Lipase mutants and uses thereof

Abstract

The application discloses a lipase mutant and application thereof, and the lipase mutant is formed by site-directed mutagenesis of multiple amino acids on the basis of a wild-type lipase, and the mutation site is selected from one of the following schemes: I, Q127M-S136A-S141P; II, Q127M-S136D-S141P; III, Q127M-S136E-S141P; and IV, Q127M-S136F-S141P. Compared with the wild-type lipase, the catalytic activity of the lipase mutant is significantly improved, which is of great significance to improving production efficiency and reducing cost, and has good practicability.

Description

Technical Field

[0001] This invention relates to the field of bioengineering technology, and in particular to a lipase mutant and its applications. Background Technology

[0002] Lipases (EC 3.1.1.3) are a class of hydrolytic enzymes that catalyze the hydrolysis of triglycerides into fatty acids, glycerol, and mono- and diglycerides. They exhibit high substrate specificity and stereoselectivity and have wide applications in industry. In aquatic product processing, they help convert lipases in fish and shrimp tissues into smaller molecules; in microbial fermentation, they can accelerate the decomposition of insect fats, facilitating the release and filtration of small protein molecules; in the food industry, lipases can be used for oil modification (such as preparing low-trans-fatty acid oils), enhancing the flavor of dairy products, and improving the quality of baked goods; in the detergent industry, as bioactive ingredients, they can effectively remove grease stains from clothing and tableware; in the pharmaceutical field, they can be used for the resolution of chiral drug intermediates, the preparation of drugs for treating obesity, and the development of clinical diagnostic reagents; in the bioenergy field, they can catalyze the key step (transesterification) in the conversion of waste oils into biodiesel.

[0003] However, wild-type lipases generally suffer from low enzyme activity, which limits their efficient application in industrial production. For example, in the biodiesel production process, low enzyme activity results in reaction cycles of 24-48 hours, requiring the addition of large amounts of enzyme preparations to maintain reaction efficiency; in detergent formulations, low enzyme activity means that lipases need to work synergistically with other enzymes (proteases, cellulases) to achieve ideal detergency, increasing formulation costs.

[0004] To address this issue, researchers have employed strategies such as directed evolution and rational design to molecularly modify lipases. However, existing methods suffer from low screening efficiency (directed evolution) and insufficient accuracy in site prediction (traditional rational design), making it difficult to efficiently obtain high-activity mutants. Therefore, developing a high-activity lipase mutant is of great significance for improving industrial production efficiency and reducing costs. Summary of the Invention

[0005] The purpose of this invention is to solve the problem of low enzyme activity of wild-type lipase in the prior art, and to provide a lipase mutant and its application. Compared with wild-type lipase, it significantly improves catalytic activity, which is of great significance for improving production efficiency and reducing costs, and has good practicality.

[0006] The technical solution adopted by this invention to solve its technical problem is:

[0007] A lipase mutant, wherein the lipase mutant is formed by site-directed mutation of multiple amino acids based on wild-type lipase, and the mutation sites are selected from one of the following schemes:

[0008] Ⅰ. Q127M-S136A-S141P: The wild-type lipase has a Q mutation at position 127 to M, an S mutation at position 136 to A, and an S mutation at position 141 to P.

[0009] II. Q127M-S136D-S141P: The wild-type lipase has a Q mutation at position 127 that is changed to M, an S mutation at position 136 that is changed to D, and an S mutation at position 141 that is changed to P.

[0010] III. Q127M-S136E-S141P: The wild-type lipase has a Q mutation at position 127 to M, an S mutation at position 136 to E, and an S mutation at position 141 to P.

[0011] IV. Q127M-S136F-S141P: The wild-type lipase has a Q mutation at position 127 to M, an S mutation at position 136 to F, and an S mutation at position 141 to P.

[0012] Preferably, the amino acid sequence of the wild-type lipase is shown in SEQ ID NO:1.

[0013] A polynucleotide encoding the lipase mutant described above.

[0014] A recombinant vector comprising the aforementioned polynucleotide.

[0015] The recombinant vector is a recombinant prokaryotic vector.

[0016] In the recombinant prokaryotic vector, the prokaryotic vector is either a pET(+) plasmid or a pGEX vector.

[0017] A recombinant genetically engineered bacterium comprising the recombinant vector or the polynucleotide.

