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 selected from any one of the following: A1) a protein obtained by changing an amino acid sequence as shown in SEQ ID NO: 1 through one or more amino acid residues; a2) protein which has more than 80% of sequence identity with the amino acid sequence of the protein limited by A1) and has the same function; wherein in A1), change is selected from at least one of substitution, deletion and addition.The lipase SALipase mutant with the catalytic activity remarkably improved is obtained by conducting site-directed mutagenesis on lipase SALipase from staphylococcus aureus through semi-rational design, and when the lipase SALipase mutant is used for preparing diglyceride, the yield of the diglyceride is remarkably improved.

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] Diglyceride oil is recognized worldwide as a green, healthy, and functional oil. Edible diester oil has positive effects, such as inhibiting fat accumulation in the body and improving blood lipid levels. Diglyceride has two isomers: 1,2- and 1,3-diglyceride. Due to the structural stability of 1,3-diglyceride, the ratio of 1,2- and 1,3-diglyceride in oils and fats is generally 3:7. Traditional diglyceride production is chemical, which typically requires very high reaction temperatures and large amounts of chemical reagents, making it environmentally unfriendly. Bio-enzyme technology for oil and fat processing is developing rapidly, gradually replacing chemical methods as the primary method of production in the oil and fat industry.

[0003] Currently, enzymatic methods for preparing diacylglycerol include the following: 1) Hydrolysis: Triacylglycerol is hydrolyzed into diacylglycerol and fatty acids by hydrolytic lipases. This hydrolysis requires the presence of water molecules. Currently, immobilized commercial lipases Novozymes 435, Lipozyme RM IM, and Lipozyme TL IM, available from Denmark, are commonly used to prepare diacylglycerol. Hydrolysis is the most direct method for preparing diacylglycerol, offering mild hydrolysis conditions and a simple process, making it feasible for industrial production. 2) Glycerolysis: Triacylglycerol and glycerol are reacted by lipases to produce diacylglycerol. Glycerolysis diacylglycerol typically appears as a byproduct of the glycerolysis process. Studies examining the effects of substrate molar ratio, reaction temperature, and water content on the glycerolysis reaction have shown that while the glycerolysis process is simple and inexpensive, high temperatures can damage the double bonds of unsaturated fatty acids, resulting in poor product quality, long reaction times, and low diacylglycerol yields. Therefore, the glycerolysis process is unsuitable for industrial production of diacylglycerol. 3) Direct esterification: This method uses a specific lipase to catalyze the esterification of fatty acids with glycerol to produce diglycerides. During the reaction, monoglycerides, triglycerides, and free fatty acids are produced as byproducts. This method produces diglycerides of relatively high purity and is easier to control. However, the immiscibility of fatty acids and glycerol limits the reaction speed of the direct esterification method. 4) Alcoholysis: Lipase-catalyzed alcoholysis of triglycerides and lower alcohols is used as the reaction substrate to produce diglycerides. Commonly used lower alcohols include methanol, ethanol, and propanol. Alcoholysis is a rapid reaction, and the product is easy to separate.

[0004] However, the lipase used in the existing enzymatic method for preparing diglyceride has the problem of poor catalytic activity. 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 of poor catalytic activity of the lipase used in the existing enzymatic method for preparing diglyceride.

[0006] Specifically, the present invention uses semi-rational design to identify the pathogens derived from Staphylococcus aureus ( Staphylococcus aureus ) was subjected to site-directed mutagenesis of the lipase SALipase (WP_113583768.1) to obtain a lipase SALipase mutant with significantly improved catalytic activity, and when diacylglycerol is prepared using the lipase SALipase mutant, the diacylglycerol yield is significantly increased.

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

[0008] The lipase SALipase provided by the present invention can be a natural, recombinant or synthetic active polypeptide. 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 SALipase is produced by introducing a recombinant vector containing its encoding gene into an expression host (e.g., E. coli BL21 (DE3)) to obtain a recombinant genetically engineered strain, and then culturing the recombinant genetically engineered strain and inducing expression to obtain the lipase SALipase.

[0010] In some embodiments, the present invention provides a nucleic acid molecule encoding the above-mentioned lipase SALipase, 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 a first aspect, the present invention provides a lipase mutant, which is selected from any one of the following: A1) a protein obtained by changing one or more amino acid residues in the amino acid sequence shown in SEQ ID NO: 1; A2) a protein that has more than 80% sequence identity with the amino acid sequence of the protein defined in A1) and has the same function; wherein, in A1), the change is selected from at least one of substitution, deletion, and addition.

[0013] In some embodiments, in A1), position 354 of the amino acid sequence shown in SEQ ID NO: 1 is mutated as follows: I354L to obtain a lipase mutant, and the lipase mutant is a protein having an amino acid sequence as shown in SEQ ID NO: 3.

