Lipase mutant as well as preparation method and application thereof

Abstract

The invention discloses a lipase mutant as well as a preparation method and application thereof, and belongs to the technical field of gene engineering. The lipase mutant is a protein obtained by carrying out one or more amino acid residue changes on at least one of the following sites of an amino acid sequence as shown in SEQ ID NO: 1: the 90th site, the 206th site and the 214th site; wherein the change is selected from at least one of substitution, deletion and addition. According to the present invention, the lipase derived from rhizopus oryzae is subjected to site-directed mutagenesis through the semi-rational design to obtain the rhizopus oryzae lipase (ROL) mutant with significantly improved catalytic activity, and the ROL mutant can significantly improve the yield of 1, 3-diglyceride when the 1, 3-diglyceride is prepared.

Description

Technical Field

[0001] The invention belongs to the technical field of genetic engineering, and specifically relates to a lipase mutant and a preparation method and application thereof. Background Art

[0002] Lipases are a class of multifunctional enzymes that can catalyze a variety of reactions, including the decomposition of triglycerides into glycerol and free fatty acids, as well as the promotion of hydrolysis, transesterification and synthesis of esters. Lipases also have enantioselectivity, which makes them important in industrial fields such as food oil processing, biodiesel, detergents, synthesis of ester-bonded compounds and synthesis of chiral drugs. Microorganisms have become the main source of industrial lipases due to their diverse lipase species, rapid reproduction ability and environmental adaptability. These enzymes can function under a wide range of pH and temperature conditions and show specificity for substrates. Among the many microorganisms that can produce lipases, genera such as Aspergillus niger, Candida, Rhizopus, Pseudomonas and Streptomyces are particularly important.

[0003] Diacylglycerol (DAG) is a compound naturally present in edible oils, consisting of glycerol and two fatty acid molecules. Due to the difference in the position of the ester bond formed by fatty acids and glycerol, it can be subdivided into two types of diacylglycerol, 1,2-diacylglycerol and 1,3-diacylglycerol. Compared with 1,2-diacylglycerol, 1,3-diacylglycerol has some unique advantages, as follows: 1) Metabolic differences: 1,3-diacylglycerol is not easily converted into stored fat during metabolism in the body, but tends to directly provide energy, which helps to control weight; 2) Health benefits: 1,3-diacylglycerol helps to lower blood lipids and reduce visceral fat accumulation, which is beneficial for preventing obesity and related diseases; 3) Bioavailability: 1,3-diacylglycerol has similar bioavailability to triglycerides, but provides lower energy density, which is suitable for weight management; 4) Safety: 1,3-diacylglycerol is recognized as a safe substance by the US FDA, with regulatory support and good safety. Due to its health benefits and production advantages, 1,3-diacylglycerol has significant market potential in many fields such as food, medicine and cosmetics.

[0004] The bioenzymatic method is an efficient way to synthesize 1,3-diacylglycerol, which is mainly completed by the specific catalysis of lipase. However, most lipases currently have poor catalytic activity when synthesizing high-purity 1,3-diacylglycerol. Summary of the invention

[0005] The purpose of the present invention is to provide a lipase mutant and a preparation method and application thereof, so as to solve the problem that the existing lipase has poor catalytic activity when synthesizing high-purity 1,3-diacerol.

[0006] Specifically, the present invention uses semi-rational design to Rhizopus oryzae ) was subjected to site-directed mutagenesis to obtain a Rhizopus oryzae lipase (ROL) mutant with significantly improved catalytic activity, and when preparing 1,3-diacylglycerol, the ROL mutant can significantly increase the yield of 1,3-diacylglycerol.

[0007] In some embodiments, the present invention provides a lipase, whose amino acid sequence is shown in SEQ ID NO: 1.

[0008] The lipase provided by the present invention can be a natural, recombinant or synthetic active polypeptide, and the active polypeptide can be a naturally purified product, a chemically synthesized product, or a product produced from a prokaryotic host (such as Escherichia coli) or a eukaryotic host (such as yeast, higher plants) using recombinant technology.

[0009] In some embodiments, the lipase is obtained by introducing a recombinant vector containing the gene encoding the lipase into an expression host (eg, Pichia pastoris GS115) to obtain a recombinant genetic engineering strain, and then culturing the recombinant genetic engineering strain and inducing expression to obtain the lipase.

