a preferred technical solution.

Herein, the term "lipase" refers to an enzyme in the class ec3.1.1 as defined by the enzyme nomenclature. It may have lipase activity (triacylglycerol lipase, ec 3.1.1.3), cutinase activity (ec 3.1.1.74), sterol esterase activity (ec 3.1.1.13) and/or wax ester hydrolase activity (ec 3.1.1.50).

The isolated polypeptide described herein refers to a polypeptide obtained by mutating the amino acid sequence shown in SEQ ID NO. 2. Herein, "isolated" means a form or substance that does not exist in nature. Non-limiting examples of isolated substances include any non-naturally occurring substance and any substance that is at least partially removed from one or more or all of the naturally occurring components associated with it in nature, including but not limited to any enzyme, variant, nucleic acid, protein, peptide, or cofactor.

Herein, mutation includes substitution, deletion, and addition. Substitution means that an amino acid occupying a position is replaced with a different amino acid; deletion means the removal of an amino acid occupying a position; and adding (or inserting) means adding an amino acid immediately after the amino acid that occupies a position.

Herein, the medium carbon chain fatty acid refers to fatty acid with 6-12 carbon atoms on a carbon chain, and mainly refers to caprylic acid and capric acid; long carbon chain fatty acids refer to fatty acids having more than 12 carbon atoms in the carbon chain.

Here, the mutations in the amino acid sequence shown in SEQ ID NO.2 include at least mutations, preferably substitution mutations, occurring at positions 11 and 167. In certain embodiments, the mutation at position 11 is a wild-type S mutation to proline or an amino acid residue belonging to the class of nonpolar hydrophobic amino acids with proline, such as alanine, phenylalanine, tryptophan, methionine, leucine, isoleucine and valine; preferably, the method comprises the steps of. The mutation is S to proline. Substitution at position 167 may be an S substitution to glycine or to an amino acid residue of the class of non-charged amino acids of the same genus as glycine, such as threonine, cysteine, tyrosine, glutamine and asparagine, or to alanine, threonine and methionine of the class of small amino acids of the same genus as glycine; preferably, the mutation is a mutation of S to glycine. An example of a mutant having substitution mutations at both positions 11 and 167 is shown in SEQ ID NO. 12.

Further, in certain embodiments, the mutation in the amino acid sequence shown in SEQ ID NO. 2 includes, in addition to the amino acid mutations at positions 11 and 167, a mutation that occurs at any one or more positions selected from positions 19, 47, 90, 156 and 232 of SEQ ID NO. 2. Preferably, the mutation is an amino acid substitution. Preferably, the amino acid substitution occurring at position 19 of SEQ ID NO. 2 is a Y substitution of C, E, H, L, W, R or T; or substituted with amino acids other than histidine, lysine and arginine to help further provide the enzymatic activity of the mutant. Preferably, the amino acid substitution at position 47 of SEQ ID NO. 2 is an E substitution A, C, D, G, K, R, S, T or V; preferably, the amino acid substitution occurring at position 90 of SEQ ID NO. 2 is an A substitution D, F, G, L, S, T, V or W; preferably, the amino acid substitution occurring at position 156 of SEQ ID NO. 2 is a G substitution A, C, D, F, G, L, P or V; preferably, the amino acid substitution occurring at position 232 of SEQ ID NO. 2 is a V substitution H, I, P, R or S.

Thus, in certain embodiments, the isolated polypeptides described herein are SEQ ID NO:2 having amino acid mutations at positions 11, 19 and 167, preferably the amino acid substitutions described previously, more preferably S11P, Y H and S167G. An exemplary amino acid sequence is shown in SEQ ID NO. 4. Compared with wild type SEQ ID NO. 2, the mutant has improved specific activity, medium-length fatty acid methyl ester hydrolysis capability and fatty acid glyceride esterification capability.

In certain embodiments, the isolated polypeptides described herein are SEQ ID NO:2 having amino acid mutations at positions 11, 47, 90 and 167, preferably the amino acid substitutions described previously, more preferably S11P, E47G, A S and S167G. An exemplary amino acid sequence is shown in SEQ ID NO. 6. Compared with wild type SEQ ID NO. 2, the mutant has improved specific activity, medium-length fatty acid methyl ester hydrolysis capability, fatty acid glycerol esterification capability and fatty acid methyl esterification performance, in particular fatty acid methyl esterification performance. Thus, such mutants are particularly useful in the preparation of biodiesel.

In certain embodiments, the isolated polypeptide described herein is SEQ ID NO. 2 having amino acid mutations at positions 11, 156, 167 and 232, preferably the amino acid substitutions described previously, more preferably S11P, G156D, S167G and V232I. An exemplary amino acid sequence is shown in SEQ ID NO. 8. Compared with wild type SEQ ID NO. 2, the mutant has improved specific activity, medium-length fatty acid methyl ester hydrolysis capability and fatty acid methyl esterification performance, in particular fatty acid methyl esterification performance. Thus, such mutants are particularly useful in the preparation of biodiesel.

In addition to mutations at the above positions, the isolated polypeptides described herein may also have amino acid mutations at other positions in SEQ ID NO. 2, including but not limited to one or more of the following positions ：1、2、3、4、5、6、7、9、10、12、16、30、31、34、36、37、39、40、42、44、51、52、53、54、56、58、59、70、71、72、73、83、88、92、93、95、96、100、101、102、104、106、109、110、112、117、119、124、125、127、128、131、132、133、134、135、137、158、159、160、161、162、163、165、166、168、170、181、182、183、189、190、192、194、196、202、210、211、212、220、225、227、228、229、230、231、233、237、238、239、240、242、246、247、248、252、259、262、264 and 269, preferably substitution mutations, as described in CN 106459939A. Preferably, substitution mutations at these positions are as described in any of the embodiments of CN106459939a, the disclosure of which is incorporated herein by reference. It will be appreciated that the mutation in SEQ ID NO. 2 should not affect its lipase activity. More preferably, the mutation results in an increase in lipase activity of the mutant relative to the wild type, i.e. SEQ ID NO. 2, e.g. by more than 1-fold, preferably by more than 2-fold, more preferably by more than 5-fold. Preferably, the number of substitutions in the polypeptides described herein is in the range of 1-40, e.g. 1-30, 1-20, 1-10 and 1-5, such as 1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39 or 40 substitutions compared to SEQ ID NO. 2. In certain embodiments, mutants having 1-30, 1-20, 1-10, 1-5, or 1-3 amino acid substitutions compared to SEQ ID NO. 4, 6, 8, or 12 are also provided herein.

Thus, in certain embodiments, mutants of SEQ ID NO. 2 provided herein have at least 80%, at least 82%, at least 84%, at least 86%, at least 88%, 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% sequence identity to SEQ ID NO. 2. In certain embodiments, also provided herein are isolated polypeptides having at least 80%, at least 82%, at least 84%, at least 86%, at least 88%, 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% sequence identity to SEQ ID nos. 4, 6,8 or 10. In a preferred embodiment, the isolated polypeptide described herein is a polypeptide isolated from Rhizomucor miehei. In general, for mutants of SEQ ID NOS 2, 4, 6,8 and 12, mutations may occur at positions 11, 19, 47, 90, 156, 167 and 232 already contained in the respective sequences described above, but substitution mutations at positions other than these are preferred. For example, the mutant of SEQ ID NO. 12 preferably has mutations at positions other than positions 11 and 167. Of course, if mutations occur at positions 11, 19, 47, 90, 156, 167 and 232, these mutations may be of the types of mutations listed previously, particularly conservative substitutions.

Sequence identity is used herein to describe the relatedness between two amino acid sequences or between two nucleotide sequences. Sequence identity can be calculated using methods well known in the art. For example, sequence identity between two amino acid sequences can be determined using the Nedel-crafts (Needleman-Wunsch) algorithm (Needleman and Wunsch,1970, journal of molecular biology, 48:443-453) implemented in the Nedel (Needle) program of the EMBOSS package (EMBOSS: european molecular biology open software suite, rice et al, 2000, genetics trend, 16:276-277). Alternatively, BLASTP at NCBI can be used to calculate sequence identity between two amino acid sequences.

The substitutions described herein are preferably conservative substitutions. Examples of conservative substitutions are within the scope of the following groups: polar positively charged amino acids (basic amino acids) include (arginine R, lysine K and histidine H), polar negatively charged amino acids (acidic amino acids) include (glutamic acid E and aspartic acid D), polar uncharged amino acids (glycine G, serine S, threonine T, cysteine C, tyrosine Y, glutamine Q and asparagine N), nonpolar hydrophobic amino acids (alanine A, proline P, phenylalanine F, tryptophan W, methionine M, leucine L, isoleucine I and valine C), aromatic amino acids (phenylalanine F, tryptophan W and tyrosine Y) and small amino acids (glycine G, alanine A, serine S, threonine T and methionine M).

