A lipase mutant, its preparation method and application

Abstract

The application discloses a lipase mutant and a preparation method and application thereof, and belongs to the technical field of genetic engineering. The lipase mutant is a protein obtained by changing one or more amino acid residues at at least one of the following positions: the 90th position, the 206th position and the 214th position 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. The application performs site-directed mutagenesis on a lipase derived from Rhizopus oryzae through semi-rational design, obtains a Rhizopus oryzae lipase (ROL) mutant with significantly improved catalytic activity, and the ROL mutant can significantly improve the yield of 1,3-glycerol diester when preparing 1,3-glycerol diester.

Description

Technical Field

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

[0002] Lipases are a class of multifunctional enzymes that catalyze a wide range of reactions, including the breakdown of triacylglycerols into glycerol and free fatty acids, as well as the hydrolysis, transesterification, and synthesis of esters. Lipases also exhibit enantioselectivity, a property that makes them important in industrial applications such as food oil processing, biodiesel, detergents, the synthesis of ester-bonded compounds, and chiral drug synthesis. Microorganisms, due to their diverse lipase profiles, rapid reproduction, and environmental adaptability, are a major source of industrial lipases. These enzymes function under a wide range of pH and temperature conditions and exhibit substrate specificity. Among the numerous microorganisms that produce lipases, genera such as Aspergillus niger, Candida albicans, Rhizopus spp., Pseudomonas spp., and Streptomyces spp. are particularly important.

[0003] Diacylglycerol (DAG) is a naturally occurring compound found in edible oils, consisting of glycerol and two fatty acid molecules. Due to the position of the ester bond between the fatty acids and glycerol, diacylglycerol can be further categorized into two types: 1,2- and 1,3-diacylglycerol. Compared to 1,2-diacylglycerol, 1,3-diacylglycerol offers several distinct advantages: 1) Metabolic differences: 1,3-diacylglycerol is less likely to be converted into stored fat during metabolism, preferring instead to directly provide energy, thus aiding weight management. 2) Health benefits: 1,3-diacylglycerol helps lower blood lipids and reduce visceral fat accumulation, potentially contributing to the prevention of obesity and related diseases. 3) Bioavailability: 1,3-diacylglycerol has similar bioavailability to triglycerides but offers a lower energy density, making it suitable for weight management. 4) Safety: 1,3-diacylglycerol has been designated as safe by the US FDA, demonstrating regulatory support and a favorable safety profile. Due to its health benefits and production advantages, 1,3-diacylglycerol has significant market potential in various sectors, including food, pharmaceuticals, and cosmetics.

[0004] The bioenzymatic method is a highly efficient pathway for synthesizing 1,3-diacylglycerol, primarily through the specific catalysis of lipases. However, most current lipases exhibit 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 its preparation method and application, so as to solve the problem that the existing lipase has poor catalytic activity when synthesizing high-purity 1,3-diglyceride.

[0006] Specifically, the present invention is to use semi-rational design to Rhizopus oryzae ) were subjected to site-directed mutagenesis to obtain a Rhizopus oryzae lipase (ROL) mutant with significantly improved catalytic activity. 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, the amino acid sequence of which 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 using recombinant technology from a prokaryotic host (such as Escherichia coli) or a eukaryotic host (such as yeast, higher plants).

