Esterase mutants and application thereof

Abstract

Provided are esterase mutants and the application thereof. The esterase mutants are SEQ ID NO: 1 mutation sequences, wherein the mutations comprise I245S; or the esterase mutants comprise an I245S mutation. Said sequences are 99% or more homologous to SEQ ID NO: 1 and have esterase catalytic activity. The esterase mutants are mutations carried out my means of a site directed mutagenesis method based on the esterase shown in SEQ ID NO: 1. The amino acid sequence of the esterase is changed, resulting in a change of protein structure and function, and then a directional screening method is used to obtain esterases having the described mutation sites. These esterase mutants have the advantage of greatly improved enzyme reaction activity and specificity, thereby greatly reducing the amount of enzyme used and the cost of industrial production. Moreover, the esterases have enhanced tolerance to organic solvents, a wide range of reaction temperatures and pH values, and stronger application advantages.

Description

Esterase mutants and their applications Technical Field

[0001] The present invention relates to the field of biotechnology, in particular to an esterase mutant and application thereof. Background Art

[0002] With the development of pharmaceuticals, pesticides, and other fine chemical industries, organic synthesis faces increasing challenges. For example, due to chiral recognition in organisms, often only one stereoisomer in a drug molecule has a therapeutic effect, while the other stereoisomers have no therapeutic effect or even have side effects. Therefore, to synthesize therapeutic stereoisomers, traditional organic synthesis often requires the introduction of protection and deprotection steps to compensate for the lack of chemical and regioselectivity. However, such operations not only increase process complexity and production costs, but also require the use of relatively large amounts of organic solvents, sometimes toxic or volatile solvents, which can easily cause environmental pollution.

[0003] Biocatalytic reactions operate under mild conditions, typically at or near neutral and room temperature. In most cases, they occur in aqueous phase, resulting in minimal environmental pollution. Furthermore, biocatalytic reactions typically exhibit high chemoselectivity, regioselectivity, and stereoselectivity, eliminating unnecessary protection and deprotection steps, significantly optimizing production processes and reducing production costs. Therefore, the application of biocatalytic technology in organic synthesis holds immense scientific significance and practical value.

[0004] Esterase is a general term for a class of enzymes that have the ability to hydrolyze ester bonds. It is widely present in animals, plants and microorganisms. It can be used in organic synthesis and is also the most studied enzyme in genetic engineering.

[0005] Although enzymes usually have good reactivity and selectivity for their natural substrates, their reactivity, stability and selectivity for non-natural substrates are generally not very good. Usually, they can be modified through directed evolution to improve their reactivity, stability and selectivity for non-natural substrates so that they can be used in production.

[0006] Summary of the Invention

[0007] The present invention aims to provide an esterase mutant and application thereof, so as to improve the reaction activity of the enzyme.

[0008] To achieve the above objectives, according to one aspect of the present invention, an esterase mutant is provided. The esterase mutant is a sequence mutated from SEQ ID NO: 1, wherein the mutation includes I245S; or the esterase mutant includes the I245S mutation and has greater than 99% homology to SEQ ID NO: 1, and has esterase catalytic activity.

[0009] Furthermore, the mutations include any one or more of the following: C33A, A46G, S48N, L65V, T71V, T71M, T87E, L131S, L131N, L131H, R184E, R210Y, K212R, K212M, S230Q, P231T, S235Q, T240F, T240L, T240C, S248D, S248M, K251T, K251L, H262A, H262S, H262C, P336C or L365M.