[0010] In some embodiments, the present invention provides a nucleic acid molecule encoding the above-mentioned lipase, whose nucleotide sequence is shown in SEQ ID NO: 2.

[0011] The nucleic acid molecules provided by the present invention can usually be obtained by PCR amplification or artificial synthesis.

[0012] In the first aspect, the present invention provides a lipase mutant, which is a protein obtained by changing one or more amino acid residues at at least one of the following sites of the amino acid sequence shown in SEQ ID NO: 1: 90, 206, and 214; wherein the change is selected from at least one of substitution, deletion, and addition.

[0013] In some embodiments, the lipase mutant is a mutation in at least one of the following positions of the amino acid sequence as shown in SEQ ID NO: 1: 1) I90C; 2) V206I; 3) F214W.

[0014] In some embodiments, the lipase mutant is a protein having an amino acid sequence as shown in SEQ ID NO: 3, SEQ ID NO: 5, or SEQ ID NO: 7.

[0015] In the present invention, the lipase mutant whose amino acid sequence is shown in SEQ ID NO: 3 is I90C (isoleucine (Ile) at position 90 of the lipase is mutated to cysteine ​​(Cys)); the lipase mutant whose amino acid sequence is shown in SEQ ID NO: 5 is V206I (valine (Val) at position 206 of the lipase is mutated to isoleucine (Ile)); and the lipase mutant whose amino acid sequence is shown in SEQ ID NO: 7 is F214W (phenylalanine (Phe) at position 214 of the lipase is mutated to tryptophan (Trp)).

[0016] In some embodiments, in the above-mentioned lipase mutant, in addition to the above-mentioned mutation occurring at at least one of positions 90, 206, and 214, it may further have conservative substitutions of amino acids at other positions, so that the mutated amino acids, such as the wild-type lipase shown in SEQ ID NO: 1, have higher catalytic efficiency. Preferably, the conservative substitution of amino acids retains the higher catalytic efficiency of the lipase mutant of the present invention. It is obvious to those skilled in the art that such substitutions can occur in regions other than the above-mentioned sites while still retaining the corresponding activity. Preferably, the conservative substitution variant has conservative substitutions of amino acids at at least one position. Examples of conservative substitutions are substitutions occurring within the following amino acid groups: basic amino acids (such as arginine, lysine, and histidine), acidic amino acids (such as glutamic acid and aspartic acid), polar amino acids (such as glutamine, asparagine), hydrophobic amino acids (such as leucine, isoleucine, and valine), aromatic amino acids (such as phenylalanine, tryptophan, and tyrosine), and small molecule amino acids (such as glycine, alanine, serine, threonine, and methionine). The most common amino acid interchanges include G to A; A to G, S; V to I, L, A, T, S; I to V, L, M; L to I, M, V; M to L, I, V; P to A, S, N; F to Y, W, H; Y to F, W, H; W to Y, F, H; R to K, E, D; K to R, E, D; H to Q, N, S; D to N, E, K, R, Q; E to Q, D, K, R, N; S to T, A; T to S, V, A; C to S, T, A; N to D, Q, H, S; Q to E, N, H, K, R, and their reverse interchanges.

[0017] The lipase mutant having a certain amino acid homology with the amino acid sequence of the above-mentioned lipase mutant preferably has a homology of 70-99%, for example, it can be 70%, 72%, 74%, 76%, 78%, 80%, 82%, 84%, 86%, 88%, 90%, 92%, 94%, 96%, 98%, 99% or a value or range between any two of these values; more preferably, the homology is between 80-99%, for example, it can be 80%, 82%, 84%, 86%, 88%, 90%, 92%, 94%, 96%, 98%, 99% or a value or range between any two of these values; further more preferably, the homology is between 90-99%, for example, it can be 90%, 92%, 94%, 96%, 98%, 99% or a value or range between any two of these values; most preferably, the homology is 99%, which also belongs to the protection scope of the present invention.

[0018] The lipase mutant provided by the present invention can be a natural, recombinant or synthetic active polypeptide, and the active polypeptide can be a naturally purified product, a chemically synthesized product, or a product produced from a prokaryotic host (such as Escherichia coli) or a eukaryotic host (such as yeast, higher plants) using recombinant technology.

