Lipase A mutant as well as preparation method and application thereof

Abstract

The invention discloses a lipase A mutant as well as a preparation method and application thereof, and belongs to the technical field of biology. The lipase A 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 94th site, the 117th site, the 121st site, the 122nd site, the 283rd site and the 431st site; wherein the change is selected from at least one of substitution, deletion and addition. Through rational design, lipase A from Candida antarctica is subjected to site-directed mutagenesis, the Candida antarctica lipase A mutant with catalytic activity and selectivity to the sn-2 site of triglyceride remarkably improved is obtained, and when 1, 3-diglyceride is prepared, the Candida antarctica lipase A mutant can remarkably improve the yield of 1, 3-diglyceride. The yield of the 1, 3-diglyceride is improved.

Description

Technical Field

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

[0002] Candida antarctica lipase A ( Candida antarctica Lipase A (CALA) has a unique position in the field of lipase research and has attracted much attention due to its obvious preference for the sn-2 site of triglycerides. This special selectivity makes it have important application value in biocatalytic processes. Compared with other types of lipases, CALA has the following significant advantages: (1) Temperature stability: CALA can still maintain its activity under high temperature conditions, which makes it more stable in industrial applications; (2) Substrate specificity: CALA can effectively hydrolyze triglycerides, especially trans-unsaturated fatty acids, which has great prospects in the food industry and biofuel production; (3) Chemical selectivity: CALA has a high degree of chemical selectivity for amino groups, which makes it play a key role in the asymmetric synthesis of amino acids and their derivatives. In addition to the above advantages, CALA also exhibits similarity to Antarctic Candida lipase B ( Candida antarctica The results suggest that CALA has similar properties to lipase B (CALB), such as high temperature stability and tolerance to organic solvents. However, CALA is more outstanding in terms of interfacial activity, which provides it with greater potential for industrial applications.

[0003] Diacylglycerol (DAG) is an important lipid molecule, which is formed by the esterification reaction between glycerol and two free fatty acid molecules, or by the replacement of one fatty acid molecule in triacylglycerol (TAG) with a hydroxyl group. DAG exists in two different stereochemical configurations: 1,2-diacylglycerol and 1,3-diacylglycerol. Recent studies have found that compared with 1,2-diacylglycerol, edible oils rich in 1,3-diacylglycerol have attracted attention due to their unique physiological activities and health benefits. As a new type of functional oil, 1,3-diacylglycerol has the following significant health benefits: (1) prevention and treatment of fatty liver; (2) reduction of the risk of cardiovascular and cerebrovascular diseases; (3) control of body weight and prevention of obesity. These characteristics of 1,3-diacylglycerol make it have a wide range of application potentials in the food industry and health fields. For example, 1,3-diacylglycerol can be used as an ingredient in healthy foods to develop low-calorie, high-nutrition value food products. In addition, 1,3-diacylglycerol also has potential applications in the medical field and may be used to develop drugs to treat related diseases.

[0004] However, the existing wild-type Candida lipase A has problems such as poor catalytic activity and poor selectivity for the sn-2 site of triglycerides when preparing 1,3-diglyceride. Summary of the invention

[0005] The purpose of the present invention is to provide a lipase A mutant and a preparation method and application thereof, so as to solve the problems that the existing wild-type Candida lipase A has poor catalytic activity and poor selectivity for the sn-2 site of triglycerides when preparing 1,3-diglyceride.

[0006] Specifically, the present invention is based on rational design to Candida antarctica ) were subjected to site-directed mutagenesis to obtain a Candida antarctica lipase A (CALA) mutant with significantly improved catalytic activity and selectivity for the sn-2 site of triglycerides, and when preparing 1,3-diacylglycerol, the CALA mutant can significantly increase the yield of 1,3-diacylglycerol.

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

[0008] The lipase A 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 A is obtained by introducing a recombinant vector containing its encoding gene into an expression host (such as 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 A.

[0010] In some embodiments, the present invention provides a nucleic acid molecule encoding the above-mentioned lipase A, 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 A 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: 94, 117, 121, 122, 283, and 431; wherein the change is selected from at least one of substitution, deletion, and addition.

[0013] In some embodiments, the lipase A 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) E94W; 2) V117L; 3) L121I; 4) D122Y; 5) F283W; 6) F431G.

[0014] In some embodiments, the lipase A mutant is a protein with an amino acid sequence as shown in SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, or SEQ ID NO: 13.