[0010] Furthermore, the mutation includes any of the following combination mutations: I245S+C33A, I245S+A46G, I245S+S48N, I245S+L65V, I245S+T71V, I245S+T71M, I245S+T87E, I245S+L131N, I245S+R184E, I245S+R210Y, I245S+K212R, I245S+S230Q, I245S+P231T, I245S+T240F, I245S+T240L, I245S+S248D, I245S+K251T, I245S+K251 L, I245S+H262A, I245S+H262S, I245S+P336C, I245S+L365M, I245S+L65 V+T71V, I245S+L65V+T71M, I245S+L65V+T87E, I245S+L65V+R210Y, I2 45S+L65V+K212R, I245S+L65V+P231T, I245S+L65V+T240F, I245S+L65V +T240L, I245S+L65V+K251T, I245S+L65V+L365M, I245S+L65V+H262A, I245S+L65V+H262S, I245S+T71V+C33A, I245S+T71V+L65V, I245S+T71V +T87E, I245S+T71V+L131N, I245S+T71V+T240F, I245S+T71V+T240L, I245S+T71V+K251T, I245S+T71V+H262A, I245S+T71V+H262S, I245S+T7 1V+L365M, I245S+L65V+H262A+T87E, I245S+L65V+H262A+R210Y, I245 S+L65V+H262A+K212R, I245S+L65V+H262A+K212M, I245S+L65V+H262A+ T240F, I245S+L65V+H262A+T240L, I245S+L65V+H262A+T240C, I245S+ L65V+H262A+S248D, I245S+L65V+H262A+S248M, I245S+L65V+H262A+K2 51T, I245S+L65V+H262A+K251L, I245S+L65V+H262A+P336C, I245S+L6 5V+H262A+L365M, I245S+T71V+T240L+C33A, I245S+T71V+T240L+L65V,I245S+T71V+T240L+T87E、I245S+T71V+T240L+L131N、I245S+T71V+T240L+S235Q、I245S+T71V+T240L+K251T、I245S+T71V+T240L+H262A、I245S+T71V+T240L+H262S、I245S+T71V+T240L+P336C、I245S+T71V+T240L+L365M、I245S+T71V+T240F+L131N+S48N、I245S+T71V+T240L+K251T+L131N、I245S+T71V+T240L+K251T+R210Y、I245S+T71V+T240L+K251T+K212R、I245S+T71V+T240L+K251T+K212M、I245S+T71V+T240L+K251T+S235Q、I245S+T71V+T240L+K251T+H262A、I245S+T71V+T240L+K251T+H262S、I245S+T71V+T240L+K251T+P336C、I245S+T71V+T240L+K251T+L365M、I245S+T71V+T240L+T87E+L65V、I245S+T71V+T240L+T87E+L65V+R210Y、I245S+T71V+T240L+T87E+L65V+K212M、I245S+T71V+T240L+T87E+L65V+K251T、I245S+T71V+T240L+T87E+L65V+H262A、I245S+T71V+T240L+T87E+L65V+H262S、I245S+T71V+T240L+T87E+L65V+H262C、I245S+T71V+T240L+T87E+L65V+K251T+K212M、I245S+T71V+T240L+T87E+L65V+K251T+H262A、I245S+T71V+T240L+T87E+L65V+K251T+L365M、I245S+L65V+H262A+T240F+L131S、I245S+L65V+H262A+T240F+L131H、I245S+L65V+H262A+T240F+L131N、I245S+L65V+H262A+T240F+R210Y、I245S+L65V+H262A+T240F+K212M、I245S+L65V+H262A+T240F+P231T、I245S+L65V+H262A+T240F+K251T or I245S+L65V+H262A+T240F+L365M.

[0011] According to another aspect of the present invention, a DNA molecule is provided, which encodes any one of the above-mentioned esterase mutants.

[0012] According to another aspect of the present invention, a recombinant plasmid is provided, wherein any one of the above-mentioned DNA molecules is connected to the recombinant plasmid.

[0013] Furthermore, the recombinant plasmids are pET-22a(+), 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(+), p ET-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-6 p-2, pBV220, pBV221, pBV222, pTrc99A, pTwin1, pEZZ18, pKK232-8, pUC-18 or pUC-19.

[0014] According to another aspect of the present invention, a host cell is provided, which contains any of the above-mentioned recombinant plasmids.

[0015] Furthermore, the host cell includes a prokaryotic cell or a eukaryotic cell; preferably, the prokaryotic cell is an Escherichia coli BL21 cell or an Escherichia coli DH5α competent cell, and the eukaryotic cell is a yeast.

[0016] According to another aspect of the present invention, a method for producing a chiral acid is provided, comprising the step of catalyzing an ester compound using an esterase, wherein the esterase is any of the above-mentioned esterase mutants.

[0017] Further, the ester compound is The reaction product is Wherein, R1 represents -CH3, -CH2CH3, -CH2CH2CH3 or -CH2CH3CH3; R2 represents one or more of a halogen atom, a nitrogen atom, an oxygen atom, a carbon chain, a heterocyclic ring with different substituents of a halogen atom, a nitrogen atom, an oxygen atom, a carbon chain or a naphthalene ring; preferably, the ester compound is

[0018] Furthermore, the pH of the reaction catalyzed by the esterase mutant is 7.0-9.0, and the reaction temperature is 20-50°C.

[0019] The esterase mutants of the present invention are obtained by mutating the esterase shown in SEQ ID NO: 1 by site-directed mutagenesis, thereby changing the amino acid sequence and achieving changes in protein structure and function. The esterases having the above-mentioned mutation sites are then obtained by targeted screening. Therefore, these esterase mutants have the advantages of greatly improved enzyme reaction activity and specificity, thereby greatly reducing the amount of enzyme used and the cost in industrial production. In addition, the esterases have enhanced tolerance to organic solvents and a wide range of reaction temperatures and pH values, thus having stronger application advantages. DETAILED DESCRIPTION

[0020] It should be noted that, in the absence of conflict, the embodiments and features in the embodiments of the present application can be combined with each other. The present invention will be described in detail below with reference to the embodiments.