[0009] In some embodiments, the lipase is produced by introducing a recombinant vector containing its encoding gene into an expression host (eg, Pichia pastoris GS115) to obtain a recombinant genetically engineered strain, and then culturing the recombinant genetically engineered 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, the nucleotide sequence of which 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 a first aspect, the present invention provides a lipase mutant, which is a protein obtained by changing one or more amino acid residues in at least one of the following sites of the amino acid sequence shown in SEQ ID NO: 1: position 90, position 206, and position 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 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 having an amino acid sequence as shown in SEQ ID NO: 3 is I90C (isoleucine (Ile) at position 90 of the lipase is mutated to cysteine ​​(Cys)); the lipase mutant having an amino acid sequence as 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 having an amino acid sequence as 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 addition to the aforementioned mutations at at least one of positions 90, 206, and 214, the lipase mutant may further comprise conservative amino acid substitutions at other positions, such that the mutated amino acids exhibit higher catalytic efficiency than the wild-type lipase shown in SEQ ID NO:1. Preferably, the conservative amino acid substitutions maintain the high catalytic efficiency of the lipase mutant of the present invention. It will be apparent to those skilled in the art that such substitutions can occur at regions other than the aforementioned positions while still retaining the corresponding activity. Preferably, the conservatively substituted variant comprises a conservative amino acid substitution at at least one position. Examples of conservative substitutions include substitutions within the following amino acid groups: basic amino acids (e.g., arginine, lysine, and histidine), acidic amino acids (e.g., glutamic acid and aspartic acid), polar amino acids (e.g., glutamine and asparagine), hydrophobic amino acids (e.g., leucine, isoleucine, and valine), aromatic amino acids (e.g., phenylalanine, tryptophan, and tyrosine), and small amino acids (e.g., 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 an homology of 70-99%, for example, 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, 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, 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 falls within the scope of protection of the present invention.

[0018] The lipase mutants provided by the present invention may be natural, recombinant or synthetic active polypeptides, and the active polypeptides may be naturally purified products, chemically synthesized products, or products produced using recombinant technology from prokaryotic hosts (e.g., Escherichia coli) or eukaryotic hosts (e.g., yeast, higher plants).

[0019] In some embodiments, the above-mentioned lipase mutant is obtained by introducing a recombinant vector containing its encoding gene into an expression host (such as Pichia pastoris GS115) to obtain a recombinant genetically engineered strain, and then culturing the recombinant genetically engineered 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; or RNA, such as mRNA or hnRNA; 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 under stringent conditions with the nucleic acid molecule defined in A1) 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" refers to the hybridization of two nucleic acid molecule fragments 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 include, for example, hybridization in 6.0×SSC at 45°C, followed by a wash step at 2×SSC at 50°C. To select the stringency, the salt concentration in the wash 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 wash step can be varied between room temperature of approximately 22°C for low stringency and 65°C for high stringency.

[0024] As used herein, the term "sequence identity" can be assessed visually or using computer software (e.g., 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 assess the identity between related sequences. A polynucleotide sequence or amino acid sequence that has a certain percentage (e.g., 90%, 95%, 98%, or 99%) of "sequence identity" with another sequence means that, when the sequences are aligned, that percentage of bases or amino acids are the same in the two sequences being compared.

[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 can be the pHBM905BDM vector.

[0027] In a fourth aspect, the present invention provides a recombinant cell comprising the aforementioned nucleic acid molecule or the aforementioned 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 to obtain 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. In addition, the methods for isolating the above-mentioned 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 that can express proteins in the art, preferably BMMY medium.

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

[0033] In a seventh aspect, the present invention provides a method for preparing 1,3-diacerol, 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-diacerol.

[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 ) were subjected to site-directed mutagenesis to obtain a Rhizopus oryzae lipase (ROL) mutant with significantly improved catalytic activity. 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 This is an SDS-PAGE image of the ROL-WT protein, ROL-I90C mutant protein, ROL-V206I mutant protein, and ROL-F214W mutant protein in Example 3 of the present invention;

[0038] Figure 2 The p-nitrophenol standard curve diagram established in Example 4 of the present invention;

[0039] Figure 3 The figure shows the enzyme activity results of ROL-WT protein, ROL-I90C mutant protein, ROL-V206I mutant protein, and ROL-F214W mutant protein against p-nitrophenolates with different carbon chain lengths in Example 4 of the present invention;

[0040] Figure 4 This 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

[0041] 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 embodiments described 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 making creative efforts are within the scope of protection of the present invention.

[0042] Experimental procedures in the examples, where specific conditions are not specified, were generally performed in accordance with conventional methods in molecular biology, including but not limited to those described in M.R. Green's Molecular Cloning: A Laboratory Manual and Robert F. Weaver's Molecular Biology, or according to the recommendations of kit and instrument manufacturers. Unless otherwise specified, reagents and biological materials used in the examples were commercially available.

