CN108251401A - Lipase and its application - Google Patents
Lipase and its application Download PDFInfo
- Publication number
- CN108251401A CN108251401A CN201710115983.7A CN201710115983A CN108251401A CN 108251401 A CN108251401 A CN 108251401A CN 201710115983 A CN201710115983 A CN 201710115983A CN 108251401 A CN108251401 A CN 108251401A
- Authority
- CN
- China
- Prior art keywords
- polypeptide
- sequence
- lipase
- seq
- nucleic acid
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Classifications
-
- 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)
- C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
- C12N9/18—Carboxylic ester hydrolases (3.1.1)
- C12N9/20—Triglyceride splitting, e.g. by means of lipase
-
- 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
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
-
- 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
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
- C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
-
- 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
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/62—Carboxylic acid esters
-
- 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
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y301/00—Hydrolases acting on ester bonds (3.1)
- C12Y301/01—Carboxylic ester hydrolases (3.1.1)
- C12Y301/01003—Triacylglycerol lipase (3.1.1.3)
-
- 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
- C12N2800/00—Nucleic acids vectors
- C12N2800/10—Plasmid DNA
- C12N2800/101—Plasmid DNA for bacteria
-
- 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
- C12N2800/00—Nucleic acids vectors
- C12N2800/10—Plasmid DNA
- C12N2800/102—Plasmid DNA for yeast
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
- Y02E50/00—Technologies for the production of fuel of non-fossil origin
- Y02E50/10—Biofuels, e.g. bio-diesel
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Genetics & Genomics (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Microbiology (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- General Chemical & Material Sciences (AREA)
- Mycology (AREA)
- Physics & Mathematics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Biophysics (AREA)
- Plant Pathology (AREA)
- Oil, Petroleum & Natural Gas (AREA)
- Medicinal Chemistry (AREA)
- Enzymes And Modification Thereof (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The present invention relates to lipase and its applications.Specifically, the present invention provides a kind of polypeptide, amino acid sequence such as SEQ ID NO:Shown in 2 or by SEQ ID NO:2 and for promoting SEQ ID NO:Amino acid sequence shown in 2 is expressed and the sequence of purifying composition.The present invention also provides the coded sequence of the polypeptide, the nucleic acid constructs containing the coded sequence, the host cell containing the nucleic acid constructs and relevant purposes.The lipase of the present invention has excellent enzymatic activity, moreover it is possible to naturally be fixed on thalline, in the industrial production with advantage.
Description
Technical field
The present invention relates to lipase and its applications.
Background technology
Lipase is a kind of enzyme with a variety of catalytic capabilities, can be catalyzed triglyceride and be hydrolyzed to glycerine and free fat
Fat acid can also be catalyzed the hydrolysis of other esters and transesterification and esters synthetic reaction, and lipase is also showed the bottom of in addition
The enantioselectivity of object.More than characteristic imparts lipase in food fats and oils processing, biodiesel, detergent, ester bond compound
Synthesis and chiral drug synthesis etc. industry in extensive use (Abhishek Kumar Singh, Mausumi
Mukhopadhyay, Overview of Fungal Lipase:A Review, 2012,166 (2):486-520).
Lipase is widely present in animals and plants and microorganism, and due to microorganism, not only type is more, breeding is fast, easily loses
The progress of disease is different, and with pH value more wider than animal, temperature range and Substratspezifitaet, therefore microbial lipase is work
The important sources of industry lipase.According to statistics, the microorganism of yielding lipase has 65 categories, is concentrated mainly on aspergillus niger, false silk ferment
Mother, head mold, pseudomonad, streptomycete etc. (Wang Zishe, Zhang Ji, Ma Junyi, Shi Rui,《Agriculture of Anhui science》, 2011,39 (7)：
3798-3800)。
The application of lipase is divided into food industry, medical and health, chemical etc..Application in chemical is included in
Fatty acid methyl ester, such as pungent methyl caprate, the application in hydrolysis.
Apply the commercialization lipase in the hydrolysis of pungent methyl caprate is most commonly used to be derived from rhizomucor miehei at present
Lipase and the lipase from antarctic candida, but due to fancy price so that the industrial applications of enzyme process by
To limitation.Therefore, the lipase that can be used for the fatty acid methyls ester hydrolysis such as pungent methyl caprate for developing a kind of high catalytic activity has
Significance.
Therefore, the present invention is directed to find novel lipase, there is the enzymatic activity for hydrolyzing pungent methyl caprate etc., meet industry
The needs of production.
Invention content
The present invention provide novel lipase, preference hydrolysis in short chain aliphatic ester, can be used for middle short chain (such as
C4-C12 length) for example pungent methyl caprate of fatty acid methyl ester hydrolysis application；The lipase active is high；And expression can be fixed on
On the thalline of the bacterial strain (such as Pichia pastoris) of the lipase.
The present invention provides a kind of polypeptide, and the polypeptide is selected from：
(1) amino acid sequence such as SEQ ID NO:Polypeptide shown in 2 and
(2) by SEQ ID NO:2 and for promoting SEQ ID NO:Amino acid sequence shown in 2 is expressed and the sequence group of purifying
Into polypeptide.
The polypeptide of the present invention is also included within SEQ ID NO:By substitution, missing or addition in amino acid sequence shown in 2
One or several amino acid, while retain SEQ ID NO：2 lipase actives having by SEQ ID NO:It is more derived from 2
Peptide.
The polypeptide of the present invention further includes and SEQ ID NO:Polypeptide shown in 2 has at least 90%, preferably at least 95%, more
Preferably at least 98%, the more preferably at least polypeptide of 99% sequence identity.
The polypeptide of the present invention, which derives from, splits pot algae.
The present invention provides a kind of polynucleotide sequence, is selected from：
(1) polynucleotide sequence of polypeptide of the present invention is encoded；
(2) complementary series of (1) described sequence；With
(3) segment of the long 10-40 base of (1) or (2) described polynucleotide sequence, preferably long 15~30 bases
Segment.
In one or more embodiments, which has SEQ ID NO:Nucleotide sequence shown in 1.
In one or more embodiments, the polynucleotide sequence is by SEQ ID NO:Nucleotide sequence group shown in 1
Into.
The present invention also provides a kind of nucleic acid constructs, which contains the polynucleotide sequence of the present invention.
In one or more embodiments, nucleic acid constructs contains the polynucleotide sequence or its for encoding polypeptide of the present invention
Complementary series.
In one or more embodiments, nucleic acid constructs contains SEQ ID NO:Nucleotide sequence shown in 1 or its
Complementary series.
In one or more embodiments, nucleic acid constructs is cloning vector or expression vector.
In one or more embodiments, nucleic acid constructs contains AOX1 promoters, α-Factor signal peptides, His4 tables
Up to frame and multiple cloning sites.
In one or more embodiments, nucleic acid constructs is using pPIC9K plasmids as skeleton.
The present invention also provides a kind of host cell, which contains the nucleic acid constructs of the present invention.
In one or more embodiments, which is plant cell.
In one or more embodiments, which is microbial cell.
In one or more embodiments, which is Pichia pastoris or Bacillus coli cells.
The present invention also provides polypeptide described herein, the complementary series of its coded sequence or the coded sequence, containing the coding
Sequence or the nucleic acid constructs of its complementary series and the host cell containing the nucleic acid constructs food industry, medical and health,
Application in chemical.
Complementary series the present invention also provides polypeptide described herein, its coded sequence or the coded sequence is in fatty acid methyl ester
Application in hydrolysis, such as the hydrolysis of pungent methyl caprate.
