CN108251401B

CN108251401B - Lipase and application thereof

Info

Publication number: CN108251401B
Application number: CN201710115983.7A
Authority: CN
Inventors: 吴伟; 戴小军; 徐正军; 曹海生; 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: 2016-12-28
Filing date: 2017-03-01
Publication date: 2022-11-04
Anticipated expiration: 2037-03-01
Also published as: CN108251401A

Abstract

The invention relates to lipase and application thereof. Specifically, the invention provides a polypeptide, the amino acid sequence of which is shown as SEQ ID NO. 2, or consists of SEQ ID NO. 2 and a sequence for promoting the expression and purification of the amino acid sequence shown as SEQ ID NO. 2. The invention also provides a coding sequence of the polypeptide, a nucleic acid construct containing the coding sequence, a host cell containing the nucleic acid construct, and related application. The lipase of the invention has excellent enzyme activity, can be naturally fixed on thalli, and has advantages in industrial production.

Description

Lipase and application thereof

Technical Field

The invention relates to lipase and application thereof.

Background

The lipase is an enzyme with various catalytic abilities, can catalyze the hydrolysis of triacylglycerol into glycerol and free fatty acid, can catalyze the hydrolysis and transesterification of other esters and the synthesis reaction of the esters, and has enantioselectivity to substrates. The characteristics endow the Lipase with wide application in industries such as food and fat processing, biodiesel, detergents, synthesis of ester bond compounds, chiral drug synthesis and the like (Abhishek Kumar Singh, mausumi Mukhopadhyay, overview of Fungal Lipase: A Review,2012, 166 (2): 486-520).

Lipases are widely present in animals, plants and microorganisms, and are important sources of industrial lipases because microorganisms are not only diverse in species, fast in propagation, and prone to genetic variation, but also have broader pH, temperature range, and substrate specificity than animals. According to statistics, the lipase-producing microorganisms belong to 65 genera, mainly including Aspergillus niger, candida, rhizopus, pseudomonas, streptomyces, etc. (King-Shao, zhang, ma Jun, shi, agricultural science of Anhui, 2011, 39 (7): 3798-3800).

The applications of lipase are divided into food industry, medicine and health, chemical industry and the like. Applications in chemistry and chemistry include the use in the hydrolysis of fatty acid methyl esters, such as methyl caprylate.

The most widely commercialized lipases currently used in the hydrolysis of methyl caprylate and caprate are those derived from Rhizomucor miehei and those derived from Candida antarctica, but due to their high price, the industrial application of the enzyme method is limited. Therefore, it is of great significance to develop a lipase with high catalytic activity for hydrolyzing fatty acid methyl esters such as octyl-decyl methyl ester.

Therefore, the present invention aims to find a novel lipase having an enzymatic activity of hydrolyzing methyl caprylate/caprate or the like, which satisfies the demand of industrial production.

Disclosure of Invention

The present invention provides novel lipases, which preferentially hydrolyze medium-short chain fatty acid esters, useful in medium-short chain (e.g., C4-C12 length) fatty acid methyl esters such as methyl caprylate-caprate; the lipase has high activity; and can be immobilized on the thallus of a strain (such as pichia pastoris) expressing the lipase.

The present invention provides a polypeptide selected from the group consisting of:

(1) A polypeptide having an amino acid sequence as shown in SEQ ID NO 2, and

(2) Polypeptide consisting of SEQ ID NO. 2 and a sequence for promoting the expression and purification of the amino acid sequence shown in SEQ ID NO. 2.

The polypeptide of the invention also comprises a polypeptide which is obtained by substituting, deleting or adding one or more amino acids in the amino acid sequence shown in SEQ ID NO. 2, and simultaneously retains the amino acid sequence shown in SEQ ID NO:2 and a polypeptide having lipase activity derived from SEQ ID No. 2.

The polypeptides of the present invention also include polypeptides having at least 90%, preferably at least 95%, more preferably at least 98%, and even more preferably at least 99% sequence identity to the polypeptide of SEQ ID NO. 2.

The polypeptide of the invention is derived from schizochytrium limacinum.

The present invention provides a polynucleotide sequence selected from:

(1) A polynucleotide sequence encoding a polypeptide of the invention;

(2) (1) the complement of said sequence; and

(3) The polynucleotide sequence of (1) or (2) which is 10 to 40 bases long, preferably 15 to 30 bases long.

In one or more embodiments, the polynucleotide sequence has the nucleotide sequence set forth in SEQ ID NO. 1.

In one or more embodiments, the polynucleotide sequence consists of the nucleotide sequence set forth in SEQ ID NO. 1.

The invention also provides a nucleic acid construct comprising a polynucleotide sequence of the invention.

In one or more embodiments, the nucleic acid construct comprises a polynucleotide sequence encoding a polypeptide of the present invention or a complement thereof.

In one or more embodiments, the nucleic acid construct comprises the nucleotide sequence set forth in SEQ ID NO. 1 or a complement thereof.

In one or more embodiments, the nucleic acid construct is a cloning vector or an expression vector.

In one or more embodiments, the nucleic acid construct comprises an AOX1 promoter, an α -Factor signal peptide, a His4 expression cassette, and a multiple cloning site.

In one or more embodiments, the nucleic acid construct is framed by a pPIC9K plasmid.

The invention also provides a host cell comprising a nucleic acid construct of the invention.

In one or more embodiments, the host cell is a plant cell.

In one or more embodiments, the host cell is a microbial cell.

In one or more embodiments, the host cell is a pichia cell or an escherichia coli cell.

The invention also provides the polypeptide, its coding sequence or its complementary sequence, the nucleic acid construction containing the coding sequence or its complementary sequence and the application of the host cell containing the nucleic acid construction in food industry, medicine and health and chemical industry.

The invention also provides the use of a polypeptide as described herein, a coding sequence thereof or a sequence complementary to the coding sequence, in the hydrolysis of fatty acid methyl esters, for example the hydrolysis of methyl caprylate/caprate.

Drawings

FIG. 1: alignment on BMMY-tributyrin plates. Wherein the left colony is a GS115 Pichia pastoris colony which is not transformed with p9K-SL4 and is used as a control, and the right colony is an SL4 expression strain; the SL4 expression strain was shown to form a clear transparent circle with lipase activity.

FIG. 2: the supernatant and the thallus sample of the Pichia pastoris transformant expressing SL4 lipase take the transformant of pPIC9K empty vector without SL4 gene as a negative control.

FIG. 3: substrate specificity of SL4 lipase.

FIG. 4 is a schematic view of: optimum temperature for SL4 lipase.

FIG. 5 is a schematic view of: the optimum pH for SL4 lipase.

FIG. 6: effect of metal ions on lipase SL4 activity.

FIG. 7: SL4 lipase was used for hydrolysis and enzyme activity was compared to commercially available lipases CALB and RML.

FIG. 8: pPIC9K plasmid map.

Detailed Description

The present invention provides a lipase having an amino acid sequence shown as SEQ ID NO. 2. The invention also includes polypeptides obtained by conservative substitution of amino acids with similar or similar properties on the basis of the amino acid sequence shown in SEQ ID NO. 2. Such conservative substitutions do not generally alter the function of the protein or polypeptide. "amino acids with similar or analogous properties" include, for example, families of amino acid residues with analogous side chains, including amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, substitution of one or more sites with another amino acid residue from the same side chain species in the polypeptide of the invention will not substantially affect its activity.

The invention thus includes the substitution, deletion or addition of one or several amino acids in the amino acid sequence shown in SEQ ID NO. 2, while preserving the amino acid sequence shown in SEQ ID NO:2 and a polypeptide having lipase activity derived from SEQ ID No. 2. The number of the units is usually 10 or less, preferably 8 or less, and more preferably 5 or less.

Knowing the sequence and biological function of SEQ ID NO. 2, one skilled in the art can determine which amino acid residues in the amino acid sequence shown in SEQ ID NO. 2 can be substituted or deleted using routine techniques. For example, by aligning sequences from different species, having the same or similar or significantly different activities, it is possible to determine which amino acid residues in these sequences can be substituted or deleted. Such sequences can be verified for enzymatic activity of the invention using methods conventional in the art, including those disclosed herein.

Furthermore, it is well known to those skilled in the art that in gene cloning procedures, it is often necessary to design appropriate cleavage sites, which may necessitate the introduction of one or more irrelevant residues at the ends of the expressed protein, which do not affect the activity of the protein of interest. Also, for example, to construct a fusion protein, to facilitate expression of a recombinant protein, to obtain a recombinant protein that is automatically secreted outside of a host cell, or to facilitate purification of a recombinant protein, it is often necessary to add some amino acids to the N-terminus, C-terminus, or other suitable regions within the recombinant protein, for example, including, but not limited to, a suitable linker peptide, signal peptide, leader peptide, terminal extension, glutathione S-transferase (GST), maltose E binding protein, protein a, or factor Xa or the proteolytic enzyme site of thrombin or enterokinase. The amino-terminus or carboxy-terminus of the amino acid sequences of the invention may also contain one or more polypeptide fragments as protein tags. Any suitable label may be used with the present invention. For example, the tag can be FLAG, HA, HA1, c-Myc, poly-His, poly-Arg, strep-TagII, AU1, EE, T7,4A6, ε, B, gE, and Ty1. These tags can be used to purify proteins. Examples of labels used include Poly-Arg, such as RRRRR (SEQ ID NO: 3); poly-His 2-10 (usually 6), such as HHHHHHHHHH (SEQ ID NO: 4); FLAG, DYKDDDDK (SEQ ID NO: 5); strep-TagII, WSHPQFEK (SEQ ID NO: 6); and C-myc, WQKLISEEDL (SEQ ID NO: 7). It is understood that the presence of these amino acid sequences does not affect the activity of the resulting polypeptide. Thus, the invention also includes polypeptides having one or more amino acids added to the C-terminus and/or N-terminus of the polypeptides of the invention, which polypeptides still have lipase activity as described herein.

Thus, the invention also encompasses amino acid sequences which have at least 90%, preferably at least 95%, more preferably at least 98%, more preferably at least 99% sequence identity with the amino acid sequence depicted in SEQ ID NO. 2. In some embodiments, such amino acid sequences are also from Schizochytrium (Schizochytrium), preferably having the same or similar lipase enzyme activity as SEQ ID NO:2 herein.

Sequence identity can be calculated for two aligned sequences using conventional means, for example, BLASTP provided by NCBI and aligned using default parameters. In certain embodiments, the polypeptide of the invention is a neutral or alkaline lipase. In certain embodiments, the polypeptides of the invention have substrate specificity as lipases, with the strongest ability to hydrolyze 4-nitrophenylbutyrate (pNPB), the second highest ability to hydrolyze 4-nitrophenyl caprylate (pNPO), 4-nitrophenyllaurate (pNPD), and the weaker ability to hydrolyze 4-nitrophenylmyristate (pNPM), 4-nitrophenylpalmitate (pNPP), 4-nitrophenylstearate (pNPS). In certain embodiments, the optimal temperature for the polypeptide of the invention is between 20-60 ℃, preferably at 35 ℃. + -. 2 ℃. In certain embodiments, the polypeptide of the invention has a pH of optimum action between 5.5 and 9, preferably between 6 and 9, more preferably between 6.1 and 7.5 or 8.5 and 9, most preferably 6 or 9. In certain embodiments, the polypeptidases of the invention are enzymatically active by ZnSO ₄ 、CuSO ₄ 、NiSO ₄ 、FeCl ₃ 、MnCl ₂ 、CaCl ₂ 、MgSO ₄ And NaCl, KCl, (NH 4) ₂ SO ₄ The enzyme activity, especially NaCl and KCl, can be activated. In certain embodiments, the polypeptides of the invention have comparable hydrolytic activity as compared to commercial enzymes. In certain embodiments, the polypeptides of the invention are used for the hydrolysis of fatty acid methyl esters, for example of fatty acid methyl esters containing medium-short chain fatty acid residues of 4 to 12 carbon atoms, in particular for the hydrolysis of methyl caprylate/caprate。

Depending on the host used in the recombinant production protocol, the polypeptide of the invention may be glycosylated or may be non-glycosylated.

The polypeptides of the invention can be naturally purified products, or chemically synthesized products, or using recombinant technology from prokaryotic or eukaryotic hosts (e.g., bacteria, yeast, higher plant, insect and mammalian cells).

The present application includes the coding sequence of the polypeptides of the invention. SEQ ID NO 1 shows one of the coding sequences for the polypeptide of the invention. "coding sequence" includes sequences that are highly homologous to SEQ ID NO 1 or to SEQ ID NO:1 or a family gene molecule that is highly homologous to the above molecules. The sequence encoding the polypeptide of the invention may be identical to SEQ ID NO:1, or a degenerate variant thereof. As used herein, "degenerate variant" means in the present invention a variant that encodes a polypeptide comprising SEQ ID NO:2, but has an amino acid sequence identical to that shown in SEQ ID NO:1, or a variant thereof.

Sequences encoding the polypeptides of the invention include: a coding sequence encoding only the mature polypeptide; the coding sequence for the mature polypeptide and various additional coding sequences; the coding sequence (and optionally additional coding sequences) as well as non-coding sequences for the mature polypeptide.

The coding sequence of the polypeptide of the present invention or a fragment thereof can be obtained by PCR amplification, recombination, or artificial synthesis. For PCR amplification, the genomic DNA of Escherichia coli or Schizochytrium (Schizochytrium) can be obtained by conventional techniques, and then primers can be designed according to the nucleotide sequences disclosed herein, especially the open reading frame sequences, to amplify the lipase gene from the genomic DNA.

Thus, the invention also includes fragments of the coding sequences of the invention, which fragments are generally 10 to 40, preferably 15 to 30 bases in length and can be used as primers or probes. "fragment" as used herein refers to a contiguous portion of the full-length sequence.

The invention also relates to nucleic acid constructs comprising a coding sequence of the invention and one or more control sequences operably linked to the coding sequence and directing the expression of the coding sequence in a host cell under suitable conditions. Polynucleotides encoding the polypeptides of the invention may be manipulated in various ways to ensure expression of the polypeptides. Manipulation of the polynucleotide sequence prior to its insertion into a vector may be desirable or necessary depending on the expression vector. Techniques for altering polynucleotide sequences using recombinant DNA methods are known in the art.

The control sequence may be an appropriate promoter sequence, a nucleotide sequence recognized by a host cell for expression of a polynucleotide encoding a polypeptide of the present invention. The promoter sequence may comprise transcriptional control sequences for the expression of the polypeptide. The promoter may be any nucleotide sequence which shows transcriptional activity in the host cell of choice 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 promoter sequences suitable for use in the present invention include the AOX1 promoter and the GAP promoter, among others.

The control sequence may also be a suitable transcription terminator sequence, a sequence recognized by a host cell to terminate transcription. The terminator sequence is operably linked to the 3' terminus of the nucleotide sequence encoding the polypeptide. Any terminator which is functional in the host cell of choice may be used in the present invention.

The control sequence may also be a suitable leader sequence, a nontranslated region of an mRNA which is important for translation by the host cell. The check-in sequence is operably linked to the 5' terminus of the nucleotide sequence encoding the polypeptide. Any terminator which is functional in the host cell of choice may be used in the present invention.

The control sequence may also be a signal peptide coding region that codes for an amino acid sequence linked to the amino acid terminus of a polypeptide and directs the encoded polypeptide into the cell's secretory pathway. The 5' end of the coding sequence of the nucleotide sequence may inherently contain a signal peptide coding region naturally linked in translation reading frame with the segment of the coding region that encodes the secreted polypeptide. Alternatively, the 5' end of the coding sequence may comprise a signal peptide coding region foreign to the coding region. Where the coding sequence does not naturally contain a signal peptide coding region, a foreign signal peptide coding region may be required. Alternatively, the foreign signal peptide coding region may simply replace the natural signal peptide coding region in order to enhance secretion of the polypeptide. However, any signal peptide coding region that directs the expressed polypeptide into the secretory pathway of a host cell of choice, i.e., into the culture medium, may be used in the present invention.

The invention also relates to cloning or expression vectors comprising a polynucleotide of the invention. These vectors may contain various regulatory sequences as described previously.

The expression vector may be any vector (e.g., a plasmid or virus) which can be conveniently subjected to recombinant DNA procedures and can bring about the expression of the nucleotide sequence of interest. The choice of the vector will generally 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 comprise any means for assuring self-replication. Alternatively, the vector may be one which, when introduced into a host cell, is integrated into the genome and replicated together with the chromosome(s) into which it has been integrated. Furthermore, a single vector or plasmid or two or more vectors or plasmids, or a transposon, which together contain the total DNA to be introduced into the genome of the host cell, may be used.

The vectors of the invention preferably comprise one or more selectable markers that allow for easy selection of transformed, transfected, transduced, or the like cells. Selectable markers are genes whose products provide resistance to antibiotics or viruses, resistance to heavy metals, prototrophy to auxotrophs, and the like.

The vectors of the present invention preferably contain elements that permit integration of the vector into the host cell genome or autonomous replication of the vector in the cell independent of the genome.

More than one copy of a polynucleotide of the invention may be inserted into a host cell to increase the yield of the gene product. An increase in the copy number of a polynucleotide can be obtained by integrating at least one additional copy of the sequence into the genome of the host cell or by including an amplifiable selectable marker gene with the polynucleotide, wherein cells containing amplified copies of the selectable marker gene and, thus, additional copies of the polynucleotide can be screened for by culturing the cells in the presence of the appropriate selectable agent.

The vectors of the present invention preferably comprise a synthetic sequence containing multiple restriction enzyme recognition sites to provide multiple sites or insertion schemes for foreign DNA. The expression vector of the invention preferably contains small peptides with 6 consecutive histidine sequences, which is beneficial to the extraction and purification of protein.

In certain embodiments, the vectors of the invention are used to express the polypeptides of the invention in E.coli. Thus, in certain embodiments, the expression vector comprises an AOX1 promoter, an α -Factor signal peptide, a His4 expression cassette, and a multiple cloning site. Preferably, the nucleic acid construct is framed by a pPIC9K plasmid.

In certain embodiments, the vectors of the invention are used to express the polypeptides of the invention in pichia pastoris. Thus, in certain embodiments, the expression vector comprises an AOX1 promoter, an α -Factor signal peptide, a His4 expression cassette, and a multiple cloning site. Preferably, the nucleic acid construct is framed by a pPIC9K plasmid.

The present invention also relates to recombinant host cells containing a polynucleotide of the present invention which are used for the recombinant production of the polypeptide. The vector comprising the polynucleotide of the present invention is introduced into a host cell so that the vector is maintained as a chromosomal integrant or as an extrachromosomal self-replicating vector as described earlier. The choice of host cell will largely depend on the gene encoding the polypeptide and its source.

The host cell may be a plant cell or a unicellular microorganism or a non-unicellular microorganism. Unicellular microorganisms such as gram-positive bacteria, including but not limited to bacillus cells, e.g., bacillus alkalophilus, bacillus amyloliquefaciens, bacillus brevis, bacillus megaterium, bacillus subtilis, bacillus licheniformis, bacillus coagulans, bacillus stearothermophilus, bacillus thuringiensis, and the like; or a streptomyces cell, such as streptomyces lividans; or gram-negative bacteria such as E.coli and Pseudomonas. In a preferred aspect, the bacterial host is a Bacillus subtilis, bacillus licheniformis, bacillus stearothermophilus, or Escherichia coli cell.

The host cell may also be a eukaryote, such as a mammalian, insect, plant, yeast, or fungal cell. In a preferred aspect, the host cell is a fungal cell, and "fungi" as used herein include Ascomycota (Ascomycota), basidiomycota (Basidiomycota), chytridiomycota (Chytridiomycota), zygomycota (Zygomyycota), and Oomycota, among others.

In a more preferred aspect, the host cell is a prokaryotic cell. "prokaryotic cells" as used herein include bacteria of the genera Pseudomonas, bacillus, enterobacter, staphylococcus, streptomyces and Escherichia. In a more preferred aspect, the host cell is a cell of the genera Pseudomonas, bacillus, streptomyces and Escherichia.

In a more preferred aspect, the host cell is Bacillus subtilis, pseudomonas fluorescens, pichia pastoris, escherichia coli, and Streptomyces lividans, among others. In a further most preferred aspect, the host cell is an Escherichia coli (Escherichia coli) cell or a Pichia pastoris (Pichia pastoris) cell.

Nucleic acid constructs comprising a polynucleotide sequence of the present invention can be transferred into host cells using conventional transfection procedures. Transfection is generally divided into transient transfection and stable transfection. The former exogenous DNA/RNA is not integrated into the host chromosome, so multiple copy numbers can be present in a host cell, resulting in high levels of expression, but usually only lasting a few days. In stable transfection, the foreign DNA may be either integrated into the host chromosome or may be present as an episome. The technical means of transfection include chemical transfection such as DEAE-dextran method, calcium phosphate method and artificial liposome method, and physical transfection such as microinjection, electroporation, gene gun, etc.

The polypeptide of the present invention may be used in food industry, medicine and chemical industry. Specifically, the polypeptide of the invention can be used for grease hydrolysis; the milk fat can be applied to dairy products for milk fat hydrolysis, the flavor of cheese, milk powder and cream can be enhanced, the maturity of cheese is promoted, and the quality of dairy products is improved; can be applied to the processing of cooked wheaten food to improve the elasticity of the cooked wheaten food, improve the taste and improve the fresh-keeping capacity of bread; can be used in chemical engineering for hydrolysis of fatty acid methyl ester, such as methyl caprylate-caprate to produce fatty acid; can be added into detergent for removing oil stain, or used for cleaning tableware and clothes, bleaching, dry cleaning, cleaning leather, cleaning eyes, cleaning industrial waste such as cosmetics and food processing, degrading organic waste of exhaust pipe and toilet bowl, etc. The invention relates in particular to the use of the polypeptides according to the invention for the hydrolysis of fatty acid methyl esters, for example of medium-short chain fatty acid methyl esters, in particular of fatty acid methyl esters containing fatty acids with a chain length of from 4 to 12, most preferably of caprylic/capric acid methyl esters. The polypeptides of the present invention can be used in the above-mentioned fields in amounts customary in the art.

Therefore, the invention provides the application of the polypeptide, the coding sequence, the nucleic acid construct and the cell in food industry, medicine and health, and chemistry and chemical industry. In particular, the invention provides the use of said polypeptides, coding sequences, nucleic acid constructs and cells in the hydrolysis of fatty acid esters, such as the hydrolysis of medium and short chain (C4-C12) fatty acid methyl esters, in particular the hydrolysis of methyl caprylate-caprate.

The invention also provides a composition which is a reaction mixture. Characterized in that the reaction mixture contains: fatty acid methyl esters, water, a polypeptide of the invention, or a host cell containing a nucleotide of the invention. Preferably, the fatty acid methyl ester is methyl caprylate.

The enzyme activity unit U is defined as the amount of enzyme 1 enzyme activity unit (U) required for the enzymatic hydrolysis of the substrate 4-nitrophenylbutyrate at a temperature of 40 ℃ and a pH of 8.0 to release 1. Mu. Mol of p-nitrophenol (pNP) per minute. Preferably, the amount of the polypeptide of the present invention is more than 5U, preferably between 5U and 10U per gram of fatty acid methyl ester; and/or preferably, water is used in an amount of 95% or more, such as between 95 and 100% or 95 and 200% by weight of fatty acid methyl esters, such as methyl caprylate/caprate.

The polypeptides of the invention may be provided as pure enzyme preparations, or may be provided in the form of compositions. The composition may be a powder composition, a liquid composition, or a paste composition. When provided in a composition, the composition may contain different adjuvants depending on the intended use of the enzyme-containing composition. Adjuvants known in the art may be added to the compositions of the present invention, including, but not limited to, one or more of sorbitol, potassium sorbate, methyl benzoate, ethyl benzoate, sucrose, mannose, trehalose, starch, sodium chloride, calcium chloride, and like stabilizers or other materials.

The present invention also provides a method for oil and fat refining, oil and fat chemical industry, improved feed, food preparation, pharmaceutical preparation using the polypeptide of the present invention, said method (and other methods of using the polypeptide of the present invention as mentioned herein) preferably being carried out under the following conditions:

(A) The temperature is 20-60 ℃, and preferably 35 +/-2 ℃; and/or

(B) The pH is 5.5-9, preferably 6-9, more preferably 6.1-7.5 or 8.5-9, most preferably 6 or 9.

The amount of the polypeptide of the present invention used in the method of the present invention can be determined in accordance with the actual circumstances.

The invention will be elucidated hereinafter by means of specific examples. Experimental procedures without specific conditions noted in the following examples, generally followed by conventional conditions such as Sambrook et al, molecular cloning: the conditions described in the Laboratory Manual (Cold Spring Harbor Laboratory Press, 1989, N.Y., USA) or according to the manufacturer's recommendations. For the use and amounts of the reagents, the conventional use and amounts are used unless otherwise indicated. Unless otherwise specified, percentages refer to weight percent.

Experimental Material

Experimental strains and plasmids

Strain: pichia pastoris GS115 (Invitrogen, cat # C181-00), escherichia coli DH5a (TAKARA: catalog #. D9057A).

Plasmid: pPIC9K plasmid (Invitrogen, cat # V175-20).

2. Culture media and solutions

LB liquid medium: 0.5% yeast extract, 1% tryptone, 1% NaCl, pH7.0.

LB solid Medium: agar was added to LB liquid medium at a concentration of 1.5%.

YPD liquid medium: 1% yeast extract, 2% peptone, 2% glucose.

YPD solid Medium: agar was added to LB liquid medium at a concentration of 2%.

MGYS solid culture medium: 1.34% Yeast Nitrogen Source base (YNB) ammonium sulfate free of amino acids, 1% glycerol, 1M sorbitol, 4X 10-5% D-Biotin, 2% agar.

And (3) screening a culture medium of tributyrin: 1.34% Yeast Nitrogen Source base (YNB) ammonium sulfate free of amino acids, 4X 10-5% D-Biotin, 0.5% methanol (added after sterilization), 2% tributyrin emulsion, 0.1M citric acid-sodium citrate buffer pH6.6,2% agar.

2% soybean lecithin emulsion: 2% tributyrin, 1% acacia, 100ml H ₂ O, homogenizing for 1min by a high-speed homogenizer at 8000 rpm.

BMGY liquid Medium: 1% yeast extract, 2% peptone, 1.34% yeast nitrogen source base (YNB) ammonium sulfate-free amino acids, 1% glycerol, 4X 10-5% D-biotin, 0.1M potassium dihydrogen phosphate-dipotassium hydrogen phosphate buffer pH6.0.

BMMY liquid medium: 1% yeast extract, 2% peptone, 1.34% yeast nitrogen alkaloid (YNB) ammonium sulfate-free amino acids, 0.3% ZnSO ₄ ·7H ₂ O,0.5% methanol (added after sterilization), 4X 10 ^-5 % D-biotin (added after sterilization), 0.1M citric acid-sodium citrate buffer pH6.6.

pNPB substrate buffer: mixing 5.3mL of solution A and 94.7mL of solution B, adding 280mL of water, adding 0.92g of sodium deoxycholate and 0.44g of Arabic gum powder, stirring for dissolution, adjusting the pH value to 8.0 with H3PO4 or NaOH, diluting to 400mL, and storing at 4 ℃.

Solution A: 0.2mol/L NaH ₂ PO ₄ Solution, 3.12g NaH was weighed ₂ PO ₄ Dissolving with distilled water and fixing volume to 100mL

And B, liquid B: 0.2mol/L Na ₂ HPO ₄ Solution 71.7g Na was weighed ₂ HPO ₄ .12H ₂ The O is dissolved in distilled water and the volume is up to 1000mL.

Substrate pNPB solution (3 mg/L): 0.030g of 4-nitrophenylbutyrate (pNPB) was weighed, dissolved by adding 10mL of isopropyl alcohol, and stored at 4 ℃.

Method for measuring lipase activity

The lipase activity was determined by colorimetric method. The method takes 4-nitrophenyl butyrate (pNPB) as a substrate and comprises the following steps: the substrate and the buffer are prepared in advance, and the preparation method is shown in experimental materials.

A reaction mixture was prepared with 1. And (3) sucking 25uL of supernatant and thallus samples, adding the supernatant and the thallus samples into 600uL of pNPB reaction mixed liquor, carrying out water bath at 40 ℃ for 15min, adding 500uL of absolute ethyl alcohol to stop the reaction, centrifuging at 12000rpm for 5min, and taking the supernatant to measure the absorbance of the samples at the wavelength of 410 nm.

Obtaining a calculation formula of lipase activity according to a p-nitrophenol standard curve:

enzyme activity (U/mL) = (a × Δ OD + b) × 10 × n × 1/15

In the formula, Δ OD: A-A0 (absorbance at 405 nm); a. b: coefficients in the pNP standard curve formula; 10: converting the enzyme activity in the enzyme solution into 1mL of enzyme activity; n: dilution factor of enzyme solution; 1/15: the reaction time was 15 minutes, which is a factor of 1 minute.

Definition of enzyme activity: the enzyme-containing sample hydrolyzed the substrate 4-nitrophenylbutyrate at a temperature of 40 ℃ and a pH of 8.0, with an enzyme activity of 1 enzyme activity unit (U) required to release 1umol of p-nitrophenol (pNP) per minute.

Note: 4-Nitrophenyl butyrate (pNPB), 4-nitrophenyl octanoate (pNPO), 4-nitrophenyllaurate (pNPD)4-Nitrophenyl myristate (pNPM), 4-nitrophenylpalmitate (pNPP) and 4-nitrophenylstearate (pNPS) (all commercially available from Sigma) were prepared as described above; restriction enzymes NotI, ecoRI, salI (from Neuro Biotechnology (Beijing) Ltd.); and (3) PCR enzyme: the TaKaRa Taq is prepared by the following steps of,

HS DNA Polymerase (available from Bao bioengineering (Dalian) Co., ltd.); t4DNA ligase (from Fulase Co., ltd.); methyl octyl decanoate (sasofol industry (hong kong, company, inc.)); calB Lipase is Novozymes Lipozyme CALB L from Novoverein; RML Lipase is Novozymes Palatase 20000L available from Novoversson.

Example 1: construction of p9K-SL4 vector

The schizochytrium limacinum lipase SL4 (SEQ ID NO: 1) is obtained by the general gene synthesis of the Comptozoon bioengineering (Shanghai) company, and is connected to the pPI C9K plasmid through restriction endonuclease sites NotI and EcoRI, so that the p9K-SL4 recombinant plasmid is obtained.

Example 2: construction and screening of Pichia pastoris strain expressing SL4 lipase

The plasmid p9K-SL4 was linearized with the restriction enzyme SalI and the linearized fragment was recovered by gel. Competent cells of Pichia pastoris GS115 strain were prepared by lithium acetate (LiAc) method, and 500ng of linearized p9K-SL4 was transformed into GS115 competent cells by electrotransformation. The transformed product was spread on MGYS plates and cultured at 30 ℃ for 3 days. And (3) selecting the monoclone on the plate, putting the monoclone on a BMMY-tributyrin screening plate, and selecting the clone with a large transparent circle, thereby obtaining the Pichia pastoris strain for expressing the SL4 lipase. GS115 without p9K-SL4 transformation and Pichia pastoris strain expressing SL4 lipase were plated to obtain a plate image as shown in FIG. 1, where the left colony is GS115 without p9K-SL4 transformation (as a control) and the right colony is SL4 expressing strain, and the results show that the SL4 expressing strain formed a clear transparent circle and had lipase activity.

Example 3: fermentation of Pichia pastoris strain expressing SL4 lipase and detection of enzyme activity distribution

A transformant of a Pichia pastoris strain expressing SL4 lipase was activated in liquid YPD, inoculated in BMGY medium, and cultured overnight at 30 ℃ with shaking at 220 rpm. Cultures were transferred to BMMY media with an initial OD600 of 6. Inducing with 2% methanol, supplementing 1% methanol after 24h and 32h respectively, supplementing 1% methanol after 48h and 56h respectively, sampling for 72h, taking 1mL of fermentation sample, centrifuging at 12000rpm for 5min, sucking out the fermentation supernatant, and then re-suspending the precipitated thalli with 1mL of sterile water to obtain a fermentation broth supernatant sample and a thalli sample, wherein a transformant of pPIC9K empty vector without SL4 gene is used as a negative control.