[0019] In some embodiments, the lipase mutant is obtained by introducing a recombinant vector containing its encoding gene into an expression host (eg, Pichia pastoris GS115) to obtain a recombinant genetic engineering strain, and then culturing the recombinant genetic engineering strain and inducing expression to obtain the lipase mutant.

[0020] In a second aspect, the present invention provides a nucleic acid molecule encoding any one of the above-mentioned lipase mutants.

[0021] The nucleic acid molecule provided by the present invention can be DNA, such as cDNA, genomic DNA or recombinant DNA; it can also be RNA, such as mRNA or hnRNA, etc.; and the nucleic acid molecule can usually be obtained by PCR amplification or artificial synthesis.

[0022] In some embodiments, the nucleic acid molecule is selected from any of the following nucleic acid molecules: A1) a nucleic acid molecule having a nucleotide sequence as shown in SEQ ID NO: 4, SEQ ID NO: 6, or SEQ ID NO: 8; A2) a nucleic acid molecule that hybridizes with the nucleic acid molecule defined in A1) under stringent conditions and encodes any of the above-mentioned lipase mutants; A3) a nucleic acid molecule that has more than 90% sequence identity with the nucleic acid molecule defined in A1) or A2) and encodes any of the above-mentioned lipase mutants.

[0023] As used herein, the term "hybridize under stringent conditions" means that two nucleic acid molecule fragments hybridize to each other under standard hybridization conditions such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (1989) (Cold Spring Lane Laboratories, New York, USA) in the section "Expression of cloned genes in Escherichia coli". Such conditions are, for example, hybridization in 6.0×SSC at 45° C., followed by a washing step in 2×SSC at 50° C. In order to select stringency, the salt concentration in the washing step can be selected, for example, between 2.0×SSC at 50° C. for low stringency and 2.0×SSC at 50° C. for high stringency. In addition, the temperature in the washing step can be varied between room temperature of about 22° C. for low stringency and 65° C. for high stringency.

[0024] As used herein, the term "sequence identity" can be evaluated by the naked eye or by computer software (such as the software program described in Ausubel et al. eds. (2007) in Current Protocols in Molecular Biology). When a position in the compared sequences is occupied by the same base or amino acid, the molecules are identical at that position. The identity between two or more sequences can be expressed as a percentage (%), which can be used to evaluate the identity between related sequences. A "sequence identity" in which a polynucleotide sequence or an amino acid sequence has a certain percentage (e.g., 90%, 95%, 98% or 99%) with another sequence means that when the sequences are aligned, the bases or amino acids in the two sequences being compared are the same.

[0025] In a third aspect, the present invention provides a recombinant vector comprising the above nucleic acid molecule.

[0026] The recombinant vectors in the present invention include cloning vectors and expression vectors. The cloning vectors are used to replicate related sequences, and the expression vectors are used to express related genes. The vector used in constructing the expression vector may be the pHBM905BDM vector.

[0027] In a fourth aspect, the present invention provides a recombinant cell comprising the above nucleic acid molecule or the above recombinant vector.

[0028] In some embodiments, the method for preparing a recombinant cell comprises the step of transforming the above-mentioned recombinant vector into an expression host cell.

[0029] In a fifth aspect, the present invention provides a method for preparing a lipase mutant, comprising the following steps: culturing the above-mentioned recombinant cells, inducing expression, and obtaining a culture; and isolating the above-mentioned lipase mutant from the above-mentioned culture.

[0030] In the present invention, there are no special requirements for the culture method and culture conditions, as long as the normal growth of the recombinant cells is ensured. And the methods for isolating the above lipase mutants from the culture are all conventional methods in the art.

[0031] In some embodiments, the culture medium used in the method for preparing the lipase mutant is a culture medium capable of expressing proteins in the art, preferably BMMY medium.

[0032] In a sixth aspect, the present invention provides use of the lipase mutant, the nucleic acid molecule, the recombinant vector, the recombinant cell or the lipase mutant prepared by the preparation method in the preparation of 1,3-diacylglycerol.

[0033] In a seventh aspect, the present invention provides a method for preparing 1,3-diacylglycerol, comprising the following steps: using the above-mentioned lipase mutant, the above-mentioned recombinant cell or the lipase mutant prepared by the above-mentioned preparation method as a catalyst to catalyze the reaction of fatty acids and glycerol to obtain 1,3-diacylglycerol.