Description of the drawings
Fig. 1：Comparison chart on BMMY- tributyrin tablets.Wherein left side bacterium colony is without conversion p9K-SL4
GS115 Pichia pastoris bacterium colony expresses bacterial strain as control, the right bacterium colony for SL4；Display SL4 expression bacterial strains form apparent saturating
Bright circle has lipase active.
Fig. 2：The Supernatant samples of Pichia pastoris transformant of SL4 lipase and thalline sample are expressed, to be free of SL4 genes
The transformant of pPIC9K empty carriers is negative control.
Fig. 3：The Substratspezifitaet of SL4 lipase.
Fig. 4：The optimum temperature of SL4 lipase.
Fig. 5：The most suitable action pH of SL4 lipase.
Fig. 6：Influence of the metal ion to lipase SL4 vigor.
Fig. 7：SL4 lipase carries out enzyme activity comparison for hydrolyzing with commercially available lipase CALB and RML.
Fig. 8：PPIC9K plasmid maps.
Specific embodiment
The present invention, which provides, has such as SEQ ID NO:The lipase of amino acid sequence shown in 2.The invention also includes in SEQ
ID NO:It is obtained when carrying out conservative replaces with amino acid with similar or analogous performance on the basis of the amino acid sequence shown in 2
Polypeptide.This conservative replaces do not usually change the function of protein or polypeptide." amino acid with similar or analogous performance "
Including for example, the family of the amino acid residue with similar side chain, these families include with basic side chain amino acid (such as
Lysine, arginine, histidine), the amino acid (such as aspartic acid, glutamic acid) with acid side-chain, with neutral
Polar side chain amino acid (such as glycine, asparagine, glutamine, serine, threonine, tyrosine, half Guang ammonia
Acid), have non-polar sidechain amino acid (such as alanine, valine, leucine, isoleucine proline, phenylalanine,
Methionine, tryptophan), the amino acid (such as threonine, valine, isoleucine) with β-branched building block and with fragrance
The amino acid (such as tyrosine, phenylalanine, tryptophan, histidine) of side chain.Therefore, it is used in polypeptide of the present invention from same
Another amino acid residue of side chain class replaces one or several sites, and will not be in substantially influences its activity.
Present invention accordingly comprises in SEQ ID NO:By substitution, missing or addition one in amino acid sequence shown in 2
Or several amino acid, while retain SEQ ID NO：2 lipase actives having by SEQ ID NO:Polypeptide derived from 2.
It is described it is several be usually 10 within, within preferably 8, within more preferable 5.
Knowing SEQ ID NO:In the case of 2 sequence and biological function, routine can be used in those skilled in the art
Technological means judge SEQ ID NO:Which amino acid residue can be substituted or delete in amino acid sequence shown in 2.For example,
By comparison from different genera, active same or like or visibly different sequence, it can be determined which ammonia in these sequences
Base acid residue is can be substituted or delete.Method (including the method disclosed in the present) verification of this field routine can be used
Whether such sequence has the enzyme activity of the present invention.
In addition, it is well known to those skilled in the art, in gene cloning operation, it is often necessary to suitable restriction enzyme site is designed,
This certainly will introduce one or more incoherent residues in expressed albumen end, and this has no effect on the work of destination protein
Property.The recombination egg being for another example secreted into automatically for construction of fusion protein, the expression of promotion recombinant protein, acquisition outside host cell
In vain or the purifying conducive to recombinant protein, it is often necessary to which some amino acid are added to the N- ends of recombinant protein, C- ends or should
In other appropriate areas in albumen, it may for example comprise but be not limited to, suitable joint peptide, signal peptide, leader peptide, end extension,
The albumen water of glutathione S-transferase (GST), maltose E binding protein, albumin A or Xa factor or fibrin ferment or enterokinase
Solve enzyme site.The aminoterminal or c-terminus of amino acid sequence of the present invention can also contain one or more polypeptide fragments, as albumen
Label.Any suitable label may be used to the present invention.For example, the label can be FLAG, HA, HA1, c-Myc,
Poly-His, Poly-Arg, Strep-TagII, AU1, EE, T7,4A6, ε, B, gE and Ty1.These labels can be used for egg
It is purified in vain.The label example used includes Poly-Arg, such as RRRRR (SEQ ID NO:3)；Poly-His 2-10 is (logical
Normal 6), such as HHHHHH (SEQ ID NO:4)；FLAG, i.e. DYKDDDDK (SEQ ID NO:5)；Strep-TagII, i.e.,
WSHPQFEK(SEQ ID NO:6)；And C-myc, i.e. WQKLISEEDL (SEQ ID NO:7).It should be understood that these amino acid sequences
Presence do not interfere with gained polypeptide activity.Therefore, the present invention is also included within the C-terminal and/or N-terminal of polypeptide of the present invention
The polypeptide obtained by one or several amino acid is added, these polypeptides still have lipase active as described herein.
Therefore, the present invention also includes and SEQ ID NO:Amino acid sequence shown in 2 has at least 90%, preferably at least
The amino acid sequence of 95%, more preferably at least 98%, more preferably at least 99% sequence identity.In some embodiments, it is such
Amino acid sequence similarly carrys out autoclasis pot algae (Schizochytrium), preferably has and this paper SEQ ID NO:2 identical or classes
As lipase activity.
The sequence identity of the two sequences of conventional means calculating ratio pair can be used, for example, being provided using NCBI
BLASTP is compared using default parameters.In certain embodiments, polypeptide of the invention is neutral or alkaline lipase.
In certain embodiments, polypeptide of the invention has Substratspezifitaet as lipase, to 4- Nitrophenyl butyrates
(pNPB) hydrolysis ability is most strong, to the hydrolysis energy of 4- nitrobenzophenones caprylate (pNPO), 4- nitrobenzophenones laurate (pNPD)
Power secondly, and to 4- nitrobenzophenones myristate (pNPM), 4- nitrobenzophenones palmitate (pNPP), 4- nitrobenzophenone stearic acid
The hydrolysis ability of ester (pNPS) is weaker.In certain embodiments, the optimum temperature of polypeptide of the present invention 20-60 DEG C it
Between, preferably at 35 DEG C ± 2 DEG C.In certain embodiments, the most suitable action pH of polypeptide of the present invention preferably exists between 5.5-9
Between 6-9, more preferably 6.1-7.5 or 8.5-9, most preferably 6 or 9.In certain embodiments, polypeptidase of the invention
It lives by ZnSO4、CuSO4、NiSO4、FeCl3、MnCl2、CaCl2、MgSO4Inhibition, and NaCl, KCl, (NH4)2SO4Can then it swash
Its enzyme activity, especially NaCl, KCl living.In certain embodiments, polypeptide of the present invention hydrolysis vigor phase compared with commercialization enzyme
When.In certain embodiments, polypeptide of the present invention is used for fatty acid methyl ester hydrolysis, such as containing in 4~12 carbon atoms
The fatty acid methyl ester hydrolysis of short chain fatty acid residues is particularly used for pungent methyl caprate hydrolysis.
According to the host used in recombinant production scheme, polypeptide of the invention can be glycosylated or can be non-sugar based
Change.
The polypeptide of the present invention can be native purified product or be chemical synthesis product or using recombinant technique from
It is generated in protokaryon or eucaryon host (for example, bacterium, yeast, higher plant, insect and mammalian cell).