The 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. In alanine scanning mutagenesis, a single alanine mutation is introduced at each residue in the molecule, and the resulting mutant molecules are tested for lipase activity to identify amino acid residues critical to the activity of the molecule. The active site of an enzyme or other biological interaction may also be determined by physical analysis of the structure, as determined by the following technique: nuclear magnetic resonance, crystallography, electron diffraction, or photoaffinity labeling, along with mutating putative contact site amino acids. The essential amino acids can also be deduced from an alignment with the relevant polypeptide.

In certain embodiments, the invention also includes fragments of the polypeptides described herein having lipase activity.

Included herein are mature polypeptides. "mature polypeptide" refers to a polypeptide in its final form after translation and any post-translational modifications such as N-terminal processing, C-terminal truncation, glycosylation, phosphorylation, and the like. Mature polypeptides are identical in amino acid sequence to the polypeptides of any of the preceding embodiments, but may introduce N-terminal and/or C-terminal modifications, and/or glycosylation and/or phosphorylation modifications during translation. It is known in the art that one host cell can produce a mixture of two or more different mature polypeptides (i.e., having different C-terminal and/or N-terminal amino acids) expressed from the same polynucleotide.

The invention also includes polynucleotide sequences that are the coding sequences for the polypeptides described herein or the complements thereof. As used herein, a coding sequence refers to a polynucleotide that directly specifies the amino acid sequence of a mutant of SEQ ID NO. 2. The boundaries of the coding sequence are generally determined by an open reading frame beginning with a start codon (e.g., ATG, GTG or TTG) and ending with a stop codon (e.g., TAA, TAG or TGA). The coding sequence may be a genomic DNA, cDNA, synthetic DNA, or a combination thereof. Exemplary coding sequences are set forth herein as SEQ ID NOs 3, 5 and 7. Also included herein are the complementary sequences of SEQ ID NOs 3, 5 and 7.

Thus, in certain embodiments, a polypeptide described herein is a polypeptide encoded by a polynucleotide that hybridizes under low stringency conditions, medium-high stringency conditions, or very high stringency conditions with (i) the coding sequence of SEQ ID NO 4, 6, 8, or 12, or the polypeptide as described, or (ii) the full length complement of (i). In certain embodiments, the polypeptides described herein are polypeptides encoded by polynucleotides having at least 80%, at least 82%, at least 84%, at least 86%, at least 88%, 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% but less than 100% sequence identity to the coding sequence set forth in SEQ ID NO 3,5 or 7.

As used herein, "low stringency conditions" refer to prehybridization and hybridization in 5 XSSPE, 0.3% SDS, 200 micrograms/ml sheared and denatured salmon sperm DNA, and 25% formamide at 42℃for 12 to 24 hours following standard southern blotting procedures for probes of at least 100 nucleotides in length. The support material was finally washed three times, 15 minutes each, using 2 XSSC, 0.2% SDS at 50 ℃. "moderately stringent conditions" means prehybridization and hybridization in 5 XSSPE, 0.3% SDS, 200 micrograms/ml sheared and denatured salmon sperm DNA, and 35% formamide at 42℃for 12 to 24 hours following standard southern blotting procedures for probes of at least 100 nucleotides in length. The support material was finally washed three times, 15 minutes each, using 2 XSSC, 0.2% SDS at 55 ℃. "Medium-high stringency conditions" means prehybridization and hybridization in 5 XSSPE, 0.3% SDS, 200 micrograms/ml sheared and denatured frog fish sperm DNA, and 35% formamide at 42℃for 12 to 24 hours following standard southern blotting procedures for probes at least 100 nucleotides long. The support material was finally washed three times, 15 minutes each, using 2 XSSC, 0.2% SDS at 60 ℃. "high stringency conditions" means prehybridization and hybridization in 5 XSSPE, 0.3% SDS, 200 micrograms/ml sheared and denatured salmon sperm DNA, and 50% formamide at 42℃for 12 to 24 hours following standard southern blotting procedures for probes at least 100 nucleotides in length. The support material was finally washed three times, 15 minutes each, using 2 XSSC, 0.2% SDS at 65 ℃. "very high stringency conditions" means prehybridization and hybridization in 5 XSSPE, 0.3% SDS, 200 micrograms/ml sheared and denatured salmon sperm DNA, and 50% formamide at 42℃for 12 to 24 hours following standard southern blotting procedures for probes of at least 100 nucleotides in length. The support material was finally washed three times, 15 minutes each, using 2 XSSC, 0.2% SDS at 70 ℃.

The polynucleotide sequences described herein, such as SEQ ID NOS 3, 5 and 7, may be used to design nucleic acid probes to identify and clone DNA encoding a parent species from lines of different genera or species according to methods well known in the art. In particular, standard southern blotting procedures can be followed, using such probes to hybridize to genomic DNA or cDNA of a cell of interest in order to identify and isolate the corresponding gene therein. Such probes may be significantly shorter than the complete sequence, but should be at least 15, such as at least 25, at least 35, or at least 70 nucleotides in length. Preferably, the nucleic acid probe is at least 100 nucleotides in length, e.g., can be within 300 nucleotides. Both DNA and RNA probes can be used. The probes will be labeled (e.g., with ³²P、³H、³⁵ S, biotin, or avidin) to detect the corresponding genes. The probes can be used to identify and obtain sequences of interest from other sources, including microorganisms isolated from nature (e.g., soil, compost, water, etc.) or DNA samples obtained directly from natural materials (e.g., soil, compost, water, etc.). Techniques for directly isolating microorganisms and DNA from the natural living environment are well known in the art. The polynucleotide of interest can then be obtained by similarly screening in a genomic DNA or cDNA library of another microorganism or mixed DNA sample. Once the polynucleotide of interest is obtained, the polynucleotide may be isolated or cloned using techniques well known to those of ordinary skill in the art (see, e.g., sambrook et al, 1989). Thus, such probes are contemplated herein. In certain embodiments, the present disclosure relates to fragments of the polynucleotide sequences of the present invention for use as probes, which fragments may range in length from 15 to 300, preferably from 25 to 300, or from 25 to 100 nucleotides.

Also provided herein is a nucleic acid construct comprising a polynucleotide sequence as described herein. Herein, a nucleic acid construct refers to a single-or double-stranded nucleic acid molecule, which is isolated from a naturally occurring gene, or which is modified to contain a segment of nucleic acid in a manner that does not otherwise exist in nature, or which is synthetic, which includes one or more control sequences. Control sequences refer to nucleic acid sequences required for expression of a polynucleotide encoding a polypeptide described herein. Each control sequence may be native (i.e., from the same gene) or exogenous (i.e., from different genes) to the coding sequence, or native or exogenous to each other. Such control sequences include, but are not limited to, a leader, polyadenylation sequence, propeptide sequence, promoter, signal peptide sequence, transcription terminator, and the like. At a minimum, the control sequences include promoters and transcriptional and translational stop signals. These control sequences may be provided with a plurality of linkers for the purpose of introducing specific restriction sites facilitating ligation of these control sequences with the coding region of the polynucleotide encoding a variant. Typically, the polynucleotide sequences described herein are operably linked to these control sequences. Operably linked refers to the positioning of a control sequence relative to a polynucleotide described herein such that the control sequence directs the expression of the coding sequence.

Thus, the nucleic acid constructs described herein comprise a polynucleotide sequence described herein operably linked to one or more control sequences that direct the expression of the polynucleotide sequence in a suitable host cell under conditions compatible with the control sequences.

The polynucleotide sequence may be manipulated in a variety of ways to provide for expression of the polypeptides described herein. Manipulation of the polynucleotide sequence prior to its insertion into the vector may be desirable or necessary depending on the expression vector. Techniques for modifying polynucleotide sequences using recombinant DNA methods are well known in the art.

The control sequence may be a promoter, i.e., a polynucleotide recognized by the host cell for expression of the polynucleotide. The promoter comprises transcriptional control sequences which mediate the expression of the polypeptides described herein. The promoter may be any polynucleotide that exhibits transcriptional activity in the host cell including mutant, 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 described herein in bacterial host cells may be obtained from any of the following genes: 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 levan sucrase gene (sacB), bacillus subtilis xylA and xylB genes, bacillus thuringiensis cryIIIA gene, E.coli lac operon, E.coli trc promoter, streptomyces coelicolor agar hydrolase gene (dagA), prokaryotic beta-lactamase gene and tac promoter.