[0034] In some embodiments, the fatty acid is a C18 fatty acid.

[0035] In the method for preparing 1,3-diacerol provided by the present invention, the lipase mutant or the recombinant cell containing the lipase mutant first catalyzes the reaction of fatty acids and glycerol to obtain triglycerides, and then hydrolyzes the triglycerides to obtain 1,3-diacerol.

[0036] The beneficial effects of the present invention are as follows: Different from the prior art, the present invention uses a semi-rational design to Rhizopus oryzae ) was subjected to site-directed mutagenesis to obtain a Rhizopus oryzae lipase (ROL) mutant with significantly improved catalytic activity, and when preparing 1,3-diacylglycerol, the ROL mutant can significantly increase the yield of 1,3-diacylglycerol. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 The SDS-PAGE image of ROL-WT protein, ROL-I90C mutant protein, ROL-V206I mutant protein, and ROL-F214W mutant protein in Example 3 of the present invention; Figure 2 The p-nitrophenol standard curve diagram established in Example 4 of the present invention; Figure 3 The results of the enzyme activities of ROL-WT protein, ROL-I90C mutant protein, ROL-V206I mutant protein, and ROL-F214W mutant protein against p-nitrophenol esters with different carbon chain lengths in Example 4 of the present invention are shown in FIG. Figure 4This is a TLC result diagram of the ROL-WT protein and the ROL-I90C mutant protein in Example 5 of the present invention after reacting with C18 fatty acid and glycerol as substrates, wherein FA: C18 fatty acid; 1,3-DAG: 1,3-diacylglycerol. DETAILED DESCRIPTION

[0038] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0039] The experimental methods without specific conditions in the examples are usually carried out according to conventional experimental methods in the field of molecular biology, including but not limited to the experimental methods described in Molecular Cloning: A Laboratory Manual by MR Green, Molecular Biology by Robert F. Weaver, etc., or according to the experimental methods recommended by the kit and instrument manufacturers. The reagents and biological materials used in the examples can be obtained from commercial channels unless otherwise specified.

[0040] Example 1 Mining of lipase and obtaining of recombinant plasmid containing lipase First, a gene from Rhizopus oryzae ( Rhizopus oryzae ) has an amino acid sequence as shown in SEQ ID NO: 1. The wild-type lipase (ROL) consists of a leader peptide and a mature peptide. The amino acid sequence of the leader peptide is as shown in positions 1-97 of SEQ ID NO: 1, and the amino acid sequence of the mature peptide is as shown in positions 98-366 of SEQ ID NO: 1. When the wild-type lipase is mutated, the amino acid sequence site of the mature peptide is mutated.

[0041] The gene encoding the wild-type lipase was codon-optimized in Pichia pastoris, and the nucleotide sequence of the gene encoding the wild-type lipase after codon optimization is shown in SEQ ID NO: 2. The gene fragment encoding the wild-type lipase after codon optimization was synthesized, and the DNA fragment shown in SEQ ID NO: 2 was used to replace the sequence between the CpoⅠ and NotⅠ restriction sites of the pHBM905BDM vector, and the other sequences remained unchanged to obtain the recombinant plasmid pHBM905BDM-ROL. The recombinant plasmid pHBM905BDM-ROL was sent for sequencing, and the results were consistent with expectations.

[0042] Example 2 Obtaining a recombinant plasmid containing a lipase mutant Conventional site-directed mutagenesis technology was used, and the recombinant plasmid pHBM905BDM-ROL obtained in Example 1 was used as a template to design amplification primers for mutants I90C (calculated based on the first site of the mature peptide in the wild-type lipase (equivalent to the 98th position of the amino acid sequence shown in SEQ ID NO: 1)), V206I, and F214W (the specific nucleotide sequences are shown in Table 1 below), respectively. After PCR amplification, recombinant plasmids pHBM905BDM-ROL-I90C, recombinant plasmids pHBM905BDM-ROL-V206I, and recombinant plasmids pHBM905BDM-ROL-F214W were obtained, respectively. The above mutant recombinant plasmids were sent for sequencing, and the results were consistent with expectations.

[0043] Table 1 Primer sequences

[0044] Example 3 Expression of lipase and its mutants The recombinant plasmid pHBM905BDM-ROL prepared in Example 1 and the recombinant plasmids pHBM905BDM-ROL-I90C, pHBM905BDM-ROL-V206I, and pHBM905BDM-ROL-F214W prepared in Example 2 were linearized using restriction endonuclease SalⅠ, and the linearized fragments were recovered by gel. The competent cells of Pichia pastoris GS115 strain were prepared by the lithium acetate (LiAc) method, and then 7 μg of the linearized recombinant plasmids were transformed into GS115 competent cells by electrotransformation. The transformants were spread on MD plates and cultured at 28°C for 3 days. Single clones on the plates were picked and inoculated on BMMY-tributyrin screening plates, respectively, and clones with large transparent circles were selected to obtain Pichia pastoris strains expressing wild-type lipase and its mutants.

[0045] The above-mentioned Pichia pastoris strains expressing wild-type lipase and its mutants were inoculated into BMGY medium, respectively, and cultured at 28°C with shaking at 220 rpm overnight until OD 600 The supernatant was removed by centrifugation, the bacterial precipitate was resuspended in half of the BMMY medium for mixed culture, and then induced with 1% methanol for 3-5 days, and centrifuged at 12000rpm for 5min to obtain the fermentation supernatant, and then the fermentation supernatant was subjected to SDS-PAGE detection. The results are as follows: Figure 1 shown.

[0046] from Figure 1 It can be seen that the molecular weights of the ROL-WT protein, ROL-I90C mutant protein, ROL-V206I mutant protein, and ROL-F214W mutant protein bands are consistent with the predicted molecular weights. The results show that the wild-type lipase and its mutants were successfully expressed.

[0047] Example 4 Determination of the enzyme activity of lipase and its mutants against p-nitrophenol esters with different carbon chain lengths The enzymatic activity of lipase and its mutants was tested by spectrophotometry. Specifically, a standard curve was first established with p-nitrophenol standard, as follows: Pipette 500μL of PBS buffer (pH 8.5) into a 1.5mL EP tube, then add 20μL of p-nitrophenol standard solution of different concentrations, react in a 40℃ water bath for 5min, and add 470μL of 1% SDS (sodium dodecyl sulfate) solution to terminate the reaction. Take 200μL of the reaction solution into a 96-well plate, measure the absorbance at 405nm using an ELISA reader, and draw a standard curve with concentration as the horizontal axis and absorbance as the vertical axis. The results are shown in the figure. Figure 2 shown.

[0048] Then, the enzymatic activity of lipase and its mutants against p-nitrophenol esters with different carbon chain lengths was tested. Specifically, 500 μL of PBS buffer (pH 8.5) was pipetted into a 1.5 mL EP tube, and 10 μL of enzyme solution (ROL-WT protein, ROL-I90C mutant protein, ROL-V206I mutant protein, ROL-F214W mutant protein) and 20 μL of p-nitrophenol esters with different carbon chain lengths (C4 (p The solution of 1% nitrophenol butyrate), C6 (p-nitrophenol hexanoate), C8 (p-nitrophenol octanoate), C10 (p-nitrophenol decanoate), C12 (p-nitrophenol laurate), C14 (p-nitrophenol myristate), C16 (p-nitrophenol palmitate), C18 (p-nitrophenol stearate) was reacted in a 40°C water bath for 5 minutes, and 470 μL of 1% SDS (sodium dodecyl sulfate) solution was added to terminate the reaction. 200 μL of the reaction solution was taken into a 96-well plate, and the absorbance at 405 nm was measured using an enzyme reader.

[0049] According to the standard curve, the concentration of p-nitrophenol, the hydrolysis product of ROL-WT protein, ROL-I90C mutant protein, ROL-V206I mutant protein, and ROL-F214W mutant protein, was calculated, thereby obtaining the enzyme activities of ROL-WT protein, ROL-I90C mutant protein, ROL-V206I mutant protein, and ROL-F214W mutant protein for p-nitrophenol esters with different carbon chain lengths. The results are shown in FIG. Figure 3 shown.

[0050] Among them, the definition of lipase activity (U) is: under certain reaction conditions, the amount of enzyme required to hydrolyze the substrate to release 1 μmol of p-nitrophenol (pNP) per minute is one enzyme activity unit, that is, 1U.

[0051] from Figure 3 It can be seen that the enzyme activity of ROL-I90C mutant protein, ROL-V206I mutant protein and ROL-F214W mutant protein is higher than that of ROL-WT protein. The results show that in the present invention, by performing site-directed mutagenesis on the wild-type ROL-WT protein, a mutant protein with significantly improved enzyme activity can be obtained.

[0052] Example 5 Preparation of 1,3-diacerol catalyzed by lipase and its mutants In a 100 mL reaction vessel, C18 fatty acid and glycerol were mixed at a mass ratio of 3:2, with a total mass of 50 g. ROL-WT and ROL-I90C enzyme solutions were added at 2% of the total mass of the substrate. The reaction was carried out for 8 hours at 40°C and 220 r / min in a constant temperature water bath shaker. After the reaction was completed, centrifuged at 12000 r / min for 5 minutes to separate the layers. 2 μL of the upper oil phase was taken and dissolved in 1 mL of ether. TLC was used for analysis and detection. The results are as follows: Figure 4 shown.

[0053] from Figure 4 It can be seen that compared with the wild-type ROL-WT protein, the ROL-I90C mutant protein can significantly increase the production of 1,3-diacylglycerol.

[0054] In summary, the present invention uses semi-rational design to Rhizopus oryzae ) was subjected to site-directed mutagenesis to obtain a Rhizopus oryzae lipase (ROL) mutant with significantly improved catalytic activity, and when preparing 1,3-diacylglycerol, the ROL mutant can significantly increase the yield of 1,3-diacylglycerol.

[0055] The above-mentioned embodiments only express the implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the invention patent. It should be pointed out that for ordinary technicians in this field, several modifications and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be based on the attached claims.

[0056] Amino acid sequence of lipase (SEQ ID NO: 1) VPVSGKSGSSTTAVSASDNSALPPLISSRCAPPSNKGSKSDLQAEPYYMQKNTEWYESHGGNTSIGKRDDNLVGGMTLDLPSDAPPISLSGSTNSASDGGKVAAATTAQIQEFTKYAGIAATAYCRSVVPGNKWDCVQCQKWVPDGKIITTFTSLLSDTNGYVLRSDKQKTIYLVFRGTNSF RSAITDIVFNFSDYKPVKGAKVHAGFLSSYEQVVNDYFPVIQEQLTANPTYKVIVTGHSLGGAQALLAGMDLYQREPRLSPKNLSIFTVGGPRVGNPTFAYYVESTGIPFQRTVHKRDIVPHVPPQSFGFLHPGVESWIKSGTSNVQICTSEIETKDCSNSIVPFTSLLDHLSYFDINEGSCL Nucleotide sequence of a codon-optimized gene encoding lipase (SEQ ID NO: 2) Amino acid sequence of lipase mutant I90C (SEQ ID NO: 3) VPVSGKSGSSTTAVSASDNSALPPLISSRCAPPSNKGSKSDLQAEPYYMQKNTEWYESHGGNTSIGKRDDNLVGGMTLDLPSDAPPISLSGSTNSASDGGKVAAATTAQIQEFTKYAGIAATAYCRSVVPGNKWDCVQCQKWVPDGKIITTFTSLLSDTNGYVLRSDKQKTIYLVFRGTNSF RSACTDIVFNFSDYKPVKGAKVHAGFLSSYEQVVNDYFPVIQEQLTANPTYKVIVTGHSLGGAQALLAGMDLYQREPRLSPKNLSIFTVGGPRVGNPTFAYYVESTGIPFQRTVHKRDIVPHVPPQSFGFLHPGVESWIKSGTSNVQICTSEIETKDCSNSIVPFTSLLDHLSYFDINEGSCL Nucleotide sequence of the gene encoding lipase mutant I90C (SEQ ID NO: 4) Amino acid sequence of lipase mutant V206I (SEQ ID NO: 5) VPVSGKSGSSTTAVSASDNSALPPLISSRCAPPSNKGSKSDLQAEPYYMQKNTEWYESHGGNTSIGKRDDNLVGGMTLDLPSDAPPISLSGSTNSASDGGKVAAATTAQIQEFTKYAGIAATAYCRSVVPGNKWDCVQCQKWVPDGKIITTFTSLLSDTNGYVLRSDKQKTIYLVFRGTNSF RSAITDIVFNFSDYKPVKGAKVHAGFLSSYEQVVNDYFPVIQEQLTANPTYKVIVTGHSLGGAQALLAGMDLYQREPRLSPKNLSIFTVGGPRVGNPTFAYYVESTGIPFQRTVHKRDIIPHVPPQSFGFLHPGVESWIKSGTSNVQICTSEIETKDCSNSIVPFTSLLDHLSYFDINEGSCL Nucleotide sequence of the gene encoding lipase mutant V206I (SEQ ID NO: 6) Amino acid sequence of lipase mutant F214W (SEQ ID NO: 7) VPVSGKSGSSTTAVSASDNSALPPLISSRCAPPSNKGSKSDLQAEPYYMQKNTEWYESHGGNTSIGKRDDNLVGGMTLDLPSDAPPISLSGSTNSASDGGKVAAATTAQIQEFTKYAGIAATAYCRSVVPGNKWDCVQCQKWVPDGKIITTFTSLLSDTNGYVLRSDKQKTIYLVFRGTNSF RSAITDIVFNFSDYKPVKGAKVHAGFLSSYEQVVNDYFPVIQEQLTANPTYKVIVTGHSLGGAQALLAGMDLYQREPRLSPKNLSIFTVGGPRVGNPTFAYYVESTGIPFQRTVHKRDIVPHVPPQSWGFLHPGVESWIKSGTSNVQICTSEIETKDCSNSIVPFTSLLDHLSYFDINEGSCL Nucleotide sequence of the gene encoding the lipase mutant F214W (SEQ ID NO: 8)