The application includes the coded sequence of polypeptide of the present invention.SEQ ID NO:1 shows the coded sequence of polypeptide of the present invention
One of." coded sequence " including with SEQ ID NO:The sequence of 1 very high homology or under strict conditions with SEQ ID NO：1 hybridization
Nucleotide sequence or the family gene molecule homologous with above-mentioned numberator height.The sequence of coding polypeptide of the present invention can be with SEQ
ID NO：Coding region sequence shown in 1 is identical or the variant of degeneracy.As used herein, " variant of degeneracy " is in this hair
Refer to that coding includes SEQ ID NO in bright：Amino acid sequence shown in 2, but with SEQ ID NO：Nucleotides sequence shown in 1 is shown
The nucleotide sequence of difference.
The sequence of coding polypeptide of the present invention includes：The coded sequence of encoding mature polypeptide；The coded sequence of mature polypeptide
With various additional coding sequences；The coded sequence (and optional additional coding sequence) and non-coding sequence of mature polypeptide.
The coded sequence of the polypeptide of the present invention or its segment can usually use PCR amplification method, recombination method or artificial synthesized
Method obtains.For PCR amplification method, can pot algae from Escherichia coli or first be split using conventional technology
(Schizochytrium) genomic DNA is obtained in, it is especially open then further according to nucleotide sequence disclosed in this invention
Reading frame sequence designs primer, for amplifying lipase gene from the genomic DNA.
Therefore, the present invention also includes the segment of coded sequence of the present invention, the segment usually long 10~40, preferably 15~30
A base can be used as primer or probe to use." segment " refers to continuous a part of sequence of full length sequence herein.
The present invention also relates to nucleic acid constructs, the nucleic acid constructs contain the present invention coded sequence and with the code sequence
Row are operatively connected one or more regulation and control sequences that the coded sequence is and guided to be expressed under suitable conditions in host cell
Row.The polynucleotides of coding polypeptide of the present invention can be operated in various ways, to ensure the expression of the polypeptide.Carrier is inserted at it
The operation of the polynucleotide sequence before may according to the expression vector but cater to the need or required.Utilize recombinant DNA side
Method is come to change the technology of polynucleotide sequence be known in the art.
Regulating and controlling sequence can be suitable promoter sequence, be by being used to express the polynucleotides for encoding polypeptide of the present invention
The nucleotide sequence of host cell identification.Promoter sequence may include the transcription regulating nucleotide sequence of polypeptide expression.Promoter can be
Any nucleotide sequence of transcriptional activity is shown in selected host cell, is started including mutation, truncated and heterozygosis
Son, and can be obtained from the gene for encoding the extracellular or intracellular polypeptide homologous or heterologous with the host cell.Suitable for this hair
Bright promoter sequence example includes AOX1 promoters and GAP promoters etc..
Regulating and controlling sequence can also be suitable transcription terminator sequences, be the sequence identified by host cell to terminate transcription
Row.3 ' ends of nucleotide sequence of the terminator sequence with encoding the polypeptide are operatively connected.Have in the host cell of selection
Any terminator of function can be used in the present invention.
Regulating and controlling sequence can also be suitable targeting sequencing, the non-translational region of the mRNA important to host cell translation.Label
It is operatively connected to sequence and 5 ' ends of the nucleotide sequence for encoding the polypeptide.Functional in the host cell of selection
What terminator can be used in the present invention.
Regulating and controlling sequence can also be the amino acid sequence that coding is connect with the amino-terminal end of polypeptide and instruct the coding
Polypeptide enter the signal peptide coding region of cell secretory pathway.It 5 ' ends of nucleotide sequence coded sequence can be inherently comprising natural
The signal peptide coding region of the translation reading frame of code area segment of the connection with coding secrete polypeptide.Alternatively, coded sequence
5 ' ends may include the signal peptide coding region with the code area external source.When signal peptide coding region is included when coded sequence non-natural,
It may need external signal peptide coding region.Alternatively, external signal peptide coding region can simply replace natural signal peptide
Code area is to enhance the secretion of polypeptide.However, the polypeptide of guidance expression enters any of the secretory pathway of the host cell of selection
Signal peptide coding region, i.e. secretion enter culture medium, can be used in the present invention.
The present invention also relates to cloning vectors or expression vector including polynucleotides of the present invention.These carriers can contain above
Each regulating and controlling sequence.
Expression vector can be easily subjected to recombinant DNA method and can lead to interested nucleotides sequence list
Any carrier (such as plasmid or virus) reached.It is thin with the host that is wherein imported into the carrier that the selection of carrier is generally dependent on carrier
The compatibility of born of the same parents.The carrier can be circular plasmids that are linear or being closed.
Carrier can be the carrier of autonomous replication, i.e., exist as extrachromosomal entity, replicate independent of chromosome
The carrier of duplication, such as plasmid, extra-chromosomal element, minichromosome or artificial chromosome.Carrier may include ensureing certainly
Any mode that I replicates.Alternatively, carrier can be when being imported into host cell, be integrated into genome and with it
The carrier that chromosome through being be integrated into replicates together.Host cell gene group will be imported into addition, can be used and include together
The single carrier of total DNA or plasmid or two or more carriers or plasmid or transposons.
The present invention carrier preferably comprise one or more allow easily select convert, transfect, transduce it is isocellular optional
Select label.Selectable label is gene, and product provides the resistance to antibiotic or virus, the resistance to heavy metal, original and supports
Type is to auxotroph etc..
The carrier of the present invention, which preferably comprises, allows the vector integration to enter host cell gene group or the carrier in cell
Independently of the element of genome autonomous replication.
The polynucleotides of the present invention of more than one copy can be inserted into host cell to increase the yield of the gene outcome.
Increasing for polynucleotide copies number can be by being integrated into host cell gene group by the sequence of at least one additional copies or leading to
It crosses and is obtained including amplifiable selectable marker gene and the polynucleotides, wherein the selectable marker gene comprising amplification copy is simultaneously
And the cell thus comprising additional copies polynucleotides can be by there are cultivate the cell during appropriate selective agent to screen.
The carrier of the present invention preferably comprises one section of artificial synthesized sequence, contains multiple limitation endonuclease recognized sites, energy
A variety of pluggable positions or interleaved plan are provided for exogenous DNA.The expression vector of the present invention is more highly preferred to include continuous 6
The small peptide of histidine sequences is conducive to the extraction and purifying of protein.
In certain embodiments, carrier of the invention is used for the polypeptide in the expression in escherichia coli present invention.Therefore, exist
In certain embodiments, the expression vector contains AOX1 promoters, α-Factor signal peptides, His4 expression cassettes and polyclonal position
Point.Preferably, the nucleic acid constructs is using pPIC9K plasmids as skeleton.
In certain embodiments, carrier of the invention is used to express the polypeptide of the present invention in Pichia pastoris.Therefore, exist
In certain embodiments, the expression vector contains AOX1 promoters, α-Factor signal peptides, His4 expression cassettes and polyclonal
Site.Preferably, the nucleic acid constructs is using pPIC9K plasmids as skeleton.
The present invention also relates to the recombinant host cells containing the polynucleotides of the present invention for being used for recombinant production polypeptide.Including
The carrier of polynucleotides of the present invention is imported into host cell so that the composition portion as chromosome of the carrier as explained earlier
Divide or maintained as extrachromosomal self-replacation carrier.The selection of host cell is heavily dependent on coding polypeptide
Gene and its source.