[0021]

[0022] First, site-directed mutagenesis was used to introduce mutations into the esterase. The mutants were then tested for activity and specificity, and those with improved activity and specificity were selected. The I245S mutant showed a significant improvement in activity and specificity compared to the starting template. Subsequently, further mutagenesis was performed using the I245S mutant as a template, aiming to identify mutants with enhanced catalytic activity and specificity.

[0023] Site-directed mutagenesis involves introducing desired changes (usually changes that indicate a favorable phenotype) into a target DNA fragment (which can be a genome or a plasmid) through methods such as the polymerase chain reaction (PCR). These changes include base additions, deletions, and point mutations. Site-directed mutagenesis can rapidly and efficiently improve the properties and characteristics of target proteins expressed by DNA, making it a very useful tool in genetic research.

[0024] The method of introducing site-directed mutagenesis using whole-plasmid PCR is simple and effective, and is currently the most widely used approach. The principle is that a pair of primers (forward and reverse) containing the mutation site are annealed to the template plasmid and then subjected to "cyclic extension" by a polymerase. (Cyclic extension refers to the polymerase extending the primer along the template, returning to the 5' end of the primer after one circle, and then undergoing repeated cycles of heating, annealing, and extension. This reaction, unlike rolling circle amplification, does not form multiple tandem copies.) The extension products of the forward and reverse primers anneal and pair to form a nicked, open-circular plasmid. The extension products are then digested by Dpn I. Since the original template plasmid, derived from conventional E. coli, is dam-methylated, it is sensitive to Dpn I and fragmented. However, the in vitro-synthesized plasmid carrying the mutation sequence, being unmethylated, is not cleaved, allowing it to be successfully transformed in subsequent transformations, resulting in cloned mutant plasmids. The mutant plasmid is transformed into E. coli cells, and the cells are then disrupted by sonication to obtain the crude enzyme.

[0025] The mutant plasmid was transformed into E. coli cells for overexpression. The crude enzyme was then obtained by ultrasonic cell disruption. The optimal conditions for esterase induction were 25°C, 0.1 mM IPTG, and 16 h of induction.

[0026] According to a typical embodiment of the present invention, an esterase mutant is provided. The esterase mutant is a sequence of a mutation of SEQ ID NO: 1, wherein the mutation includes I245S; or the esterase mutant includes the I245S mutation and has a homology of greater than 80% with SEQ ID NO: 1, preferably a homology of greater than 85%, 86%, 87%, 88%, or 89%, and more preferably a homology of greater than 95%, 96%, 97%, 98%, or 99%.

[0027] Preferably, the sites where amino acid mutations occur also include any one or more of the following: C33A, A46G, S48N, L65V, T71V, T71M, T87E, L131S, L131N, L131H, R184E, R210Y, K212R, K212M, S230Q, P231T, S235Q, T240F, T240L, T240C, S248D, S248M, K251T, K251L, H262A, H262S, H262C, P336C or L365M.

