Lipase variants and their use

Abstract

The present invention provides a lipase variant and its use, specifically, based on the amino acid sequence shown in SEQ ID NO:1, one or more mutations selected from the group consisting of A262H, A338V, V364I, A158P / V and I159N occur, and compared with the parent lipase, the lipase variant has a modified protein structure and function, improved stereoselectivity, a reduced amount of enzyme used to a certain extent, and reduced difficulty in post-treatment, making it suitable for industrial production.

Description

Technical Field

[0001] The present invention relates to the field of industrial enzymes, and specifically to lipase variants and their use.

Background Art

[0002] Currently, as methods commonly used in the industrial application of chiral drug synthesis, there are chemical catalysis methods and enzyme catalysis methods. In chemical catalysis, an alkali metal, which is inexpensive, has a mature process, and is relatively easy for large-scale production, is used as a catalyst. However, the reaction of this method has a certain degree of randomness, poor selectivity, no specificity in the product, many by-products, and low yield. In contrast, enzyme catalysis in the enzyme method has advantages such as mild catalysis conditions, being eco-friendly and environmentally friendly, and fewer by-products in the reaction. For example, when resolving acid, alcohol, and ester-based chiral drugs, generally, a chemical method is used to synthesize a racemic compound such as the corresponding methyl ester, ethyl ester, or propyl ester, and then stereoselective hydrolysis is performed using lipase or esterase to obtain a chiral unit with a single enantiomeric conformation.

[0003] Lipase is an enzyme widely present in animals, plants, and microorganisms, and can catalyze reactions such as ammonolysis, alcoholysis, esterification, transesterification, and the reverse synthesis of esters. Commonly used lipases include porcine pancreatic lipase, Candida lipase, Pseudomonas lipase, and Mucor lipase (Use of biocatalysts in chiral drug synthesis [J]. Amino Acids & Biotic Resources, 2013, 35(4): 39-42). For example, the invention patent application with the application disclosure number CN108642025A discloses that the 1,3-position selectivity of the mutants M8A, L10A, and T9A derived from Humicola lanuginosa lipase is 4.75 times higher than that of the wild-type enzyme. The mutant was applied to the catalytic reaction of human milk fat substitutes, and significant effects were obtained.

[0004] Microbial lipases have advantages such as high stereoselectivity, a wide catalytic temperature range, high transformation efficiency, and few by-products. They are commonly used in the preparation of organic synthesis intermediates such as the resolution of catalytic chiral, single chiral alcohol systems, amine systems, and ester systems (Research progress on the immobilization of lipase and its chiral resolution [J]. Applied Chemical Industry, 2011, 40(10): 1823 - 1827). For example, the herbicide (R)-α-phenoxypropyl and the anti-inflammatory agent (S)-phenylpropyl alcohol are both transformed into single active phenoxypropyl by the stereoselectivity of lipase.

[0005] However, current existing commercial lipases have the problem of high cost. In industrial production, especially when the substrate exists in the form of a racemic compound, the stereoselectivity of most wild-type lipases is poor, and it is almost impossible to directly obtain wild-type industrial enzymes that catalyze single-conformation substrates. Moreover, many wild-type enzymes have disadvantages such as low catalytic efficiency and weak stability. Therefore, there are not many stereoselective lipases that can be actually widely used.

Summary of the Invention

Problems to be Solved by the Invention

[0006] The main object of the present invention is to provide a lipase mutant and its use to solve the problem in the prior art that the stereoselectivity of industrial lipases is low.

Means for Solving the Problems

[0007] To achieve the above object, according to one aspect of the present invention, a lipase mutant is provided. The lipase mutant has the following amino acid mutations based on SEQ ID NO: 1.

Table 1

[0008] Furthermore, the amino acid sequence of the lipase mutant has a homology of 90% or more, preferably 95% or more, more preferably 99% or more with the amino acid sequence where the mutation occurred, and the lipase mutant is derived from Pseudomonas putida and has lipase activity.