Host cell can be plant cell or unicellular microorganism or non-unicellular micro-organism.Unicellular microorganism is for example
Gram-positive bacterium, including but not limited to bacillus cell, for example, Alkaliphilic bacillus, bacillus amyloliquefaciens, short bud
Spore bacillus, bacillus megaterium, bacillus subtilis, bacillus licheniformis, bacillus coagulans, bacillus stearothermophilus and
Bacillus thuringiensis etc.；Or streptomyces cell, such as money Streptomyces glaucoviolaceus；Or gramnegative bacterium, such as Escherichia coli
And pseudomonas.Bacterial host is bacillus subtilis, bacillus licheniformis, stearothermophilus gemma bar in a preferred aspect,
Bacterium and Bacillus coli cells.
Host cell can also be eucaryote, such as mammal, insect, plant, yeast or fungal cell.Preferred
Aspect, host cell is fungal cell, and as used herein " fungi " includes Ascomycota (Ascomycota), Basidiomycota
(Basidiomycota), chytridiomycota (Chytridiomycota), Zygomycota (Zygomycota) and oomycota etc..
At preferred aspect, host cell is prokaryotic cell." prokaryotic cell " is including pseudomonad as used herein
Belong to (Pseudomonas), bacillus (Bacillus), Enterobacter (Enterobacter), staphylococcus
(Staphylococcus), the bacterium of streptomyces (Streptomyces) and Escherichia (Escherichia).More
For preferred aspect, host cell is the cell of pseudomonas, bacillus, streptomyces and Escherichia.
At preferred aspect, host cell is bacillus subtilis (Bacillus subtilis), Pseudomonas fluorescens
(Pseudomonas fluorescens), Pichia pastoris (Pichia pastoris), Escherichia coli (Escherichia
) and muta lead mycillin (Streptomyces lividans) etc. coli.In addition most preferably aspect, host cell is big
Enterobacteria (Escherichia coli) cell or Pichia pastoris (Pichia pastoris) cell.
Conventional transfection method can be used the nucleic acid constructs containing polynucleotide sequence of the present invention is transferred in host cell.
Transfection is generally divided into transient transfection and stable transfection.In the former exogenous DNA/RNA unconformity to host chromosome, therefore a place
Multiple copy numbers may be present in chief cell, generate high-caliber expression, but usually only last for several days.In stable transfection, exogenous DNA
Both it can be integrated into host chromosome, it is also possible to exist as a kind of episome.The technological means of transfection includes chemical transfection
And physical transfection, the former such as DEAE- glucans method, calcium phosphate method and artificial liposome method, the latter such as microinjection, electroporation and
Particle gun etc..
The polypeptide of the present invention can be used in food industry, medical and health, chemical.Specifically, polypeptide of the present invention can
For grease hydrolysis；Progress butterfat hydrolysis in dairy products is can be applicable to, the flavor of cheese, milk powder, cream can be enhanced, promote cheese
Maturation improves the quality of dairy products；It can be applied in wheaten food processing, so that wheaten food elasticity improves, improve sense of food, improve bread
Preservativity；Fatty acid methyl ester hydrolysis, such as the hydrolysis of pungent methyl caprate can be used in chemical, to produce aliphatic acid；
It can be added in detergent, for removing degreasing or the cleaning, bleaching, dry-cleaning for tableware and clothing, leather cleaning is stealthy
Eyes clean, the cleaning of the industrial wastes such as cosmetics and food processing, degradation of exhaust pipe and water closet debirs etc..This
Invention more particularly to polypeptide of the present invention particularly contain in fatty acid methyl ester hydrolysis, such as Short-Chain Fatty Acids first ester hydrolysis
There is the fatty acid methyl ester hydrolysis of C4~C12 chain length fatty acids, most preferably the application in the hydrolysis of pungent methyl caprate.It can be with ability
The dosage of domain routine is by the polypeptide of the present invention in above-mentioned each field.
Therefore, the present invention provide polypeptide of the present invention, coded sequence, nucleic acid constructs and cell food industry,
Application in medical and health, chemical.Especially, the present invention provides the polypeptide, coded sequence, nucleic acid constructs and thin
Born of the same parents are in aliphatic acid ester hydrolysis, such as middle short chain (C4~C12) fatty acid methyl ester hydrolysis, particularly pungent methyl caprate hydrolysis
Application.
The present invention also provides a kind of composition, is a kind of reaction mixture.It is characterized in that, the reaction mixture contains
Have：Fatty acid methyl ester, water, the polypeptide of the present invention or the host cell containing nucleotide of the invention.Preferably, the fatty acid methyl
Ester is pungent methyl caprate.
Enzyme activity unit U is defined as under conditions of 40 DEG C of temperature, pH value 8.0, enzyme hydrolysis substrate 4- nitrobenzophenone fourths
Acid esters, the enzyme amount per minute released needed for 1 μm of ol p-nitrophenol (pNP) are 1 enzyme activity unit (U).Preferably, this hair
The dosage of bright polypeptide is more than every gram of fatty acid methyl ester 5U, preferably between 5U~10U；And/or preferably, the dosage of water is
Fatty acid methyl ester, such as more than the 95% of pungent methyl caprate weight, such as between 95~100% or between 95~200%.
The present invention polypeptide can be pure enzyme preparation form provide, can also composition form provide.Composition can
To be powder composition or liquid composition or paste composition.When being provided with composition forms, enzyme is contained according to this
The different purposes of composition, the composition contain different auxiliary materials.Auxiliary material known in the art can be added to the present invention's
In composition, this kind of auxiliary material includes but not limited to sorbierite, potassium sorbate, methyl benzoate, ethyl benzoate, sucrose, sweet dew
It is one or more in the stabilizers such as sugar, trehalose, starch, sodium chloride, calcium chloride or other substances.
The present invention also provides a kind of using polypeptide of the present invention progress oil and fat refining, oil and fat chemical, improvement feed, food system
Method prepared by standby, drug, the method (and other methods mentioned in this article using polypeptide of the present invention) preferably with
It is carried out under the conditions of lower：
(A) temperature is 20 DEG C -60 DEG C, preferably 35 ± 2 DEG C；And/or
(B) pH be 5.5-9, more preferably preferably 6-9,6.1-7.5 or 8.5-9, most preferably 6 or 9.
The amount of polypeptide of the present invention used in the method for the present invention can be determined according to actual conditions.
Hereafter the present invention will be illustrated in a manner of specific embodiment.The experiment side of actual conditions is not specified in the following example
Method, such as Sambrook usually according to normal condition,《Molecular cloning：Lab guide》(New York, United States：Cold spring harbor laboratory
Publishing house (Cold Spring Harbor Laboratory Press), 1989) condition described in or according to institute of manufacturer
It is recommended that condition carry out.For the usage and dosage of reagent, unless otherwise stated, making according to conventional usage and dosage
With.Unless otherwise instructed, percentage refers to weight percent.
Experiment material
Experimental strain and plasmid
Bacterial strain：Pichia pastoris GS115 (Invitrogen, article No. C181-00), escherichia coli DH5a (TAKARA:
Catalog#.D9057A)。
Plasmid：PPIC9K plasmids (Invitrogen, article No. V175-20).
2. culture medium and solution
LB fluid nutrient mediums：0.5% yeast extract, 1% tryptone, 1%NaCl, pH7.0.
LB solid mediums：Agar concentration 1.5% is added in LB fluid nutrient mediums.
YPD fluid nutrient mediums：1% yeast extract, 2% peptone, 2% glucose.
YPD solid mediums：Agar concentration 2% is added in LB fluid nutrient mediums.