[0043] Example 1: Discovery of lipase and acquisition of recombinant plasmid containing lipase

[0044] First, a gene from Rhizopus oryzae ( Rhizopus oryzae ) has a wild-type lipase, the amino acid sequence of which is 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 shown in positions 1-97 of SEQ ID NO: 1, and the amino acid sequence of the mature peptide is 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.

[0045] The gene encoding wild-type lipase was codon-optimized in Pichia pastoris. The nucleotide sequence of the codon-optimized gene encoding wild-type lipase is shown in SEQ ID NO: 2. The above-described codon-optimized gene fragment encoding wild-type lipase was synthesized by whole gene synthesis, and the DNA fragment shown in SEQ ID NO: 2 was used to replace the sequence between the Cpo I and Not I enzyme cutting sites of the pHBM905BDM vector, and the remaining sequence was kept unchanged, to obtain the recombinant plasmid pHBM905BDM-ROL. The recombinant plasmid pHBM905BDM-ROL was sequenced, and the result was consistent with the expectation.

[0046] Example 2. Obtaining of a recombinant plasmid containing a lipase mutant

[0047] Using conventional site-directed mutagenesis technology, the recombinant plasmid pHBM905BDM-ROL obtained in Example 1 was used as a template, and amplification primers for mutants I90C (calculated from the first site of the mature peptide in wild-type lipase (equivalent to position 98 of the amino acid sequence shown in SEQ ID NO: 1)), V206I, and F214W (the specific nucleotide sequences are shown in Table 1 below) were designed. After PCR amplification, the recombinant plasmid pHBM905BDM-ROL-I90C, the recombinant plasmid pHBM905BDM-ROL-V206I, and the recombinant plasmid pHBM905BDM-ROL-F214W were obtained, respectively. The above-described mutant recombinant plasmids were sequenced, and the results were consistent with the expectation.

[0048] Table 1. Primer sequences

[0049]

[0050] Example 3. Expression of lipase and its mutants

[0051] 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 the restriction enzyme Sal I, and the linearized fragments were recovered by gel. The competent cells of the Pichia pastoris GS115 strain were prepared using the lithium acetate (LiAc) method, and 7 μg of the linearized recombinant plasmids were transformed into the GS115 competent cells by electroporation. The transformants were spread on MD plates and cultured at 28°C for 3 days. Single colonies on the plates were picked and inoculated on BMMY-tri-butyrin selection plates, and clones with large transparent circles were selected, thereby obtaining Pichia pastoris strains expressing wild-type lipase and its mutants.

[0052] The above-mentioned Pichia pastoris strains expressing wild-type lipase and its mutants were inoculated into BMGY medium, and cultured at 28°C with shaking at 220 rpm overnight until the OD 600 The supernatant was removed by centrifugation and the bacterial pellet was resuspended in half of the BMMY medium for mixed culture. After induction with 1% methanol for 3-5 days, the supernatant was obtained by centrifugation at 12000 rpm for 5 minutes. The supernatant was then subjected to SDS-PAGE detection. The results were as follows: Figure 1 shown.

[0053] 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.

[0054] Example 4 Determination of the Enzyme Activity of Lipase and Its Mutants on p-Nitrophenol Esters with Different Carbon Chain Lengths

[0055] The enzymatic activity of lipase and its mutants was tested by spectrophotometry. Specifically, a standard curve was first established using a p-nitrophenol standard as follows:

[0056] 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. Incubate in a 40℃ water bath for 5 minutes, 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 and measure the absorbance at 405nm using a microplate reader. Plot a standard curve with concentration as the horizontal axis and absorbance as the vertical axis. The results are shown in the figure below. Figure 2 shown.