[0028] More preferably, the sites where amino acid mutations occur include any of the following combination mutation sites: I245S+C33A, I245S+A46G, I245S+S48N, I245S+L65V, I245S+T71V, I245S+T71M, I245S+T87E, I245S+L131N, I245S+R184E, I245S+R210Y, I245S+K212R, I245S+S230Q, I245S+P231T, I245S+T240F, I245S+T240L, I245S+S248D, I245S+K251T, I245S+K251L, I245S+H262A , I245S+H262S, I245S+P336C, I245S+L365M, I245S+L65V+T71V, I245S+L65 V+T71M, I245S+L65V+T87E, I245S+L65V+R210Y, I245S+L65V+K212R, I245S +L65V+P231T, I245S+L65V+T240F, I245S+L65V+T240L, I245S+L65V+K251T , I245S+L65V+L365M, I245S+L65V+H262A, I245S+L65V+H262S, I245S+T71V+ C33A, I245S+T71V+L65V, I245S+T71V+T87E, I245S+T71V+L131N, I245S+T7 1V+T240F, I245S+T71V+T240L, I245S+T71V+K251T, I245S+T71V+H262A, I2 45S+T71V+H262S, I245S+T71V+L365M, I245S+L65V+H262A+T87E, I245S+L6 5V+H262A+R210Y, I245S+L65V+H262A+K212R, I245S+L65V+H262A+K212M, I2 45S+L65V+H262A+T240F, I245S+L65V+H262A+T240L, I245S+L65V+H262A+T 240C, I245S+L65V+H262A+S248D, I245S+L65V+H262A+S248M, I245S+L65V+ H262A+K251T, I245S+L65V+H262A+K251L, I245S+L65V+H262A+P336C, I245 S+L65V+H262A+L365M, I245S+T71V+T240L+C33A, I245S+T71V+T240L+L65V,I245S+T71V+T240L+T87E、I245S+T71V+T240L+L131N、I245S+T71V+T240L+S235Q、I245S+T71V+T240L+K251T、I245S+T71V+T240L+H262A、I245S+T71V+T240L+H262S、I245S+T71V+T240L+P336C、I245S+T71V+T240L+L365M、 I245S+T71V+T240F+L131N+S48N、I245S+T71V+T240L+K251T+L131N、I245S+T71V+T240L+K251T+R210Y、I245S+T71V+T240L+K251T+K212R、I245S+T71V+T240L+K251T+K212M、I245S+T71V+T240L+K251T+S235Q、I245S+T71V+T240L+K251T+H262A、I245S+T71V+T240L+K251T+H262S、I245S+T71V+T240L+K251T+P336C、I245S+T71V+T240L+K251T+L365M、I245S+T71V+T240L+T87E+L65V、I245S+T71V+T240L+T87E+L65V+R210Y、I245S+T71V+T240L+T87E+L65V+K212M、I245S+T71V+T240L+T87E+L65V+K251T、I245S+T71V+T240L+T87E+L65V+H262A、I245S+T71V+T240L+T87E+L65V+H262S、I245S+T71V+T240L+T87E+L65V+H262C、I245S+T71V+T240L+T87E+L65V+K251T+K212M、I245S+T71V+T240L+T87E+L65V+K251T+H262A、I245S+T71V+T240L+T87E+L65V+K251T+L365M、I245S+L65V+H262A+T240F+L131S、I245S+L65V+H262A+T240F+L131H、I245S+L65V+H262A+T240F+L131N、I245S+L65V+H262A+T240F+R210Y、I245S+L65V+H262A+T240F+K212M、I245S+L65V+H262A+T240F+P231T、I245S+L65V+H262A+T240F+K251T or I245S+L65V+H262A+T240F+L365M.

[0029] The esterase mutants of the present invention are obtained by mutating the esterase shown in SEQ ID NO: 1 by site-directed mutagenesis, thereby changing the amino acid sequence and achieving changes in protein structure and function. The esterases having the above-mentioned mutation sites are then obtained by targeted screening. Therefore, these esterase mutants have the advantages of greatly improved enzyme reaction activity and specificity, thereby greatly reducing the amount of enzyme used and the cost in industrial production. In addition, the esterases have enhanced tolerance to organic solvents and a wide range of reaction temperatures and pH values, thus having stronger application advantages.

[0030] According to a typical embodiment of the present invention, a DNA molecule is provided. The esterase encoded by the DNA has improved enzyme activity and stability, which can reduce the amount of enzyme added and the difficulty of post-processing in the industrial production of amino acids.

[0031] The above-mentioned DNA molecules of the present invention can also exist in the form of "expression cassettes". "Expression cassettes" refer to linear or circular nucleic acid molecules, covering DNA and RNA sequences that can direct the expression of specific nucleotide sequences in appropriate host cells. Generally speaking, it includes a promoter that is effectively connected to the target nucleotide, which is optionally effectively connected to a termination signal and / or other regulatory elements. The expression cassette can also include sequences required for the correct translation of the nucleotide sequence. The coding region usually encodes the target protein, but also encodes a target functional RNA in the sense or antisense direction, such as antisense RNA or non-translated RNA. The expression cassette containing the target polynucleotide sequence can be chimeric, meaning that at least one of its components is heterologous to at least one of its other components. The expression cassette can also be naturally occurring, but obtained in an effective recombinant formation for heterologous expression.

[0032] According to a typical embodiment of the present invention, a recombinant plasmid is provided. The recombinant plasmid contains any of the above-mentioned DNA molecules. The DNA molecules in the recombinant plasmid are placed in appropriate positions in the recombinant plasmid so that the DNA molecules can be correctly and smoothly replicated, transcribed, or expressed.

[0033] Although the present invention uses the qualifier "comprising" when defining the aforementioned DNA molecules, this does not mean that other sequences unrelated to their function can be arbitrarily added to either end of the DNA sequence. Those skilled in the art will recognize that, in order to meet the requirements of recombinant operations, it is necessary to add appropriate restriction endonuclease sites or additional start and stop codons to both ends of the DNA sequence. Therefore, using closed-ended definitions will not truly cover these situations.

[0034] The term "plasmid" as used in the present invention includes any plasmid, cosmid, phage or Agrobacterium binary nucleic acid molecule in double-stranded or single-stranded linear or circular form, preferably a recombinant expression plasmid, which can be a prokaryotic expression plasmid or a eukaryotic expression plasmid, but preferably a prokaryotic expression plasmid. In certain embodiments, the recombinant plasmid is selected from pET-22a(+), 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-2 9a(+), 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(+), pQE 2. 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 or pUC-19. More preferably, the recombinant plasmid is pET-22b(+).