[0014] In the present invention, the lipase mutant having an amino acid sequence as shown in SEQ ID NO: 3 is I354L (isoleucine (Ile) at position 354 of the lipase is mutated to leucine (Leu)).

[0015] In some embodiments, in addition to the aforementioned mutation at position 354, 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 higher 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 positions 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.

[0016] 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.

[0017] 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).

[0018] 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 E. coli BL21 (DE3)) to obtain a recombinant genetically engineered strain, and then culturing the recombinant genetically engineered strain and inducing expression to obtain the lipase mutant.

[0019] In a second aspect, the present invention provides a nucleic acid molecule encoding any one of the above-mentioned lipase mutants.

[0020] 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.

[0021] In some embodiments, the nucleic acid molecule is selected from any of the following: B1) a nucleic acid molecule having a nucleotide sequence as shown in SEQ ID NO: 4; B2) a nucleic acid molecule that hybridizes under stringent conditions with the nucleic acid molecule defined in B1) and encodes any of the above-mentioned lipase mutants; B3) a nucleic acid molecule that has a sequence identity of more than 90% with the nucleic acid molecule defined in B1) or B2) and encodes any of the above-mentioned lipase mutants.

[0022] 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.

[0023] 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.

[0024] In a third aspect, the present invention provides a recombinant vector comprising the above nucleic acid molecule.

[0025] 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 vectors used to construct the expression vectors can be at least one of pET-26b(+), pET28a, and pET23a vectors.

[0026] In a fourth aspect, the present invention provides a recombinant cell comprising the aforementioned nucleic acid molecule or the aforementioned recombinant vector.

[0027] 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.

[0028] In the present invention, the expression host cell is a conventional host cell in the art, as long as the recombinant vector can stably replicate itself and the genes it carries can be effectively expressed. It can be a prokaryotic cell or a eukaryotic cell, such as Escherichia coli, yeast, etc. Escherichia coli can be, for example, E. coli BL21 (DE3), Rosetta (DE3), BL21 (DE3) plysS. In the present invention, E. coli BL21 (DE3) is preferred as the expression host.

[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 LB 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 diglycerides.

[0033] In a seventh aspect, the present invention provides a method for preparing diglyceride, 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 hydrolysis reaction of soybean oil to obtain diglyceride.

[0034] In some embodiments, the hydrolysis reaction specifically includes: the reaction temperature is 30-50°C, for example, it can be 30°C, 35°C, 40°C, 45°C, 50°C or other values ​​within this range; the time is 8-24h, for example, it can be 8h, 12h, 16h, 20h, 24h or other values ​​within this range.

[0035] In some embodiments, soybean oil accounts for 20-50% (V / V) of the hydrolysis reaction system, for example, 20%, 25%, 30%, 35%, 40%, 45%, 50% or other values ​​within this range.

[0036] In some embodiments, the pH value of the hydrolysis reaction is 7-10, for example, 7, 7.5, 8, 8.5, 9, 9.5, 10 or other values ​​within the range.

[0037] The lipase mutants provided herein can be used in the form of whole engineered bacterial cells, unpurified crude enzymes, partially purified enzymes, or completely purified enzymes. The lipase mutants of the present invention can also be prepared into catalysts in the form of immobilized enzymes or immobilized cells using immobilization techniques known in the art.

[0038] The beneficial effects of the present invention are as follows: Different from the prior art, the present invention uses a semi-rational design to Staphylococcus aureus ) was subjected to site-directed mutagenesis of the lipase SALipase (WP_113583768.1) to obtain a lipase SALipase mutant with significantly improved catalytic activity, and when diacylglycerol is prepared using the lipase SALipase mutant, the diacylglycerol yield is significantly increased. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 This is an SDS-PAGE image of the SALipase-WT protein and the SALipase-I354L mutant protein in Example 3 of the present invention, wherein lane M is a protein marker, lanes 1-3 are the whole cells, supernatant, and precipitate of the recombinant E. coli BL21 (DE3) / pET-23a-SALipase, and lanes 4-6 are the whole cells, supernatant, and precipitate of the recombinant E. coli BL21 (DE3) / pET-23a-SALipase-I354L, respectively; Figure 2 The liquid chromatograms of the diglyceride standard in Example 4 of the present invention, wherein (A) is the liquid chromatogram of the 1,2-diglyceride standard, and (B) is the liquid chromatogram of the 1,3-diglyceride standard; Figure 3 This is a liquid chromatogram of the product obtained after the reaction of pure water and soybean oil in Example 4 of the present invention; Figure 4 This is a liquid chromatogram of the product obtained after the SALipase-WT protein and soybean oil are mixed and reacted in Example 4 of the present invention; Figure 5 This is a liquid chromatogram of the product obtained after the SALipase-I354L mutant protein and soybean oil are mixed and reacted in Example 4 of the present invention. DETAILED DESCRIPTION

[0040] 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.

