Esterase variants and their use

Abstract

Provided is an esterase variant and its use. The esterase variant obtained through rational design and multiple rounds of enzyme evolution and screening based on the amino acid sequence shown in SEQ ID NO: 1 has a modified protein structure and function compared to wild-type esterase, and in practical applications, the catalytic activity and / or stereoselectivity of the esterase variant is significantly improved, and when a specific organic solubilizing agent is contained in the system, the catalytic activity and / or stereoselectivity is still relatively stable. In addition, the improved catalytic activity and / or stereoselectivity of the esterase variant allows the amount of enzyme used to be reduced to a certain extent, and the difficulty of post-treatment is also reduced, making it suitable for industrialized production.

Description

【Technical Field】 【0001】 The present invention relates to the field of preparation of industrial enzymes, and specifically to esterase variants and their use. 【Background Art】 【0002】 Organic synthesis is facing increasingly large challenges in the development processes of pharmaceuticals, agrochemicals, and the fine chemical industry. In drug molecules, often only one stereoisomer has a therapeutic effect, while the other stereoisomers have no therapeutic effect or even have side effects. In conventional organic synthesis, protection and deprotection steps are often introduced to compensate for the lack of chemical and regioselectivity of the reaction. On the other hand, biocatalytic reactions with enzymes often have relatively high catalytic activity and / or stereoselectivity, are often carried out under neutral and room temperature conditions, and have less environmental pollution. Therefore, the application of biocatalytic technology with enzymes in organic synthesis has very great practical value. 【0003】 Esterase is a general term for enzymes that catalyze the hydrolysis of esters and is a hydrolase widely present in plants, animals, and microorganisms. Animal pancreatic esterase and microbial esterase are the main sources, mainly fungi, and then bacteria. Esterases from different sources have different catalytic characteristics and catalytic activities. Esterases are commercialized and play an important role in fields such as food, pharmaceuticals, and the chemical industry. 【0004】 CN 107058362 A discloses the esterase gene est816 and its recombinant esterase, which are expressed efficiently and solublely in E. coli and Pichia yeast expression systems, and the recombinant esterase has a strong degrading effect on pyrethroid pesticides (including cyhalothrin, cypermethrin, fenvalerate, and deltamethrin), with a high degradation rate of over 90%, indicating broad potential applications for pyrethroid pesticide residues. CN 106929493 A discloses a lactonase having an amino acid sequence in which the 167th amino acid of the sequence of Sequence ID No. 5 is mutated from valine to histidine, improving the degradation efficiency against α-zearalenol. 【0005】 When biocatalysts are used in organic synthesis, they are often non-natural substrates. However, non-natural substrates often have poor reaction activity, stability, and selectivity, and therefore, there are not many esterases that are widely used in practice. [Overview of the Initiative] [Problems that the invention aims to solve] 【0006】 The main objective of the present invention is to provide esterase variants and their use in order to solve the problems in the prior art of low esterase activity and / or low stereoselectivity. [Means for solving the problem] 【0007】 To achieve the above objective, according to one aspect of the present invention, an esterase mutant is provided, the amino acid sequence of the esterase mutant is based on SEQ ID NO: G19S, G19S+H86S, G19S+H86A, G19S+H86Q, G19S+H86M, G19S+H86T, G19S+H86C, G19S+H86N, G19S+F88N, G19S+F88R, G19S+F88Y, G19S+F 88K, G19S+S111T, G19S+S111V, G19S+M113I, G19S+M113L, G19S+M113V, G19S+F125Y, G19S+F125S, G19S+Y128 