[0009] To achieve the above object, according to a second aspect of the present invention, there is provided a DNA molecule encoding the above lipase mutant.

[0010] According to a third aspect of the present invention, there is provided a recombinant plasmid to which the above DNA molecule is ligated.

[0011] Furthermore, the recombinant plasmid is any one selected from the group consisting of pET-21b(+), pET-22b(+), pET-3a(+), pET-3d(+), pET-11a(+), pET-12a(+), pET-14b, pET-15b(+), pET-16b(+), pET-17b(+), pET-19b(+), pET-20b(+), pET-21a(+), pET-23a(+), pET-23b(+), pET-24a(+), pET-25b(+), pET-26b(+), pET-27b(+), pET-28a(+), pET-29a(+), pET-30a(+), pET-31b(+), pET-32a(+), pET-35b(+), pET-38b(+), pET-39b(+), pET-40b(+), pET-41a(+), pET-41b(+), pET-42a(+), pET-43a(+), pET-43b(+), pET-44a(+), pET-49b(+), pQE2, pQE9, pQE30, pQE31, pQE32, pQE40, pQE70, pQE80, pRSET-A, pRSET-B, pRSET-C, pGEX-5X-1, pGEX-6p-1, pGEX-6p-2, pBV220, pBV221, pBV222, pTrc99A, pTwin1, pEZZ18, pKK232-8, pUC-18 and pUC-19.

[0012] According to a fourth aspect of the present invention, there is provided a non-plant host cell containing any one of the above recombinant plasmids.

[0013] Furthermore, the host cell is a prokaryotic cell or a eukaryotic cell, and the eukaryotic cell is a yeast cell.

[0014] Furthermore, the host cell is a competent cell.

[0015] Furthermore, the competent cell is Escherichia coli BL21 cell or Escherichia coli W3110.

[0016] According to the fifth aspect of the present invention, a method for preparing a chiral compound is provided, and the preparation method includes catalyzing the hydrolysis of an ester compound represented by Formula I into an acid compound represented by Formula II and an alcohol compound represented by Formula III by using any one of the above lipase mutants.

Chemical formula

[0017] Furthermore, the ester compound is

Chemical formula

[0018] Furthermore, the lipase mutant catalyzes the hydrolysis reaction of the ester compound represented by Formula I at a temperature of 15°C to 30°C.

[0019] Furthermore, the mass ratio of the amount of the lipase mutant bacterial sludge to the ester compound is 1:10 to 1:1.

[0020] Furthermore, the reaction system contains any one organic solvent selected from the group consisting of DMSO, acetone, dimethyltetrahydrofuran, isopropyl alcohol and n-propanol.

[0021] Furthermore, the volume percentage content of the organic solvent in the reaction system is 5% to 10%.

Advantages of the Invention

[0022] When the technical solution of the present invention is applied, based on the amino acid sequence shown in SEQ ID NO: 1, the lipase mutant obtained by reasonable design and several rounds of enzyme evolution and screening has its protein structure and function changed. In actual application, the stereoselectivity of the lipase mutant is extremely improved. In addition, because the stereoselectivity of the lipase mutant is improved, the amount of enzyme used is reduced to a certain extent, and the difficulty of post-treatment is also reduced, which is suitable for industrial production.

Embodiments for Carrying Out the Invention

[0023] In addition, the embodiments and features of the embodiments in this application can be combined with each other as long as they do not conflict. Hereinafter, the present invention will be described in detail with reference to the embodiments.

[0024] As mentioned in the background art, the stereoselectivity of existing industrial lipases is low. To improve this situation, the present invention obtained a series of lipase mutants with improved stereoselectivity through reasonable design and several rounds of enzyme evolution and screening. Based on this, the applicant submitted the solution of this application.