[0041] 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.

[0042] Example 1: Discovery of lipase and acquisition of recombinant plasmid containing lipase First, a gene from Staphylococcus aureus ( Staphylococcus aureus The wild-type lipase SALipase (WP_113583768.1) from the wild-type strain was synthesized. The amino acid sequence is shown in SEQ ID NO: 1, and the nucleotide sequence of the gene encoding this wild-type lipase SALipase is shown in SEQ ID NO: 2. The gene fragment encoding the wild-type lipase SALipase was fully synthesized. The DNA fragment shown in SEQ ID NO: 2 was used to replace the sequence between the EcoRI and HindIII restriction sites in the pET23a vector, while the remaining sequence remained unchanged. This yielded the recombinant plasmid pET23a-SALipase. The recombinant plasmid pET23a-SALipase was sequenced, and the results were consistent with expectations.

[0043] Example 2 Obtaining a recombinant plasmid containing a lipase mutant Conventional site-directed mutagenesis was used, using the recombinant plasmid pET23a-SALipase obtained in Example 1 as a template, to design amplification primers for the lipase SALipase mutant I354L (the specific nucleotide sequence is shown in Table 1 below). After PCR amplification, the recombinant plasmid pET23a-SALipase-I354L was obtained. The mutant recombinant plasmid pET23a-SALipase-I354L was sequenced, and the results were consistent with expectations.

[0044] Table 1 Primer sequences

[0045] Example 3 Expression of lipase and its mutants The recombinant plasmid pET23a-SALipase prepared in Example 1 and the recombinant plasmid pET23a-SALipase-I354L prepared in Example 2 were respectively transformed into E. coli BL21 (DE3) competent cells, and the transformants were spread on LB solid medium containing 50 μg / mL ampicillin for screening to obtain recombinant bacteria E. coli BL21 (DE3) / pET-23a-SALipase and E. coli BL21 (DE3) / pET-23a-SALipase-I354L expressing wild-type lipase and its mutants.

[0046] The recombinant E. coli BL21 (DE3) / pET-23a-SALipase and E. coli BL21 (DE3) / pET-23a-SALipase-I354L were inoculated into 100 mL LB liquid medium containing 50 μg / mL ampicillin, and cultured at 37 ° C and 220 rpm until the OD 600 The cell suspension was centrifuged at 6000 rpm for 10 minutes, and the cell pellet was collected. The cell pellet was resuspended in 10 mM Tris-HCl buffer with a pH of 8.0 and then disrupted with a high-pressure sterilizer. 1% PMSF was added to the resuspended cell pellet before disruption. The cell pellet was centrifuged at 12000 rpm for 30 minutes to obtain the supernatant. The results were analyzed by SDS-PAGE. Figure 1 shown.

[0047] from Figure 1 It can be seen that the molecular weights of the SALipase-WT protein and SALipase-I354L mutant protein bands are consistent with the predicted molecular weights, and the expression level of the SALipase-I354L mutant protein is significantly higher than that of the SALipase-WT protein. The results show that the wild-type lipase and its mutants are successfully expressed.

[0048] Example 4 Preparation of diglyceride catalyzed by lipase and its mutants 30 mL of the supernatant containing the SALipase-WT protein and the SALipase-I354L mutant protein prepared in Example 2 were mixed with 20 mL of soybean oil, respectively, and the pH value of the solution was adjusted to 8. The reaction was carried out at a temperature of 30° C. and a rotation speed of 200 rpm for 8 h. The above protein supernatant was replaced with an equal amount of pure water as a blank control. After the reaction was completed, the mixture was allowed to stand or centrifuged at 1000 rpm. The upper layer of oil was taken and dissolved in isopropanol and acetonitrile (isopropanol:acetonitrile = 1:1). After filtration, the diacylglycerol content was detected by high performance liquid chromatography. The liquid chromatograms were as shown below. Figure 2-5 shown.

[0049] Among them, a Thermo Fisher U3000 high performance liquid chromatograph was used for detection, and the chromatographic conditions were as follows: C18 chromatographic column (4.6×150 mm); mobile phase was acetonitrile and isopropanol; flow rate was 0.8 mL / min; UV detector, wavelength was 275 nm; column temperature was 30°C; all samples were filtered through a 0.22 μm filter membrane, and the sample volume was 10 μL.

[0050] from Figure 2-5 It can be seen that both SALipase-WT protein and SALipase-I354L mutant protein can catalyze the reaction using soybean oil as substrate to produce diacylglycerol. Furthermore, calculations found that the conversion rate of soybean oil by SALipase-I354L mutant protein (48.9%) was significantly higher than that of SALipase-WT protein (35%). In addition, compared with SALipase-WT protein, SALipase-I354L mutant protein can catalyze the production of diacylglycerol (1,3-diacylglycerol and 1,2-diacylglycerol) with significantly increased content.