F, G19S+L157V, G19S+M166L, G19S+L187V, G19S+L187I, G19S+S218Y, G19S+S218H, G19S+S218F, G19S+S218N, G19S+H86S+S111T, G19S+H86S+S111V, G19S+H86S+M113A, G19S+H86S+M113G, G19S+H86S+M113 I, G19S+H86S+M113L, G19S+H86S+M113V, G19S+H86S+M113I+L157A, G19S+H86S+M113I+L157G, G 19S+H86S+M113I+L157V, G19S+H86A+S111T, G19S+H86A+S111V, G19S+H86A+M113A, G19S+H86A +M113G, G19S+H86A+M113I, G19S+H86A+M113L, G19S+H86A+M113V, G19S+H86A+M113I+S218E, G1 9S+H86A+M113I+S218F, G19S+H86A+M113I+S218I, G19S+H86A+M113I+S218L, G19S+H86A+M113 I+S218M, G19S+H86A+M113I+S218T, G19S+H86A+M113I+S218V, G19S+H86A+M113I+S218Y, G19S+ H86A+M113I+S218F+A86C, G19S+H86A+M113I+S218F+A86M, G19S+H86A+M113I+S218F+A86N, G19 S+H86A+M113I+S218F+A86Q, G19S+H86A+M113I+S218F+A86S, G19S+H86A+M113I+S218F+M137F,G19S+H86A+M113I+S218F+M137L, G19S+H86A+M113I+S218F+M137Y, G19S+H86A+M113I+S218F+M137E, G19S+H86A+M113I+S218F+M137W , G19S+H86A+M113I+S218F+F219Y, G19S+H86A+M113I+S218F+F219L, G19S+H86A+M113I+S218F+F219T, G19S+H86A+M113I+S218F+F219Q The amino acid mutations G19S+H86A+M113I+S218F+F219Y+M137F, G19S+H86A+M113I+S218F+F219Y+M137L, G19S+H86A+M113I+S218F+F219Y+M137W, G19S+H86A+M113I+S218F+F219Y+M137Y, or G19S+H86A+M113I+S218F+F219Y+M137S have occurred, or the sequence has 80% or more homology to the amino acid sequence in which the mutation occurred. 【0008】 Furthermore, the amino acid sequence of the esterase mutant has 90% or more homology, preferably 95% or more, and more preferably 99% or more, with the amino acid sequence in which the mutation occurred. 【0009】 Furthermore, the esterase variant originates from Rauvolfia serpentina. 【0010】 According to a second aspect of the present application, a DNA molecule encoding the above-mentioned esterase variant is provided. 【0011】 According to a third aspect of the present application, a recombinant plasmid is provided in which the above-mentioned DNA molecules are linked. 【0012】 Furthermore, the recombinant plasmids are 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-2 1a(+), 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(+), pE T-43b(+), pET-44a(+), pET-49b(+), pQE2, pQE9, pQE30, pQE31, pQE32, pQE40, pQE70, pQ It is one selected from the group consisting of E80, 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. 【0013】 According to a second aspect of the present application, a non-plant host cell containing any one of the above recombinant plasmids is provided. 【0014】 Furthermore, the host cell can be a prokaryotic or eukaryotic cell, and a eukaryotic cell is a yeast cell. 【0015】 Furthermore, the host cells are competent cells. 【0016】 Furthermore, the competent cells are either E. coli BL21 cells or E. coli W3110 cells. 【0017】 According to the second aspect of the present application, a method for preparing a chiral compound is provided, and the preparation method includes using the above esterase variant 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】 (In the formula, n = 1, 2, 3 or 4, X = C, O or S, R1 = CH3, CH2CH3, CH2-CH2CH3 or CHCH3CH3, R2 = H, F, Cl, Br, CH3 or CH2CH3.) 【0018】 Furthermore, the ester compound is 【Chemical formula】 , or 【Chemical formula】 any one of them. 【0019】 Furthermore, the esterase variant catalyzes the hydrolysis reaction of the ester compound represented by Formula I at a temperature of 20°C to 40°C. 【0020】 Furthermore, the ester compound and the esterase are dissolved in a potassium phosphate buffer solution to form a catalytic reaction system, where the concentration of the potassium phosphate buffer solution is 0.1M to 1M and the pH value is 6.0 to 7.5. 【0021】 Furthermore, the mass ratio of the esterase variant to the ester compound is 0.2mg to 2mg:20mg. 【0022】 Furthermore, the mass ratio of the esterase variant to the ester compound is 0.1g to 0.5g:10g. 【0023】 Furthermore, the catalytic reaction system also includes a solubilizing agent selected from the group consisting of DMSO, DCM, and 2-MeTHF. 