[0015] In the present invention, the lipase A mutant whose amino acid sequence is shown in SEQ ID NO: 3 is E94W (glutamic acid (Glu) at position 94 of lipase A is mutated to tryptophan (Trp)); the lipase A mutant whose amino acid sequence is shown in SEQ ID NO: 5 is V117L (valine (Val) at position 117 of lipase A is mutated to leucine (Leu)); the lipase A mutant whose amino acid sequence is shown in SEQ ID NO: 7 is L121I (leucine (Leu) at position 121 of lipase A is mutated to isoleucine (Ile)); the lipase A mutant whose amino acid sequence is shown in SEQ ID NO: 9 is D122Y (aspartic acid (Asp) at position 122 of lipase A is mutated to tyrosine (Tyr)); the lipase A mutant whose amino acid sequence is shown in SEQ ID NO: 11 is F283W (phenylalanine (Phe) at position 283 of lipase A is mutated to tryptophan (Trp)); the The lipase A mutant shown in 13 is F431G (phenylalanine (Phe) at position 431 of lipase A is mutated to glycine (Gly)).

[0016] In some embodiments, in the above-mentioned lipase A mutant, in addition to the above-mentioned mutation occurring at at least one of positions 94, 117, 121, 122, 283, and 431, it may further have conservative substitutions of amino acids at other positions, so that the mutated amino acids, such as the wild-type lipase A 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 A 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 A mutant having a certain amino acid homology with the amino acid sequence of the above-mentioned lipase A 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 A 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 A 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 genetic engineering strain, and then culturing the recombinant genetic engineering strain and inducing expression to obtain the lipase A mutant.

[0020] In a second aspect, the present invention provides a nucleic acid molecule encoding any one of the above-mentioned lipase A 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, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, or SEQ ID NO: 14; 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 A 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 A 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 A mutant, comprising the following steps: culturing the above-mentioned recombinant cells, inducing expression, and obtaining a culture; and isolating the above-mentioned lipase A 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-mentioned lipase A mutant 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 A 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 A mutant, the nucleic acid molecule, the recombinant vector, the recombinant cell or the lipase A 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 A mutant, the above-mentioned recombinant cell or the lipase A mutant prepared by the above-mentioned preparation method as a catalyst to catalyze the reaction of soybean oil to obtain 1,3-diacylglycerol.

[0034] The beneficial effects of the present invention are as follows: Different from the prior art, the present invention uses rational design to treat Candida australis ( Candida antarctica ) were subjected to site-directed mutagenesis to obtain a Candida antarctica lipase A (CALA) mutant with significantly improved catalytic activity and selectivity for the sn-2 site of triglycerides, and when preparing 1,3-diacylglycerol, the CALA mutant can significantly increase the yield of 1,3-diacylglycerol. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 It is an SDS-PAGE image of the CALA-WT protein and the CALA-E94W, CALA-V117L, CALA-L121I, CALA-D122Y, CALA-F283W, and CALA-F431G mutant proteins 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 It is a diagram showing the enzyme activity results of the CALA-WT protein and the CALA-E94W, CALA-V117L, CALA-L121I, CALA-D122Y, CALA-F283W, and CALA-F431G mutant proteins in Example 4 of the present invention; Figure 4 This is a graph showing the results of thin layer chromatography analysis of the CALA-WT protein and the CALA-V117L mutant protein in Example 5 of the present invention after reacting with soybean oil as a substrate, wherein MAG: monoglyceride; TAG: triglyceride; FA: fatty acid; 1,2-DAG: 1,2-diaceryl; 1,3-DAG: 1,3-diaceryl. DETAILED DESCRIPTION

[0036] 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.

[0037] 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.

[0038] Example 1 Obtaining a recombinant plasmid containing lipase A The whole gene synthesis was derived from wild-type Candida antarctica ( Candida antarctica ) of the gene fragment of lipase A (CALA, whose nucleotide sequence is shown in SEQ ID NO: 2). The DNA fragment shown in SEQ ID NO: 2 was seamlessly cloned by T5 nuclease method to replace the sequence between CpoⅠ and NotⅠ restriction sites of pHBM905BDM vector, and the other sequences remained unchanged to obtain the recombinant plasmid pHBM905BDM-CALA. The recombinant plasmid pHBM905BDM-CALA was sent for sequencing, and the results were consistent with expectations.