Claims

1. A lipase mutant, characterized in that: The lipase mutant is a protein obtained by changing one or more amino acid residues at at least one of the following positions: position 90, position 206, and position 214 of the amino acid sequence shown in SEQ ID NO: 1; Wherein, the change is selected from at least one of substitution, deletion and addition.

2. The lipase mutant according to claim 1, characterized in that The lipase mutant is a mutation in at least one of the following positions of the amino acid sequence shown in SEQ ID NO: 1: 1) I90C; 2) V206I; 3) F214W.

3. The lipase mutant according to claim 2, characterized in that The lipase mutant is a protein having an amino acid sequence as shown in SEQ ID NO: 3, SEQ ID NO: 5, or SEQ ID NO:

7.

4. A nucleic acid molecule encoding the lipase mutant according to any one of claims 1 to 3.

5. The nucleic acid molecule according to claim 4, characterized in that The nucleic acid molecule is selected from any of the following nucleic acid molecules: A1) a nucleic acid molecule having a nucleotide sequence as shown in SEQ ID NO: 4, SEQ ID NO: 6, or SEQ ID NO: 8; A2) a nucleic acid molecule that hybridizes with the nucleic acid molecule defined in A1) and encodes the lipase mutant according to any one of claims 1 to 3; A3) A nucleic acid molecule having a sequence identity of 90% or more with the nucleic acid molecule defined in A1) or A2) and encoding the lipase mutant according to any one of claims 1 to 3.

6. A recombinant vector, characterized in that: Comprising the nucleic acid molecule according to claim 4 or 5.

7. A recombinant cell, characterized in that Comprising the nucleic acid molecule according to claim 4 or 5 or the recombinant vector according to claim 6.

8. A method for preparing a lipase mutant, characterized in that: The steps include: Cultivating the recombinant cell according to claim 7, inducing expression, and obtaining a culture; The lipase mutant according to any one of claims 1 to 3 is isolated from the culture.

9. Use of the lipase mutant according to any one of claims 1 to 3, the nucleic acid molecule according to claim 4 or 5, the recombinant vector according to claim 6, the recombinant cell according to claim 7 or the lipase mutant prepared by the preparation method according to claim 8 in the preparation of 1,3-diaglyceride.

10. A method for preparing 1,3-diacerol, characterized in that: The steps include: The lipase mutant according to any one of claims 1 to 3, the recombinant cell according to claim 7 or the lipase mutant prepared by the preparation method according to claim 8 is used as a catalyst to catalyze the reaction of fatty acids and glycerol to obtain 1,3-diacerol.

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