MGYS solid mediums：1.34% yeast nitrogen alkali (YNB) liquid containing ammonium sulfate is free of amino acid, 1% glycerine, 1M sorbs
Alcohol, 4 × 10-5%D- biotins, 2% agar.
Tributyrin screening and culturing medium：1.34% yeast nitrogen alkali (YNB) liquid containing ammonium sulfate is free of amino acid, 4 × 10-
5%D- biotins, 0.5% methanol (add in) after sterilizing, and 2% tributyrin emulsion, 0.1M citric acid-sodium citrates are delayed
Fliud flushing pH6.6,2% agar.
2% soybean lecithin emulsion：2% tributyrin, 1% Arabic gum, 100ml H2O, with high-speed homogenization machine
8000rpm is homogenized 1min.
BMGY fluid nutrient mediums：1% yeast extract, 2% peptone, 1.34% yeast nitrogen alkali (YNB) liquid containing ammonium sulfate
Without amino acid, 1% glycerine, 4 × 10-5%D- biotins, 0.1M potassium dihydrogen phosphates-dipotassium hydrogen phosphate pH of buffer 6.0.
BMMY fluid nutrient mediums：1% yeast extract, 2% peptone, 1.34% yeast nitrogen alkali (YNB) liquid containing ammonium sulfate
Without amino acid, 0.3%ZnSO4·7H2O, 0.5% methanol (add in) after sterilizing, and 4 × 10-5%D- biotins (add after sterilizing
Enter), 0.1M citric acid-sodium citrate buffer solutions pH6.6.
PNPB substrate buffer solutions：5.3mL A liquid and 94.7mL B liquid are taken, about 280mL water is added in after mixing, adds in 0.92g
NaTDC, 0.44g gum arabic powders, stirring and dissolving with H3PO4 or NaOH adjusting pH value to 8.0, are settled to 400mL,
4 DEG C of preservations.
A liquid：0.2mol/L NaH2PO4Solution weighs 3.12g NaH2PO4With distillation water dissolution and it is settled to 100mL
B liquid：0.2mol/L Na2HPO4Solution weighs 71.7g Na2HPO4.12H2O distills water dissolution and is settled to
1000mL。
Substrate pNPB solution (3mg/L)：4- Nitrophenyl butyrates (pNPB) 0.030g is weighed, 10mL isopropanols is added in and stirs
Mix dissolving, 4 DEG C of preservations.
The assay method of lipase activity
Using colorimetric method for determining lipase activity.With 4- Nitrophenyl butyrates (pNPB) for substrate, specific method is as follows：
Substrate and buffer solution are pre-configured with, preparation method is shown in experiment material.
By substrate and buffer solution with 1:9 (v/v) are made into reaction mixture.The sample for drawing 25uL supernatant thalline adds in
In 600uL pNPB reaction mixtures, 40 DEG C of water-bath 15min add in 500uL absolute ethyl alcohols and terminate reaction, 12000rpm centrifugations
5min takes the absorbance of supernatant determination sample under 410nm wavelength.
According to p-nitrophenol standard curve, the calculation formula of lipase activity is obtained：
Enzyme activity (U/mL)=(a × Δ OD+b) × 10 × n × 1/15
In formula, Δ OD：A-A0 (light absorption value at 405nm)；a、b：Coefficient in pNP calibration curve formulas；10：By enzyme solution
In enzyme activity conversion for 1mL enzyme activity；n：The extension rate of enzyme solution；1/15：Reaction time is 15 minutes, and it is 1 minute to convert
Coefficient.
Enzyme activity defines：It is 40 DEG C in temperature, under conditions of pH value 8.0, enzyme-containing sample hydrolysis substrate 4- nitrobenzophenone butyric acid
Ester, the enzyme amount per minute released needed for 1umol p-nitrophenols (pNP) are 1 enzyme activity unit (U).
Note：4- Nitrophenyl butyrates (pNPB), 4- nitrobenzophenones caprylate (pNPO), 4- nitrobenzophenone laurates
(pNPD), 4- nitrobenzophenones myristate (pNPM), 4- nitrobenzophenones palmitate (pNPP), 4- nitrobenzophenone stearates
(pNPS) it is for example above-mentioned that manner of formulation (is purchased to Sigma)；Restriction enzyme NotI, EcoRI, SalI are (purchased from knob Great Britain biology
Technology (Beijing) Co., Ltd)；PCR enzymes：TaKaRa Taq,HS DNA Polymerase are (purchased from precious raw
Object engineering (Dalian) Co., Ltd)；T4DNA ligases (are purchased from Fu Meitaisi Co., Ltds)；Pungent methyl caprate (Sha Suofeng benefit alcohol
Industrial (Lianyun Harbour) Co., Ltd)；CalB lipase is available from the Novozymes Lipozyme CALB L of Novozymes Company；
RML lipase is available from the Novozymes Palatase 20000L of Novozymes Company.
Embodiment 1：P9K-SL4 vector constructions
Commission Sangon Biotech (Shanghai) Co., Ltd.) limited company will split pot algae lipase SL4 (SEQ ID NO.:1) pass through
The mode of full-length genome synthesis obtains, and passes through restriction enzyme restriction enzyme site NotI, and EcoRI is connected on pPIC9K plasmids,
So as to obtain p9K-SL4 recombinant plasmids.
Embodiment 2：Express Pichi strain structure and the screening of SL4 lipase
P9K-SL4 plasmids restriction enzyme SalI is linearized, linearized fragment is recycled by gel.Utilize acetic acid
Lithium (LiAc) method prepares the competent cell of Pichia pastoris GS115 bacterial strain, then the p9K- for linearizing 500ng by electricity conversion
SL4 is converted to GS115 competent cells.Conversion product is coated on MGYS tablets, 30 DEG C are cultivated 3 days.List on picking tablet
Clone until in BMMY- tributyrin screening flat boards, chooses the big clone of transparent circle, so as to obtain expression SL4 lipase
Pichi strain.The Pichi strain of the GS115 of no conversion p9K-SL4 and expression SL4 lipase are applied into tablet, obtained
Lithograph as shown in Figure 1, wherein left side bacterium colony are without converting the GS115 of p9K-SL4 (as control), and the right bacterium colony is
SL4 expresses bacterial strain, as a result shows that SL4 expression bacterial strains form apparent transparent circle, has lipase active.
Embodiment 3：Express the fermentation of the Pichi strain of SL4 lipase and enzyme activity distribution detection
The Pichi strain transformant of expression SL4 lipase is taken, is first activated in liquid YPD, is then inoculated in BMGY
In culture medium, at 30 DEG C, 220rpm shaken cultivations are stayed overnight.Culture is gone in BMMY culture mediums, initial OD 600 is 6.With
2% methanol is induced, for 24 hours with respectively add 1% methanol after 32h, respectively add 1% methanol after 48h and 56h, 72h samplings take
The fermented sample 12000rpm centrifugation 5min of 1mL are sucked out after fermentation supernatant and precipitation thalline are resuspended with the sterile water of 1mL, so as to obtain
Fermented liquid supernatant sample and thalline sample, using be free of SL4 genes pPIC9K empty carriers transformant as negative control.
The lipase activity of fermented liquid supernatant sample and thalline sample is calculated, as a result sees Fig. 2, it can be found that 99.3%
Lipase active all in thalline sample, and Supernatant samples are almost without lipase active, and in addition empty carrier transformant is born pair
According to display thalline sample and Supernatant samples all without lipase active, and in intracellular, thalline must crush just if SL4
Lipase enzymatic activity can be detected, so to sum up showing that expressed SL4 lipase 99.3% is all fixed on Pichia pastoris thalline
On, natural immobilised enzymes is formd, and substantially without lipase activity in fermented liquid supernatant.