[0057] 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-nitrophenol esters)) were added. A solution of 1,4-dinitrophenol (4-nitrophenol butyrate), C6 (4-nitrophenol hexanoate), C8 (4-nitrophenol octanoate), C10 (4-nitrophenol decanoate), C12 (4-nitrophenol laurate), C14 (4-nitrophenol myristate), C16 (4-nitrophenol palmitate), and C18 (4-nitrophenol stearate) was reacted in a 40°C water bath for 5 min. The reaction was terminated by adding 470 μL of 1% sodium dodecyl sulfate (SDS) solution. 200 μL of the reaction solution was transferred to a 96-well plate, and the absorbance at 405 nm was measured using a microplate reader.

[0058] According to the above 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 towards p-nitrophenol esters with different carbon chain lengths. The results are shown in FIG. Figure 3 shown.

[0059] 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.

[0060] 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, mutant proteins with significantly improved enzyme activity can be obtained.

[0061] Example 5 Preparation of 1,3-diacerol catalyzed by lipase and its mutants

[0062] 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 a concentration equivalent to 2% of the total mass of the substrate. The reaction was carried out in a constant temperature water bath shaker at 40°C and 220 r / min for 8 h. After the reaction was completed, the mixture was centrifuged at 12000 r / min for 5 min to separate the layers. 2 μL of the upper oil phase was taken and dissolved in 1 mL of ether. The results were analyzed and detected by TLC. Figure 4 shown.

[0063] 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.

[0064] In summary, the present invention is to use semi-rational design to Rhizopus oryzae ) were subjected to site-directed mutagenesis to obtain a Rhizopus oryzae lipase (ROL) mutant with significantly improved catalytic activity. When preparing 1,3-diacylglycerol, the ROL mutant can significantly increase the yield of 1,3-diacylglycerol.

[0065] The above-described embodiments merely illustrate the implementation methods of the present invention. While the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the patent. It should be noted that a person skilled in the art would be able to make various modifications and improvements without departing from the spirit of the present invention, all of which fall within the scope of protection of the present invention. Therefore, the scope of protection of the patent for this invention shall be determined by the appended claims.

[0066] Amino acid sequence of lipase (SEQ ID NO: 1)

[0067] VPVSGKSGSSTTAVSASDNSALPPLISSRCAPPSNKGSKSDLQAEPYYMQKNTEWYESHGGNLTSIGKRDDNLVGGMTLDLPSDAPPISLSGSTNSASDGGKVAAATTAQIQEFTKYAGIAATAYCRSVVPGNKWDCVQCQKWVPDGKIITTFTSLLSDTNGYVLRSDKQKTIYLVFRGTNSFRSAITDIVFNFSDYKPVKGAKVHAGFLSSYEQVVNDYFPVIQEQLTANPTYKVIVTGHSLGGAQALLAGMDLYQREPRLSPKNLSIFTVGGPRVGNPTFAYYVESTGIPFQRTVHKRDIVPHVPPQSFGFLHPGVESWIKSGTSNVQICTSEIETKDCSNSIVPFTSLLDHLSYFDINEGSCL

[0068] Nucleotide sequence of the codon-optimized gene encoding the lipase (SEQ ID NO: 2)

[0069]

[0070] Amino acid sequence of lipase mutant I90C (SEQ ID NO: 3)

[0071] VPVSGKSGSSTTAVSASDNSALPPLISSRCAPPSNKGSKSDLQAEPYYMQKNTEWYESHGGNTSIGKRDDNLVGGMTLDLPSDAPPISLSGSTNSASDGGKVAAATTAQIQEFTKYAGIAATAYCRSVVPGNKWDCVQCQKWVPDGKIITTFTSLLSDTNGYVLRSDKQKTIYLVFRGTNSF RSACTDIVFNFSDYKPVKGAKVHAGFLSSYEQVVNDYFPVIQEQLTANPTYKVIVTGHSLGGAQALLAGMDLYQREPRLSPKNLSIFTVGGPRVGNPTFAYYVESTGIPFQRTVHKRDIVPHVPPQSFGFLHPGVESWIKSGTSNVQICTSEIETKDCSNSIVPFTSLLDHLSYFDINEGSCL