Suitable promoters for directing transcription of the nucleic acid constructs of the invention in a filamentous fungal host cell may be promoters obtained from any of the following genes: aspergillus nidulans acetamidase, aspergillus niger neutral alpha-amylase, aspergillus niger or Aspergillus awamori glucoamylase (glaA), aspergillus oryzae TAKA amylase, aspergillus oryzae alkaline protease, aspergillus oryzae triose phosphate isomerase, fusarium oxysporum trypsin-like protease, fusarium venenatum amyloglucosidase, fusarium venenatum Daria (Fusarium venenatum Daria), fusarium venenatum Quinn (Fusarium venenatum Quinn), rhizomucor miehei (Rhizomucor miehei) lipase, rhizomucor miehei aspartic proteinase, trichoderma reesei beta-glucosidase, trichoderma reesei cellobiohydrolase I, trichoderma reesei cellobiohydrolase II, trichoderma reesei endo-dextranase I, trichoderma reesei endo-dextranase II, trichoderma reesei endo-dextranase III, trichoderma reesei endo-dextranase IV, trichoderma reesei endo-dextranase V, trichoderma reesei xylanase I, trichoderma reesei beta-xylosidase II, trichoderma reesei beta-xylosidase, and promoter 2-tzeingii.

In yeast hosts, useful promoters may be obtained from any of the following genes: saccharomyces cerevisiae enolase (ENO-1), saccharomyces cerevisiae galactokinase (GAL 1), saccharomyces cerevisiae alcohol dehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH 1, ADH 2/GAP), saccharomyces cerevisiae Triose Phosphate Isomerase (TPI), saccharomyces cerevisiae metallothionein (CUP 1), and Saccharomyces cerevisiae 3-phosphoglycerate kinase.

The control sequence may also be a transcription terminator which is recognized by a host cell to terminate transcription. The terminator sequence is operably linked to the 3' terminus of the polynucleotide encoding the polypeptide described herein. Any terminator which is functional in the host cell may be used.

For example, preferred terminators for bacterial host cells may be those obtained from genes for Bacillus clausii alkaline protease (aprH), bacillus licheniformis alpha-amylase (amyL), and E.coli ribosomal RNA (rrnB). Preferred terminators for filamentous fungal host cells may be obtained from the following genes: aspergillus nidulans anthranilate synthase, aspergillus niger glucoamylase, aspergillus niger alpha-glucosidase, aspergillus oryzae TAKA amylase, and Fusarium oxysporum trypsin-like protease. Preferred terminators for yeast host cells may be obtained from any of the following genes: saccharomyces cerevisiae enolase, saccharomyces cerevisiae cytochrome C (CYC 1), and Saccharomyces cerevisiae glyceraldehyde-3-phosphate dehydrogenase.

The control sequence may also be an mRNA stabilizing region downstream of the promoter and upstream of the gene coding sequence, which increases the expression of the gene. Suitable mRNA stable regions can be obtained from the following genes: the bacillus thuringiensis cryIIIA gene and the bacillus subtilis SP82 gene.

The control sequence may also be a leader sequence, which is a region of untranslated mRNA that is important for translation by the host cell. The leader sequence is operably linked to the 5' end of the polynucleotide encoding the polypeptide described herein. Any leader that is functional in the host cell may be used. For example, preferred leaders for filamentous fungal host cells may be obtained from the genes for Aspergillus oryzae TAKA amylase and Aspergillus nidulans triose phosphate isomerase. The leader for a yeast host cell can be obtained from any of the following genes: saccharomyces cerevisiae enolase (ENO-1), saccharomyces cerevisiae 3-phosphoglycerate kinase, saccharomyces cerevisiae alpha-factor, and Saccharomyces cerevisiae alcohol dehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH 2/GAP).

The control sequence may also be a polyadenylation sequence, a sequence which is operably linked to the 3' terminus of the variant coding sequence and which, when transcribed, is recognized by the host cell as a signal to add polyadenosine residues to transcribed mRNA. Any polyadenylation sequence which is functional in the host cell may be used. For example, preferred polyadenylation sequences for filamentous fungal host cells may be obtained from any of the genes: aspergillus nidulans anthranilate synthase, aspergillus niger glucoamylase, aspergillus niger alpha-glucosidase, aspergillus oryzae TAKA amylase, and Fusarium oxysporum trypsin-like protease. Polyadenylation sequences for yeast host cells are described in mol. Cellular biol.,1995, 15:5983-5990.

The control sequence may also be a signal peptide coding region that encodes a signal peptide linked to the N-terminus of a polypeptide described herein and directs the polypeptide into the secretory pathway of a cell. The 5' end of the polynucleotide may inherently contain a signal peptide coding sequence naturally linked in translation reading frame with the segment of the coding sequence encoding the polypeptide described herein. Alternatively, the 5' end of the coding sequence may comprise a signal peptide coding sequence that is foreign to the coding sequence. In cases where the coding sequence does not naturally comprise a signal peptide coding sequence, an exogenous signal peptide coding sequence may be required. Alternatively, the foreign signal peptide coding sequence may simply replace the natural signal peptide coding sequence in order to increase secretion of the variant. However, any signal peptide coding sequence that directs the expressed variant into the secretory pathway of a host cell may be used.

Effective signal peptide coding sequences for bacterial host cells can be obtained from any of the following genes: bacillus NCIB 11837 maltogenic amylase, bacillus licheniformis subtilisin, bacillus licheniformis beta-lactamase, bacillus stearothermophilus alpha-amylase, bacillus stearothermophilus neutral protease (nprT, nprS, nprM), and Bacillus subtilis prsA. Effective signal peptide coding sequences for filamentous fungal host cells may be obtained from any of the following genes: aspergillus niger neutral amylase, aspergillus niger glucoamylase, aspergillus oryzae TAKA amylase, humicola insolens cellulase, humicola insolens endoglucanase V, humicola lanuginosa lipase, and Mucor miehei aspartic proteinase. The signal peptide for a yeast host cell can be obtained from any of the following genes: saccharomyces cerevisiae alpha-factor and Saccharomyces cerevisiae invertase.

The control sequence may also be a propeptide coding sequence that codes for a propeptide positioned at the N-terminus of a polypeptide described herein. The resulting polypeptide is referred to as a precursor enzyme or a pro-polypeptide. A pro-polypeptide is typically inactive and can be converted to an active polypeptide by catalytic or autocatalytic cleavage of a propeptide from the pro-polypeptide. The propeptide coding sequence may be obtained from any one of the following genes: bacillus subtilis alkaline protease (aprE), bacillus subtilis neutral protease (nprT), myceliophthora thermophila laccase (WO 95/33836), rhizomucor miehei aspartic proteinase, and Saccharomyces cerevisiae alpha factor.

In the case where both a signal peptide sequence and a propeptide sequence are present, the propeptide sequence is positioned next to the N-terminus of a polypeptide described herein and the signal peptide sequence is positioned next to the N-terminus of the propeptide sequence.

In certain embodiments, the nucleic acid constructs described herein further comprise a regulatory system that modulates expression of the polypeptides described herein relative to growth of the host cell. Examples of regulatory systems include sequences that cause the expression of a gene to be turned on or off in response to a chemical or physical stimulus. Regulatory systems in prokaryotic systems include the lac, tac, and trp operator systems. In yeast, the ADH2 system or GAL1 system may be used. In filamentous fungi, the Aspergillus niger glucoamylase promoter, the Aspergillus oryzae TAKA alpha-amylase promoter, and the Aspergillus oryzae glucoamylase promoter may be used.

Also provided herein are recombinant expression vectors comprising polynucleotides encoding the polypeptides described herein, promoters, and transcriptional and translational stop signals. The different nucleotide and control sequences may be joined together to produce a recombinant expression vector, which may include one or more convenient restriction sites to allow for insertion or substitution of a polynucleotide encoding a polypeptide described herein at these 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 generating the expression vector, the coding sequence is located in the vector such that the coding sequence is operably linked to the appropriate control sequences for expression.

The recombinant expression vector may be any vector (e.g., a plasmid or virus) that is capable of conveniently performing a recombinant DNA procedure and that is capable of causing expression of the polynucleotide. The choice of 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 which 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 element for ensuring self-replication. Alternatively, the vector may be an integrating vector, i.e. when it is introduced into the host cell, is integrated into the genome and replicates together with one or more chromosomes into which it has been integrated. Furthermore, a single vector or plasmid or two or more vectors or plasmids which together contain the total DNA to be introduced into the genome of the host cell or transposons may be used.

The vector preferably comprises one or more selectable markers that allow convenient selection of cells, such as transformed cells, transfected cells, transduced cells, or the like. A selectable marker is a gene whose product provides for biocide or viral resistance, heavy metal resistance, prototrophy to an auxotroph, and the like.