The lipase activities of the fermentation broth supernatant sample and the thallus sample are obtained by calculation, and the result is shown in fig. 2, 99.3% of lipase activity is found in the thallus sample, but the supernatant sample has almost no lipase activity, and in addition, the negative control of the empty vector transformant shows that the thallus sample and the supernatant sample have no lipase activity, and if SL4 is in cells, the thallus must be broken to detect the lipase activity, so the conclusion shows that 99.3% of the expressed SL4 lipase is fixed on the Pichia pastoris thallus to form a natural immobilized enzyme, and the fermentation broth supernatant has almost no lipase activity.

Example 4: detection of enzymatic Properties of SL4 Lipase

1. Substrate specificity

Preparing 4-nitrophenyl butyrate (pNPB), 4-nitrophenyl caprylate (pNPO), 4-nitrophenyl laurate (pNPD), 4-nitrophenyl myristate (pNPM), 4-nitrophenyl palmitate (pNPP) and 4-nitrophenyl stearate (pNPS) (dissolving by isopropanol), detecting the activity of lipase according to a standard pNPB method (replacing the pNPM with a corresponding substrate), and calculating the relative enzyme activity under other substrates by using the enzyme activity with the enzyme activity measured by measuring the highest substrate as 100%. As can be seen from FIG. 3, the SL4 lipase has the strongest ability to hydrolyze pNPB, the second ability to hydrolyze pNPO and pNPD, and the very poor ability to hydrolyze pNPM, pNPP and pNPS.

2. Optimum temperature of action

And (3) determining the activity of the lipase at different temperatures (20-70 ℃) according to a pNPD method, and calculating the relative activity (%) at other temperatures by determining the activity of the lipase when the activity is the highest as 100%. As can be seen from FIG. 4, the optimum temperature for the enzyme activity of SL4 is 35 ℃, and 60% of the enzyme activity is still active at 50 ℃, which proves that the applicable temperature range is wide.

3. Optimum pH for action

Respectively adding the suspended bacteria into buffers with different pH values (3.0-11.0) (pH 3.0-6.0 is acetic acid-NaOH buffer solution, pH6.0-6.5 is MES buffer solution, pH6.5-9 is Tris-HCl buffer solution, pH9-11 is glycine-NaOH buffer solution), measuring the lipase activity at 35 ℃ according to a pNPD method, and calculating the relative activity (%) of the enzyme at other pH values by taking the enzyme activity when the activity is the highest as 100%. As can be seen from FIG. 5, SL4 lipase can show higher lipase enzyme activity at pH 6-9.

4. Effect of Metal ions on Lipase Activity

Preparation of ZnSO ₄ 、MnCl ₂ 、CoCl ₂ 、CaCl ₂ 、MgSO ₄ 、CuSO ₄ 、KCl、(NH ₄ ) ₂ SO ₄ 、NaCl、NiSO ₄ And FeCl ₃ And the like. According to the pNPD method, the reaction mixture is first prepared, 600uL of the mixture is packed into each reaction tube, and a stock solution of an inorganic salt is added to a final concentration of 5mmol/L, and the activity is measured according to a standard method. The enzyme activity was recorded as 100% with water added as a control, and the relative activity of the other groups was calculated. As shown in FIG. 6, znSO ₄ 、CuSO ₄ 、NiSO ₄ 、FeCl ₃ 、MnCl ₂ 、CaCl ₂ 、MgSO ₄ All have strong inhibitory action on SL4 lipase, but NaCl, KCl and (NH) ₄ ) ₂ SO ₄ Then has obvious activating effect on SL4 lipase, especially NaCl and KCl, and probably has activating effect on SL4 lipase because the content of sodium and potassium ions in the ocean where schizochytrium limacinum grows is relatively high.

Example 5: comparison of enzyme Activity of SL4 Lipase with that of commercial enzyme

Taking 50mL shake flask fermentation liquor for expressing SL4 lipase, centrifuging at 12000rpm for 5min, washing the thalli with sterile water for 2 times, and then suspending in 5mL sterile water to obtain the SL4 lipase finished product. Control commercial enzyme preparations were the lipases Candida antarctica lipase B (Novozymes Lipozyme C ALB L) and Rhizomucor miehei lipase RML (Novozymes Palatase 20000L) which hydrolyze medium-chain fatty acid methyl esters.

After diluting SL4 lipase finished product, novozymes Lipozyme CALB L and Novozymes Palatase 20000L by 1000 times respectively, 25 μ L of sample is sucked and added into 600 μ L of pNPB reaction mixed solution, water bath is carried out for 15min at 35 ℃, 500 μ L of absolute ethyl alcohol is added to stop reaction, centrifugation is carried out for 5min at 12000rpm, and the supernatant is taken out to measure the absorbance of the sample under the wavelength of 410 nm. The results are shown in FIG. 7, and the enzyme activities of the added enzyme preparations are as follows: the enzyme activity of the finished product SL4 lipase is 106U/mL, the enzyme activity of Novozymes Lipozyme CALB L is 116U/mL, and the enzyme activity of Nov ozymes Palatase 20000L is 81U/mL; the activity level of the finished SL4 lipase expressed by the non-optimized pichia pastoris is basically equivalent to that of candida antarctica lipase B and rhizomucor miehei lipase purchased from Novitin, and the feasibility of applying the SL4 lipase to production on the aspect of enzyme activity expression is proved.