Embodiment 4：The zymologic property detection of SL4 lipase
1. Substratspezifitaet
Prepare 4- Nitrophenyl butyrates (pNPB), 4- nitrobenzophenones caprylate (pNPO), 4- nitrobenzophenone laurates
(pNPD), 4- nitrobenzophenones myristate (pNPM), 4- nitrobenzophenones palmitate (pNPP), 4- nitrobenzophenone stearates
(pNPS) (isopropanol dissolving), according to the vigor of standard pNPB methods (being changed to corresponding substrate) detection lipase, with enzyme activity determination
Enzyme activity measured by highest substrate is 100%, calculates the enzyme activity under other substrates.As seen from Figure 3, SL4 fat
Enzyme hydrolysis pNPB abilities are most strong, hydrolyze pNPO, pNPD ability secondly, and excessively poor to the hydrolysis ability of pNPM, pNPP, pNPSD.
2. optimum temperature
Lipase activity is measured according to pNPD methods at different temperature (20-70 DEG C), to measure enzyme during enzyme activity highest
Vigor is 100%, calculates the opposite enzyme activity (%) at a temperature of other.From fig. 4, it can be seen that the enzyme activity optimum temperature of SL4 is 35
DEG C, and still having at 50 DEG C 60% enzyme activity, it was demonstrated that its applicable temperature range is wider.
3. most suitable action pH
Suspension thalline is added in respectively in the buffer solution of different pH (3.0-11.0) (pH3.0-6.0 be acetic acid-NaOH buffering
Liquid, pH6.0-6.5 are MES buffer solutions, pH6.5-9 is Tris-HCl buffer solutions, pH9-11 is glycine-NaOH buffer), 35
Lipase activity is measured by pNPD methods at DEG C, using enzyme activity when measuring vigor highest as 100%, calculates the phase of enzyme under other pH
To vigor (%).As seen from Figure 5, SL4 lipase can show higher lipase activity in pH value 6-9.
4. influence of the metal ion to lipase activity
Prepare ZnSO4、MnCl2、CoCl2、CaCl2、MgSO4、CuSO4、KCl、(NH4)2SO4、NaCl、NiSO4And FeCl3
Wait inorganic salts storage liquid.According to pNPD methods, reaction mixture is prepared first, and 600uL mixed liquors are dispensed into each reaction tube,
And the inorganic salts storage liquid of final concentration of 5mmol/L is added, measure activity according still further to standard method.To add water as control enzyme activity
100% is denoted as, calculates the relative activity of other each groups.As shown in Figure 6, ZnSO4、CuSO4、NiSO4、FeCl3、MnCl2、
CaCl2、MgSO4There are high inhibition effect, but NaCl, KCl, (NH to SL4 lipase4)2SO4Then have significantly to SL4 lipase
Activation, especially NaCl, KCl, may due to split pot algae growth ocean in, the content of sodium and potassium ion is relatively high,
So there is activation to SL4 lipase.
Embodiment 5：The enzyme activity of SL4 lipase and the comparison of commercialization enzyme
Take the shake flask fermentation liquid of 50mL expression SL4 lipase, 12000rpm centrifugation 5min, after sterile water washing thalline 2 times,
It is resuspended in 5mL sterile waters, so as to obtain SL4 lipase finished products.Commercialization enzyme preparation is compareed, is hydrolyzable Short-Chain Fatty Acids
The lipase candida antarctica lipase B (Novozymes Lipozyme CALB L) and rhizomucor miehei lipase of methyl esters
RML(Novozymes Palatase 20000L)。
By SL4 lipase finished product, Novozymes Lipozyme CALB L and Novozymes Palatase20000L,
It after diluting 1000 times respectively, draws 25 μ L samples and adds in 600 μ L pNPB reaction mixtures, 35 DEG C of water-bath 15min add in 500
μ L absolute ethyl alcohols terminate reaction, and 12000rpm centrifuges 5min, takes the absorbance of supernatant determination sample under 410nm wavelength.As a result
As shown in fig. 7, the enzyme activity of the enzyme preparation added is as follows：SL4 lipase finished products enzyme activity is 106U/mL, Novozymes
Lipozyme CALB L enzyme activity is that 116U/mL and Novozymes Palatase 20000L enzyme activity is 81U/mL；Without excellent
The finished product SL4 lipase of the Pichia anomala expression of change believes the candida antarctica lipase B and rhizomucor miehei of purchase with Novi
The enzyme activity level of lipase is substantially suitable, it was demonstrated that SL4 lipase has the feasibility applied to production in enzyme activity performance.
Embodiment 6：SL4 lipase is applied to pungent methyl caprate hydrolysising experiment
The pungent methyl caprates of 30g are weighed, 30g water is added in, 1.5mL SL4 finished product bacterium solutions is stirring evenly and then adding at 35 DEG C
(5%, v/m, the ratio between the SL4 enzyme solutions volumetric quantities in terms of mL and the numerical value of pungent methyl caprate in terms of g), such as example 5 above
Described in, which is 106U/mL, is 56mbar with diaphragm pump setting system vacuum degree, reaches certain reaction time
Afterwards, stratification (if layering unobvious, are detached by way of centrifugation), AV is measured by sampling in upper strata, calculates percent hydrolysis.
AV0Refer to pungent methyl caprate raw material acid value, AVtRefer to certain time interval t sampling acid values, AVTheoretical valueIt is complete to refer to pungent methyl caprate
Acid value during hydrolysis.In this experiment, AV0It is 0, AVTheoretical valueFor 327.3mgKOH/g.
The results are shown in Table 1, the results show that SL4 has ideal hydrolysis effect to pungent methyl caprate.
1 SL4 lipase of table is to the percent hydrolysis of pungent methyl caprate
Thalline were collected by centrifugation, re-starts hydrolysising experiment, so as to verify the repeatable profit of the SL4 lipase of natural immobilization
It with property, the results are shown in Table shown in 2, after having reused 6 times, SL4 lipase still keeps good hydrolysis effect.