Examples of bacterial selectable markers are the bacillus licheniformis or bacillus subtilis dal genes, or markers conferring antibiotic resistance (e.g., ampicillin, chloramphenicol, kanamycin, neomycin, spectinomycin, or tetracycline resistance). Suitable markers for yeast host cells include, but are not limited to, ADE2, HIS3, LEU2, LYS2, MET3, TRP1, and URA3. Selectable markers for use in a filamentous fungal host cell include, but are not limited to, amdS (acetamidase), argB (ornithine carbamoyltransferase), bar (phosphinothricin acetyltransferase), hph (hygromycin phosphotransferase), niaD (nitrate reductase), pyrG (orotidine-5' -phosphate decarboxylase), sC (sulfate adenyltransferase), and trpC (anthranilate synthase), along with equivalents thereof. Preferred for use in Aspergillus cells are the Aspergillus nidulans or Aspergillus oryzae amdS and pyrG genes and the Streptomyces hygroscopicus bar gene.

The vector preferably contains one or more elements that allow the vector to integrate into the genome of the host cell or the vector to autonomously replicate in the cell independently of the genome.

For integration into the host cell genome, the vector may rely on the polynucleotide sequences encoding the polypeptides described herein or any other element of the vector for integration into the genome by homologous or non-homologous recombination. Alternatively, the vector may comprise additional polynucleotides for directing integration by homologous recombination into one or more precise locations in one or more chromosomes in the host cell genome. To increase the likelihood of integration at a precise location, these integration elements should contain a sufficient number of nucleic acids, for example 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 with the corresponding target sequence to increase the likelihood of homologous recombination. These integrational elements may be any sequence that is homologous with the target sequence in the genome of the host cell. Furthermore, these integrational elements may be non-encoding polynucleotides 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 autonomously replicate in the host cell of interest. The origin of replication may be any plasmid replicon that functions in a cell to mediate autonomous replication. Examples of bacterial origins of replication include, for example, origins of replication of plasmids pBR322, pUC19, pACYC177, and pACYC184, which allow replication in E.coli, and origins of replication of plasmids pUB110, pE194, pTA1060, and pAM beta 1, which allow replication in Bacillus. Examples of origins of replication for use in yeast host cells include the 2 micron origins of replication ARS1, ARS4, a combination of ARS1 and CEN3, and a combination of ARS4 and CEN 6. Origins of replication for use in the filamentous fungal cell include AMA1 and ANS1.

More than one copy of a polynucleotide described herein may be inserted into a host cell to increase production of a polypeptide of interest.

Procedures for ligating the elements described above to construct recombinant expression vectors of the invention are well known to those of ordinary skill in the art, see, e.g., sambrook et al, 1989.

Also described herein are recombinant host cells comprising a polynucleotide encoding a polypeptide described herein operably linked to one or more control sequences that direct the expression of the polypeptide. In certain embodiments, the recombinant host cells described herein express a polypeptide described herein. Introducing a nucleic acid construct or vector comprising a polynucleotide as described herein into a host cell such that the nucleic acid construct or vector is maintained as a chromosomal integrant or as an autonomously replicating extra-chromosomal vector. The term "host cell" encompasses any progeny of a parent cell that is different from the parent cell due to mutations that occur during replication. The choice of host cell will depend to a large extent on the gene encoding the variant and its source.

The host cell may be any cell useful in recombinantly producing variants, such as a prokaryotic cell or a eukaryotic cell. 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, marine bacillus, staphylococcus, streptococcus, and streptomyces. Gram negative bacteria include, but are not limited to: campylobacter, escherichia coli, flavobacterium, fusobacterium, helicobacter, mudacter, neisseria, pseudomonas, salmonella, and ureaplasma. The bacterial host cell may be any bacillus cell including, but not limited to: bacillus alcalophilus, 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 bacterial host cell may also be any streptococcus cell including, but not limited to: streptococcus equi, streptococcus pyogenes, streptococcus uberis, and streptococcus equi subsp zooblast cells. The bacterial host cell may also be any Streptomyces cell including, but not limited to, streptomyces chromogenes, streptomyces avermitilis, streptomyces coelicolor, streptomyces griseus, and Streptomyces lividans cells. The host cell may also be a eukaryotic cell, such as a mammalian, insect, plant, or fungal cell. The host cell may be a fungal cell, which may be a yeast cell. The yeast host cell may be a candida, hansenula, kluyveromyces, pichia, saccharomyces, schizosaccharomyces, or yarrowia cell, such as a kluyveromyces lactis, karst, saccharomyces cerevisiae, saccharifying yeast, dag's yeast, kluyveromyces, nod's yeast, oval yeast, or yarrowia lipolytica cell. The fungal host cell may be a filamentous fungal cell. The filamentous fungal host cell may be a plant of the genus Acremonium, aspergillus, aureobasidium, tuber, ceriporiopsis, chrysosporium, coprinus the genus Zygomyces, ceripomoea, and chrysosporium genus, coprinus genus New Meibacterium, neurospora, paecilomyces, penicillium Phanerochaete, pelebia (Phlebia), ruminox, pot fungus Phanerochaete, phlebia (Phlebia) the genus Ruminous Pot fungus.

The nucleic acid construct or vector may be introduced into the host cell using methods well known in the art, including but not limited to protoplast transformation, competent cell transformation, electroporation, conjugation, transduction, and the like. The method of introduction for verification may be selected according to the host cell.

Also provided herein is a method of producing a polypeptide described herein, comprising: culturing a host cell described herein under conditions suitable for expression of the polypeptide, and recovering the polypeptide.

These host cells may be cultured in a nutrient medium suitable for producing the polypeptide using methods known in the art. For example, the cells may be cultured by shake flask culture, or small-scale or large-scale fermentation (including continuous fermentation, batch fermentation, fed-batch fermentation, or solid state fermentation) in a laboratory or industrial fermentor under conditions allowing the polypeptide to be expressed and/or isolated. The culturing takes place in a suitable nutrient medium comprising carbon and nitrogen sources and inorganic salts using procedures known in the art. Suitable media are available from commercial suppliers or may be prepared according to published compositions (e.g., in catalogues of the American type culture Collection). If the variant is secreted into the nutrient medium, the variant can be recovered directly from the medium. If the variant is not secreted, it can be recovered from the cell lysate.

The polypeptides may be detected using methods known in the art that are specific for these polypeptides. These detection methods include, but are not limited to, the use of specific antibodies, the formation of enzyme products, or the disappearance of enzyme substrates.

The polypeptides described herein can be recovered using methods known in the art. For example, the polypeptides described herein may be recovered from the nutrient medium by a variety of conventional procedures including, but not limited to, collection, centrifugation, filtration, extraction, spray drying, evaporation, or precipitation.

The polypeptides described herein can be purified to obtain substantially pure the polypeptides by a variety of procedures known in the art, including, but not limited to, chromatography (e.g., ion exchange chromatography, affinity chromatography, hydrophobic interaction chromatography, chromatofocusing, and size exclusion chromatography), electrophoresis procedures (e.g., preparative isoelectric focusing), differential solubility (e.g., ammonium sulfate precipitation), SDS-PAGE, or extraction.

The invention also provides a composition comprising a polypeptide as described herein. The composition may be a fermentation broth or a lysate of a cell expressing a polypeptide described herein, or a concentrate of said fermentation broth or lysate.

The compositions herein are useful for cleaning purposes. Thus, in certain embodiments, provided herein is a cleaning composition comprising a polypeptide as described herein and various additives commonly used in the cleaning arts, including, but not limited to, one or more of surfactants, builders, chelating agents, dye transfer inhibiting agents, dispersants, enzymes, and enzyme stabilizers, catalytic materials, bleach activators, hydrogen peroxide, sources of hydrogen peroxide, preformed peracids, polymeric dispersing agents, clay removal/anti-redeposition agents, brighteners, suds suppressors, dyes, hueing dyes, perfumes, perfume delivery systems, structure elasticizing agents, fabric softeners, carriers, hydrotropes, processing aids, solvents and/or pigments. For further examples of additives, see CN106459939a.

The cleaning compositions described herein are useful in laundry, hard surface cleaning, dishwashing applications, and cosmetic applications (e.g., denture, tooth, hair, and skin). The cleaning compositions of the present invention may be solid or liquid, such as regular, compressed or concentrated liquids; gel; paste; a soap bar; regular or compressed powder; a particulate solid; a homogeneous or multilayer tablet having two or more layers (same or different phases); a pouch having one or more compartments; single or multiple compartment unit dosage forms; or any combination thereof.

In certain embodiments, the cleaning compositions of the present invention are in the form of lipase particles. In lipase particles, the polypeptides described herein are contained in a water-soluble film. These lipase particles may comprise one or more additional enzymes, such as hemicellulases, peroxidases, proteases, cellulases, xylanases, lipases, phospholipases, esterases, cutinases, pectinases, mannanases, pectin lyases, keratinases, reductases, oxidases, phenol oxidases, lipoxygenases, xanthanases, ligninases, pullulanases, tannase, pentosanases, ma Lana enzymes, beta-glucanases, arabinosidases, hyaluronidase, chondroitinase, laccase, chlorophyllase and amylase. When present in the composition, the aforementioned additional enzyme may be present at a level of from 0.00001wt% to 2wt%, from 0.0001wt% to 1wt%, or from 0.001wt% to 0.5wt% enzyme protein by weight of the composition. The lipase particles may be lipase crystals, lipase precipitated, sprayed or freeze-dried lipase or any form of granular lipase, as a suspension in a powder or liquid.