[0039] Example 2 Obtaining a recombinant plasmid containing a lipase A mutant Conventional site-directed mutagenesis technology was used, and the recombinant plasmid pHBM905BDM-CALA obtained in Example 1 was used as a template to design amplification primers for mutants E94W, V117L, L121I, D122Y, F283W, and F431G (the specific nucleotide sequences are shown in Table 1 below), respectively. After PCR amplification, recombinant plasmids pHBM905BDM-CALA-E94W, recombinant plasmid pHBM905BDM-CALA-V117L, recombinant plasmid pHBM905BDM-CALA-L121I, recombinant plasmid pHBM905BDM-CALA-D122Y, recombinant plasmid pHBM905BDM-CALA-F283W, and recombinant plasmid pHBM905BDM-CALA-F431G were obtained, respectively. The above mutant recombinant plasmids were sent for sequencing, and the results were consistent with expectations.

[0040] Table 1 Primer sequences

[0041] Example 3 Expression and purification of lipase A and its mutants The recombinant plasmid pHBM905BDM-CALA prepared in Example 1 and the recombinant plasmids pHBM905BDM-CALA-E94W, recombinant plasmid pHBM905BDM-CALA-V117L, recombinant plasmid pHBM905BDM-CALA-L121I, recombinant plasmid pHBM905BDM-CALA-D122Y, recombinant plasmid pHBM905BDM-CALA-F283W and recombinant plasmid pHBM905BDM-CALA-F431G prepared in Example 2 were respectively digested with restriction endonuclease SalⅠ, and the solution was recovered to obtain linearized fragments, which were then electrotransformed into Pichia pastoris GS115 competent cells respectively; the transformation solution was spread on an MD plate and cultured at 30°C until a single colony grew, and positive clones were screened to obtain the target strain.

[0042] The target strains obtained above were cultured in shake flasks, inoculated into 50 mL of BMGY medium, and cultured in a constant temperature shaker at 30°C and 220 rpm until OD 600 =2-6 (about 16-18h), transfer the fermentation broth to a sterile 50mL centrifuge tube, centrifuge at 3000g for 5min, remove the supernatant, resuspend the bacteria with 25mL BMMY medium and pour it back into a 250mL Erlenmeyer flask, continue to culture at 30℃, 220rpm constant temperature shaker, add 1% methanol for induction every 24h, and the optimal induction time is 4-5d. Centrifuge at 3000g for 5min, and the supernatant is the crude enzyme solution of CALA-WT and the crude enzyme solution of mutants.

[0043] Add 2 mL of Ni-NTA agarose resin to the gravity protein purification column, and rinse three times with 5 mL of purification buffer (50 mM Tris-HCl, pH 8.5, 50 mM NaCl) containing a final concentration of 10 mM imidazole to activate the Ni-NTA agarose resin; filter the crude enzyme solution with a 0.45 µm filter membrane, add it to the purification column, seal it, and incubate it in a silent mixer at 4°C for 1 h; collect the flow-through, rinse the Ni-NTA agarose resin with 10 mL of purification buffer containing final concentrations of 10 mM, 20 mM, 50 mM, 100 mM, and 200 mM imidazole, respectively, and collect the eluate each time. The eluate is detected by G250, and each gradient is eluted until there is no color change on G250. The eluate is subjected to SDS-PAGE detection, and the component with a relatively single target protein band is concentrated using a 30 kDa ultrafiltration tube, and the storage buffer (50 mM Replace the solution with Tris-HCl, pH 8.5, 50 mM NaCl, 1 mM EDTA, 5% Glycerol) and concentrate to 2 mL.

[0044] The protein obtained above was subjected to SDS-PAGE detection. The results were as follows Figure 1 As shown. Figure 1 It can be seen that CALA-WT protein and CALA mutant proteins (CALA-E94W, CALA-V117L, CALA-L121I, CALA-D122Y, CALA-F283W, CALA-F431G mutant proteins) have protein bands of consistent size.

[0045] Example 4 Determination of enzyme activity of lipase A and its mutants The enzymatic activity of lipase A and its mutants was tested by spectrophotometry. Specifically, a standard curve was first established with p-nitrophenol standard, as follows: Pipette 710μL of Tris-HCl buffer (50mM, pH 8.5) into a 1.5mL EP tube, preheat in a 40℃ water bath for 5min, then add 90μL of p-nitrophenol standard solution of different concentrations, react in a 40℃ water bath for 10min, and add 200μL of 10% 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 enzyme 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.

[0046] from Figure 2 It can be seen that the linear regression equation of the standard curve is y=0.0093x+0.05, and the correlation coefficient R 2 =0.9992.

[0047] Then, the enzyme activity of lipase A and its mutants was tested. Specifically, 700 μL of Tris-HCl buffer (50 mM, pH 8.5) was aspirated into a 1.5 mL EP tube, and 10 μL of enzyme solution (CALA-WT protein and CALA mutant protein) was added respectively and preheated in a 40°C water bath for 5 min, and then 90 μL of p-nitrophenol palmitate solution (20 mM, solvent is isopropanol) was added, and the reaction was carried out in a 40°C water bath for 10 min. 200 μL of 10% SDS (sodium dodecyl sulfate) solution was added to terminate the reaction. 200 μL of the reaction solution was placed in a 96-well plate, and the absorbance at 405 nm was measured using a microplate reader. The concentration of p-nitrophenol, the hydrolysis product of CALA-WT protein and CALA mutant protein, was calculated based on the standard curve, thereby obtaining the enzyme activity of CALA-WT protein and CALA mutant protein (CALA-E94W, CALA-V117L, CALA-L121I, CALA-D122Y, CALA-F283W, CALA-F431G mutant protein). The results are shown in Figure 3 shown.

[0048] 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.

[0049] from Figure 3 It can be seen that the CALA-E94W, CALA-V117L, CALA-L121I, CALA-D122Y, CALA-F283W, and CALA-F431G mutant proteins have higher enzyme activity than the CALA-WT protein. The results show that in the present invention, by performing site-directed mutagenesis on the wild-type CALA-WT protein, mutant proteins with significantly improved enzyme activity can be obtained.

[0050] Example 5 Preparation of 1,3-diacerol catalyzed by lipase A and its mutants Soybean oil and Tris-HCl buffer (50 mM, pH 8.5) were mixed in a volume ratio of 3:2 to a total volume of 50 mL. The mixture was then transferred to a 150 mL conical flask, and CALA-WT protein and CALA-V117L mutant protein accounting for 2% of the total mass of soybean oil were added respectively; the mixture was reacted for 12 h under the conditions of a constant temperature water bath shaker at 40 ° C and 220 r / min; after the reaction, the aqueous phase and the oil phase were separated by centrifugation at 12000 rpm for 5 min, and 2 μL of the upper oil phase was taken. The proportion of 1,3-diacylglycerol in the oil phase was detected by thin layer chromatography. The results are as follows: Figure 4 shown.

[0051] from Figure 4 It can be seen that compared with the wild-type CALA-WT protein, the CALA-V117L mutant protein can improve the selectivity for the sn-2 site of triglycerides, thereby increasing the production of 1,3-diacylglycerol.

[0052] In summary, the present invention is to Candida antarctica ) were subjected to site-directed mutagenesis to obtain a Candida antarctica lipase A (CALA) mutant with significantly improved catalytic activity and selectivity for the sn-2 site of triglycerides, and when preparing 1,3-diacylglycerol, the CALA mutant can significantly increase the yield of 1,3-diacylglycerol.

[0053] It should be noted that the above embodiments all belong to the same inventive concept, and the description of each embodiment has its own focus. For matters that are not described in detail in some embodiments, reference may be made to the description in other embodiments.

[0054] The above-mentioned embodiments only express the implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations 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 subject to the attached claims.

Claims

1. A lipase A mutant, characterized in that: The lipase A mutant is a protein obtained by changing one or more amino acid residues at at least one of the following positions: 94, 117, 121, 122, 283, 431 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 A mutant according to claim 1, characterized in that The lipase A mutant is a mutation in at least one of the following positions of the amino acid sequence shown in SEQ ID NO: 1: 1) E94W; 2) V117L; 3) L121I; 4) D122Y; 5) F283W; 6) F431G.

3. The lipase A mutant according to claim 2, characterized in that The lipase A mutant is a protein with an amino acid sequence as shown in SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, and SEQ ID NO:

13.

4. A nucleic acid molecule encoding the lipase A 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, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, or SEQ ID NO: 14; A2) a nucleic acid molecule that hybridizes with the nucleic acid molecule defined in A1) and encodes the lipase A mutant according to any one of claims 1 to 3; A3) A nucleic acid molecule having a sequence identity of more than 90% with the nucleic acid molecule defined in A1) or A2) and encoding the lipase A 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 A mutant, characterized in that: The steps include: Cultivating the recombinant cell according to claim 7, inducing expression, and obtaining a culture; The lipase A mutant according to any one of claims 1 to 3 is isolated from the culture.

9. Use of the lipase A 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 A 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 A mutant according to any one of claims 1 to 3, the recombinant cell according to claim 7 or the lipase A mutant prepared by the preparation method according to claim 8 is used as a catalyst to catalyze the reaction of soybean oil to obtain 1,3-diacerol.

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