【0024】 Furthermore, the volume percentage content of the solubilizer in the catalytic reaction system is ≤20%. [Effects of the Invention] 【0025】 By applying the technical solution of the present invention, the esterase mutant obtained through rational design based on the amino acid sequence shown in Sequence ID No. 1 and several enzyme evolution screenings exhibits a modified protein structure and function compared to wild-type esterase. In actual application, the catalytic activity and / or stereoselectivity of the esterase mutant is greatly improved, and it still maintains relatively stable catalytic activity and / or stereoselectivity when a specific organic solubilizer is included in the system. Furthermore, the improved catalytic activity and / or stereoselectivity of the esterase mutant reduces the amount of enzyme used to some extent and lowers the difficulty of post-processing, making it suitable for industrial production. [Modes for carrying out the invention] 【0026】 The embodiments and features of the embodiments described herein can be combined with each other as long as they do not contradict each other, and the present invention will be described in detail below with reference to the embodiments. 【0027】 In a typical example, an esterase mutant is provided, the amino acid sequence of the esterase mutant being G19S, G19S+H86S, G19S+H86A, G19S+H86Q, G19S+H86M, G19S+H86T, G19S+H86C, G19S+H86N, G19S+F88N, G19S+F88R, G19S+F88Y, G19S+F88K, G19S+S111T, G19S+S111V, G19S+M113I, G19S+M113L, G19S+M113V, G19S+F125Y, G 19S+F125S, G19S+Y128F, G19S+L157V, G19S+M166L, G19S+L187V, G19S+L1 87I, G19S+S218Y, G19S+S218H, G19S+S218F, G19S+S218N, G19S+H86S+S111 T, G19S+H86S+S111V, G19S+H86S+M113A, G19S+H86S+M113G, G19S+H86S+M 113I, G19S+H86S+M113L, G19S+H86S+M113V, G19S+H86S+M113I+L157A, G19 S+H86S+M113I+L157G, G19S+H86S+M113I+L157V, G19S+H86A+S111T, G19S +H86A+S111V, G19S+H86A+M113A, G19S+H86A+M113G, G19S+H86A+M113I, G1 9S+H86A+M113L, G19S+H86A+M113V, G19S+H86A+M113I+S218E, G19S+H86A +M113I+S218F, G19S+H86A+M113I+S218I, G19S+H86A+M113I+S218L, G19S+ H86A+M113I+S218M, G19S+H86A+M113I+S218T, G19S+H86A+M113I+S218V, G19S+H86A+M113I+S218Y, G19S+H86A+M113I+S218F+A86C, G19S+H86A+M11 3I+S218F+A86M, G19S+H86A+M113I+S218F+A86N, G19S+H86A+M113I+S218 F+A86Q, G19S+H86A+M113I+S218F+A86S, G19S+H86A+M113I+S218F+M137F,G19S+H86A+M113I+S218F+M137L, G19S+H86A+M113I+S218F+M137Y, G19S+H86A+M113I+S 218F+M137E, G19S+H86A+M113I+S218F+M137W, G19S+H86A+M113I+S218F+F219Y, G19S+H 86A+M113I+S218F+F219L, G19S+H86A+M113I+S218F+F219T, G19S+H86A+M113I+S218F+F 219Q, G19S+H86A+M113I+S218F+F219Y+M137F, G19S+H86A+M113I+S218F+F219Y+M137L, The amino acid sequence is one in which a mutation has occurred in the following order: G19S+H86A+M113I+S218F+F219Y+M137W, G19S+H86A+M113I+S218F+F219Y+M137Y, or G19S+H86A+M113I+S218F+F219Y+M137S, or an amino acid sequence in which a mutation site exists in the amino acid sequence in which the esterase mutant occurred, and which has 80% or more (preferably 90% or more, or 95% or more, or 99% or more) homology to the amino acid sequence in which the mutation occurred. 【0028】 The amino acid sequences of the above esterase mutants are those in which mutations have occurred in the amino acid sequence shown in Sequence ID No. 1, and the important sites where the relevant mutations occurred are G19S, H86S, H86A, H86Q, H86M, H86T, H86C, H86N, F88N, F88R, F88Y, F88K, S111T, S111V, M113A, M113G, M113I, M113L, M113V, F125Y, F125S, and Y1 This includes, but is not limited to, one or more of the following parts: 28F, M137F, M137L, M137Y, M137E, M137W, M137S, L157V, L157A, L157G, M166L, L187V, L187I, S218Y, S218H, S218F, S218N, S218E, S218I, S218L, S218M, S218T, S218V, F219Y, F219L, F219T, and F219Q. 【0029】 The above esterase mutant was obtained through rational design based on the amino acid sequence shown in Sequence ID No. 1 and several enzyme evolution screenings, resulting in changes to the protein structure and function compared to the parent esterase. In actual application, the catalytic activity and / or stereoselectivity of the esterase mutant is greatly improved, and it still exhibits relatively stable catalytic activity and / or stereoselectivity when certain organic solubilizers are included in the system. Furthermore, the improved catalytic activity and / or stereoselectivity of the esterase mutant reduces the amount of enzyme used to some extent and lowers the difficulty of post-processing, making it suitable for industrial production. 【0030】 The amino acid sequence of Sequence ID No. 1 (derived from Rauvolfia serpentina) is as follows: [ka] 【0031】 This invention performs codon optimization on E. coli based on the protein sequence shown in Sequence ID No. 1, and the specific nucleic acid sequence (i.e., CDS, including the terminal codon taa) is Sequence ID No. 2. [ka] 【0032】 The rational design and specific methods or steps for enzyme evolution and screening described above include, but are not limited to, those exemplified below. 【0033】 First, a mutation site is introduced into Sequence ID No. 1 using the Flu Plasmid PCR method, and the activity and selectivity of the mutants are detected. A mutant with improved activity and selectivity is then selected. 【0034】 Using Sequence ID No. 1 as a template, site-directed mutation primers are designed, and using site-directed mutation techniques, a mutant plasmid containing the target gene is obtained using pET-22b(+) as the expression carrier. 【0035】 Here, site-directed mutation (SMU) refers to introducing a necessary change (usually a change that characterizes a favorable direction) into a target DNA fragment (which may be a genome or a plasmid) using methods such as polymerase chain reaction (PCR). This includes base addition, deletion, and site mutations. Site-directed mutation can rapidly and efficiently improve the properties and characteristics of the target protein expressed by the DNA, making it an extremely useful tool in genetic research. 【0036】 The method of introducing site-directed mutations using Flu plasmid PCR is simple, effective, and currently widely used. The principle is as follows: After annealing a pair of primers (forward and reverse) containing the mutation site and a template plasmid, "circular extension" is performed using polymerase. This so-called circular extension involves the polymerase extending the primers one full circle according to the template, stopping when it returns to the 5' end of the primer, and then repeating the heating, annealing, and extension cycle. Unlike rolling circle amplification, this reaction does not form multiple tandem copies. After annealing, the extension products of the forward and reverse primers are paired to form an open circular plasmid with a cleavage site. The extension products are cleaved with Dpn I enzyme. Since the original template plasmid is derived from common E. coli, it is methylated and sensitive to Dpn I, and is therefore shredded. However, the plasmid with the mutant sequence synthesized in vitro is not methylated and is therefore not cleaved, thus allowing for successful subsequent transformation and obtaining a clone of the mutant plasmid. 【0037】 Based on obtaining mutants with improved properties through single-site mutations, it is possible to obtain mutants with even better properties by combining beneficial amino acid sites. 【0038】 After obtaining esterase mutants with significantly improved activity and enantiomer selectivity, even higher-performance mutants are obtained using a saturation mutation method. 【0039】 Saturated mutation is a method for rapidly obtaining mutants in which the target amino acids are replaced by 19 other amino acids by modifying the coding gene of a target protein. This method is not only a powerful tool for targeted protein modification but also an important means of studying protein structure-function relationships. Saturated mutation often yields more ideal evolutionary forms than single-site mutations. These problems that cannot be solved by site-directed mutation methods are unique advantages of saturated mutation. 【0040】 As described above, the mutant plasmid is transformed into E. coli cells and overexpressed within the E. coli. The crude enzyme is then obtained by sonication of the cells. The optimal conditions for esterase induction expression are 0.06 mM IPTG and overnight induction expression at 25°C. 【0041】 The screened mutants of this invention were validated through extensive experiments, demonstrating that when the substrate, present in racemic compound form, is not excessively transformed, the catalytic activity and / or stereoselectivity of the enzyme-catalyzed reaction is significantly improved (e.g., ee improves from the initial <10% to at least 80%), thus greatly meeting the needs of industrial production. 