[0035] According to a typical embodiment of the present invention, a host cell is provided, comprising any of the above-described recombinant plasmids. Suitable host cells for use in the present invention include, but are not limited to, prokaryotic cells or eukaryotic cells. Preferably, the prokaryotic cells are Escherichia coli BL21 cells or Escherichia coli DH5α competent cells, and the eukaryotic cells are yeast.

[0036] According to an exemplary embodiment of the present invention, a method for producing a chiral acid is provided. The method comprises the step of catalyzing an ester compound using an esterase, wherein the esterase is any of the aforementioned esterase mutants. Because the esterases of the present invention have improved specificity and even higher enzymatic activity, using the esterase mutants of the present invention to prepare chiral acids not only reduces production costs but also results in higher ee values ​​for the resulting amino acids.

[0037] According to a typical embodiment of the present invention, the ester compound is The reaction product is wherein R1 represents -CH3, -CH2CH3, -CH2CH2CH3 or -CH2CH3CH3; R2 represents one or more of a halogen atom, a nitrogen atom, an oxygen atom, a carbon chain, a heterocyclic ring having different substituents of a halogen atom, a nitrogen atom, an oxygen atom, a carbon chain, or a naphthalene ring;

[0038] Preferably, the ester compound is

[0039] Preferably, the pH range of the esterase mutant catalyzed reaction is 7.0-9.0, and the reaction temperature is 20-50°C.

[0040] The beneficial effects of the present invention will be further illustrated below with reference to specific embodiments.

[0041] Example 1

[0042] To a 1 mL reaction system, 5 mg of substrate 1, substrate 2, substrate 3, substrate 4, substrate 5, or substrate 6 (dissolved in 0.2 mL DMSO) was added, along with 5 mg of esterase. The reaction was incubated at 40°C in 0.3 M potassium phosphate buffer (pH 7.5) for 2 h. After this, 3 mL of anhydrous ethanol was added to the 0.5 mL reaction system, the mixture was thoroughly shaken, and centrifuged at 12,000 rpm for 3 min. The supernatant was collected for liquid chromatography analysis to determine conversion and ee. The results are shown in Table 1.

[0043] Table 1

[0044] The activity (the conversion rate represents the enzyme activity) is reduced or increased by folds compared to the parent: --- reduced by 10-50 times, -- reduced by 5-10 times, - reduced by 1-5 times, + increased by 1-5 times, ++ increased by 5-10 times, +++ increased by 10-50 times, ++++ increased by more than 50 times, and +++++ increased by more than 70 times.

[0045] The ee value is less than 0%*, the ee value is between 0-50%**, the ee value is between 50-95%***, and the ee value is greater than 95%****.

[0046] Example 2

[0047] Continue mutation to improve enzyme activity and product ee value.

[0048] To a 1 mL reaction system, 5 mg of substrate 1, substrate 2, substrate 3, substrate 4, substrate 5, or substrate 6 (dissolved in 0.2 mL DMSO) was added, along with 2 mg of esterase. The reaction was allowed to proceed at 40°C for 2 h in 0.3 M KPB pH 7.5 buffer. Afterward, 3 mL of anhydrous ethanol was added to the 0.5 mL reaction system, which was thoroughly shaken and centrifuged at 12,000 rpm for 3 min. The supernatant was collected for liquid chromatography analysis to determine conversion and ee. The results are shown in Table 2.

[0049] Table 2

[0050] The activity (the conversion rate represents the enzyme activity) is reduced or increased by folds compared to the parent: --- reduced by 10-50 times, -- reduced by 5-10 times, - reduced by 1-5 times, + increased by 1-5 times, ++ increased by 5-10 times, +++ increased by 10-50 times, ++++ increased by more than 50 times, and +++++ increased by more than 70 times.

[0051] The ee value is less than 0%*, the ee value is between 0-50%**, the ee value is between 50-95%***, and the ee value is greater than 95%****.

[0052] Example 3

[0053] Continue to perform mutations to improve enzyme activity and product ee value, as well as increase substrate concentration and reduce reaction volume.

[0054] To a 0.5 mL reaction system, 5 mg of substrate 1, substrate 2, substrate 3, substrate 4, substrate 5, or substrate 6 (dissolved in 0.1 mL DMSO) and 1 mg of esterase were added. 0.3 M KPB pH 7.5 buffer was added and reacted at 40°C for 2 h. Then, 3 mL of anhydrous ethanol was added to the 0.5 mL reaction system, the mixture was thoroughly shaken, and centrifuged at 12,000 rpm for 3 min. The supernatant was collected for liquid chromatography analysis to determine conversion and ee. The results are shown in Table 3.

