Lipase mutant as well as preparation method and application thereof
By mutating and recombining lipases, chiral (S)-p-chloro-β-phenylpropanol was synthesized using lipase mutant catalysts, solving the problems of high synthesis cost and low efficiency in existing technologies, and achieving industrial production with high yield and high purity.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-12-01
- Publication Date
- 2026-03-10
AI Technical Summary
Existing technologies for synthesizing the key intermediate p-chloro-β-phenylalanine in cappicetil suffer from high costs, dangerous and complex operations, large solvent consumption, and difficulty in industrial production. Furthermore, they exhibit low resolution efficiency, low yield, and difficulty in product purification.
Using lipase mutants as biocatalysts, high-optical-purity chiral (S)-p-chloro-β-phenylalanine was prepared by single- or double-site mutations at arginine position 138 and tyrosine position 251. The catalytic reaction was carried out using recombinant expression vectors and engineered bacteria, avoiding the use of expensive resolving agents.
The yield was increased to over 45%, the ee value reached 99.7%, the cost was reduced, the requirements for industrial production were met, and the green environmental protection standards were met, achieving an efficient and easy-to-operate synthesis process.
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Figure CN121628876A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of organic synthesis and enzyme catalysis technology, specifically relating to a lipase mutant, its preparation method and application. Background Technology
[0002] Capiste is the world's first pan-AKT inhibitor (First in Class), indicated in combination with fulvestrant for adult patients with metastatic locally advanced or metastatic breast cancer who have progressed after at least one prior endocrine therapy, or who have relapsed during or within 12 months of completing adjuvant therapy, and who are hormone receptor (HR) positive, human epidermal growth factor receptor 2 (HER2) negative, and have one or more PIK3CA / AKT1 / PTEN alterations.
[0003] - p-Chloro-β-phenylpropanol is a key intermediate in the synthesis of cappicrite. The main synthesis method is as follows: p-chlorobenzaldehyde is condensed with malonic acid to obtain 3-amino-3-(4-chlorophenyl)propionic acid, which is then esterified to obtain methyl 3-amino-3-(4-chlorophenyl)propionate. After resolution, the S-type methyl 3-amino-3-(4-chlorophenyl)propionate is obtained, and then reduced to obtain the target product. This method has low resolution efficiency, high cost, and is difficult to industrialize.
[0004] The synthesis route is as follows: The current process has the following main problems: 1) Due to its high cost, dangerous operation, complexity and large solvent consumption, it is difficult to obtain satisfactory results in industrial preparation; 2) The raw material cost of this route is high, the yield is low, and the product purification is difficult. In particular, in the chiral resolution process, more than 3 resolutions are required to reach an ee value of more than 97%, and the yield is only 33%, which wastes a lot of resolving agent; 3) It simply cannot meet the requirements of industrial production and the market. Summary of the Invention
[0005] In view of the current technical deficiencies, the present invention provides a lipase mutant, its preparation method and application. The present invention uses the lipase mutant or its recombinant expression vector or its recombinant engineered bacteria as active enzymes to provide a powerful biocatalyst for the synthesis of (S)-p-chloro-β-phenylpropanol.
[0006] The technical solution adopted in this invention is as follows: A lipase mutant is obtained by performing single-site or double-site mutations at arginine position 138 and tyrosine position 251, based on the lipase with nucleotide sequence SEQ ID No. 1 and amino acid sequence SEQ ID No. 2.
[0007] As a preferred embodiment, the arginine at position 138 is mutated to serine, the nucleotide sequence of the lipase mutant is SEQ ID No. 3, and the amino acid sequence of the lipase mutant is SBQ ID No. 4.
[0008] As a preferred embodiment, the tyrosine at position 251 is mutated to phenylalanine, the nucleotide sequence of the lipase mutant is SBQ ID No. 5, and the amino acid sequence of the lipase mutant is SBQ ID No. 6.