[0072] Nucleotide sequence of the gene encoding lipase mutant I90C (SEQ ID NO: 4)

[0073]

[0074] Amino acid sequence of lipase mutant V206I (SEQ ID NO: 5)

[0075] VPVSGKSGSSTTAVSASDNSALPPLISSRCAPPSNKGSKSDLQAEPYYMQKNTEWYESHGGNTSIGKRDDNLVGGMTLDLPSDAPPISLSGSTNSASDGGKVAAATTAQIQEFTKYAGIAATAYCRSVVPGNKWDCVQCQKWVPDGKIITTFTSLLSDTNGYVLRSDKQKTIYLVFRGTNSF RSAITDIVFNFSDYKPVKGAKVHAGFLSSYEQVVNDYFPVIQEQLTANPTYKVIVTGHSLGGAQALLAGMDLYQREPRLSPKNLSIFTVGGPRVGNPTFAYYVESTGIPFQRTVHKRDIIPHVPPQSFGFLHPGVESWIKSGTSNVQICTSEIETKDCSNSIVPFTSLLDHLSYFDINEGSCL

[0076] Nucleotide sequence of the gene encoding lipase mutant V206I (SEQ ID NO: 6)

[0077]

[0078] Amino acid sequence of lipase mutant F214W (SEQ ID NO: 7)

[0079] VPVSGKSGSSTTAVSASDNSALPPLISSRCAPPSNKGSKSDLQAEPYYMQKNTEWYESHGGNTSIGKRDDNLVGGMTLDLPSDAPPISLSGSTNSASDGGKVAAATTAQIQEFTKYAGIAATAYCRSVVPGNKWDCVQCQKWVPDGKIITTFTSLLSDTNGYVLRSDKQKTIYLVFRGTNSF RSAITDIVFNFSDYKPVKGAKVHAGFLSSYEQVVNDYFPVIQEQLTANPTYKVIVTGHSLGGAQALLAGMDLYQREPRLSPKNLSIFTVGGPRVGNPTFAYYVESTGIPFQRTVHKRDIVPHVPPQSWGFLHPGVESWIKSGTSNVQICTSEIETKDCSNSIVPFTSLLDHLSYFDINEGSCL

[0080] Nucleotide sequence of the gene encoding the lipase mutant F214W (SEQ ID NO: 8)

[0081]

Claims

1. A lipase mutant, characterized in that: The lipase mutant is a protein with an amino acid sequence as shown in SEQ ID NO: 3, SEQ ID NO: 5, or SEQ ID NO:

7.

2. A nucleic acid molecule encoding the lipase mutant according to claim 1.

3. The nucleic acid molecule according to claim 2, characterized in that The nucleic acid molecule is selected from any one of the following nucleic acid molecules: A1) A nucleic acid molecule having the nucleotide sequence shown in SEQ ID NO: 4, SEQ ID NO: 6, or SEQ ID NO:

8.

4. A recombinant vector, characterized in that Comprising the nucleic acid molecule according to claim 2 or 3.

5. A recombinant cell, characterized in that Comprising the nucleic acid molecule according to claim 2 or 3 or the recombinant vector according to claim 4.

6. A method for preparing a lipase mutant, characterized in that: The steps include: Cultivating the recombinant cell according to claim 5 and inducing expression to obtain a culture; The lipase mutant according to claim 1 is isolated from the culture.

7. Use of the lipase mutant according to claim 1, the nucleic acid molecule according to claim 2 or 3, the recombinant vector according to claim 4, the recombinant cell according to claim 5, or the lipase mutant prepared by the preparation method according to claim 6 in the preparation of 1,3-diglyceride.

8. A method for preparing 1,3-diglyceride, characterized in that: The steps include: The lipase mutant according to claim 1, the recombinant cell according to claim 5, or the lipase mutant prepared by the preparation method according to claim 6 is used as a catalyst to catalyze the reaction of fatty acids and glycerol to obtain 1,3-diglyceride.

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