[0055] Table 3

[0056] The activity (the conversion rate represents the enzyme activity) is reduced or increased by folds compared to the parent: --- reduced by 10-50 times, -- reduced by 5-10 times, - reduced by 1-5 times, + increased by 1-5 times, ++ increased by 5-10 times, +++ increased by 10-50 times, ++++ increased by more than 50 times, and +++++ increased by more than 70 times.

[0057] The ee value is less than 0%*, the ee value is between 0-50%**, the ee value is between 50-95%***, and the ee value is greater than 95%****.

[0058] Example 4

[0059] Based on the reaction conditions, the reaction system was optimized.

[0060] In a 0.5 mL reaction system, 5 mg of substrate 1 (dissolved in 0.1 mL DMSO) and 1 mg of esterase were added, using 0.3 M KPB pH 7.5 buffer. Based on the reaction conditions, the reaction system was optimized, using varying solubility of the cosolvent DMSO (0% to 25%), varying buffer concentrations (0.1 M to 1 M KPB pH 7.5), varying pH buffers (0.3 M KPB pH 7-8, 0.3 M Tris-HCl pH 8-9), and a reaction temperature (20°C to 50°C). After 2 h of reaction, 3 mL of anhydrous ethanol was added to the 0.5 mL reaction system, the mixture was thoroughly shaken, and centrifuged at 12,000 rpm for 3 min. The supernatant was collected for liquid chromatography to determine conversion and ee. Results are shown in Tables 4-7.

[0061] Table 4

[0062] Table 5

[0063] Table 6

[0064] Table 7

[0065] The activity (the conversion rate represents the enzyme activity) is reduced or increased by folds compared to the parent: --- reduced by 10-50 times, -- reduced by 5-10 times, - reduced by 1-5 times, + increased by 1-5 times, ++ increased by 5-10 times, +++ increased by 10-50 times, ++++ increased by more than 50 times, and +++++ increased by more than 70 times.

[0066] The ee value is less than 0%*, the ee value is between 0-50%**, the ee value is between 50-95%***, and the ee value is greater than 95%****.

[0067] Example 5

[0068] According to the optimized reaction system, an amplification reaction of 1g of substrate was carried out.

[0069] Based on the optimized reaction system, substrate 1 was amplified. To a 20 mL reaction system, 1 g of substrate 1 (dissolved in 4 mL of DMSO) was added, along with 50 mg of esterase (I245S+L65V+H262A+T240F) and 0.3 M Tris-HCl pH 8.0 buffer. The reaction was carried out at 40°C, and samples were taken for testing over the reaction time. The pH was adjusted to around 8.0 until one of the substrate configurations was completely converted. Sampling and testing: 100 μL of the reaction system was added to 3 mL of anhydrous ethanol and thoroughly shaken. The mixture was centrifuged at 12,000 rpm for 3 minutes, and the supernatant was collected for liquid chromatography to detect the conversion rate and ee value. The results are shown in Table 8.

[0070] Table 8

[0071] With the reaction time, the conversion rate gradually increases, + conversion rate 0-10%, ++ conversion rate 10-45%, +++ conversion rate 45%-50%.

[0072] The ee value is less than 0%*, the ee value is between 0-50%**, the ee value is between 50-95%***, and the ee value is greater than 95%****.

[0073] Example 6

[0074] Beneficial mutation sites are further combined to further increase substrate concentration and reduce reaction volume.

[0075] To a 0.5 mL reaction system, 10 mg of substrate 2, substrate 3, substrate 4, substrate 5, or substrate 6 (dissolved in 0.1 mL DMSO) and 1 mg of esterase were added in 0.3 M KPB pH 7.5 buffer. After reacting at 40°C for 2 h, 3 mL of anhydrous ethanol was added to the 0.5 mL reaction system, which was thoroughly shaken and centrifuged at 12,000 rpm for 3 min. The supernatant was collected for liquid chromatography analysis to determine conversion and ee. The results are shown in Table 9.

[0076] Table 9

[0077] The activity (the conversion rate represents the enzyme activity) is reduced or increased by folds compared to the parent: --- reduced by 10-50 times, -- reduced by 5-10 times, - reduced by 1-5 times, + increased by 1-5 times, ++ increased by 5-10 times, +++ increased by 10-50 times, ++++ increased by more than 50 times, and +++++ increased by more than 70 times.

[0078] The ee value is less than 0%*, the ee value is between 0-50%**, the ee value is between 50-95%***, and the ee value is greater than 95%****.

[0079] Example 7

[0080] Based on the reaction conditions, the reaction system was optimized.