[0009] As a preferred embodiment, arginine at position 138 is mutated to serine and tyrosine at position 251 is mutated to phenylalanine. The nucleotide sequence of the lipase mutant is SEQ ID No. 7, and the amino acid sequence of the lipase mutant is SEQ ID No. 8.
[0010] The present invention also provides a method for preparing the above-mentioned lipase mutant, wherein the recombinant expression transformant of the lipase mutant is cultured and recombinant lipase mutant protein is induced.
[0011] Preferably, the recombinant expression transformant is inoculated into LB medium containing kanamycin and cultured until the OD of the culture medium reaches a certain level. 600 When the concentration reaches 0.5–0.7, a recombinant lipase mutant is obtained under the induction of a final concentration of 0.1–1.0 mM isopropyl-BD-thiogalactopyranoside; the recombinant expression transformant is recombinant Escherichia coli.
[0012] This invention also provides the application of the above-mentioned lipase mutants in the preparation of recombinant expression vectors. The recombinant expression vectors contain the nucleotide sequence of any of the above-mentioned lipase mutants.
[0013] Preferably, the recombinant expression vector is converted into a host microorganism to prepare a genetically engineered bacterium expressing a recombinant lipase mutant. This is obtained by converting any of the above-mentioned recombinant expression vectors into a host microorganism.
[0014] Preferably, the lipase mutant, recombinant expression vector, or genetically engineered bacteria expressing the recombinant lipase mutant is used to prepare chiral (S)-p-chloro-β-phenylalanine.
[0015] Preferably, methyl 3-amino-3-(4-chlorophenyl)propionate is used as a substrate, and lipase mutants, recombinant expression vectors, or genetically engineered bacteria are used as catalysts. The reaction is carried out in a transformation reaction system consisting of a buffer solution with pH 5.5 to 10. After the reaction is complete, the reaction solution is separated and purified to obtain S-type 3-amino-3-(4-chlorophenyl)propionic acid, which is then reduced to obtain chiral (S)-p-chloro-β-phenylpropanol.
[0016] Typical chemical resolution methods yield between 30% and 35%, with an ee value of around 97%. The presence of isomers often leads to the final product, making purification difficult. Using the method of this invention, the yield can be increased to over 45%, with an ee value of 99.7%. This increased yield avoids the use of expensive resolving agents, reducing costs and fully meeting product quality requirements, while also aligning with green and environmentally friendly principles.
[0017] Compared with the prior art, the present invention has the following beneficial effects: 1. A lipase mutant with significantly enhanced catalytic activity and its nucleotide sequence are provided, as well as a recombinant expression vector and recombinant engineered bacteria containing the corresponding mutant gene. High-optical-purity chiral (S)-p-chloro-β-phenylpropanol can be prepared by asymmetric reduction of these lipase mutants or recombinant cells containing the corresponding mutant protein.
[0018] 2. The lipase mutant or recombinant cells containing the mutant protein can be used to prepare chiral (S)-p-chloro-β-phenylalanine by asymmetric reduction. This method has high catalytic activity and high stereoselectivity, and can synthesize chiral (S)-p-chloro-β-phenylalanine with high optical purity (ee>99%).
[0019] 3. In the synthesis of chiral (S)-p-chloro-β-phenylpropanol, the present invention has the advantages of easy preparation, mild reaction conditions, wide substrate adaptability, and environmental friendliness. Attached Figure Description
[0020] Figure 1 This is a graph showing the colony PCR identification results of this invention; Figure 2 This is a liquid chromatography spectrum of the chiral purity of the product of this invention; Figure 3 This is the NMR spectrum of the product of this invention; Figure 4 This is a liquid phase spectrum of the purity of the product of this invention. Detailed Implementation
[0021] The technical solution of the present invention will be further described in detail below through embodiments. These embodiments are for illustrative purposes only and are not intended to limit the present invention. Based on the embodiments in this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.