[0018] The host bacteria of the recombinant engineered bacteria include Escherichia coli. Preferably, it is Escherichia coli BL21(DE3).

[0019] A method for preparing the lipase mutant, the method comprising: inoculating the recombinant genetically engineered bacteria into a fermentation medium, fermenting, and then centrifuging to collect the precipitate to obtain a crude product of the lipase mutant. Preferably, the fermentation medium comprises LB medium; fermentation is carried out until the OD600 is 0.4-0.8.

[0020] The invention relates to the application of the described lipase mutant, polynucleotide, recombinant vector, or recombinant genetically engineered bacteria in food processing, enzymatic fermentation, detergent production, biodiesel preparation, or pharmaceutical intermediate synthesis. The food processing includes noodle processing, dairy processing, and oil processing. In enzymatic fermentation applications, it can serve as an important component, working synergistically with proteases, oxidases, and probiotics to extract small molecule peptides. In detergent production applications, this invention can be used as a core ingredient in enzyme-added laundry detergents, liquid laundry detergents, and dishwasher detergents.

[0021] The beneficial effects of this invention are:

[0022] This invention utilizes machine learning algorithms (combined with lipase amino acid sequence databases and three-dimensional structural data) to predict mutation sites in wild-type lipases, screening for mutations at the following positions: glutamine (Gln, Q) at position 127 is mutated to methionine (Met, M); serine (Ser, S) at position 136 is mutated to alanine (Ala, A), aspartic acid (Asp, D), glutamic acid (Glu, E), or alanine (Phe, F); and serine (Ser, S) at position 141 is mutated to proline. Four lipase mutants (named Q127M-S136A-S141P, Q127M-S136D-S141P, Q127M-S136E-S141P, and Q127M-S136F-S141P, abbreviated as Q127M-S136(A / D / E / F)-S141P) were constructed using acid (Pro, P). Compared with the wild-type lipase, the catalytic activity was significantly improved, which is of great significance for improving production efficiency and reducing costs.

[0023] This invention uses an Escherichia coli expression system to express lipase mutants. The E. coli cells have strong metabolic capabilities and rich cellular mechanisms, enabling them to efficiently synthesize and fold proteins. Compared with other expression systems, the E. coli expression system is simple to operate and low in cost. The recombinant engineered bacteria culture and protein expression cycle of this invention is relatively short, making it suitable for rapid large-scale production. Attached Figure Description

[0024] Figure 1 This is a schematic diagram of a lipase structure; the active site is shown in blue. Residues representing the catalytic triplet are shown in pink.

[0025] Figure 2 The mutation sites are listed in red and those of this invention in blue.

[0026] Figure 3The graph shows the expression of wild-type lipase (Origin) and its mutant (Q127M-S136(A / D / E / F)-S141P); M is the protein marker; Ctrl is the control E. coli containing the empty pET28a(+) plasmid; Lys is the whole-cell lysate containing the expressed enzyme Origin and (Q127M-S136(A / D / E / F)-S141P); Sup is the protein in the supernatant after centrifugation at 4 °C; Pel is the precipitate or insoluble protein fraction after centrifugation at 4 °C.

[0027] Figure 4 The purified Origin, Q127M-S136(A / D / E / F)-S141P protein.

[0028] Figure 5 The specific activity diagram of Origin, Q127M-S136(A / D / E / F)-S141P enzyme.

[0029] Figure 6 This is a schematic diagram of the wild-type (Origin) and mutant (Q127M) lipase.

[0030] Figure 7 This is a schematic diagram of the wild-type lipase (Origin) and the mutant (S136(A / D / E / F)).

[0031] Figure 8 This is a schematic diagram of the wild-type (Origin) and mutant (S141P) lipase.

[0032] Figure 9 The image shows the thermal stability of Origin Q127M-S136(A / D / E / F)-S141P after heat treatment for 30 minutes. Detailed Implementation

[0033] The technical solution of the present invention will be further described in detail below through specific embodiments.

[0034] In this invention, unless otherwise specified, all raw materials and equipment used are commercially available or commonly used in the field. The methods described in the following embodiments are conventional methods in the field, unless otherwise specified.

[0035] Example 1: Prediction and Acquisition of Lipase Mutants

[0036] Machine learning-assisted computation methods utilize bioinformatics data from multiple protein sequences to predict one or more amino acid sequences. These sequences can be substituted based on ancestor or consensus homology to generate stable mutants, which are experimentally attractive in enzyme engineering research. This embodiment uses machine learning-assisted computation methods to predict and design lipase mutants of wild-type lipase (Ideonella sakaiensis accession number: ADZ30931.1) in order to obtain lipase mutants with higher enzyme activity. The amino acid sequence of the wild-type lipase is as follows:

[0037] MNFPRASRLMQAAVLGGLMAVSAAATAQTNPYARGPNPTAASLEASAGPFTVRSFTVSRPSGYGAGTVYYPTNAGGTVGAIAIVPGYTARQSSIKWWGPRLASHGFVVITIDTNSTLDQPSSRSSQQMAALRQVASLNGTSSSPIYG KVDTARMGVMGWSMGGGGSLISAANNPSLKAAAPQAPWDSSTNFSSVTVPTLIFACENDSIAPVNSSALPIYDSMSRNAKQFLEINGGSHSCANSGNSNQALIGKKGVAWMKRFMDNDTRYSTFACENPNSTRVSDFRTANCS (SEQ IDNO:1).

[0038] The specific steps are as follows:

[0039] The crystal structure of the lipase (PDB ID: 7SH6) was constructed using PyMol, in which the target-substituted amino acids (before and after substitution) and active sites form a network. The enzyme active site was determined using the online protein surface morphology computer mapping (CASTp) program (http: / / sts.bioe.uic.edu / castp / ). To improve the catalytic activity of the lipase, machine-aided computational methods using FireProt (http: / / loschmidt.chemi.muni.cz / fireprot) and a web server (http: / / pmlabstack.pythonanywhere.com / SCMTPP) were used to predict and design lipase mutants with potential to enhance enzyme activity.

[0040] Enzyme activity enhancement mutation criteria:

[0041] Correlation score ≥ 0.5 (high correlation with activity);

[0042] Conserved sites (Conservation ≤ 6);

[0043] FoldX ΔΔG is not significantly positive (to avoid damaging the structure);

[0044] The higher the SCMTTP level, the better, as it comprehensively represents stability and activity potential.

[0045] The specific mutation results are shown in Table 1. Through software analysis and machine learning, the correlation scores of Q127M, S136(A / D / E / F), and S141P were 0.58, 1.49, and 0.69, respectively, indicating that the variation of amino acid residues at these three sites is strongly positively correlated with the catalytic activity of lipase. The mutation can directly enhance the binding ability of the enzyme to substrates (such as triglycerides and p-nitropalmitate) or the reaction efficiency of the catalytic group. ΔΔG (FoldX) were -3.05, 0.04, and -2.88, respectively, indicating that the mutation did not cause a significant change in the folding free energy of the lipase protein, avoiding the deformation or misfolding of the enzyme's three-dimensional structure caused by the mutation, and ensuring the conformational integrity of the enzyme's catalytic active site (the active site Ser-His-Asp triplet). The conserved sites were 5, 2, and 4, respectively. The mutation did not interfere with the conserved functional regions of the lipase, and at the same time, the change in the amino acid side chain (serine hydroxyl group → alanine methyl group) reduced steric hindrance and increased the rate at which the substrate entered the active site. Taking all the above into consideration, we constructed a mutant with three site mutations in Q127M-S136(A / D / E / F)-S141P.

[0046] Table 1. Predictive analysis results of different lipase mutants

[0047] .

[0048] We searched and compiled existing literature and found that several mutation sites of this lipase are associated with its catalytic activity and stability (Table 2). The active site of the lipase is occupied by amino acids Y87, W159, S160, M161, W185, D206, and H237, among which S160, D206, and H237 form a catalytic triad. Lipases are known to form an open groove, and some variants possess a cap to regulate the entry and hydrolysis of bulk lipid substrates. Furthermore, the cap plays a crucial role in interfacial activation, allowing the enzyme to act on insoluble substrates forming oil-water interfaces or emulsions, inducing and regulating the conformational changes required to expose the active site to bulk insoluble droplets in the matrix. The structure of the active site is shown in Table 2. Figure 1 As shown, an open groove is revealed, allowing the substrate to directly enter the catalytic site. This invention utilizes machine learning / computational prediction to identify three mutation sites in Q127M-S136(A / D / E / F)-S141P; no related research reports on these mutants have been found in the published literature to date.Figure 2 ).

[0049] Table 2. Enzyme catalytic activity and stability of different lipase mutants reported in the literature.

[0050] .

[0051] Example 2. Construction of the recombinant vector (Origin, Q127M-S136(A / D / E / F)-S141P)

[0052] In this invention, the LB liquid culture medium formula is: 10 g / L tryptone, 5 g / L yeast extract, and 10 g / L NaCl; the LB solid culture medium formula is: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L NaCl, and 20 g / L agar.

[0053] In Example 1 of the whole-genome synthesis by Nanjing Genscript Biotech Co., Ltd., the wild-type (Origin) and mutant (Q127M-S136(A / D / E / F)-S141P) sequences of the above five genes were modified by adding BamHI and EcoRI restriction sites to the 5' and 3' ends of the sequences, respectively, to obtain the nucleotide sequences of the above five genes with BamHI and EcoRI restriction sites. These nucleotide sequences were then constructed into the pET28a(+) plasmid, denoted as pET28a(+)-Origin and pET28a(+)-Q127M-S136(A / D / E / F)-S141P. E. coli were transformed using pET28a(+)-Origin and pET28a(+)-Q127M-S136(A / D / E / F)-S141P, respectively. The specific steps are as follows:

[0054] Remove competent E. coli BL21(DE3) cells from a -80℃ freezer and thaw them on ice. Add 5 μL of each of the five plasmids mentioned above, and gently tap the bottom of the EP tube to mix. Place the tubes on ice for 30 minutes, incubate at 42℃ for 45 seconds, and then immediately place them on ice for two minutes. After incubation, add 700 μL of preheated LB liquid medium in a clean bench, mix well, and shake at 37℃ and 200 rpm for 2 hours to revive the cells. Then, centrifuge at 10,000 rpm for one minute at room temperature to collect the cells. Discard 600 μL of supernatant. Mix the remaining supernatant with the cells and spread the mixture on LB solid medium containing 50 μg / mL kanamycin. Incubate overnight at 37℃ to obtain recombinant E. coli transformed with the five plasmids mentioned above, namely recombinant E. coli expressing Origin, Q127M-S136(A / D / E / F)-S141P.

[0055] Example 3. Large-scale culture of Escherichia coli expressing Origin, Q127M-S136(A / D / E / F)-S141P

[0056] (1) The recombinant Escherichia coli expressing Origin, Q127M-S136(A / D / E / F)-S141P obtained in Example 2 were transferred to LB solid medium containing 50 μg / mL kanamycin and cultured overnight at 37°C. After the culture was completed, the single colonies in the medium were transferred to 5 mL of LB liquid medium containing 50 μg / mL kanamycin and cultured overnight at 37°C and 200 rpm. After the culture was completed, 3 mL of recombinant Escherichia coli was inoculated into 300 mL of LB liquid medium containing 50 μg / mL kanamycin and grown at 37°C and 200 rpm for about 3 h until the bacterial OD 600 It reaches between 0.4 and 0.8.

[0057] (2) Place the LB liquid medium after the culture in step (1) on ice for 20 minutes, add isopropyl β-D-1-thiogalactopyranoside (IPTG) to it to a final concentration of 0.4 mM, and culture at 25°C with shaking at 180 rpm for 16-20 h (overnight) to induce Origin, Q127M-S136(A / D / E / F)-S141P expression. After the induction, centrifuge the liquid medium at 3000 rpm and 4°C for 20 minutes and discard the supernatant to obtain the cell precipitate cultured at 25°C. Store it at -80°C for later use.

[0058] (3) Thaw the cell precipitate obtained in step (2) and resuspend it in 10 mL Tris-HCl (50 mM, pH 8.0). Then, centrifuge at 3000 rpm and 4℃ for 20 minutes, discard the supernatant, collect the cells, and wash the cells twice with 50 mM Tris-HCl with a pH of 8.0.

[0059] The bacterial cell resuspended in 20 mL of Tris-HCl buffer containing 1 mM PMSF (200 μL) and lysed on ice using an ultrasonic cell disruptor for 30 min, followed by alternating sonication for 6 s and intermittent cooling for 6 s to obtain a solution containing cell pellet (total protein). The total protein was centrifuged at 13000 rpm for 30 min at 4 °C to obtain the supernatant and pellet. The supernatant was transferred to a new EP tube, and the pellet was resuspended in 50 mM pH 8.0 Tris-HCl. SDS-PAGE was performed on the total protein, supernatant, and pellet to analyze recombinant protein expression and solubility. The results are shown below. Figure 3As shown in the figure. The results indicate that under 25℃ induction conditions, Origin, Q127M-S136(A / D / E / F)-S141P cell lysates (total protein), supernatant, and precipitate all showed significant bands at 38 kDa, indicating that all five recombinant proteins were successfully expressed; and the presence of significant bands in the supernatant indicates that they are soluble.

[0060] Example 4. Origin, Q127M-S136(A / D / E / F)-S141P protein Ni-NTA nickel column purification

[0061] The supernatant containing Origin, Q127M-S136(A / D / E / F)-S141P obtained in Example 3 was purified using a Ni-NTA column. The specific steps are as follows:

[0062] Add 10 mL of nickel removal buffer (Shanghai Sangon Biotech) to the column and wash twice until the column turns from blue to white. Wash the column twice with 10 mL of deionized water. Then, slowly add 10 mL of NiSO4 buffer (Sinopharm Group) to the column, cover with both caps, and incubate in a chromatography cabinet at 4 °C for 30 min by rotation. Open the bottom cap to allow excess NiSO4 to flow out, and then wash once with 10 mL of deionized water and once with 10 mL of PBS buffer (pH 7.4).

[0063] Add 4 mL of cell lysed Origin and the supernatant of Q127M-S136(A / D / E / F)-S141P to the column, respectively, and incubate at 4 °C for 2 h. Open the bottom cap to allow unbound protein samples to flow out, and wash again with 15 mL of PBS buffer. After washing, elute with 2 mL of 150 mM, 250 mM, and 450 mM imidazole, respectively. Prepare protein samples for loading, and determine the purification results by SDS-PAGE. The results are shown below. Figure 4 As shown. From Figure 4 As can be seen from the data, Origin, Q127M-S136(A / D / E / F)-S141P has a significant band at 38 kDa and relatively few impurities, indicating that soluble Origin, Q127M-S136(A / D / E / F)-S141P has been successfully purified.

[0064] Example 5: Origin, Q127M-S136(A / D / E / F)-S141P enzyme activity detection

[0065] Lipases catalyze the hydrolysis of p-nitrophenylacetic acid (p-NPA) to produce p-nitrophenol (pNP) and acetic acid. Under alkaline or neutral conditions, the product pNP is yellow and has a characteristic absorption peak at 410 nm. The rate of pNP formation can be calculated by real-time monitoring with a spectrophotometer or by measuring the rate of increase in absorbance at 410 nm using an endpoint method, thus determining the catalytic activity of the lipase. The spectrophotometer sample chamber temperature was set to 40 °C, and the required volumes of PBS buffer and substrate solution were preheated in a 40 °C water bath for at least 10 minutes. 970 μL of preheated PBS buffer (40 °C) and 20 μL (1.2 mg / mL) of active enzyme solution were added to cuvettes. 10 μL of preheated 10 mM p-NPA substrate solution was rapidly added to each of the two cuvettes, and the mixture was immediately stirred with a pipette (avoiding air bubbles), while timing was started simultaneously. After reacting precisely for 10 minutes, immediately add 100 μL of 1 M NaOH solution to the cuvette to terminate the reaction (this step can be omitted if the reaction solution itself is alkaline and linear, and the measurement can be performed directly). After mixing, zero the instrument using a blank tube and read the absorbance value of the measurement tube at a wavelength of 410 nm.

[0066] The results are as follows Figure 5 The enzyme activities of the Q127M-S136(A / D / E / F)-S141P mutant shown are 2.32, 2.18, 2.41, and 2.65 times that of the wild-type Origin, respectively. This indicates that the mutation of glutamine (Q) at position 127 to methionine (M); the mutation of serine (S) at position 136 to alanine (A), aspartic acid (D), glutamic acid (E), or alanine (F) are site-directed mutations; and the mutation of serine (S) at position 151 to proline (P) significantly increases the enzyme's catalytic activity.