2 SL4 lipase of table is to the repeatable usability of pungent methyl caprate
Sequence table
<110>Feng Yi（Shanghai）Co., Ltd of biotechnology research and development centre
<120>Lipase and its application
<130> 2016SL4
<160> 7
<170> PatentIn version 3.5
<210> 1
<211> 1221
<212> DNA
<213>Artificial sequence
<400> 1
aaagaggtcg tcgtctttgt tcatggtttg gccggttttg gtgatgatga attgggtccc 60
attgattatt gggccgagtt ggatgaattt cctcgtgatc gtttcgatgt tttcgttgcc 120
tctgttggtc ctgtttcttc taactgggat cgtgcctgcg aattgtatgc ccagattaaa 180
ggtacccgtg ttgattatgg tcctgcccat tcttctacct attcccatga acgttacggt 240
cgtgattatt ctggtcaggg cttttatcct tcttggtcta ccaccaaacg tattcacttg 300
atcggtcatt ctatgggtgg tcagaccatt cgtcagttgg aagttatgtt gcaggaaggt 360
gttcctgccg aagtttctgc cggttctacc tctcctttgt ttgccggttt gggcaacatg 420
attaaatccg tcaccaccat ttctacccct catgatggta cccctttgat tgatgttttg 480
ggtgaggata tcgtcgaatt gatcaaggat ttgatcttgg gttttgccgg tattaccggt 540
aacaccttcg ttgatttgat ctacgacttc gacttggacc attttggttt gggtcgtcag 600
cctggtgaat cttttggtga ttacgttgaa cgtgtttgcg ccaacgccat ttttgatatg 660
ggtttccgtg atttggcccc ttatgatttg tctattgacg gttctcgtga attgcagcag 720
caaggtcagc aggcctattc taacaccttc tactttggta tggctaccga acagaccgtt 780
cagaacgttc cttggtgcga atggggttat ggttgcggtt tgatttctgt tgccgaaacc 840
accatgggtt tgttgttgca gcctttcgcc aacattattg gttctttgcg tgttgcctct 900
gatttgcgta aaaacgatgg tttggtccct tttgagtctt ctaagtgccc taagatcggt 960
tattctaacg gtgccaacgg ttgcaccaaa tttaccggta cctgggcacc tggtcgttgg 1020
tattggatgg aagttgacag agatcacttg caggtcattg gtttcacctt cttgtatgcc 1080
ttgaccaacg atgccatcta ttctaaccat gccgaacgta tttggggtat ttctggtggt 1140
aacacctatg ttggttcttc caacgttggt gatggtaccg atgaagtttc tgaacctgaa 1200
cagacctcta actctggtta g 1221
<210> 2
<211> 406
<212> PRT
<213>Artificial sequence
<400> 2
Lys Glu Val Val Val Phe Val His Gly Leu Ala Gly Phe Gly Asp Asp
1 5 10 15
Glu Leu Gly Pro Ile Asp Tyr Trp Ala Glu Leu Asp Glu Phe Pro Arg
20 25 30
Asp Arg Phe Asp Val Phe Val Ala Ser Val Gly Pro Val Ser Ser Asn
35 40 45
Trp Asp Arg Ala Cys Glu Leu Tyr Ala Gln Ile Lys Gly Thr Arg Val
50 55 60
Asp Tyr Gly Pro Ala His Ser Ser Thr Tyr Ser His Glu Arg Tyr Gly
65 70 75 80
Arg Asp Tyr Ser Gly Gln Gly Phe Tyr Pro Ser Trp Ser Thr Thr Lys
85 90 95
Arg Ile His Leu Ile Gly His Ser Met Gly Gly Gln Thr Ile Arg Gln
100 105 110
Leu Glu Val Met Leu Gln Glu Gly Val Pro Ala Glu Val Ser Ala Gly
115 120 125
Ser Thr Ser Pro Leu Phe Ala Gly Leu Gly Asn Met Ile Lys Ser Val
130 135 140
Thr Thr Ile Ser Thr Pro His Asp Gly Thr Pro Leu Ile Asp Val Leu
145 150 155 160
Gly Glu Asp Ile Val Glu Leu Ile Lys Asp Leu Ile Leu Gly Phe Ala
165 170 175
Gly Ile Thr Gly Asn Thr Phe Val Asp Leu Ile Tyr Asp Phe Asp Leu
180 185 190
Asp His Phe Gly Leu Gly Arg Gln Pro Gly Glu Ser Phe Gly Asp Tyr
195 200 205
Val Glu Arg Val Cys Ala Asn Ala Ile Phe Asp Met Gly Phe Arg Asp
210 215 220
Leu Ala Pro Tyr Asp Leu Ser Ile Asp Gly Ser Arg Glu Leu Gln Gln
225 230 235 240
Gln Gly Gln Gln Ala Tyr Ser Asn Thr Phe Tyr Phe Gly Met Ala Thr
245 250 255
Glu Gln Thr Val Gln Asn Val Pro Trp Cys Glu Trp Gly Tyr Gly Cys
260 265 270
Gly Leu Ile Ser Val Ala Glu Thr Thr Met Gly Leu Leu Leu Gln Pro
275 280 285
Phe Ala Asn Ile Ile Gly Ser Leu Arg Val Ala Ser Asp Leu Arg Lys
290 295 300
Asn Asp Gly Leu Val Pro Phe Glu Ser Ser Lys Cys Pro Lys Ile Gly
305 310 315 320
Tyr Ser Asn Gly Ala Asn Gly Cys Thr Lys Phe Thr Gly Thr Trp Ala
325 330 335
Pro Gly Arg Trp Tyr Trp Met Glu Val Asp Arg Asp His Leu Gln Val
340 345 350
Ile Gly Phe Thr Phe Leu Tyr Ala Leu Thr Asn Asp Ala Ile Tyr Ser
355 360 365
Asn His Ala Glu Arg Ile Trp Gly Ile Ser Gly Gly Asn Thr Tyr Val
370 375 380
Gly Ser Ser Asn Val Gly Asp Gly Thr Asp Glu Val Ser Glu Pro Glu
385 390 395 400
Gln Thr Ser Asn Ser Gly
405
<210> 3
<211> 5
<212> PRT
<213>Artificial sequence
<400> 3
Arg Arg Arg Arg Arg
1 5
<210> 4
<211> 6
<212> PRT
<213>Artificial sequence
<400> 4
His His His His His His
1 5
<210> 5
<211> 8
<212> PRT
<213>Artificial sequence
<400> 5
Asp Tyr Lys Asp Asp Asp Asp Lys
1 5
<210> 6
<211> 8
<212> PRT
<213>Artificial sequence
<400> 6
Trp Ser His Pro Gln Phe Glu Lys
1 5
<210> 7
<211> 10
<212> PRT
<213>Artificial sequence
<400> 7
Trp Gln Lys Leu Ile Ser Glu Glu Asp Leu
1 5 10
Claims (10)
1. a kind of polypeptide, is selected from：
(1) amino acid sequence such as SEQ ID NO:Polypeptide shown in 2 and
(2) by SEQ ID NO:2 and for promoting SEQ ID NO:The sequence composition that amino acid sequence shown in 2 is expressed and purified
Polypeptide.
2. a kind of polynucleotide sequence, is selected from：
(1) polynucleotide sequence of polypeptide described in claim 1 is encoded；
(2) polynucleotide sequence of the polynucleotide sequence complementation and described in (1)；With
(3) segment of the long 10-40 base of the polynucleotide sequence described in (1) or (2).
3. polynucleotide sequence as claimed in claim 2, which is characterized in that the polynucleotide sequence has SEQ ID NO:
Nucleotide sequence shown in 1 or by SEQ ID NO:Nucleotide sequence composition shown in 1.
4. a kind of nucleic acid constructs, which is characterized in that the nucleic acid constructs contains：
(a) claim 2 (1) or the polynucleotide sequence described in (2) item；Or
(b) polynucleotide sequence described in claim 3.
5. nucleic acid constructs as claimed in claim 4, which is characterized in that the nucleic acid constructs is that cloning vector or expression carry
Body；
Preferably, the nucleic acid constructs contains AOX1 promoters, α-Factor signal peptides, His4 expression cassettes and polyclonal position
Point；It is highly preferred that the nucleic acid constructs is using pPIC9K plasmids as skeleton.
6. a kind of host cell, which is characterized in that the host cell contains the nucleic acid constructs described in claim 4 or 5；
Preferably, the host cell is microbial cell, preferably Pichia pastoris or Bacillus coli cells.
7. a kind of composition, which is characterized in that the composition contains described in polypeptide described in claim 1 or claim 6
Host cell, also optionally contain auxiliary material；Preferably, the auxiliary material is selected from activated carbon, aluminium oxide, diatomite, porous pottery
The sorbing material of porcelain, cellular glass.