[0025] In a typical embodiment, a lipase mutant is provided, and the amino acid sequence of the lipase mutant is obtained by mutating the amino acid sequence shown in SEQ ID NO: 1 as follows,

Table 2

[0026] The amino acid sequence of the lipase variant has mutations in the amino acid sequence shown in SEQ ID NO: 1, and the important sites where related mutations occur include, but are not limited to, one or more sites among A262, A338, V364, A158, I159, I245, L65, F66, S67, C68, R123, T160, V226, L234, T236, R237, P243, A244, N263, H335, P336, G337, Y363 and L365.

[0027] In addition, the inventors screened the catalysis of more than 100 wild-type lipases derived from different sources against the substrate of this type (for example, Substrate 1), and found that the e.e. values for catalyzing selectivity were all very low (<5%). In contrast, the e.e. value of the catalytic reaction with the lipase of SEQ ID NO: 1 (derived from Pseudomonas putida) was the best. Nevertheless, since its e.e. value was not sufficiently ideal, it was selected to perform evolution based on the lipase of SEQ ID NO: 1.

[0028] The above lipase variant was obtained by reasonable design and several rounds of enzyme evolution and screening based on the amino acid sequence shown in SEQ ID NO: 1, realizing changes in protein structure and function. In actual application, the stereoselectivity of the lipase was greatly improved. In addition, due to the improvement of the stereoselectivity of the lipase variant, the amount of enzyme used decreased to a certain extent, and the difficulty of post-treatment also decreased, making it suitable for industrial production.

[0029] The amino acid sequence of SEQ ID NO: 1 is specifically as follows.

Chemical formula

[0030] The corresponding nucleic acid sequence SEQ ID NO: 2 is as follows.

Chemical formula

[0031] The above-mentioned reasonable design and specific methods or steps of enzyme evolution and screening include, but are not limited to, those exemplified below.

[0032] First, by means of full plasmid PCR, mutation sites are introduced into SEQ ID NO: 1, the activities and selectivities of mutants are detected, and mutants with improved activities and selectivities are selected.

[0033] Using SEQ ID NO: 1 as a template, site-directed mutagenesis primers (refer to Table 1 for site-directed mutagenesis sites) are designed, and site-directed mutagenesis means are utilized to obtain a mutant plasmid carrying the target gene with pET-22b(+) as the expression vector.

[0034] Here, site-directed mutagenesis refers to introducing necessary changes (usually changes characterizing the advantageous direction) into a target DNA fragment (which can be a genome or a plasmid) by methods such as polymerase chain reaction (PCR), including base addition, deletion, site mutation, etc. Site-directed mutagenesis can rapidly and efficiently improve the properties and characteristics of the target protein expressed by DNA and is a very useful means in gene research work.

[0035] The method of introducing site-directed mutations using full plasmid PCR is simple and effective and is a commonly used means at present. The principle is as follows. After annealing a pair of primers (forward and reverse) containing the mutation site and the template plasmid, "circular extension" is carried out using polymerase. So-called circular extension means that the polymerase extends the primer around the template once, and when it returns to the 5' end of the primer, it ends, and then the cycle of heating, annealing, and extension is repeated. This reaction, different from rolling circle amplification, does not form multiple tandem copies. The extension products of the forward and reverse primers are paired after annealing to become a nicked open circular plasmid. The extension products are cleaved with Dpn I enzyme. Since the original template plasmid is derived from common Escherichia coli, it is dam-methylated and sensitive to Dpn I and is cleaved. However, the plasmid with the mutated sequence synthesized in vitro is not methylated and is not cleaved. Therefore, subsequent transformation is successful, and clones of the mutated plasmid can be obtained.

[0036] Based on obtaining mutants with improved properties by single-site mutations, beneficial amino acid sites can be combined to obtain mutants with more excellent properties.

[0037] After obtaining a lipase mutant with significantly improved activity and enantioselectivity, it is randomly mutated using the error-prone PCR method to construct a high-quality mutant library, and an appropriate high-throughput screening method is developed to screen the library to obtain mutants with further improved stereoselectivity.

[0038] Error-prone PCR refers to PCR under error-prone conditions, that is, a PCR technique in which errors are likely to occur in the copied DNA sequence, and is also called mismatch PCR or biased error PCR. Specifically, by using a low-fidelity Taq DNA polymerase and changing the PCR reaction conditions, the fidelity of DNA copying is reduced, the number of base mismatches in the synthesis process of the new DNA strand is increased, and thereby, a method for in vitro inducing DNA sequence mutations with multiple-site mutations occurring in the amplification product is provided.

[0039] Error-prone PCR is currently the simplest and most effective in vitro random mutagenesis technique for genes, and its principle is as follows. Mismatches are made possible by the isomerization of bases. Each of the four bases that make up DNA has tautomers. Here, the three oxygen-containing bases, guanine (G), cytosine (C), and thymine (T), have two tautomers, the keto form and the enol form. The two nitrogen-containing bases, adenine (A) and thymine, have two tautomers, the amine form and the imine form. G, C, and T mainly exist in the keto form structure, and the proportion of the enol form structure is extremely low. The nitrogen atoms on the two nitrogen-containing bases of A and T mainly exist in the amino group (NH2) state, and the proportion of the imino group (NH) state is extremely low. Due to the difference in the position of hydrogen atoms between different isomers and the difference in the direction of the shift of the electron cloud at the same position, the base pairing form can be changed, and in this way, there is a possibility of mismatch occurring in the daughter strand after copying. For example, when thymine exists in the keto form structure, it pairs with adenine, and when it exists in the enol form structure, it pairs with guanine. In this way, an unstable base pair in which A can pair with C and T can pair with G appears, thereby causing a mismatch.

[0040] Among several known heat-resistant DNA polymerases, Taq DNA polymerase has the highest mismatch rate. Taq DNA polymerase is one of the heat-resistant DNA polymerases with the highest activity. It has 5'-3' exonuclease activity but no 3'-5' exonuclease activity. Therefore, during synthesis, it has no correction function for mismatches of some single nucleotides. As a result, the probability of mismatches occurring is higher than that of DNA polymerases with 3'-5' proofreading activity. The use of four dNTPs with different concentrations, the addition of Mn 2+ , the improvement of Mg 2+ concentration, and other various methods can reduce the fidelity of DNA polymerase. There are several mutagenesis methods, and the mechanisms of base mutations for amplifying DNA strands are different. MnC12 is a mutagenesis factor for DNA polymerase. When Mn 2+ is added, the specificity of the polymerase for the template decreases, the mismatch rate increases, and the probability of incorrect base incorporation due to the imbalance of the four dNTPs concentrations is improved, thus realizing mismatches. Mg 2+ has the effect of activating Taq enzyme. By increasing the Mg 2+ concentration beyond the normal usage amount, non-complementary base pairs can be stabilized. By increasing the usage amount of Taq DNA polymerase and the elongation time of each cycle, the elongation probability of the mismatch end can be increased. When the concentration of the starting template is decreased, the ratio of mutant templates in subsequent PCR cycles increases.

[0041] By screening the mutant library constructed for error-prone PCR, lipase mutants with further improved activity and enantioselectivity are obtained. Also, to obtain mutants with significantly improved activity and enantioselectivity, saturated mutagenesis primers are designed to further evolve the mutants.

[0042] Saturation mutagenesis is a method that can obtain mutants in which the amino acids at the target site are each substituted with 19 other types of amino acids in a short period of time by modifying the coding gene of the target protein. This method is not only a powerful tool for directed modification of proteins but also an important means for studying protein structure-function relationships. Saturation mutagenesis can often obtain more ideal evolved forms than single-site mutagenesis. These problems that cannot be solved by site-directed mutagenesis methods are the unique features of saturation mutagenesis methods.

[0043] As described above, the mutant plasmid is transformed into E. coli cells and overexpressed in E. coli. Then, crude enzyme is obtained by a method of disrupting cells with ultrasonic waves. The optimal conditions for inducing lipase expression are to induce expression with 0.06 mM IPTG at 20 °C for 16 h.

[0044] The screened mutants of the present application have been verified by a large number of experiments. When the substrate existing in the racemic compound form is not overly transformed, finally, it is proved that the enantiomeric excess (e.e.) of the S-type product of the enzyme-catalyzed reaction has improved from less than 5% initially to at least 80% or more, greatly meeting the needs of industrial production.

[0045] In a typical embodiment of the present invention, further provided is a DNA molecule encoding any one of the above lipase mutants. The encoded lipase mutant has the advantage of high selectivity.

[0046] In a typical embodiment of the present invention, further provided is a recombinant plasmid to which the above DNA molecule is ligated. The DNA molecule can encode any one of the above highly selective lipase mutants. The specific sequence is selected from the sequences in Tables 1 to 5, or nucleotide sequences in which mutations such as substitution, addition, or deletion have occurred in the amino acid sequences of other sites on the premise of maintaining the amino acid site changes in these sequences.

[0047] Among the above recombinant plasmids, any recombinant plasmid that can be used to express the DNA molecule of the above lipase is suitable for the present invention. In a preferred embodiment of the present invention, the recombinant plasmid is one selected from the group consisting of pET-22b(+), pET-21b(+), pET-3a(+), pET-3d(+), pET-11a(+), pET-12a(+), pET-14b, pET-15b(+), pET-16b(+), pET-17b(+), pET-19b(+), pET-20b(+), pET-21a(+), pET-23a(+), pET-23b(+), pET-24a(+), pET-25b(+), pET-26b(+), pET-27b(+), pET-28a(+), pET-29a(+), pET-30a(+), pET-31b(+), pET-32a(+), pET-35b(+), pET-38b(+), pET-39b(+), pET-40b(+), pET-41a(+), pET-41b(+), pET-42a(+), pET-43a(+), pET-43b(+), pET-44a(+), pET-49b(+), pQE2, pQE9, pQE30, pQE31, pQE32, pQE40, pQE70, pQE80, pRSET-A, pRSET-B, pRSET-C, pGEX-5X-1, pGEX-6p-1, pGEX-6p-2, pBV220, pBV221, pBV222, pTrc99A, pTwin1, pEZZ18, pKK232-8, pUC-18 and pUC-19.

[0048] In a typical embodiment of the present invention, further provided is a non-plant host cell containing any one of the above recombinant plasmids. The specific host cell may be a prokaryotic cell or a eukaryotic cell. Preferably, the eukaryotic cell is a yeast cell. More preferably, the above host cell is a competent cell, and even more preferably, the competent cell is Escherichia coli BL21 cell or Escherichia coli W3110.

[0049] In a typical embodiment of the present invention, further provided is a method for preparing a chiral compound, the preparation method including using any one of the lipase mutants to catalyze the hydrolysis of an ester compound represented by Formula I into an acid compound represented by Formula II and an alcohol compound represented by Formula III.

Chemical formula

[0050] In a preferred embodiment, the ester compound is

Chemical formula

[0051] In a preferred embodiment, the lipase mutant catalyzes the hydrolysis reaction of the ester compound represented by Formula I at a temperature of 15°C to 30°C.

[0052] In a preferred embodiment, the mass ratio of the amount of the lipase mutant cell sludge to the ester compound is 1:10 to 1:1. The transformation efficiencies of different mutants are somewhat different, and the mass ratios used to achieve the same transformation e.e. value are also different.

[0053] In a preferred embodiment, the reaction system further contains an organic solvent which is any one selected from the group consisting of DMSO, acetone, dimethyltetrahydrofuran, isopropyl alcohol and n-propanol.

[0054] In a preferred embodiment, the volume percentage content of the organic solvent in the reaction system is 5% - 10%. When the volume percentage content of the organic solvent is within this range, all the variants of the present application still have high stereoselectivity.

[0055] Hereinafter, with reference to specific examples, the beneficial effects of the present application will be further described. The substrates used in the following examples include the following.

Chemical formula

[0056] (Example 1) Take 10 mg each of Substrate 1 / Substrate 2 / Substrate 3, and sequentially add to the reaction system 1 mg of resuspended lipase or its mutant cell sludge, and 0.3 M KPB Buffer (54.91 g of K2HPO4·3H2O, 8.08 g of KH2PO4, add 0.9 L of ultrapure water and dissolve, adjust the pH to 7.5, and then make up to 1 L) until the system reaches 1000 μL. React at a constant temperature of 30 °C at 200 rpm for 1 h. Add 60 μL of 6 M dilute hydrochloric acid to the system, mix uniformly to terminate the reaction, then add 1 mL of n-hexane for extraction, shake well, centrifuge at 12000 rpm for 2 min, take the upper layer into a sample flask and perform normal-phase HPLC to detect the e.e. value of the S-type product. The reaction characteristics of some mutants are as shown in Table 1 below.

Table 3

[0057] The high selectivity e.e. of the S-type product is represented by *, "-" represents that the selectivity of the parent and the mutant corresponding to the parent is 0 - 5%, * represents that the e.e. is 5% - 10%, ** represents that the e.e. is 15 - 20%, *** represents that the e.e. is 20 - 50%, **** represents that the e.e. is 50 - 80%, and ***** represents that the e.e. ≥ 80%.

[0058] (Example 2) Take 10 mg each of Substrate 1 and Substrate 4, and sequentially add 1 mg of the resuspended lipase or its mutant bacterial sludge and 0.3 M KPB Buffer with pH 7.5 to the reaction system, and supplement until the system reaches 1000 μL. React at 200 rpm and 30 °C for 1 h under constant temperature. Add 60 μL of 6 M dilute hydrochloric acid to the system, mix uniformly to terminate the reaction, then add 1 mL of n - hexane for extraction, shake well, centrifuge at 12000 rpm for 2 min, take the upper layer into a sample flask and perform normal - phase HPLC to detect the e.e. value of the S - type product. The reaction characteristics of some mutants are as shown in Table 2 below.

Table 4

[0059] The selectivity e.e. height of the S - type product is represented by *, “ - ” indicates that the selectivity of the parent is 0 - 5%, * indicates that the e.e. is 5% - 10%, ** indicates that the e.e. is 15 - 20%, *** indicates that the e.e. is 20 - 50%, **** indicates that the e.e. is 50 - 80%, and ***** indicates that the e.e. ≥ 80%.

[0060] (Example 3) Take 10 mg each of Substrate 1 / Substrate 2 / Substrate 3 / Substrate 4 / Substrate 5 / Substrate 6, and sequentially add 1 mg of the resuspended lipase or its mutant bacterial sludge and 0.3 M KPB Buffer with pH 7.5 to the reaction system, and supplement until the system reaches 1000 μL. React at 200 rpm and 30 °C for 1 h under constant temperature. Add 60 μL of 6 M dilute hydrochloric acid to the system, mix uniformly to terminate the reaction, then add 1 mL of n - hexane for extraction, shake well, centrifuge at 12000 rpm for 2 min, take the upper layer into a sample flask and perform normal - phase HPLC to detect the e.e. value of the S - type product. The reaction characteristics of some mutants are as shown in Table 3 below.

Table 5

[0061] The selectivity e.e. height of the S-type product is represented by *, "-" indicates that the selectivity of the parent and the variant corresponding to the parent is 0-5%, * indicates that the e.e. is 5%-10%, ** indicates that the e.e. is 15-20%, *** indicates that the e.e. is 20-50%, **** indicates that the e.e. is 50-80%, and ***** indicates that the e.e. ≥ 80%.

[0062] (Example 4) Take 10 mg / 100 mg / 1 g of Substrate 2 / Substrate 3 / Substrate 6 respectively, and sequentially add 1 mg of the resuspended lipase or its variant cell sludge and 0.3 M KPB Buffer with pH 7.5 to the reaction system, and supplement until the system reaches 1000 μL. React at a constant temperature of 30 °C and 200 rpm for 1 h. Add 60 μL of 6 M dilute hydrochloric acid to the system, mix uniformly to terminate the reaction, then add 1 mL of n-hexane for extraction, shake well, centrifuge at 12,000 rpm for 2 min, take the upper layer into a sample flask and perform normal-phase HPLC to detect the e.e. value of the S-type product. Three relatively good variants were selected and amplified stepwise from 10 mg to 100 mg and then to 1 g. The e.e. of the S-type product was clearly improved in all cases. The specific reaction characteristics are as shown in Table 4 below.

Table 6

[0063] The selectivity e.e. height of the S-type product is represented by *, "-" indicates that the selectivity of the parent is 0-5%, * indicates that the e.e. is 5%-10%, ** indicates that the e.e. is 15-20%, *** indicates that the e.e. is 20-50%, **** indicates that the e.e. is 50-80%, and ***** indicates that the e.e. ≥ 80%.

[0064] (Example 5) Take 10 mg each of Substrate 2 / Substrate 5, and sequentially add the resuspended lipase or its mutant cell paste and 0.3 M KPB Buffer at pH 7.5 to the reaction system, and supplement until the system reaches 1000 μL. React at different reaction temperatures of 15 °C / 20 °C / 30 °C at 200 rpm for 1 h, and investigate the effect of temperature on the selectivity e.e. of the S-type product.

[0065] Also, add DMSO, acetone, dimethyltetrahydrofuran, isopropyl alcohol, and n-propanol with volume percentages of 5% and 10% respectively to the system, add 0.3 M KPB Buffer at pH 7.5, and supplement until the system reaches 1000 μL. React at a constant temperature of 20 °C for 1 h, and investigate the effect of different additives on the selectivity e.e. of the S-type product.

[0066] Add 60 μL of 6 M dilute hydrochloric acid to the above system, mix uniformly to terminate the reaction, then add 1 mL of n-hexane for extraction, shake well, centrifuge at 12000 rpm for 2 min, take the upper layer into a sample flask and perform normal-phase HPLC to detect the e.e. value of the S-type product. The specific reaction characteristics are as shown in Table 5 below.

Table 7

[0067] The height of the selectivity e.e. of the S-type product is represented by *, "-" indicates that the selectivity of the parent is 0 - 5%, * indicates that the e.e. is 5% - 10%, ** indicates that the e.e. is 15 - 20%, *** indicates that the e.e. is 20 - 50%, **** indicates that the e.e. is 50 - 80%, and ***** indicates that the e.e. ≥ 80%.

[0068] As can be seen from the above description, the following technical effects have been achieved by the above embodiments of the present invention. Through reasonable design and a series of evolution and screening for the amino acid sequence of lipase represented by SEQ ID NO: 1, lipase mutants with changed structures and functions are obtained. When these lipase mutants are used in catalytic reactions, the enantiomeric excess (e.e.) value of the stereoselectivity for producing S-type products is significantly improved. Thus, from the initial situation where it is almost impossible to produce selective S-type products using the enzyme-catalyzed method, when controlled to a certain conversion rate, S-type products with a selectivity greater than at least 80% can be obtained, greatly meeting the needs of industrial production.

[0069] The above description is only a preferred embodiment of the present invention and does not limit the present invention. For those skilled in the art, the present invention can be subject to various modifications and changes. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should all be included in the protection scope of the present invention.

Claims

1. A lipase mutant in which the following amino acid mutations have occurred based on SEQ ID NO: 1, 【Table 1】 or an amino acid sequence having a mutation site in the amino acid sequence in which the mutation has occurred, having 90% or more identity with the amino acid sequence in which the mutation has occurred, derived from Pseudomonas putida, and having lipase activity, characterized in that it is a lipase mutant.

2. An amino acid sequence having 95% or more identity with the amino acid sequence in which the mutation has occurred, derived from Pseudomonas putida, and having lipase activity, characterized in that it is the lipase mutant according to Claim 1.

3. An amino acid sequence having 99% or more identity with the amino acid sequence in which the mutation has occurred, derived from Pseudomonas putida, and having lipase activity, characterized in that it is the lipase mutant according to Claim 1.

4. An amino acid sequence having 99.5% or more identity with the amino acid sequence in which the mutation has occurred, derived from Pseudomonas putida, and having lipase activity, characterized in that it is the lipase mutant according to Claim 1.

5. Encoding the lipase mutant according to any one of Claims 1 to 4, characterized in that it is a DNA molecule.

6. Characterized in that the DNA molecule according to Claim 5 is ligated, characterized in that it is a recombinant plasmid.

7. The recombinant plasmid is any one selected from the group consisting of pET-21b(+), pET-22b(+), pET-3a(+), pET-3d(+), pET-11a(+), pET-12a(+), pET-14b, pET-15b(+), pET-16b(+), pET-17b(+), pET-19b(+), pET-20b(+), pET-21a(+), pET-23a(+), pET-23b(+), pET-24a(+), pET-25b(+), pET-26b(+), pET-27b(+), pET-28a(+), pET-29a(+), pET-30a(+), pET-31b(+), pET-32a(+), pET-35b(+), pET-38b(+), pET-39b(+), pET-40b(+), pET-41a(+), pET-41b(+), pET-42a(+), pET-43a(+), pET-43b(+), pET-44a(+), pET-49b(+), pQE2, pQE9, pQE30, pQE31, pQE32, pQE40, pQE70, pQE80, pRSET-A, pRSET-B, pRSET-C, pGEX-5X-1, pGEX-6p-1, pGEX-6p-2, pBV220, pBV221, pBV222, pTrc99A, pTwin1, pEZZ18, pKK232-8, pUC-18 and pUC-19. The recombinant plasmid according to claim 6, characterized in that.

8. A plant-free host cell containing the recombinant plasmid according to claim 6 or 7. Characterized by being a non-plant host cell.

9. It is a prokaryotic cell or a eukaryotic cell, and the eukaryotic cell is a yeast cell. The host cell according to claim 8, characterized in that.

10. The host cell is a competent cell. The host cell according to claim 8, characterized in that.

11. The competent cell is Escherichia coli BL21 cell or Escherichia coli W3110. The host cell according to claim 10, characterized in that.

12. Using the lipase mutant according to any one of claims 1 to 4, catalyzing the hydrolysis of the ester compound represented by formula I into the acid compound represented by formula II and the alcohol compound represented by formula III. 【Chemical 1】 Here, R １ is any one selected from CH ３ , CH ２ CH ３ , CH ２ -CH ２ CH ３ or CHCH ３ CH ３ and is any one selected therefrom, R ２ 、R ３ 、R ４ and R ５ are each independently any one selected from H, F, Cl, Br, I, OH, CH ３ or CH ２ CH ３ and is one of the selections, The cyclohexane ring may or may not have a double bond. If present, the double bond is between R ３ and R ４ between R ５ and R ６ between R ７ and R ８ between R ９ and R １０ and is formed in any one or more of the intervals between them. A method for preparing a chiral compound, characterized in that.

13. The ester compound is. 【Chemical Formula 2】 Any one of. The preparation method according to claim 12, characterized in that.

14. The lipase variant catalyzes the hydrolysis reaction of the ester compound represented by Formula I at a temperature of 15°C to 30°C. The preparation method according to claim 12, characterized in that.

15. The mass ratio of the amount of bacterial sludge of the lipase variant to the ester compound is 1:10 to 1:

1. The preparation method according to claim 12, characterized in that.

16. The reaction system further contains any one organic solvent selected from the group consisting of DMSO, acetone, dimethyltetrahydrofuran, isopropyl alcohol and n-propanol. The preparation method according to claim 12, characterized in that.

17. The volume percentage content of the organic solvent in the reaction system is 5% to 10%. The preparation method according to claim 16, characterized in that.

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