[0081] In a 0.5 mL reaction system, 5 mg of substrate 2 or substrate 4 (dissolved in 0.1 mL DMSO) and 0.5 mg of esterase were added, using 0.3 M KPB pH 7.5 buffer. Based on the reaction conditions, the reaction system was optimized, using different solubility cosolvents (0% to 25%), different buffer concentrations (0.1 M to 1 M KPB pH 7.5), different pH buffers (0.3 M KPB pH 7-8, 0.3 M Tris-HCl pH 8-9), and a reaction temperature (20°C to 50°C). After 2 hours of reaction, 3 mL of anhydrous ethanol was added to the 0.5 mL reaction system, the mixture was thoroughly shaken, centrifuged at 12,000 rpm for 3 minutes, and the supernatant was collected for liquid chromatography to determine the conversion and ee value. The results are shown in Tables 10-13.

[0082] Table 10

[0083] Table 11

[0084] Table 12

[0085] Table 13

[0086] Activity (the conversion rate represents the enzyme activity) is reduced and increased by folds compared to the parent, --- reduced by 10-50 times, -- reduced by 5-10 times, - reduced by 1-5 times, + increased by 1-5 times, ++ increased by 5-10 times, +++ increased by 10-50 times, ++++ increased by more than 50 times, +++++ increased by more than 70 times, ++++++ increased by more than 100 times,

[0087] The ee value is less than 0%*, the ee value is between 0-50%**, the ee value is between 50-95%***, and the ee value is greater than 95%****.

[0088] Example 8

[0089] According to the optimized reaction system, an amplification reaction of 1g of substrate was carried out.

[0090] To a 20 mL reaction system, add 1 g of substrate 2 (dissolved in 4 mL of DMSO), 25 mg of esterase (I245S+T71V+T240L+T87E+L65V+K251T+K212M), and 0.3 M Tris-HCl pH 8.0 buffer. The reaction was carried out at 40°C, with sampling and testing following the reaction time. The pH was adjusted to around 8.0 until one of the substrate configurations was completely converted. For sampling and testing, 100 μL of the reaction system was added to 3 mL of anhydrous ethanol and thoroughly shaken. Centrifuge at 12,000 rpm for 3 minutes, and the supernatant was collected for liquid chromatography to determine the conversion rate and ee value. The results are shown in Table 14.

[0091] Table 14

[0092] With the reaction time, the conversion rate gradually increases, + conversion rate 0-10%, ++ conversion rate 10-45%, +++ conversion rate 45%-50%.

[0093] The ee value is less than 0%*, the ee value is between 0-50%**, the ee value is between 50-95%***, and the ee value is greater than 95%****.

[0094] Example 9

[0095] According to the optimized reaction system, an amplification reaction of 1g of substrate was carried out.

[0096] To a 20 mL reaction system, add 1 g of substrate 4 (dissolved in 4 mL of DMSO), 25 mg of esterase (I245S+T71V+T240L+T87E+L65V+K251T+K212M), and 0.3 M Tris-HCl pH 8.0 buffer. The reaction was carried out at 40°C, with sampling and testing following the reaction time. The pH was adjusted to around 8.0 until one of the substrate configurations was completely converted. For sampling and testing, 100 μL of the reaction system was added to 3 mL of anhydrous ethanol and thoroughly shaken. Centrifuge at 12,000 rpm for 3 minutes, and the supernatant was collected for liquid chromatography to determine the conversion rate and ee value. The results are shown in Table 15.

[0097] Table 15

[0098] With the reaction time, the conversion rate gradually increases, + conversion rate 0-10%, ++ conversion rate 10-45%, +++ conversion rate 45%-50%.

[0099] The ee value is less than 0%*, the ee value is between 0-50%**, the ee value is between 50-95%***, and the ee value is greater than 95%****.

[0100] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are intended to be within the scope of protection of the present invention.

Claims

1. An esterase mutant, characterized in that The esterase mutant is a mutated sequence of SEQ ID NO: 1, wherein the mutation includes I245S; or the esterase mutant includes the I245S mutation and has more than 99% homology with SEQ ID NO: 1, and has esterase catalytic activity.

2. The esterase mutant according to claim 1, characterized in that The mutations also include any one or more of the following: C33A, A46G, S48N, L65V, T71V, T71M, T87E, L131S, L131N, L131H, R184E, R210Y, K212R, K212M, S230Q, P231T, S235Q, T240F, T240L, T240C, S248D, S248M, K251T, K251L, H262A, H262S, H262C, P336C or L365M.