[0022] Unless otherwise specified, the experimental methods described in the embodiments are conventional methods; unless otherwise specified, the reagents and materials are commercially available.
[0023] This invention discloses a lipase mutant, which is obtained by single-site or double-site mutations at arginine position 138 and tyrosine position 251 based on the amino acid sequence of the lipase, SEQ ID No. 2. The nucleotide sequence of this lipase is shown in SEQ ID No. 1 of the sequence listing.
[0024] The mutation involves altering the arginine at position 138 to serine. The nucleotide sequence of the mutant is shown in SEQ ID No. 3 of the sequence listing, and its encoded amino acid sequence is shown in SBQ ID No. 4 of the sequence listing.
[0025] The mutation at position 251, tyrosine, is phenylalanine. The nucleotide sequence of the mutant is shown in SBQ ID No. 5 of the sequence listing, and its encoded amino acid sequence is shown in SBQ ID No. 6 of the sequence listing.
[0026] The mutant is created by mutating arginine at position 138 to serine and tyrosine at position 251 to phenylalanine. The nucleotide sequence of the mutant is shown in SEQ ID No. 7 of the sequence listing, and its encoded amino acid sequence is shown in SEQ ID No. 8 of the sequence listing. Any deletion, insertion, or substitution of one or more amino acids in the above mutant amino acid sequence still falls within the protection scope of this invention.
[0027] This invention discloses a recombinant expression vector containing the nucleotide sequence of a mutant lipase described above. The recombinant expression vector can be constructed by linking the nucleotide sequence of the lipase mutant of this invention to various vectors using conventional methods in the art. The vector can be any conventional vector in the art, such as various plasmids, bacteriophages, or viral vectors, preferably pET-28a.
[0028] This invention discloses a genetically engineered bacterium expressing a lipase mutant, obtained by converting a recombinant expression vector described above into a host microorganism. The host microorganism can be any conventional host microorganism in the art, as long as the recombinant expression vector can stably self-replicate and the lipase mutant gene carried by it can be effectively expressed. *Escherichia coli* is preferred, and *Escherichia coli* is more preferred. E.coil BL21(DE3).
[0029] The application of the aforementioned lipase mutant, recombinant expression vector, or genetically engineered bacterium expressing the recombinant lipase mutant in the synthesis of chiral (S)-p-chloro-β-phenylalanine. Specifically, using Formula I as the substrate, and the purified lipase mutant, recombinant expression vector, or genetically engineered bacterium as the catalyst, the reaction is carried out in a transformation reaction system composed of a buffer solution at pH 5.5-10. After the reaction is complete, the reaction solution is separated and purified to obtain the final product. I The initial concentration of the potential substrate in this transformation reaction system is 5–300 mmol / L; the concentration of lipase is 0.1–2.0 mg / mL, or the amount of engineered bacteria containing the lipase mutant is 10–400 g / L based on the wet weight of the bacteria. Preferably, the reaction is carried out in a buffer solution at pH 7.5.
[0030] The conversion reaction system also includes an alcohol or sugar at a mass concentration of 1–50% as a co-substrate. The sugar is glucose, with a mass concentration of 5% (w / w).
[0031] The above-mentioned method for separating and purifying the conversion reaction solution is as follows: After the reaction is completed, the conversion reaction solution is centrifuged, and the supernatant is extracted with an equal volume of ethyl acetate. The organic layer contains the crude product of the corresponding chiral (S)-p-chloro-β-phenylalanine. The crude product is then purified to obtain the corresponding chiral (S)-p-chloro-β-phenylalanine. The method for purifying the crude product is a well-known technique in the art, and typically involves organic solvent extraction, chromatographic separation, and adsorption separation.
[0032] (S)-p-chloro-β-phenylalanine is reduced to (S)-p-chloro-β-phenylalanine.
[0033] 40 kg of tetrahydrofuran and 3.2 kg of zinc chloride were added to the reactor. Sodium borohydride (total 0.89 kg) was added in four batches, with an interval of 10 min between each batch. The mixture was stirred at 20 ± 5 °C for 1 h. 5 kg of (S)-p-chloro-β-phenylalanine was added dropwise at 15 ± 5 °C. After the addition was complete, the temperature was gradually increased to 50 ± 5 °C and stirred for 4 h under control. The HPLC raw material concentration was ≤ 0.5%. The temperature was lowered to 15 ± 5 °C, and 10.49 kg of 10% hydrochloric acid aqueous solution was added dropwise until the solution was clear. The mixture was stirred for 30 min, and 15 kg of dichloromethane was added to extract impurities. The dichloromethane was washed with 5 kg of 10% hydrochloric acid. The aqueous phases were combined, and then 35 kg of dichloromethane was added to the aqueous phase. 6.25 kg of ammonia was added dropwise until the white turbid liquid became clear. The mixture was stirred for 30 min, and the layers were separated. The layers were extracted, and then extracted again with 35 kg of dichloromethane. The organic phases were combined, washed with 25 kg of saturated brine, dried, and concentrated to obtain the product.
[0034] The above-mentioned method for preparing a lipase mutant includes the following steps: culturing a recombinant expression transformant of the lipase mutant and inducing the production of recombinant lipase mutant protein; wherein the culture medium used for culturing the recombinant expression transformant is LB medium, comprising: 10 g / L peptone, 5 g / L yeast extract, 10 g / L sodium chloride, pH 7.2; and inoculating recombinant Escherichia coli into LB medium containing kanamycin and culturing until the OD of the culture medium reaches a certain level. 600When the concentration reaches 0.5–0.7, it is obtained by induction with a final concentration of 0.1–1.0 mM isopropyl-BD-thiogalactopyranoside (IPTG). The above reaction conditions can be selected according to conventional conditions used in the art, as long as the transformant can grow and produce lipase mutant protein.
[0035] In the text, Arg138 refers to arginine at position 138, Tyr251 refers to phenylalanine at position 251, A138S indicates a mutant by mutating arginine at position 138 to serine, Y251F indicates a mutant by mutating tyrosine at position 251 to phenylalanine, and A138S / Y251F indicates a mutant by a combination of two-site mutations: arginine at position 138 is mutated to serine, and tyrosine at position 251 is mutated to phenylalanine.
[0036] Example 1 Construction of lipase mutants: Using oligonucleotide fragments containing mutation points as primers, as shown in Table 1, the pBT-28a recombinant plasmid containing the lipase gene was amplified using the QuickChange™ method.
[0037] Table 1 The underlined 'a' indicates a mutation site.
[0038] PCR reaction system: upstream primer 10m, 1.0uL; downstream primer 10m, 1.0uL; recombinant plasmid template, 10ng; PrimerSTAR Max DNA Polymerase (2X), 12.5uL; add ddH2O to a total volume of 25uL.
[0039] PCR program: Step (1) 98℃, 1min; Step (2) 98℃, 10s; Step (3) 55℃, 10s; Step (4) 72℃, 6min. After cycling (2)-(4) 15 times, cool to 4℃. Obtain PCR product.
[0040] After washing, the PCR products were digested using the restriction endonuclease DpnI, which specifically recognizes methylation sites, to degrade the template plasmid. The enzyme digestion reaction system and conditions were as follows: 17 μL of washed PCR product, 2.0 μL of 10× buffer, 1.0 μL of restriction endonuclease DpnI, and incubated at 37°C for 1 h.