A kind of 8. method of hydrolyzed fat acid methyl esters, which is characterized in that the method includes make the ingredient of fatty acids methyl esters with
The step of composition contact described in host cell or claim 7 described in polypeptide described in claim 1 or claim 6
Suddenly；
Preferably, the fatty acid methyl ester is pungent methyl caprate；And/or
Preferably, the contact conditions include：
(A) temperature is 20 DEG C -60 DEG C, preferably 35 ± 2 DEG C；And/or
(B) pH is 5.5-9, preferably 6-9.
9. a kind of hydrolysis reaction mixture, which is characterized in that the hydrolysis reaction mixture contains：
Fatty acid methyl ester；
Water；With
Polypeptide described in claim 1；
Preferably, the fatty acid methyl ester is the fatty acid methyl ester containing Short-Chain Fatty Acids, more preferably described fatty acid methyl
Ester is the fatty acid methyl ester containing C4~C12 chain length fatty acids, and most preferably the fatty acid methyl ester is pungent methyl caprate；With/
Or
Preferably, the dosage of the polypeptide needed for every gram of fatty acid methyl ester is more than 5U, preferably between 5U~10U；
And/or
Preferably, the dosage of water is more than the 90% of the weight of fatty acid methyl ester, such as between 90%~200%；
Enzyme activity unit U is defined as under conditions of 40 DEG C of temperature, pH value 8.0, enzyme hydrolysis substrate 4- Nitrophenyl butyrates, often
The enzyme amount that minute releases needed for 1 μm of ol p-nitrophenol (pNP) is 1 enzyme activity unit (U).
10. described in the polynucleotide sequence, claim 4 or 5 described in polypeptide described in claim 1, Claims 2 or 3
The composition described in host cell or claim 7 described in nucleic acid constructs, claim 6 is in fats and oils processing, oil product
Application in work, food industry, medical and health, chemical or answering in hydrolyzed fat acid methyl esters, the pungent methyl caprate of hydrolysis
With.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201611232848 | 2016-12-28 | ||
| CN2016112328482 | 2016-12-28 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN108251401A true CN108251401A (en) | 2018-07-06 |
| CN108251401B CN108251401B (en) | 2022-11-04 |
Family
ID=62721355
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710115983.7A Active CN108251401B (en) | 2016-12-28 | 2017-03-01 | Lipase and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN108251401B (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113122521A (en) * | 2019-12-31 | 2021-07-16 | 丰益国际有限公司 | Polypeptides having lipase activity and uses thereof |
| CN113302310A (en) * | 2018-12-18 | 2021-08-24 | 帕西昂奥地利有限两合公司 | Single step biocatalytic amidation |
| CN114317491A (en) * | 2022-01-04 | 2022-04-12 | 西南科技大学 | Novel lipase AjLip970 and application thereof |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20060168687A1 (en) * | 2003-02-27 | 2006-07-27 | Basf Plant Science Gmbh | Method for the production of polyunsaturated fatty acids |
-
2017
- 2017-03-01 CN CN201710115983.7A patent/CN108251401B/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20060168687A1 (en) * | 2003-02-27 | 2006-07-27 | Basf Plant Science Gmbh | Method for the production of polyunsaturated fatty acids |
Non-Patent Citations (4)
| Title |
|---|
| LIU ET AL.: "Production of Structured Triacylglycerols Containing Palmitic Acids at sn-2 Position and Docosahexaenoic Acids at sn-1, 3 Positions", 《J OLEO SCI》 * |
| WANG ET AL.: "From microalgae oil to produce novel structured triacylglycerols enriched with unsaturated fatty acids", 《BIORESOUR TECHNOL》 * |
| 兰君等: "利用伯克霍尔德氏菌脂肪酶富集裂壶藻油脂中的二十二碳六烯酸", 《食品科学》 * |
| 张娟梅等: "裂殖壶菌Schizochytrium sp.FJU-512细胞油脂的研究", 《福建师范大学学报(自然科学版)》 * |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113302310A (en) * | 2018-12-18 | 2021-08-24 | 帕西昂奥地利有限两合公司 | Single step biocatalytic amidation |
| CN113302310B (en) * | 2018-12-18 | 2024-05-03 | 帕西昂奥地利有限两合公司 | Single step biocatalytic amidation |
| CN113122521A (en) * | 2019-12-31 | 2021-07-16 | 丰益国际有限公司 | Polypeptides having lipase activity and uses thereof |
| CN113122521B (en) * | 2019-12-31 | 2024-05-14 | 丰益国际有限公司 | Polypeptides having lipase activity and uses thereof |
| CN114317491A (en) * | 2022-01-04 | 2022-04-12 | 西南科技大学 | Novel lipase AjLip970 and application thereof |
| CN114317491B (en) * | 2022-01-04 | 2023-05-23 | 西南科技大学 | Novel lipase AjLip970 and application thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN108251401B (en) | 2022-11-04 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Choi et al. | Characterization and heterologous gene expression of a novel esterase from Lactobacillus casei CL96 | |
| Quyen et al. | High-level formation of active Pseudomonas cepacia lipase after heterologous expression of the encoding gene and its modified chaperone in Escherichia coli and rapid in vitro refolding | |
| Liu et al. | Cloning, expression and characterization of a lipase gene from the Candida antarctica ZJB09193 and its application in biosynthesis of vitamin A esters | |
| CA2202553A1 (en) | Lipase, microorganism producing same, method for preparing said lipase and uses thereof | |
| US10738288B2 (en) | Efficient phospholipase C mutant that does not rely on zinc ions | |
| CN101993861A (en) | Recombinant expression of carboxyl esterase | |
| US20040142441A1 (en) | Enzymes with lipase/acyltransferase activity, nucleic acids encoding the same and methods of use thereof | |
| CN108239648B (en) | Method for efficiently expressing rhizomucor miehei lipase | |
| CN108251401A (en) | Lipase and its application | |
| JP3333521B2 (en) | Nucleic acid encoding a polypeptide having abscisdia lipase activity | |
| CN108251400B (en) | Lipase and application thereof | |
| CN102965355A (en) | Carboxylesterase and application thereof in degradation of pesticides malathion and carbaryl | |
| JP5438259B2 (en) | Yeast presenting Candida antarctica-derived lipase B on the cell surface | |
| Fu et al. | A novel cold-adapted phospholipase A1 from Serratia sp. xjF1: Gene cloning, expression and characterization | |
| JP7063807B2 (en) | Polypeptide with excellent stability and lipase activity | |
| KR20130018202A (en) | Amphipathic peptide-lipase conjugate having advanced lipase activity and use thereof | |
| CN101085984A (en) | Low-temperature lipase and its gene sequence | |
| US6221821B1 (en) | Haloperoxidases with altered pH profiles | |
| CN106635941B (en) | A kind of thermophilic esterase and its functional verification from Aquifex aeolicus bacterial strain | |
| CA2667064A1 (en) | Clonation, expression and use of acid lysophospholipases | |
| Nagao et al. | C-terminal peptide of Fusarium heterosporum lipase is necessary for its increasing thermostability | |
| Lock et al. | Lipases produced by yeasts: powerful biocatalysts for industrial purposes | |
| US20040161836A1 (en) | Recombinant porcine liver esterases, their use and a method for the production thereof | |
| WO2004055177A2 (en) | Artificial esterases | |
| CN113122520A (en) | Novel glycerol mono-diacyl ester lipase |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |