Lipase a mutant, its preparation method and application

Abstract

The application discloses a lipase A mutant and a preparation method and application thereof, and belongs to the technical field of biology. The lipase A mutant is a protein obtained by changing one or more amino acid residues at at least one of the following positions: 94th, 117th, 121st, 122nd, 283rd and 431st in 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 mutation on the lipase A derived from Candida antarctica by rational design, obtains a Candida antarctica lipase A mutant with significantly improved catalytic activity and selectivity to the sn-2 site of triglyceride, and the Candida antarctica lipase A mutant can significantly improve the yield of 1,3-glycerol diester when preparing the 1,3-glycerol diester.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a lipase A mutant, its preparation method, and its application. Background Technology

[0002] Antarctic Candida lipase A ( Candida antarctica Lipase A (CALA) holds a unique position in the field of lipase research, attracting significant attention due to its distinct preference for the sn-2 site of triglycerides. This special selectivity makes it valuable for applications in biocatalysis. Compared with other types of lipases, CALA has several significant advantages: (1) Temperature stability: CALA maintains its activity under high temperature conditions, making it more stable in industrial applications; (2) Substrate specificity: CALA can effectively hydrolyze triglycerides, especially trans-unsaturated fatty acids, which has great potential in the food industry and biofuel production; (3) Chemoselectivity: CALA has high chemoselectivity for amino groups, enabling it to 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 Candida antarcticis lipase B (Candida antarcticis lipase B). Candida antarctica CALA exhibits similar properties to lipase B (CALB), such as high-temperature stability and resistance to organic solvents. However, CALA demonstrates superior interfacial activity, offering greater potential for industrial applications.

[0003] Diacylglycerol (DAG) is an important lipid molecule formed by the esterification of glycerol with two free fatty acid molecules, or by the substitution of a fatty acid molecule in triacylglycerol (TAG) with a hydroxyl group. DAG exists in two different stereoconfigurations: 1,2-diacylglycerol and 1,3-diacylglycerol. Recent studies have found that edible oils rich in 1,3-diacylglycerol have attracted attention due to their unique physiological activities and health benefits compared to 1,2-diacylglycerol. As a novel 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) weight control and prevention of obesity. These properties of 1,3-diacylglycerol make it a promising candidate for applications in the food industry and health sector. For example, 1,3-diacylglycerol can be used as an ingredient in health foods to develop low-calorie, high-nutritional-value food products. In addition, 1,3-glycerol also has potential applications in the pharmaceutical 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-diglycerides. Summary of the Invention

[0005] The purpose of this invention is to provide a lipase A mutant, its preparation method, and its applications. This addresses the problems of poor catalytic activity and poor selectivity for the sn-2 site of triglycerides in the preparation of 1,3-diglycerides by existing wild-type Candida lipase A.

[0006] Specifically, this invention, through rational design, targets Candida albicans derived from Antarctica (… Candida antarctica Site-directed mutagenesis was performed on lipase A in Candida antarctica to obtain a CALA mutant with significantly enhanced catalytic activity and selectivity for the sn-2 site of triglycerides. Furthermore, the CALA mutant significantly increased the yield of 1,3-diglycerides during the preparation of 1,3-diglycerides.

[0007] In some embodiments, the present invention provides a lipase A, the amino acid sequence of which 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. The active polypeptide can be a naturally purified product, a chemically synthesized product, or a product produced from a prokaryotic host (e.g., Escherichia coli) or a eukaryotic host (e.g., 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 (e.g., Pichia pastoris GS115) to obtain a recombinant genetically engineered strain, then culturing the recombinant genetically engineered strain and inducing expression to obtain lipase A.

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

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

[0012] In a 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 in the amino acid sequence shown in SEQ ID NO: 1: position 94, position 117, position 121, position 122, position 283, and position 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 at at least one of the following positions of the amino acid sequence set forth 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 having an amino acid sequence set forth 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 application, the lipase A mutant having an amino acid sequence set forth in SEQ ID NO: 3 is E94W (glutamic acid (Glu) at position 94 of the lipase A is mutated to tryptophan (Trp)); the lipase A mutant having an amino acid sequence set forth in SEQ ID NO: 5 is V117L (valine (Val) at position 117 of the lipase A is mutated to leucine (Leu)); the lipase A mutant having an amino acid sequence set forth in SEQ ID NO: 7 is L121I (leucine (Leu) at position 121 of the lipase A is mutated to isoleucine (lie)); the lipase A mutant having an amino acid sequence set forth in SEQ ID NO: 9 is D122Y (aspartic acid (Asp) at position 122 of the lipase A is mutated to tyrosine (Tyr)); the lipase A mutant having an amino acid sequence set forth in SEQ ID NO: 11 is F283W (phenylalanine (Phe) at position 283 of the lipase A is mutated to tryptophan (Trp)); and the lipase A mutant having an amino acid sequence set forth in SEQ ID NO: 13 is F431G (phenylalanine (Phe) at position 431 of the lipase A is mutated to glycine (Gly)).

[0016] In some embodiments, in addition to the mutations at at least one of positions 94, 117, 121, 122, 283, 431, the above-described lipase A mutants can further have conservative substitutions of amino acids at other positions, such that the mutated amino acids of the wild-type lipase A as shown in SEQ ID NO: 1 have higher catalytic efficiency. Preferably, the conservative substitutions of amino acids retain the higher catalytic efficiency of the lipase A mutants of the present application. It will be apparent to those skilled in the art that such substitutions can occur in regions other than those described above, while retaining the corresponding activity. Preferably, the conservatively substituted variants have conservative substitutions of amino acids at at least one position. Examples of conservative substitutions are substitutions within groups of amino acids that include the following: acidic amino acids (e.g., aspartic acid and glutamic acid); basic amino acids (e.g., lysine, arginine, and histidine); polar amino acids (e.g., glutamine and asparagine); hydrophobic amino acids (e.g., leucine, isoleucine, valine, alanine, and methionine); 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 exchanges are the following: amino acids 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 inverse exchanges.

[0017] Lipase A mutants having a certain amino acid homology to the amino acid sequence of the above-described lipase A mutants, preferably a homology of between 70 and 99%, such as, for example, 70%, 72%, 74%, 76%, 78%, 80%, 82%, 84%, 86%, 88%, 90%, 92%, 94%, 96%, 98%, 99%, or a value or a range between any two of these values; more preferably a homology of between 80 and 99%, such as, for example, 80%, 82%, 84%, 86%, 88%, 90%, 92%, 94%, 96%, 98%, 99%, or a value or a range between any two of these values; even more preferably a homology of between 90 and 99%, such as, for example, 90%, 92%, 94%, 96%, 98%, 99%, or a value or a range between any two of these values; and most preferably a homology of 99%, also belong to the scope of the present application.

[0018] The lipase A mutant provided by the present application can be a natural, recombinant or synthetic active polypeptide, which can be a natural purified product, a chemically synthesized product, or a product produced using a recombinant technique from a prokaryotic host (e.g., Escherichia coli) or a eukaryotic host (e.g., yeast, higher plants).

[0019] In some embodiments, the lipase A mutant is obtained by introducing a recombinant vector containing a gene encoding the same into an expression host (e.g., Pichia pastoris GS115), culturing the recombinant genetically engineered strain, and inducing expression to obtain the lipase A mutant.

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

[0021] The nucleic acid molecule provided by the present application can be DNA, such as cDNA, genomic DNA or recombinant DNA; or RNA, such as mRNA or hnRNA, etc.; and the nucleic acid molecule can generally 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 set forth in SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14; A2) a nucleic acid molecule that hybridizes under stringent conditions to the nucleic acid molecule defined in A1) and encodes any of the above-mentioned lipase A mutants; A3) a nucleic acid molecule that has a sequence identity of 90% or more to 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 "hybridizes under stringent conditions" means that two nucleic acid molecule fragments hybridize to each other under standard hybridization conditions as described in the section "Expression of cloned genes in E. coli" of Sambrook et al., Molecular Cloning: A Laboratory Manual (1989) (Cold Spring Harbor Laboratory Press, New York, USA). Such conditions are, for example, hybridization in 6.0x SSC at 45°C, followed by a washing step in 2x SSC at 50°C. To select the stringency, the salt concentration in the washing step can be chosen, for example, between 2.0x SSC at 50°C for low stringency and 2.0x SSC at 50°C for high stringency. In addition, the temperature in the washing step can vary 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 programs described in Ausubel et al. eds. (2007) in Current Protocols in Molecular Biology. When a position in compared sequences is occupied by the same base or amino acid, then the molecules are identical at that position. A degree of identity between two or more sequences can be expressed as a percentage (%) which can be used to evaluate the identity between related sequences. A polynucleotide sequence or an amino acid sequence has a certain percentage (e.g., 90%, 95%, 98%, or 99%) of "sequence identity" to another sequence if the percentage of bases or amino acids in the two sequences that are identical when the sequences are aligned.

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

[0026] The recombinant vector in the present application includes a cloning vector for replicating the relevant sequence and an expression vector for expressing the relevant gene, wherein the vector used in constructing the expression vector can be a pHBM905BDM vector.