[0041] The PCR product obtained from the above enzyme digestion treatment was transformed into Escherichia coli BL21 ( E. coli Recombinant Escherichia coli were obtained from BL21 and DE3, plated on agar plates containing kanamycin, and incubated overnight at 37°C. Single colonies were randomly selected for colony PCR identification and sequencing verification. The colony PCR identification results are as follows: Figure 1 As shown, the results indicate that the recombinant expression vector containing the lipase mutant gene was successfully transformed into the expression host. E. coli In BL21(DE3), the nucleotide sequences of mutants A138S, Y251F, and A138S / Y251F were finally obtained. The sequencing results are shown in SBQ ID No. 3, SEQ ID No. 5, and SBQ ID No. 7 in the sequence listing, respectively, and the corresponding amino acid sequences of the encoded proteins are shown in SEQ ID No. 4, SEQ ID No. 6, and SEQ ID No. 8 in the sequence listing.
[0042] Example 2 Induced expression of lipase mutants The engineered bacteria constructed in Example 1 were inoculated into LB liquid medium containing 50 μg / mL kanamycin and cultured overnight at 37°C. Then, at a 1% inoculation rate (v / v), they were inoculated into 50 mL of LB medium containing 50 μg / mL kanamycin and cultured at 37°C and 200 rpm until the bacterial concentration reached 0 D. 600 When the concentration reaches approximately 0.6, add isopropyl-β-D-thiogalactoside (IPTG) to a final concentration of 0.1 mM. After inducing culture at 30°C for 6 h, collect the bacterial cells by centrifugation at 4°C and 4000 rpm for 10 min, and store at -80°C for later use.
[0043] Example 3 Isolation and purification of lipase mutant enzymes The bacterial cells collected in Example 2 were suspended in 10 mL of 100 mM Na2HPO4-NaH2PO4 buffer (pH 8.0), shaken well, and then sonicated for 10 min. The lysate was centrifuged at 12,000 rpm for 15 min to remove cell debris, and the supernatant was collected as the crude enzyme solution for subsequent enzyme separation and purification. A Ni-NTA column with a packing volume of 5 mL was used for purification. The Ni-NTA column was first equilibrated with loading equilibration buffer (20 mM sodium phosphate, 500 mM NaCl, and 20 mM imidazole, pH 7.4). The crude enzyme solution was loaded at a rate of 5 mL / min, and eluted with loading equilibration buffer to remove unadsorbed protein. Finally, the target protein was collected by elution with elution buffer (20 mM sodium phosphate, 500 mM NaCl, and 500 mM imidazole, pH 7.4). The enzyme solution was desalted using a HiTrap desalting column. The desalting buffer was 100 mM Na2HP04-NaH2PO4, pH 7.5 buffer. The resulting pure enzyme solution of the lipase mutant was stored at 4°C for later use.
[0044] Example 4 Specific activity of lipases and their mutants The total volume of the reaction system was 1.0 mL, comprising: 10 mM lipid compound, 5% (w / w) glucose, and 1.0 mM Co. 2+ The enzyme contains 100 mM Na₂HPO₄-NaH₂PO₄ buffer, pH 7.5, and an appropriate amount of purified enzyme. The enzyme activity unit (U) is defined as: The amount of enzyme required to catalyze 1 μmol of substrate per minute at 35°C; specific activity is the enzyme activity U / mg per milligram of protein. The substrate is methyl 3-amino-3-(4-chlorophenyl)propionate. The specific activities and stereoselectivities of lipases (WT) and their mutants (A138S, Y251F, A138S / Y251F) catalyzing the corresponding substrates are shown in Table 2.
[0045] Table 2 Catalytic activity of lipases and their mutants As shown in Table 2, compared with lipase, the specific activity of the lipase mutant is higher than that of lipase, indicating that the catalytic activity of the mutant is significantly improved, and the stereoselectivity is not affected.
[0046] Example 5 Kinetic parameters of lipases and their mutants Under standard conditions, enzyme activity was determined by varying the substrate concentration in the reaction system, and the corresponding kinetic constants were calculated using the double reciprocal plot method. The substrate used in the kinetic constant calculation was methyl 3-amino-3-(4-chlorophenyl)propionate. The substrate concentration ranged from 2.5 to 20 mM. Apparent kinetic parameters of lipase (WT) and its mutants catalyzing the corresponding substrates: Michaelis constant. K m (mM), catalytic constant Kcat (min) -1 ), catalytic efficiency of enzymes K cat / K m(min) -1 mM -1 As shown in Table 3.
[0047] Table 3 Apparent kinetic parameters of lipases and their mutant lipolytic enzymes The results showed that the lipase mutant exhibited a lower Km (Michaelis constant) value than the lipase, indicating an increased affinity between the substrate and the reductase mutant. Compared to the lipase, the lipase mutant showed significantly higher Kcat / Km values, indicating a significant improvement in catalytic efficiency.
[0048] Example 6 Lipase and its mutant A138S / Y251F convert high concentrations of methyl 3-amino-3-(4-chlorophenyl)propionate. The total volume of the reaction system was 10.0 mL, including: 0.4 g wet bacterial cells, 100 mM methyl 3-amino-3-(4-chlorophenyl)propionate, 5% (w / w) glucose, and 1.0 mM Co. 2+ 10.0 mL of 100 mM Na₂HPO₄-NaH₂PO₄ buffer, pH 7.5, was used. The reaction was carried out at 35°C and 200 rpm. The catalytic activity of the whole-cell lipase-recombinant *E. coli* (lipase reaction group) was significantly lower than that of the mutant A138S / Y251F. After 5.5 h of reaction, the whole-cell catalytic yield of the mutant A138S / Y251F reached over 46%, with an ee value of 99.7%, while the yield of the lipase reaction group was only 38.5% and 99.2%. After 8 h of reaction, the whole-cell catalytic rate of the recombinant *E. coli* lipase decreased significantly, and by 13 h, the conversion rate reached only 35%, and the ee value also showed a decreasing trend. However, the mutant A138S / Y251F of this invention could still maintain a stable high conversion rate and high selectivity. In addition, the mutant A138S / Y251F still exhibited good stereoselectivity when catalyzing the conversion of high-concentration substrates, and the product ee value remained above 99.7%.
[0049] Example 7 Lipase and its mutant A138S convert high concentrations of methyl 3-amino-3-(4-chlorophenyl)propionate. The total volume of the reaction system was 10.0 mL, including: 0.4 g wet bacterial cells, 100 mM methyl 3-amino-3-(4-chlorophenyl)propionate, 5% (w / w) glucose, and 1.0 mM Co. 2+ The reaction was carried out at 35℃ and 200 rpm using 10.0 mL of 100 mM Na₂HPO₄-NaH₂PO₄ buffer, pH 7.5. The catalytic activity of the whole-cell lipase group (recombinant *E. coli* lipase reaction group) was significantly lower than that of the mutant A138S. After 7 h of reaction, the whole-cell catalytic yield of the mutant A138S reached over 45%, with an ee value of over 99.7%, while the yield of the lipase reaction group was only 34.2% and 99.1%. After 8.5 h of reaction, the conversion rate of the whole-cell lipase group no longer increased, reaching only 28% by 13 h. Furthermore, the mutant A138S still exhibited good stereoselectivity when catalyzing high-concentration substrate conversion, maintaining an ee value of over 99.7% and a yield of over 45%.
[0050] The chiral purity liquid chromatography spectrum of mutant A138S converted to high concentration of methyl 3-amino-3-(4-chlorophenyl)propionate is shown in the figure below. Figure 2 As shown, the NMR spectrum is as follows Figure 3 As shown, the liquid phase spectrum is as follows Figure 4 As shown; from Figure 2 It can be seen that the purity of the product ee is above 99.7%; from Figure 3 It can be seen that the mutant A138S of this invention successfully transformed high-concentration methyl 3-amino-3-(4-chlorophenyl)propionate, yielding the target product. Furthermore, the high purity of the product can be observed from NMR spectroscopy. Figure 4 It can be seen that the product has a very high purity and almost no other impurities.
[0051] Example 8 Lipase and its mutant Y251F convert high concentrations of methyl 3-amino-3-(4-chlorophenyl)propionate. The total volume of the reaction system was 10.0 mL, including: 0.4 g wet bacterial cells, 100 mM methyl 3-amino-3-(4-chlorophenyl)propionate, 5% (w / w) glucose, and 1.0 mM Co. 2+ The reaction was carried out at 35℃ and 200 rpm using 10.0 mL of 100 mM Na2HPO4-NaH2PO4 buffer at pH 7.5. The whole-cell catalytic activity of the recombinant E. coli lipase was significantly lower than that of the mutant Y251F. After 1 h of reaction, the whole-cell catalytic yield of the mutant Y251F reached over 48%, while the yield of the lipase group was only 41.3%. After 2.5 h of reaction, the conversion rate of the whole-cell catalytic reaction of the recombinant E. coli lipase reached 38%. Furthermore, the mutant Y251F still exhibited good stereoselectivity when catalyzing high-concentration substrate conversion, with an ee value of over 99.7% and a yield of over 48%.
[0052] The above embodiments are only used to illustrate the technical solutions of this application, and are not intended to limit it. Although this application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of this application.
Claims
1. A lipase mutant, characterized in that, Based on the lipase with the nucleotide sequence of SEQ ID No. 1 and the amino acid sequence of SEQ ID No. 2, single-point mutation or double-point mutation is carried out at the 138th arginine and the 251st tyrosine to obtain a lipase mutant.
2. The lipase mutant according to claim 1, characterized in that, The 138th arginine is mutated to serine, the nucleotide sequence of the lipase mutant is SEQ ID No. 3, and the amino acid sequence of the lipase mutant is SEQ ID No.
4.
3. The lipase mutant according to claim 1, characterized in that, The 251st tyrosine is mutated to phenylalanine, the nucleotide sequence of the lipase mutant is SEQ ID No. 5, and the amino acid sequence of the lipase mutant is SEQ ID No.
6.
4. The lipase mutant according to claim 1, characterized in that, The 138th arginine is mutated to serine and the 251st tyrosine is mutated to phenylalanine, the nucleotide sequence of the lipase mutant is SEQ ID No. 7, and the amino acid sequence of the lipase mutant is SEQ ID No.
8.
5. A process for the production of the lipase mutant according to any one of claims 1 to 4, characterized in that, The recombinant expression transformant of the lipase mutant is cultured, and the recombinant lipase mutant protein is induced and obtained.
6. The method of claim 5, wherein the lipase mutant is prepared by, The recombinant expression transformant was inoculated into LB medium containing kanamycin and cultured, and when the OD 600 of the culture solution reached 0.5-0.7, the recombinant lipase mutant was obtained under induction of isopropyl-B-D-thiogalactopyranoside at a final concentration of 0.1-1.0 mM; the recombinant expression transformant was recombinant Escherichia coli.
7. Use of a lipase mutant according to any one of claims 1 to 4, characterized in that, A recombinant expression vector is prepared.
8. Use of a lipase mutant according to claim 7, characterized in that, The recombinant expression vector is transformed into a host microorganism to obtain a genetically engineered bacterium expressing a recombinant lipase mutant.
9. Use of a lipase mutant according to claim 8, characterized in that, The lipase mutant, the recombinant expression vector or the genetically engineered bacterium expressing the recombinant lipase mutant is used for preparing chiral (S)-p-chloro-β-phenylalaninol.
10. Use of a lipase mutant according to claim 9, characterized in that, 3-Amino-3-(4-chlorophenyl)propionic acid methyl ester is used as a substrate, and the lipase mutant, the recombinant expression vector or the genetically engineered bacterium is used as a catalyst to react in a conversion reaction system composed of a buffer solution with pH 5.5-10, and after the reaction is completed, the reaction solution is separated and purified to obtain S-type 3-amino-3-(4-chlorophenyl)propionic acid, and then chiral (S)-p-chloro-β-phenylalaninol is obtained by reduction.