Example 6: SL4 lipase applied to octyl-decyl methyl ester hydrolysis experiment

Weighing 30g of methyl caprylate/caprate, adding 30g of water, stirring uniformly at 35 ℃, adding 1.5mL of SL4 finished bacteria liquid (5%, v/m, the ratio of the volume value of the SL4 enzyme liquid in mL to the value of the methyl caprylate/caprate in g), setting the enzyme activity of the SL4 enzyme liquid to be 106U/mL and the vacuum degree of a system to be 56mbar by using a diaphragm pump as described in the embodiment 5, standing for layering (if the layering is not obvious, separating by a centrifugal mode), sampling the upper layer, measuring AV, and calculating the hydrolysis rate.

AV ₀ Acid value of methyl finger octyl decanoate _t Means that the acid value, AV, is sampled at a time interval t _{Theoretical value} Acid value when methyl caprylate/caprate is completely hydrolyzed. In this experiment, AV ₀ Is 0,AV _{Theoretical value} It was 327.3mgKOH/g.

The results are shown in Table 1 and show that SL4 has a desirable hydrolysis effect on methyl octyldecanoate.

TABLE 1 hydrolysis ratio of methyl caprylate/caprate by SL4 Lipase

The bacterial cells were collected by centrifugation and the hydrolysis experiment was repeated to verify the reusability of the naturally immobilized SL4 lipase, and the results are shown in Table 2, wherein after 6 times of reuse, the SL4 lipase still maintains a good hydrolysis effect.

TABLE 2 reusability of octyl decanoate methyl ester by SL4 lipase

Sequence listing

<110> Fengyi (Shanghai) Biotechnology research and development center, inc

<120> lipase and use thereof

<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

1. A polypeptide selected from the group consisting of:

(1) A polypeptide having an amino acid sequence as shown in SEQ ID NO. 2, and

2. A polynucleotide molecule which is a polynucleotide sequence encoding the polypeptide of claim 1.

3. The polynucleotide molecule of claim 2, wherein said polynucleotide molecule is the nucleotide sequence set forth in SEQ ID No. 1.

4. A nucleic acid construct comprising:

(a) The polynucleotide molecule of claim 2; or

(b) The polynucleotide molecule of claim 3.

5. The nucleic acid construct of claim 4, wherein said nucleic acid construct is a cloning vector or an expression vector.

6. The nucleic acid construct of claim 5, wherein said nucleic acid construct comprises an AOX1 promoter, an alpha-Factor signal peptide, a His4 expression cassette, and a multiple cloning site.

7. The nucleic acid construct of claim 6, wherein the nucleic acid construct is a backbone of a pPIC9K plasmid.

8. A host cell comprising the nucleic acid construct of any one of claims 4-7.

9. The host cell of claim 8, wherein the host cell is a microbial cell.

10. The host cell of claim 9, wherein the host cell is a pichia cell or an escherichia coli cell.

11. A composition comprising the polypeptide of claim 1 or the host cell of any one of claims 8 to 10, optionally together with an adjuvant.

12. The composition of claim 11, wherein the adjunct is an adsorbent material selected from the group consisting of activated carbon, alumina, diatomaceous earth, porous ceramics, and porous glass.

13. A method for hydrolyzing fatty acid methyl esters, said method comprising the step of contacting a component comprising a fatty acid methyl ester with the polypeptide of claim 1 or the host cell of any one of claims 8-10 or the composition of any one of claims 11-12, said fatty acid methyl ester being a fatty acid methyl ester comprising a C4 to C12 chain length fatty acid.

14. The method of claim 13, wherein the fatty acid methyl ester is methyl caprylate decanoate; and/or

The contact conditions include:

(A) The temperature is 20-60 ℃; and/or

(B) The pH value is 5.5-9.

15. The process of claim 14, wherein under said contacting conditions, (a) the temperature is 35 ± 2 ℃ and (B) the pH is 6 to 9.

16. A hydrolysis reaction mixture, comprising:

fatty acid methyl esters;

water; and

the polypeptide of claim 1;

the fatty acid methyl ester is a fatty acid methyl ester containing a fatty acid having a chain length of C4 to C12.

17. The hydrolysis reaction mixture of claim 16,

the dosage of the polypeptide required by each gram of the fatty acid methyl ester is more than 5U; and/or

The dosage of the water is more than 90 percent of the weight of the fatty acid methyl ester;

the enzyme activity unit U is defined as that 1 enzyme activity unit U is required for enzyme hydrolysis of 4-nitrophenyl butyrate of the substrate under the conditions of 40 ℃ and 8.0 of pH value, and 1 micromole of p-nitrophenol is released per minute.

18. The hydrolysis reaction mixture of claim 17,

the dosage of the polypeptide required by each gram of the fatty acid methyl ester is 5U-10U; and/or

The dosage of the water is 90-200% of the weight of the fatty acid methyl ester.

19. The hydrolysis reaction mixture of claim 18, wherein said fatty acid methyl ester is methyl caprylate decanoate.

20. Use of the polypeptide of claim 1, the polynucleotide molecule of claim 2 or 3, the nucleic acid construct of any one of claims 4 to 7, the host cell of any one of claims 8 to 10, or the composition of claim 11 or 12 in the food industry, chemistry and chemical industry.

21. The use of claim 20, wherein the chemical application is the hydrolysis of fatty acid methyl esters, wherein the fatty acid methyl esters are fatty acid methyl esters containing fatty acids having a chain length of C4 to C12.

22. The use of claim 21, wherein the fatty acid methyl ester is methyl caprylate/caprate.