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

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

[0029] In a fifth aspect, the present application provides a method for preparing a lipase A mutant, comprising the steps of: culturing the above-mentioned recombinant cell, inducing expression to obtain a culture; and isolating the above-mentioned lipase A mutant from the culture.

[0030] In the present application, the culture method and culture conditions are not particularly required, as long as the normal growth of the recombinant cell is ensured. Moreover, the method for isolating the above-mentioned lipase A mutant from the culture is a conventional method in the art.

[0031] In some embodiments, the culture medium used in the method for preparing the lipase A mutant is a culture medium that can express proteins in the art, preferably a BMMY culture medium.

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

[0033] In a seventh aspect, the present application provides a method for preparing 1,3-diglyceride, comprising the following step: using the lipase A mutant prepared by the above-mentioned method, the above-mentioned recombinant cell or the above-mentioned method as a catalyst to catalyze soybean oil to obtain 1,3-diglyceride.

[0034] The present application has the following beneficial effects: different from the prior art, the present application performs site-directed mutation on the lipase A derived from Candida antarctica by rational design, obtains a Candida antarctica lipase A (CALA) mutant with significantly improved catalytic activity and selectivity for the sn-2 site of triglyceride, and the CALA mutant can significantly improve the yield of 1,3-diglyceride in the preparation of 1,3-diglyceride. Candida antarctica BRIEF DESCRIPTION OF DRAWINGS

[0035] Figure 1 FIG. 1 is an SDS-PAGE diagram of the CALA-WT protein and the CALA-E94W, CALA-V117L, CALA-L121I, CALA-D122Y, CALA-F283W and CALA-F431G mutant protein in Example 3 of the present application;

[0036] Figure 2 FIG. 4 is a standard curve diagram of p-nitrophenol established in Example 4 of the present application;

[0037] Figure 3 FIG. 5 is an enzyme activity result diagram of the CALA-WT protein and the CALA-E94W, CALA-V117L, CALA-L121I, CALA-D122Y, CALA-F283W and CALA-F431G mutant protein in Example 4 of the present application;

[0038] Figure 4 FIG. 6 is a thin layer chromatography analysis result diagram of the CALA-WT protein and the CALA-V117L mutant protein after reacting with soybean oil as a substrate in Example 5 of the present application, wherein MAG represents monoglyceride; TAG represents triglyceride; FA represents fatty acid; 1,2-DAG represents 1,2-diglyceride; and 1,3-DAG represents 1,3-diglyceride. DETAILED DESCRIPTION

[0039] The technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the embodiments of the present application. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments of the present application. Based on the embodiments in the present application, all other embodiments obtained by a person of ordinary skill in the art without creative labor fall within the scope of protection of the present application.

[0040] ​Experimental methods not specifically described in the examples are generally performed according to conventional experimental methods in the field of molecular biology, including but not limited to those described in *Molecular Cloning: A Laboratory Manual* by M.R. Green and *Molecular Biology* by Robert F. Weaver, or according to the experimental methods recommended by the reagent kit and instrument manufacturers. Unless otherwise specified, all reagents and biological materials used in the examples are commercially available.

[0041] Example 1: Obtaining a recombinant plasmid containing lipase A

[0042] The entire genome was synthesized from wild-type Candida antarctica ( Candida antarctica The gene fragment of lipase A (CALA, whose nucleotide sequence is shown in SEQ ID NO: 2) was cloned seamlessly using the T5 exonuclease method. The sequence between the CpoⅠ and NotⅠ restriction sites of the pHBM905BDM vector was replaced, while the rest of the sequence remained unchanged, resulting in the recombinant plasmid pHBM905BDM-CALA. The recombinant plasmid pHBM905BDM-CALA was sent for sequencing, and the results were consistent with expectations.

[0043] Example 2: Obtaining a recombinant plasmid containing a lipase A mutant

[0044] Using conventional site-directed mutagenesis techniques, and with the recombinant plasmid pHBM905BDM-CALA obtained in Example 1 as a template, amplification primers for mutants E94W, V117L, L121I, D122Y, F283W, and F431G were designed (specific nucleotide sequences are shown in Table 1 below). After PCR amplification, recombinant plasmids pHBM905BDM-CALA-E94W, pHBM905BDM-CALA-V117L, pHBM905BDM-CALA-L121I, pHBM905BDM-CALA-D122Y, pHBM905BDM-CALA-F283W, and pHBM905BDM-CALA-F431G were obtained, respectively. The above-mentioned mutant recombinant plasmids were sent for sequencing, and the results were consistent with expectations.