The cleaning compositions of the present invention may also be in the form of a water-soluble film. Water-soluble films, optional ingredients for use therein, and methods of making them are well known in the art. In certain embodiments, the water-soluble film comprises PVOH.

The compositions herein are useful in the production of biodiesel, hydrolysis of esters, esterification of fatty acids. Thus, in certain embodiments, the compositions herein contain various additives commonly used in the art of polypeptide and enzyme formulation described herein, including, but not limited to, adsorption materials, protectants, preservatives, dispersants, and the like.

The invention will be illustrated by way of specific examples. It should be understood that these examples are illustrative only and are not intended to limit the scope of the invention. The methods and reagents used in the examples, unless otherwise indicated, are those well known and conventional in the art.

Embodiment one: mutant acquisition

Error-prone PCR

Error-prone PCR was performed on the mature peptide region to obtain mutants. The primer is as follows:

RMLHindU: aaaAAGCTTccatcgacggaggtattag（SEQ ID NO:9）；

RMLEcoRD：aaaGAATTCttaagtacacaaaccggtgttaat（SEQ ID NO:10）。

The PCR was performed using Takara rTaq enzyme, and the reaction system was prepared according to the instructions, but MnCl _2. PCR was additionally added at a final concentration of 0.3mM, and the reaction was circulated for 33 times at an annealing temperature of 52℃and an extension time of 1min. The PCR amplified product was purified by the kit, and was double-digested overnight with HindIII and EcoRI restriction enzymes (Takara Co., ltd.), and recovered for use.

A pmAO-2pro/RML vector was constructed according to the method mentioned in the example of CN108239648A, and the aox1 promoter in the vector was replaced with a constitutive promoter by using a conventional method to obtain a vector pSPI-2P-RML plasmid, and the SPI promoter sequence was the promoter sequence described in SEQ ID NO:2 in CN 201210390049.3. The pSPI-2P-RML plasmid was double-digested with Takara HindIII and EcoRI restriction enzymes overnight, separated by agarose gel electrophoresis, and the vector backbone portion of about 8.1k was recovered by tapping.

② Connection

The vector and PCR enzyme cut were ligated overnight using Takara T4 DNA ligase according to the instructions of use.

③ Obtaining library nucleic acids

The ligation transformed DH 5. Alpha. Competent cells were screened by plating with ampicillin plates. The next day water scrapes off the resistant positive bacteria on the plate, extracts the plasmid, and obtains the mutant vector library. The plasmid was linearized with SalI restriction enzyme and recovered by Omega PCR product purification kit. The resulting plasmid was a mixture containing multiple RML mutants.

④ Acquisition of mutant Yeast libraries

Pichia pastoris GS115 competence is prepared according to a literature method, a linearized mutant library is transferred into yeast by an electric shock method, and MD plates are coated for histidine auxotrophy marking and screening positive clones.

The HIS4+ strain is selected into an active plate, and the formula comprises 2% peptone, 1% yeast powder, 2% glycerol, 0.1M sodium citrate buffer solution pH6.5,2.5% lipid substrate (4% polyvinyl alcohol emulsified olive oil and biodiesel mixture 1:2), 2.5% agar, rhodamine B, biotin, histidine and kanamycin. The colonies were cultured in a 28℃incubator for 48 hours and checked for viability.

The experimental procedure and results are shown in figure 1. And selecting a colony with dark color, fermenting by using a BMGY liquid culture medium to obtain crude enzyme liquid, and testing the characteristics of the crude enzyme liquid, wherein one strain R9 has better characteristics. The detecting content comprises the following steps:

a、PNPP：

The PNPP method adopts p-nitrophenol palmitate as a substrate for lipase hydrolysis, and is a common substrate for lipase activity detection. The chain length of the substrate fatty acid is 16, and the substrate fatty acid and the main fatty acid C16 and C18 in common animal and vegetable oil belong to the same long-chain fatty acid, so PNPP can reflect the hydrolysis performance of lipase on 16-carbon and 18-carbon fatty acids. The invention adopts PNPP hydrolysis to detect the extensive hydrolytic capacity of RML mutant.

20. Mu.l of the enzyme solution, which was appropriately diluted, was mixed with 500. Mu.l of PNPP substrate (1 ml, 3mg/ml PNPP isopropyl alcohol solution, 9ml, 50mM Tris-HCl buffer pH 7.5), reacted at 1300rpm at 40℃for 15min on an Eppendorf shaker, and 500. Mu.l of absolute ethanol was added to terminate the reaction. After centrifugation of the reaction at 12000rpm for 2min, the supernatant was taken to determine the light absorption at 410 nm.

B. esterification:

Methyl esterification properties: methyl esterification is the core reaction of biodiesel. The biodiesel generally utilizes the esterification of fatty acid components in waste animal and vegetable oil and fat with methanol, namely the methyl esterification reaction of mainly 16-carbon and 18-carbon fatty acids. Among these substrate materials, palm oil deodorized distillate PFAD is the most expensive, low pretreatment-requiring material for biodiesel industry. The invention tests PFAD methyl esterification performance of RML mutant and biodiesel industrial application performance.

Using 20ul of enzyme solution, it was reacted overnight with 80 ul of water, 100 ul of fatty acid methyl ester, 300 ul of palm oil deodorized distillate PFAD,40 ul of methanol (10 ul/hr added 4 times total) at 40℃in an eppendorf shaker at 1300 rpm. After centrifugation at 12000rpm for 2min, the acid value of the upper oil phase was measured according to the method of GB 5009.229-2016, and the content of residual free fatty acid in the post-reaction system was calculated according to the fatty acid content (%) =1/2 acid value estimation equation. Comparing the amounts of fatty acids converted by mutant and wild type RML, the less fatty acid residues, the better the esterification performance.

Fatty acid glycerol esterification performance: esterification of fatty acids with glycerol is a potential alternative to the deacidification step in the refining of edible oils, and free fatty acids that would otherwise be waste materials can be converted to edible fatty acid glycerides. The invention uses the fatty acid glycerol esterification performance to test the performance of the RML mutant in the edible oil enzymatic deacidification industry.

Using 20ul of the enzyme solution, it was reacted with 80. Mu.l of water, 300. Mu.l of oleic acid, 160. Mu.l of glycerol overnight at 40℃at 1300rpm on an eppendorf shaker. After centrifugation at 12000rpm for 2min, the acid value of the upper oil phase was measured according to the method of GB 5009.229-2016, and the content of residual free fatty acid in the post-reaction system was calculated according to the fatty acid content (%) =1/2 acid value estimation equation. Comparing the amounts of fatty acids converted by mutant and wild type RML, the less fatty acid residues, the better the esterification performance.

C. methyl ester hydrolysis:

C8/C10 methyl ester is a byproduct of coconut oil refining, is a main source of C8 acid and C10 acid of medium carbon chain fatty acid, and is hydrolyzed by a chemical method or an enzymatic method to obtain free acid which is used as a raw material of carbon chain fatty acid triglyceride (MCT) in fat-reducing oil. The invention uses hydrolysis of C8/C10 methyl esters to test the performance of RML mutants for this industrial use.

Using 20ul of the enzyme solution, it was reacted with 400 ul of 200 ul l C of 8/10 fatty acid methyl ester in water at 1300rpm at 40℃on an Eppendorf shaker overnight. After centrifugation at 12000rpm for 2min, the acid value of the upper oil phase was determined according to the method GB 5009.229-2016. Or all reactants were directly transferred into 10ml of isopropanol and the amount of fatty acids therein was titrated with a base. The amounts of free fatty acids produced by hydrolysis of methyl esters by each RML were compared. The more fatty acids are produced, the better the hydrolytic methyl ester properties.

The results of the measurements are shown in table 1 below, the R9 mutant is shown to have better performance than the wild-type gene.

The RML sequence of R9 was sequenced, resulting in the RML mutant form (S11P/S167G). Using the sequence as a template, random mutation was again performed, colonies with large hydrolysis circles were selected on the olive oil plate, and these colonies were subjected to a further round of thermal stability plate screening to obtain several mutant bacteria. The results are shown in FIG. 2.

Embodiment two: mutant sequence analysis and verification

(1) Construction of mutant partial purity, subcloning sequencing, recombinant Yeast

Pichia pastoris transformed by electric shock generally contains a plurality of copies of exogenous genes, and in order to determine mutation sites, RML fragments are amplified and separated into single genes for transformation verification again.

(A) And (2) PCR: the RML HINDIII-6HisEcoRI fragment was amplified from the above yeast colonies using KOD-FX enzyme. Primer was used:

RMLHindU: aaaAAGCTTccatcgacggaggtattag（SEQ ID NO:9）；

RML6HisD：aaaGAATTCtcaatgatgatgatgatgatggtcgacagtacacaa（SEQ ID NO:11）。

The PCR was amplified using Toyobo KOD-FX enzyme, and the reaction system was prepared according to the instructions. PCR was cycled 33 times, annealing temperature 55℃and extension time 1min. The PCR amplified product was purified by kit and double-cut overnight with HindIII and EcoRI restriction enzymes of Takara, inc., and purified. Enzyme cutting sites are introduced at two ends of the RML fragment by PCR, and meanwhile, his-tag is introduced at the N end of the RML for later purification. Wild-type RML was constructed using the same strategy.

(B) The RML fragment of each mutant was ligated into pSPI-2pro/RML vector. DH 5. Alpha. Was transformed and analyzed by sequencing, and the results are shown in Table 2.

(C) Re-transformation

The plasmid containing the mutant clone was extracted, and the entire expression cassette sequence was amplified using the SPI promoter 5' segment primer and the HIS4 gene termination region primer. PCR was performed using the Toyobo KOD-FX enzyme, procedure 32 cycles of 2-step amplification: denaturation at 98℃for 30s and 65℃for 7min. After purification of PCR products, GS115 was transformed by electric shock, MD plates were coated, and the plates were incubated at 28℃for new culture. And (3) picking up the transformant on the MD plate into YPD-biodiesel-rhodamine B active plates, picking up 2 bacterial colonies with large hydrolysis circles respectively, and carrying out fermentation detection.

A single colony was inoculated into BYPD medium (0.1M citrate buffer pH6.5,2% peptone, 1% yeast powder, 2% glycerol, 50ml of 250ml baffle-flask liquid volume) and cultured at 30℃for 5 days with shaking table 240 rpm. 1ml of 50% glycerol was supplemented daily starting on day 3.

① Enzyme solution acquisition

The fermentation broth was transferred to a 50ml centrifuge tube and centrifuged for 10min with a 4000rpm horizontal rotor. The supernatant was microfiltered with a 0.22uM filter membrane, then with a 10kDa ultrafiltration tube, and subjected to centrifugal ultrafiltration at 4000rpm for 30min in a centrifuge at 4℃until all the enzyme solutions were concentrated to about 2ml, and washed twice with deionized water.

② Purification

200 Μl of Qiagen Ni-NTA resin was centrifuged at 2000rpm for 1min to remove supernatant, and 1ml of deionized water was added to wash 2 times to remove ethanol from the product. The volume of the enzyme solution after ultrafiltration was measured, 1/10 of 50mM Tris-HCl buffer, pH7.5, naCl to a final concentration of 300mM, imidazole mother liquor to a final concentration of 5mM, NTA gel beads were added to the enzyme solution after ultrafiltration, and the mixture was stirred and mixed for 30 minutes under ice bath conditions. NTA beads were washed 3 times with 1ml each using a wash solution (20 mM imidazole, 300mM NaCl,50mM pH7.5 Tris-HCl). Finally, the eluate (20 mM imidazole, 300mM NaCl,50mM pH7.5 Tris-HCl) was used 3 times, 1ml each. After filtration through a 0.22um filter, the total eluate was concentrated to 100. Mu.l using a 10kDa ultrafiltration tube, washed 3 times with 400. Mu.l of deionized water and 2 times with 50mM sodium phosphate buffer, pH 6.5. Finally, the pure enzyme solution is stored at 4 ℃.

③ Protein assay

The purified protein was diluted 10-fold with PBS and 10. Mu.l was used to measure protein content using Shanghai Bradford reagent. According to the retest result, each protein is diluted to 0.01mg/ml, PNPP activity in a short time is measured, and specific activity of the mutant is characterized. The protein is diluted to 0.1mg/ml, and a methyl ester hydrolysis experiment, a glycerol esterification experiment and a fatty acid methyl esterification experiment are carried out, so that the transformation capacity of the mutant under long-time reaction is represented, and the comprehensive expression of activity and stability is realized.

The results are shown in Table 2 below. The following values are the biological averages of two colonies of the same gene.

Note that: the nucleotide and amino acid sequences of the wild type, T13, T14 and T15 are respectively shown as SEQ ID NO 1-2, SEQ ID NO 3-4, SEQ ID NO 5-6 and SEQ ID NO 7-8, and the amino acid sequence of R9 is shown as SEQ ID NO 12.

From Table 2, it can be seen that R9, T13, T14 have one more S167G mutation than T9 in common, and that their PNPP specific activity is increased compared with T9, indicating that S167G contributes to the increase of specific activity and the hydrolysis ability of long chain fatty acid is improved. Ser is one hydroxyl more than Gly and belongs to nucleophilic groups, so it is speculated that mutation of Ser to nucleophilic or electronegative groups may help to improve methyl esterification properties such as aspartic acid Asp, histidine His, tyrosine Tyr, cysteine Cys, etc. Mutation to an amino acid other than aspartic acid, histidine, tyrosine, or cysteine may increase the ability of RML to hydrolyze long chain fatty acid esters.

T13 has more Y19H mutation than R9, the long carbon chain fatty acid ester hydrolytic capacity, the glycerol esterification capacity and the biodiesel reaction capacity are reduced, and only the medium carbon chain fatty acid ester hydrolytic capacity is unchanged. It was shown that Y19H did not improve the stability of the mutant and the like, which had a negative effect on the viability of the RML. Mutation of Thr19 to the amino acid of the evidence charged group has a negative effect, thus it is inferred that mutation to amino acids other than histidine, lysine and arginine may contribute to the improvement of RML activity.

The T14 mutant has more E47G/A90S mutation than R9, the specific activity is not changed greatly, but the capacity of the fatty glyceride is reduced, and the biodiesel reaction performance is greatly improved, so that the E47G/A90S mutation is helpful for the biodiesel reaction application of RML.

T15 has more G156D/V232I mutation than R9, and RML has reduced hydrolysis capability of long-chain fatty acid ester, hydrolysis capability of medium-chain fatty acid ester and glycerinum esterification performance of long-chain fatty acid, but has improved methyl esterification performance of long-chain fatty acid.

Sequence listing

<110> Feng Yi (Shanghai) Biotechnology research and development center Co., ltd

<120> Lipase mutant, composition and use thereof

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tccatcgacg gaggtattag agccgctact tctcaggaaa tcaacgaact tacttactat 60

acaactttgt cagctaattc ttactgtaga actgttattc ctggtgctac ttgggattgc 120

atacattgtg acgccactga agatttaaag ataattaaaa cctggtctac tttgatttac 180

gacactaacg ctatggttgc tagaggagat tccgagaaga ctatttatat cgtgtttaga 240

ggttcttcat ctattcgtaa ttggatcgct gatttgacat tcgttccagt ctcttaccct 300

ccagtttctg gtactaaggt tcacaaagga tttcttgatt cttatggtga agttcaaaac 360

gagttggttg ctactgtctt ggatcagttt aaacaatacc catcttataa ggttgctgtc 420

actggtcact ctttgggagg tgctactgcc ttgctgtgtg ctttaggttt ataccagaga 480

gaggaaggat tgtcttcaag taacctattc ttgtacactc aaggtcagcc tagagttgga 540

gatccagcat ttgctaatta tgtggtttct actggtattc catatagacg tactgttaac 600

gaaagagaca tagtaccaca cttgcctcca gctgccttcg gatttctgca tgccggtgaa 660

gagtactgga tcacagataa ttctcctgaa accgttcaag tgtgtacatc tgatttagag 720

acttccgact gctctaacag tattgttcca tttacttcag ttcttgatca tttgtcttat 780

tttggaatta acaccggttt gtgtacttaa 810

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<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 2

Ser Ile Asp Gly Gly Ile Arg Ala Ala Thr Ser Gln Glu Ile Asn Glu

1 5 10 15

Leu Thr Tyr Tyr Thr Thr Leu Ser Ala Asn Ser Tyr Cys Arg Thr Val

20 25 30

Ile Pro Gly Ala Thr Trp Asp Cys Ile His Cys Asp Ala Thr Glu Asp

35 40 45

Leu Lys Ile Ile Lys Thr Trp Ser Thr Leu Ile Tyr Asp Thr Asn Ala

50 55 60

Met Val Ala Arg Gly Asp Ser Glu Lys Thr Ile Tyr Ile Val Phe Arg

65 70 75 80

Gly Ser Ser Ser Ile Arg Asn Trp Ile Ala Asp Leu Thr Phe Val Pro

85 90 95

Val Ser Tyr Pro Pro Val Ser Gly Thr Lys Val His Lys Gly Phe Leu

100 105 110

Asp Ser Tyr Gly Glu Val Gln Asn Glu Leu Val Ala Thr Val Leu Asp

115 120 125

Gln Phe Lys Gln Tyr Pro Ser Tyr Lys Val Ala Val Thr Gly His Ser

130 135 140

Leu Gly Gly Ala Thr Ala Leu Leu Cys Ala Leu Gly Leu Tyr Gln Arg

145 150 155 160

Glu Glu Gly Leu Ser Ser Ser Asn Leu Phe Leu Tyr Thr Gln Gly Gln

165 170 175

Pro Arg Val Gly Asp Pro Ala Phe Ala Asn Tyr Val Val Ser Thr Gly

180 185 190

Ile Pro Tyr Arg Arg Thr Val Asn Glu Arg Asp Ile Val Pro His Leu

195 200 205

Pro Pro Ala Ala Phe Gly Phe Leu His Ala Gly Glu Glu Tyr Trp Ile

210 215 220

Thr Asp Asn Ser Pro Glu Thr Val Gln Val Cys Thr Ser Asp Leu Glu

225 230 235 240

Thr Ser Asp Cys Ser Asn Ser Ile Val Pro Phe Thr Ser Val Leu Asp

245 250 255

His Leu Ser Tyr Phe Gly Ile Asn Thr Gly Leu Cys Thr

260 265

<210> 3

<211> 810

<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 3

tccatcgacg gaggtattag agccgctact cctcaggaaa tcaacgaact tactcactat 60

acaactttgt cagctaattc ttactgtaga actgttattc ctggtgctac ttgggattgc 120

atacattgtg acgccactga agatttaaag ataattaaaa cctggtctac tttgatttac 180

gacacaaacg ctatggttgc tagaggagat tccgagaaga ctatttatat cgtgtttaga 240

ggttcttcat ctatccgtaa ttggatcgct gatttgacat tcgttccagt ctcttaccct 300

ccagtttctg gtactaaggt tcacaaagga tttcttgatt cttatggtga ggttcaaaac 360

gagttggttg ctactgtctt ggatcagttt aaacaatacc catcttataa ggttgctgtc 420

actggtcact ctttgggagg tgctactgcc ttgctgtgtg ctttaggttt ataccagaga 480

gaggaaggat tgtcttcagg taacctattc ttgtacactc aaggtcagcc tagagttgga 540

gatccagcat ttgctaatta tgtggtttct actggtattc catatagacg tactgttaac 600

gaaagagaca tagtaccaca cttgcctcca gctgccttcg gatttctgca tgccggtgag 660

gagtactgga tcacagataa ttctcctgaa accgttcaag tgtgtacatc tgatttagag 720

acttccgact gctctaacag tattgttcca tttacttcag ttcttgatca tttgtcttat 780

tttggaatta acaccggttt gtgtacttaa 810

<210> 4

<211> 269

<212> PRT

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 4

Ser Ile Asp Gly Gly Ile Arg Ala Ala Thr Pro Gln Glu Ile Asn Glu

1 5 10 15

Leu Thr His Tyr Thr Thr Leu Ser Ala Asn Ser Tyr Cys Arg Thr Val

20 25 30

Ile Pro Gly Ala Thr Trp Asp Cys Ile His Cys Asp Ala Thr Glu Asp

35 40 45

Leu Lys Ile Ile Lys Thr Trp Ser Thr Leu Ile Tyr Asp Thr Asn Ala

50 55 60

Met Val Ala Arg Gly Asp Ser Glu Lys Thr Ile Tyr Ile Val Phe Arg

65 70 75 80

Gly Ser Ser Ser Ile Arg Asn Trp Ile Ala Asp Leu Thr Phe Val Pro

85 90 95

Val Ser Tyr Pro Pro Val Ser Gly Thr Lys Val His Lys Gly Phe Leu

100 105 110

Asp Ser Tyr Gly Glu Val Gln Asn Glu Leu Val Ala Thr Val Leu Asp

115 120 125

Gln Phe Lys Gln Tyr Pro Ser Tyr Lys Val Ala Val Thr Gly His Ser

130 135 140

Leu Gly Gly Ala Thr Ala Leu Leu Cys Ala Leu Gly Leu Tyr Gln Arg

145 150 155 160

Glu Glu Gly Leu Ser Ser Gly Asn Leu Phe Leu Tyr Thr Gln Gly Gln

165 170 175

Pro Arg Val Gly Asp Pro Ala Phe Ala Asn Tyr Val Val Ser Thr Gly

180 185 190

Ile Pro Tyr Arg Arg Thr Val Asn Glu Arg Asp Ile Val Pro His Leu

195 200 205

Pro Pro Ala Ala Phe Gly Phe Leu His Ala Gly Glu Glu Tyr Trp Ile

210 215 220

Thr Asp Asn Ser Pro Glu Thr Val Gln Val Cys Thr Ser Asp Leu Glu

225 230 235 240

Thr Ser Asp Cys Ser Asn Ser Ile Val Pro Phe Thr Ser Val Leu Asp

245 250 255

His Leu Ser Tyr Phe Gly Ile Asn Thr Gly Leu Cys Thr

260 265

<210> 5

<211> 810

<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 5

tccatcgacg gaggtattag agccgctact cctcaggaaa tcaacgaact tacttactat 60

acaactttgt cagctaattc ttactgtaga actgttattc ctggtgctac ttgggattgc 120

atacattgtg acgccactgg agatttaaag ataattaaaa cctggtctac tttgatttac 180

gacacaaacg ctatggttgc tagaggagat tccgagaaga ctatttatat cgtgtttaga 240

ggttcttcat ctattcgtaa ttggatctct gatttgacat tcgttccagt ctcttaccct 300

ccagtttctg gtactaaggt tcacaaagga tttcttgatt cttatggtga ggttcaaaac 360

gagttggttg ctactgtctt ggatcagttt aaacaatacc catcttataa ggttgctgtc 420

actggtcact ctttgggagg tgctactgcc ttgctgtgtg ctttaggttt ataccagaga 480

gaggaaggat tgtcttcagg taacctattc ttgtacactc aaggtcagcc tagagttgga 540

gatccagcat ttgctaatta tgtggtttct actggtattc catatagacg tactgttaac 600

gaaagagaca tagtaccaca cttgcctcca gctgccttcg gatttctgca tgccggtgag 660

gagtactgga tcacagataa ttctcctgaa accgttcaag tgtgtacatc tgatttagag 720

acttccgact gctctaacag tattgttcca tttacttcag ttcttgatca tttgtcttat 780

tttggaatta acaccggttt gtgtacttaa 810

<210> 6

<211> 269

<212> PRT

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 6

Ser Ile Asp Gly Gly Ile Arg Ala Ala Thr Pro Gln Glu Ile Asn Glu

1 5 10 15

Leu Thr Tyr Tyr Thr Thr Leu Ser Ala Asn Ser Tyr Cys Arg Thr Val

20 25 30

Ile Pro Gly Ala Thr Trp Asp Cys Ile His Cys Asp Ala Thr Gly Asp

35 40 45

Leu Lys Ile Ile Lys Thr Trp Ser Thr Leu Ile Tyr Asp Thr Asn Ala

50 55 60

Met Val Ala Arg Gly Asp Ser Glu Lys Thr Ile Tyr Ile Val Phe Arg

65 70 75 80

Gly Ser Ser Ser Ile Arg Asn Trp Ile Ser Asp Leu Thr Phe Val Pro

85 90 95

Val Ser Tyr Pro Pro Val Ser Gly Thr Lys Val His Lys Gly Phe Leu

100 105 110

Asp Ser Tyr Gly Glu Val Gln Asn Glu Leu Val Ala Thr Val Leu Asp

115 120 125

Gln Phe Lys Gln Tyr Pro Ser Tyr Lys Val Ala Val Thr Gly His Ser

130 135 140

Leu Gly Gly Ala Thr Ala Leu Leu Cys Ala Leu Gly Leu Tyr Gln Arg

145 150 155 160

Glu Glu Gly Leu Ser Ser Gly Asn Leu Phe Leu Tyr Thr Gln Gly Gln

165 170 175

Pro Arg Val Gly Asp Pro Ala Phe Ala Asn Tyr Val Val Ser Thr Gly

180 185 190

Ile Pro Tyr Arg Arg Thr Val Asn Glu Arg Asp Ile Val Pro His Leu

195 200 205

Pro Pro Ala Ala Phe Gly Phe Leu His Ala Gly Glu Glu Tyr Trp Ile

210 215 220

Thr Asp Asn Ser Pro Glu Thr Val Gln Val Cys Thr Ser Asp Leu Glu

225 230 235 240

Thr Ser Asp Cys Ser Asn Ser Ile Val Pro Phe Thr Ser Val Leu Asp

245 250 255

His Leu Ser Tyr Phe Gly Ile Asn Thr Gly Leu Cys Thr

260 265

<210> 7

<211> 810

<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 7

tccatcgacg gaggtattag agccgctact cctcaggaaa tcaacgaact tacttactat 60

acaactttgt cagctaattc ttactgtaga actgttattc ctggtgctac ttgggattgc 120

atacattgtg acgccactga agatttaaag ataattaaaa cctggtctac tttgatttac 180

gacacaaacg ctatggttgc tagaggagat tccgagaaga ctatttatat cgtgtttaga 240

ggttcttcat ctattcgtaa ttggatcgct gatttgacat tcgttccagt ctcttaccct 300

ccagtttctg gtactaaggt tcacaaagga tttcttgatt cttatggtga ggttcaaaac 360

gagttggttg ctactgtctt ggatcagttt aaacaatacc catcttataa ggttgctgtc 420

actggtcact ctttgggagg tgctactgcc ttgctgtgtg ctttagattt ataccagaga 480

gaggaaggat tgtcttcagg taacctattc ttgtacactc aaggtcagcc tagagttgga 540

gatccagcat ttgctaatta tgtggtttct actggtattc catatagacg tactgttaac 600

gaaagagaca tagtaccaca cttgcctcca gctgccttcg gatttctgca tgccggtgag 660

gagtactgga tcacagataa ttctcctgaa accattcaag tgtgtacatc tgatttagag 720

acttccgact gctctaacag tattgttcca tttacttcag ttcttgatca tttgtcttat 780

tttggaatta acaccggttt gtgtacttaa 810

<210> 8

<211> 269

<212> PRT

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 8

Ser Ile Asp Gly Gly Ile Arg Ala Ala Thr Pro Gln Glu Ile Asn Glu

1 5 10 15

Leu Thr Tyr Tyr Thr Thr Leu Ser Ala Asn Ser Tyr Cys Arg Thr Val

20 25 30

Ile Pro Gly Ala Thr Trp Asp Cys Ile His Cys Asp Ala Thr Glu Asp

35 40 45

Leu Lys Ile Ile Lys Thr Trp Ser Thr Leu Ile Tyr Asp Thr Asn Ala

50 55 60

Met Val Ala Arg Gly Asp Ser Glu Lys Thr Ile Tyr Ile Val Phe Arg

65 70 75 80

Gly Ser Ser Ser Ile Arg Asn Trp Ile Ala Asp Leu Thr Phe Val Pro

85 90 95

Val Ser Tyr Pro Pro Val Ser Gly Thr Lys Val His Lys Gly Phe Leu

100 105 110

Asp Ser Tyr Gly Glu Val Gln Asn Glu Leu Val Ala Thr Val Leu Asp

115 120 125

Gln Phe Lys Gln Tyr Pro Ser Tyr Lys Val Ala Val Thr Gly His Ser

130 135 140

Leu Gly Gly Ala Thr Ala Leu Leu Cys Ala Leu Asp Leu Tyr Gln Arg

145 150 155 160

Glu Glu Gly Leu Ser Ser Gly Asn Leu Phe Leu Tyr Thr Gln Gly Gln

165 170 175

Pro Arg Val Gly Asp Pro Ala Phe Ala Asn Tyr Val Val Ser Thr Gly

180 185 190

Ile Pro Tyr Arg Arg Thr Val Asn Glu Arg Asp Ile Val Pro His Leu

195 200 205

Pro Pro Ala Ala Phe Gly Phe Leu His Ala Gly Glu Glu Tyr Trp Ile

210 215 220

Thr Asp Asn Ser Pro Glu Thr Ile Gln Val Cys Thr Ser Asp Leu Glu

225 230 235 240

Thr Ser Asp Cys Ser Asn Ser Ile Val Pro Phe Thr Ser Val Leu Asp

245 250 255

His Leu Ser Tyr Phe Gly Ile Asn Thr Gly Leu Cys Thr

260 265

<210> 9

<211> 28

<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 9

aaaaagcttc catcgacgga ggtattag 28

<210> 10

<211> 33

<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 10

aaagaattct taagtacaca aaccggtgtt aat 33

<210> 11

<211> 45

<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 11

aaagaattct caatgatgat gatgatgatg gtcgacagta cacaa 45

<210> 12

<211> 269

<212> PRT

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 12

Ser Ile Asp Gly Gly Ile Arg Ala Ala Thr Pro Gln Glu Ile Asn Glu

1 5 10 15

Leu Thr Tyr Tyr Thr Thr Leu Ser Ala Asn Ser Tyr Cys Arg Thr Val

20 25 30

Ile Pro Gly Ala Thr Trp Asp Cys Ile His Cys Asp Ala Thr Glu Asp

35 40 45

Leu Lys Ile Ile Lys Thr Trp Ser Thr Leu Ile Tyr Asp Thr Asn Ala

50 55 60

Met Val Ala Arg Gly Asp Ser Glu Lys Thr Ile Tyr Ile Val Phe Arg

65 70 75 80

Gly Ser Ser Ser Ile Arg Asn Trp Ile Ala Asp Leu Thr Phe Val Pro

85 90 95

Val Ser Tyr Pro Pro Val Ser Gly Thr Lys Val His Lys Gly Phe Leu

100 105 110

Asp Ser Tyr Gly Glu Val Gln Asn Glu Leu Val Ala Thr Val Leu Asp

115 120 125

Gln Phe Lys Gln Tyr Pro Ser Tyr Lys Val Ala Val Thr Gly His Ser

130 135 140

Leu Gly Gly Ala Thr Ala Leu Leu Cys Ala Leu Gly Leu Tyr Gln Arg

145 150 155 160

Glu Glu Gly Leu Ser Ser Gly Asn Leu Phe Leu Tyr Thr Gln Gly Gln

165 170 175

Pro Arg Val Gly Asp Pro Ala Phe Ala Asn Tyr Val Val Ser Thr Gly

180 185 190

Ile Pro Tyr Arg Arg Thr Val Asn Glu Arg Asp Ile Val Pro His Leu

195 200 205

Pro Pro Ala Ala Phe Gly Phe Leu His Ala Gly Glu Glu Tyr Trp Ile

210 215 220

Thr Asp Asn Ser Pro Glu Thr Val Gln Val Cys Thr Ser Asp Leu Glu

225 230 235 240

Thr Ser Asp Cys Ser Asn Ser Ile Val Pro Phe Thr Ser Val Leu Asp

245 250 255

His Leu Ser Tyr Phe Gly Ile Asn Thr Gly Leu Cys Thr

260 265

Claims

1. An isolated polypeptide, wherein the amino acid sequence of the polypeptide is shown in SEQ ID NO. 4, 6, 8 or 12.

2. A polynucleotide molecule having a polynucleotide sequence selected from the group consisting of:

(1) A polynucleotide sequence encoding the isolated polypeptide of claim 1;

(2) The complement of the polynucleotide sequence of (1).

3. The polynucleotide molecule of claim 2, wherein the polynucleotide sequence is set forth in SEQ ID No. 3,5 or 7.

4. A nucleic acid construct comprising the polynucleotide molecule of claim 2 or 3.

5. The nucleic acid construct of claim 4, wherein the nucleic acid construct is a vector.

6. The nucleic acid construct of claim 5, wherein the vector is an expression vector or a cloning vector.

7. A host cell comprising a polynucleotide molecule according to claim 2 or 3 or a nucleic acid construct according to any one of claims 4 to 6 and/or expressing a polypeptide according to claim 1.

8. The host cell of claim 7, wherein the host cell is escherichia coli or yeast or aspergillus.

9. An isolated polypeptide, wherein the isolated polypeptide is expressed by the host cell of claim 7 or 8.

10. A composition comprising the polypeptide of claim 1 or 9.

11. Use of the polypeptide of claim 1 or 9, the polynucleotide molecule of claim 2 or 3, the nucleic acid construct of any one of claims 4-6, the host cell of claim 7 or 8, or the composition of claim 10 in the hydrolysis of medium-long carbon chain fatty acid esters.

12. Use of an isolated polypeptide having the amino acid sequence shown in SEQ ID No. 4 or 12, a polynucleotide molecule encoding said isolated polypeptide or its complement, a nucleic acid construct comprising said polynucleotide molecule, a host cell expressing said isolated polypeptide or a composition comprising said isolated polypeptide in the esterification of fatty acids.

13. Use of an isolated polypeptide having the amino acid sequence shown in SEQ ID No. 6 or 8, a polynucleotide molecule encoding said isolated polypeptide or its complement, a nucleic acid construct comprising said polynucleotide molecule, a host cell expressing said isolated polypeptide or a composition comprising said isolated polypeptide in the enzymatic synthesis of biodiesel.