[0051] In summary, the present invention uses semi-rational design to Staphylococcus aureus ) was subjected to site-directed mutagenesis of the lipase SALipase (WP_113583768.1) to obtain a lipase SALipase mutant with significantly improved catalytic activity, and when diacylglycerol is prepared using the lipase SALipase mutant, the diacylglycerol yield is significantly increased.

[0052] 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.

[0053] Amino acid sequence of wild-type lipase SALipase (SEQ ID NO: 1) MANQVQPLNKYPVVFVHGFLGLVGDNAPALYPNYWGGNKFKVIEELRKQGYNVHQASVSAFGSNYDRAVELYYYIKGGRVDYGAAHAAKYGHERYGKTYKGIMPNWEPGKKVHLVGHSMGGQTIRLMEEFLRNGNKEEIAYHKAHGGEISPLFTGGHNNMVASITTLATPHNGSQAADKFGNTEAVRKIMFALNRFMG NKYSNIDLGLTQWGFKQLPNESYIDYIKRVSKSKIWTSDDNAAYDLTLDGSAKLNNMTSMNPNITYTTYTGVSSHTGPLGYENPDLGTFFLMDTTSRIIGHDAREEWRKNDGVVPVISSLHPSNQPFVNVTNDEPATRRGIWQVKPIIQGWDHVDFIGVDFLDFKRKGAELANFYTGIINDLLRVEATESKGTQLKAS Nucleotide sequence of the gene encoding wild-type lipase SALipase (SEQ ID NO: 2) Amino acid sequence of lipase SALipase mutant I354L (SEQ ID NO: 3) MANQVQPLNKYPVVFVHGFLGLVGDNAPALYPNYWGGNKFKVIEELRKQGYNVHQASVSAFGSNYDRAVELYYYIKGGRVDYGAAHAAKYGHERYGKTYKGIMPNWEPGKKVHLVGHSMGGQTIRLMEEFLRNGNKEEIAYHKAHGGEISPLFTGGHNNMVASITTLATPHNGSQAADKFGNTEAVRKIMFALNRFMG NKYSNIDLGLTQWGFKQLPNESYIDYIKRVSKSKIWTSDDNAAYDLTLDGSAKLNNMTSMNPNITYTTYTGVSSHTGPLGYENPDLGTFFLMDTTSRIIGHDAREEWRKNDGVVPVISSLHPSNQPFVNVTNDEPATRRGIWQVKPIIQGWDHVDFLGVDFLDFKRKGAELANFYTGIINDLLRVEATESKGTQLKAS Nucleotide sequence of the gene encoding lipase SALipase mutant I354L (SEQ ID NO: 4)

Claims

1. A lipase mutant, characterized in that: The lipase mutant is selected from any one of the following: A1) a protein obtained by changing one or more amino acid residues in the amino acid sequence shown in SEQ ID NO: 1; A2) a protein that has an amino acid sequence identity of more than 80% with the protein defined in A1) and has the same function; Wherein, in A1), the change is selected from at least one of substitution, deletion and addition.

2. The lipase mutant according to claim 1, characterized in that A1), the 354th position of the amino acid sequence as shown in SEQ ID NO: 1 is mutated as follows: I354L, to obtain the lipase mutant, and the lipase mutant is a protein with an amino acid sequence as shown in SEQ ID NO:

3.

3. A nucleic acid molecule encoding the lipase mutant according to any one of claims 1 to 2.

4. The nucleic acid molecule according to claim 3, characterized in that The nucleic acid molecule is selected from any one of the following: B1) a nucleic acid molecule having a nucleotide sequence as shown in SEQ ID NO: 4; B2) a nucleic acid molecule that hybridizes with the nucleic acid molecule defined in B1) and encodes the lipase mutant according to any one of claims 1 to 2; B3) A nucleic acid molecule having a sequence identity of 90% or more with the nucleic acid molecule defined in B1) or B2) and encoding the lipase mutant according to any one of claims 1 to 2.

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

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

7. A method for preparing a lipase mutant, characterized in that: The steps include: Cultivating the recombinant cell according to claim 6, inducing expression, and obtaining a culture; The lipase mutant according to any one of claims 1 to 2 is isolated from the culture.

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

9. A method for preparing diglyceride, characterized in that: The steps include: The lipase mutant according to any one of claims 1 to 2, the recombinant cell according to claim 6 or the lipase mutant prepared by the preparation method according to claim 7 is used as a catalyst to catalyze the hydrolysis reaction of soybean oil to obtain diglyceride.

10. The method for preparing diglyceride according to claim 9, characterized in that: The hydrolysis reaction specifically includes: the reaction temperature is 30-50° C. and the reaction time is 8-24 hours.

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