[0045] Table 1 Primer Sequences

[0046]

[0047] Example 3 Expression and purification of lipase A and its mutants

[0048] The recombinant plasmid pHBM905BDM-CALA prepared in Example 1 and the recombinant plasmid pHBM905BDM-CALA-E94W, the recombinant plasmid pHBM905BDM-CALA-V117L, the recombinant plasmid pHBM905BDM-CALA-L121I, the recombinant plasmid pHBM905BDM-CALA-D122Y, the recombinant plasmid pHBM905BDM-CALA-F283W, and the recombinant plasmid pHBM905BDM-CALA-F431G prepared in Example 2 were respectively digested with a restriction endonuclease Sal I, and linearized fragments were recovered from the solution, and then were respectively electroporated into competent cells of Pichia pastoris GS115; the transformation liquid was spread on MD plates, and was incubated at 30°C until single colonies were grown, and positive clones were screened to obtain target strains.

[0049] The target strains obtained above were respectively subjected to shake flask culture, and single colonies were inoculated into 50 mL BMGY medium, and were incubated at 30°C and 220 rpm on a constant temperature shaker until OD 600 =2-6 (about 16-18 h), the fermentation liquid was transferred to a sterile 50 mL centrifuge tube, and was centrifuged at 3000 g for 5 min, the supernatant was removed, and the bacterial body was resuspended with 25 mL BMMY medium, and was poured back into a 250 mL flask, and was continuously incubated at 30°C and 220 rpm on a constant temperature shaker, 1% methanol was added every 24 h for induction, and the optimal induction time was 4-5 d. The liquid was centrifuged at 3000 g for 5 min, and the supernatant was the crude enzyme liquid of CALA-WT and the mutant.

[0050] Ni-NTA agarose resin was added to a gravity type protein purification column, and was washed with 5 mL of purification Buffer (50 mM Tris-HCl, pH 8.5, 50 mM NaCl) containing 10 mM imidazole to activate the Ni-NTA agarose resin; the crude enzyme liquid above was filtered with a 0.45 µm filter membrane, was added to the purification column, and was incubated at 4°C for 1 h in a silent mixer; the flow-through liquid was collected, the Ni-NTA agarose resin was washed with 10 mL of purification Buffer containing 10 mM, 20 mM, 50 mM, 100 mM, and 200 mM imidazole respectively, and the eluate of each time was collected, the eluate was detected with G250, and each gradient was eluted until there was no color change of G250, and the eluate above was subjected to SDS-PAGE detection, and the component with a relatively single protein band was concentrated with a 30 kDa ultrafiltration tube, and was replaced with storage Buffer (50 mM Tris-HCl, pH 8.5, 50 mM NaCl, 1 mM EDTA, 5% glycerol) to a volume of 2 mL.

[0051] The protein obtained above was subjected to SDS-PAGE detection, and the results were as follows:Figure 1 As shown in Figure 1 As can be seen from

[0052] Example 4 Enzymatic activity determination of lipase A and its mutants

[0053] The enzymatic activity of lipase A and its mutants was tested by spectrophotometry. Specifically, a standard curve was first established using a p-nitrophenol standard, as follows:

[0054] Tris-HCl buffer (50 mM, pH 8.5) 710 μL was taken in a 1.5 mL EP tube and preheated in a 40°C water bath for 5 min, then 90 μL of different concentrations of p-nitrophenol standard solution was added, and the reaction was carried out in a 40°C water bath for 10 min, and 200 μL of 10% SDS (sodium dodecyl sulfate) solution was added to terminate the reaction. 200 μL of the reaction solution was taken in a 96-well plate, and the absorbance value at 405 nm was measured using an enzyme marker. The standard curve was plotted with concentration as the abscissa and absorbance value as the ordinate, and the results are shown in Figure 2 .

[0055] As can be seen from Figure 2 The linear regression equation of the standard curve is y = 0.0093x + 0.05, and the correlation coefficient R 2 = 0.9992.

[0056] The enzymatic activity of lipase A and its mutants was tested by spectrophotometry. Specifically, a standard curve was first established using a p-nitrophenol standard, as follows:

[0054] Tris-HCl buffer (50 mM, pH 8.5) 710 μL was taken in a 1.5 mL EP tube and preheated in a 40°C water bath for 5 min, then 90 μL of different concentrations of p-nitrophenol standard solution was added, and the reaction was carried out in a 40°C water bath for 10 min, and 200 μL of 10% SDS (sodium dodecyl sulfate) solution was added to terminate the reaction. 200 μL of the reaction solution was taken in a 96-well plate, and the absorbance value at 405 nm was measured using an enzyme marker. The standard curve was plotted with concentration as the abscissa and absorbance value as the ordinate, and the results are shown in Figure 2 .

[0055] As can be seen from Figure 2 The linear regression equation of the standard curve is y = 0.0093x + 0.05, and the correlation coefficient R 2 = 0.9992.

[0056] The enzymatic activity of lipase A and its mutants was tested by spectrophotometry. Specifically, a standard curve was first established using a p-nitrophenol standard, as follows:

[0054] Tris-HCl buffer (50 mM, pH 8.5) 710 μL was taken in a 1.5 mL EP tube and preheated in a 40°C water bath for 5 min, then 90 μL of different concentrations of p-nitrophenol standard solution was added, and the reaction was carried out in a 40°C water bath for 10 min, and 200 μL of 10% SDS (sodium dodecyl sulfate) solution was added to terminate the reaction. 200 μL of the reaction solution was taken in a 96-well plate, and the absorbance value at 405 nm was measured using an enzyme marker. The standard curve was plotted with concentration as the abscissa and absorbance value as the ordinate, and the results are shown in Figure 2 .Figure 3 As shown.

[0057] Lipase activity (U) is defined as the amount of enzyme required to release 1 μmol of p-nitrophenol (pNP) per minute by hydrolyzing the substrate under certain reaction conditions. This amount is called one enzyme activity unit, or 1U.

[0058] from Figure 3 As can be seen, the mutant proteins CALA-E94W, CALA-V117L, CALA-L121I, CALA-D122Y, CALA-F283W, and CALA-F431G have higher enzyme activities than the CALA-WT protein. The results indicate that, in this invention, by performing site-directed mutations on the wild-type CALA-WT protein, mutant proteins with significantly enhanced enzyme activities can be obtained.

[0059] Example 5: Preparation of 1,3-diglyceride catalyzed by lipase A and its mutants

[0060] Soybean oil and Tris-HCl buffer (50 mM, pH 8.5) were mixed at a volume ratio of 3:2, with a total volume of 50 mL. The mixture was then transferred to a 150 mL Erlenmeyer flask, and CALA-WT protein and CALA-V117L mutant protein (2% of the total soybean oil mass) were added separately. The mixture was reacted at 40 °C and 220 rpm in a water bath for 12 h. After the reaction, the aqueous and oil phases were separated by centrifugation at 12000 rpm for 5 min. 2 μL of the upper oil phase was collected, and the proportion of 1,3-glycerol diester in the oil phase was determined by thin-layer chromatography. The results are as follows: Figure 4 As shown.

[0061] from Figure 4 As can be seen, 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-diglycerides.

[0062] In summary, this invention provides a method for treating Candida albicans (Antarctic strain) Candida antarctica Site-directed mutagenesis was performed on lipase A in Candida antarctica to obtain a CALA mutant with significantly enhanced catalytic activity and selectivity for the sn-2 site of triglycerides. Furthermore, the CALA mutant significantly increased the yield of 1,3-diglycerides during the preparation of 1,3-diglycerides.

[0063] It should be noted that all the above embodiments belong to the same inventive concept, and the descriptions of each embodiment have different focuses. Where the description in a particular embodiment is not detailed, please refer to the description in other embodiments.

[0064] The above embodiments only express the implementation of the present application, and the description is more specific and detailed, but it cannot be understood as a limitation on the scope of the patent. It should be noted that for ordinary skilled in the art, without departing from the concept of the present application, a number of modifications and improvements can be made, which are within the scope of the present application. Therefore, the scope of protection of the present application patent should be subject to the appended claims.

Claims

1. A lipase A mutant, characterized in that, The lipase A mutant is a protein having an amino acid sequence as shown in SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO:

13.

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

3. The nucleic acid molecule of claim 2, wherein, The nucleic acid molecule is selected from any one of the following nucleic acid molecules: A1) a nucleic acid molecule having a nucleotide sequence as shown in SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO:

14.

4. A recombinant vector, characterized in that, comprising the nucleic acid molecule of claim 2 or 3.

5. A recombinant cell, characterized in that, comprising the nucleic acid molecule of claim 2 or 3 or the recombinant vector of claim 4.

6. A method for producing a lipase A mutant, characterized by, comprising the following steps: culturing the recombinant cell of claim 5, inducing expression to obtain a culture; isolating the lipase A mutant of claim 1 from the culture.

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

8. A method for producing 1,3-diglyceride, characterized by, comprising the following steps: using the lipase A mutant of claim 1, the recombinant cell of claim 5 or the lipase A mutant prepared by the preparation method of claim 6 as a catalyst to catalyze the reaction of soybean oil to obtain 1,3-diglyceride.

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