3. The esterase mutant according to claim 1, characterized in that The mutations include any of the following combined mutations: I245S+C33A, I245S+A46G, I245S+S48N, I245S+L65V, I245S+T71V, I245S+T71M, I245S+T87E, I245S+L131N, I245S+R184E, I245S+R210Y, I245S+K212R, I245S+S230Q, I245S+P231T, I245S+T240F, I245S+T240L, I245S+S248D, I245S+K251T, I245S+K251L, I245S+H262A, I245S+H2 62S, I245S+P336C, I245S+L365M, I245S+L65V+T71V, I245S+L65V+T71M, I 245S+L65V+T87E, I245S+L65V+R210Y, I245S+L65V+K212R, I245S+L65V+P2 31T, I245S+L65V+T240F, I245S+L65V+T240L, I245S+L65V+K251T, I245S+ L65V+L365M, I245S+L65V+H262A, I245S+L65V+H262S, I245S+T71V+C33A, I 245S+T71V+L65V, I245S+T71V+T87E, I245S+T71V+L131N, I245S+T71V+T2 40F, I245S+T71V+T240L, I245S+T71V+K251T, I245S+T71V+H262A, I245S+T 71V+H262S, I245S+T71V+L365M, I245S+L65V+H262A+T87E, I245S+L65V+H 262A+R210Y, I245S+L65V+H262A+K212R, I245S+L65V+H262A+K212M, I245S +L65V+H262A+T240F, I245S+L65V+H262A+T240L, I245S+L65V+H262A+T24 0C, I245S+L65V+H262A+S248D, I245S+L65V+H262A+S248M, I245S+L65V+H2 62A+K251T, I245S+L65V+H262A+K251L, I245S+L65V+H262A+P336C, I245S +L65V+H262A+L365M, I245S+T71V+T240L+C33A, I245S+T71V+T240L+L65V,I245S+T71V+T240L+T87E、I245S+T71V+T240L+L131N、I245S+T71V+T240L+S235Q、I245S+T71V+T240L+K251T、I245S+T71V+T240L+H262A、I245S+T71V+T240L+H262S、I245S+T71V+T240L+P336C、I245S+T71V+T240L+L365M、I245S+T71V+T240F+L131N+S48N、I245S+T71V+T240L+K251T+L131N、I245S+T71V+T240L+K251T+R210Y、I245S+T71V+T240L+K251T+K212R、I245S+T71V+T240L+K251T+K212M、I245S+T71V+T240L+K251T+S235Q、I245S+T71V+T240L+K251T+H262A、I245S+T71V+T240L+K251T+H262S、I245S+T71V+T240L+K251T+P336C、I245S+T71V+T240L+K251T+L365M、I245S+T71V+T240L+T87E+L65V、I245S+T71V+T240L+T87E+L65V+R210Y、I245S+T71V+T240L+T87E+L65V+K212M、I245S+T71V+T240L+T87E+L65V+K251T、I245S+T71V+T240L+T87E+L65V+H262A、I245S+T71V+T240L+T87E+L65V+H262S、I245S+T71V+T240L+T87E+L65V+H262C、I245S+T71V+T240L+T87E+L65V+K251T+K212M、I245S+T71V+T240L+T87E+L65V+K251T+H262A、I245S+T71V+T240L+T87E+L65V+K251T+L365M、I245S+L65V+H262A+T240F+L131S、 I245S+L65V+H262A+T240F+L131H、I245S+L65V+H262A+T240F+L131N、I245S+L65V+H262A+T240F+R210Y、I245S+L65V+H262A+T240F+K212M、I245S+L65V+H262A+T240F+P231T、I245S+L65V+H262A+T240F+K251T or I245S+L65V+H262A+T240F+L365M.

4. A DNA molecule, characterized in that The DNA molecule encodes the esterase mutant according to any one of claims 1 to 3.

5. A recombinant plasmid, characterized in that: The recombinant plasmid is connected to the DNA molecule according to claim 4.

6. The recombinant plasmid according to claim 5, characterized in that The recombinant plasmids are pET-22a(+), 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(+), pE T-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-6 p-2, pBV220, pBV221, pBV222, pTrc99A, pTwin1, pEZZ18, pKK232-8, pUC-18 or pUC-19.

7. A host cell, characterized in that The host cell contains the recombinant plasmid according to claim 5 or 6.

8. The host cell according to claim 7, characterized in that The host cell includes a prokaryotic cell or a eukaryotic cell; preferably, the prokaryotic cell is an Escherichia coli BL21 cell or an Escherichia coli DH5α competent cell, and the eukaryotic cell is a yeast.

9. A method for producing a chiral acid, comprising the step of catalyzing an ester compound with an esterase, characterized in that: The esterase is the esterase mutant according to any one of claims 1 to 3.

10. The method according to claim 9, characterized in that The ester compound is The reaction product is Wherein, R1 represents -CH3, -CH2CH3, -CH2CH2CH3 or -CH2CH3CH3; R2 represents one or more of a halogen atom, a nitrogen atom, an oxygen atom, a carbon chain, a heterocyclic ring having different substituents of a halogen atom, a nitrogen atom, an oxygen atom, a carbon chain or a naphthalene ring; Preferably, the ester compound is 11. The method according to claim 10, characterized in that The pH of the esterase mutant catalytic reaction is 7.0-9.0, and the reaction temperature is 20-50°C.

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