[0067] The Q127M-S136(A / D / E / F)-S141P triple mutation of this invention is located in the α3–β5 short ring and its adjacent region. By synergistically reconstructing the hydrogen bond network, hydrophobicity, and electric field environment at the active channel entrance, it primarily improves catalytic efficiency rather than significantly altering overall thermal stability. Q127 is changed from a polar amide to a hydrophobic methionine (M), removing unfavorable hydrogen bonds and enhancing local hydrophobic packaging, making the active channel entrance drier and more pre-organized. S141P restricts short ring oscillation with a rigid proline backbone, reducing the conformational entropy of the unfolded state and slightly improving high-temperature residual activity while maintaining structural integrity. S136 serves as the channel "bottleneck site." Different substitutions of A / D / E / F respectively expand substrate entry into the channel and optimize transition state electric field stability by removing the hydrogen bond between Ser-OH and Asp177, changing the local charge, introducing aromatic ring π-π interactions, and hydrophobic interactions. In particular, S136F constructs an aromatic channel, endowing it with the highest substrate binding and catalytic activity. Figure 6-8 ).

[0068] Example 6: Thermal and storage stability of Origin, Q127M-S136(A / D / E / F)-S141P

[0069] This embodiment examined the thermostability of Origin, Q127M-S136(A / D / E / F)-S141P to evaluate the stability of the lipase mutant, as detailed below:

[0070] The purified Origin, Q127M-S136(A / D / E / F)-S141P was placed at 20, 30, 40, 50, 60, 70, and 80 °C for 30 minutes to detect its heat resistance. Enzyme activity was determined according to the method described in Example 5, with the highest enzyme activity value taken as 100%. Other enzyme activity values ​​were compared with the highest enzyme activity value, and curves of relative activity at different temperatures were plotted. The test results are shown below. Figure 9 As shown.

[0071] Figure 9 The results show that, compared to the wild type (Origin), the mutant Q127M-S136(A / D / E / F)-S141P did not show a significant increase in stability at 40, 50, 60, and 70℃, indicating that the above site modifications mainly achieve high activity through fine-tuning of the epistatic effect of the active center, while having a limited impact on overall thermal stability.

[0072] In summary, the four lipase mutants obtained in this invention have higher enzyme catalytic activity compared with the wild type, which is of great significance for improving production efficiency and reducing costs, and has good practicality.

[0073] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the present invention in any way. Other variations and modifications are possible without departing from the technical solutions described in the claims.

Claims

1. A lipase mutant, characterized in that, The lipase mutant was formed by site-directed mutation of multiple amino acids based on wild-type lipase, with the mutation sites selected from one of the following schemes: Ⅰ. Q127M-S136A-S141P: The wild-type lipase has a Q mutation at position 127 to M, an S mutation at position 136 to A, and an S mutation at position 141 to P. II. Q127M-S136D-S141P: The wild-type lipase has a Q mutation at position 127 that is changed to M, an S mutation at position 136 that is changed to D, and an S mutation at position 141 that is changed to P. III. Q127M-S136E-S141P: The wild-type lipase has a Q mutation at position 127 to M, an S mutation at position 136 to E, and an S mutation at position 141 to P. IV. Q127M-S136F-S141P: The wild-type lipase has a Q mutation at position 127 to M, an S mutation at position 136 to F, and an S mutation at position 141 to P. The amino acid sequence of the wild-type lipase is shown in SEQ ID NO:

1.

2. A polynucleotide, characterized in that, It encodes the lipase mutant of claim 1.

3. A recombinant vector, characterized in that, It contains the polynucleotide as described in claim 2.

4. The recombinant vector according to claim 3, characterized in that, The recombinant vector is a recombinant prokaryotic vector.

5. The recombinant vector according to claim 4, characterized in that, In the recombinant prokaryotic vector, the prokaryotic vector is either a pET(+) plasmid or a pGEX vector.

6. A recombinant genetically engineered bacterium, characterized in that, It comprises the recombinant vector as described in any one of claims 3-5 or the polynucleotide as described in claim 2.

7. The recombinant genetically engineered bacteria according to claim 6, characterized in that, The host bacteria of the recombinant genetically engineered bacteria include Escherichia coli.

8. A method for preparing the lipase mutant of claim 1, characterized in that, The method includes: inoculating the recombinant genetically engineered bacteria of claim 6 or 7 into a fermentation medium, fermenting and culturing, and then centrifuging to collect the precipitate to obtain the crude product of the lipase mutant.

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