【0042】 In a typical embodiment of the present invention, a DNA molecule encoding any one of the above-mentioned esterase variants is further provided. The encoded esterase variant has the advantage of high catalytic activity and / or high stereoselectivity. 【0043】 In a typical embodiment of the present invention, a recombinant plasmid is provided in which the above-mentioned DNA molecules are linked. The DNA molecules can encode an esterase variant that has high catalytic activity and / or high stereoselectivity of any one of the above-mentioned esterases. 【0044】 Any recombinant plasmid from the above recombinant plasmids that can be used to express the above esterase DNA molecule is suitable for the present invention. In preferred embodiments of the present invention, the recombinant plasmids are pET-22b(+), pET-21b(+), pET-3a(+), pET-3d(+), pET-11a(+), pET-12a(+), pET-14b, pET-15b(+), pET-16b(+), pET-17b(+), pET-19b(+), pET-20 b(+), pET-21a(+), pET-23a(+), pET-23b(+), pET-24a(+), pET-25b(+), pET-26b(+), pE T-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-43 a(+), pET-43b(+), pET-44a(+), pET-49b(+), pQE2, pQE9, pQE30, pQE31, pQE32, pQE40, pQ It is one selected from the group consisting of E70, 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. 【0045】 In a typical embodiment of the present invention, a non-plant host cell containing any one of the above recombinant plasmids is further provided. The specific host cell may be a prokaryotic cell or a eukaryotic cell, preferably a yeast cell. More preferably, the host cell is a competent cell, and even more preferably, the competent cell is an E. coli BL21 cell or an E. coli W3110 cell. 【0046】 In a typical embodiment of the present invention, a method for preparing a chiral compound is provided, which includes catalyzing the hydrolysis of an ester compound represented by formula I to an acid compound represented by formula II and an alcohol compound represented by formula III using any one of the aforementioned esterase variants. 【0047】 In preferred examples, the ester compound is one of the following: [ka] 【0048】 In preferred examples, the esterase mutant exhibits relatively high catalytic activity or relatively high stereoselectivity at temperatures of 20°C to 40°C, and can therefore catalyze the hydrolysis reaction of ester compounds represented by formula I. 【0049】 In preferred examples, potassium phosphate buffer, an ester compound, and an esterase form a catalytic reaction system, where the concentration of potassium phosphate buffer is preferably 0.1 M to 1 M, and the pH is preferably 6.0 to 7.5. Under these conditions, different substrates can be catalyzed, and the reaction exhibits relatively high activity and / or relatively high stereoselectivity. 【0050】 In preferred examples, the mass ratio of the esterase variant to the ester compound is 0.2 mg to 2 mg:20 mg, which is suitable for small reaction volumes (e.g., 0.6 mL), and when the corresponding substrate dose is 20 mg, the enzyme mass is in the range of 2 mg to 0.2 mg. 【0051】 In another preferred embodiment, the mass ratio of the esterase variant to the ester compound is 0.1g to 0.5g:10g. This dose ratio is particularly suitable for large reaction systems (e.g., 100mL), where the enzyme mass is in the range of 0.1g to 0.5g when the corresponding substrate dose is 10g. 【0052】 The mutants obtained by evolution and screening in this invention can not only catalyze the hydrolysis of substrates under appropriate buffer conditions, but also exhibit relatively high stability, catalytic activity, and / or stereoselectivity in the presence of specific solubilizers. In preferred examples, the reaction system further includes a solubilizer selected from the group consisting of DMSO, DCM, and 2-MeTHF. In the reaction system in the presence of these solubilizers, the catalytic activity and / or stereoselectivity of the mutants of this invention remain relatively stable. 【0053】 In preferred embodiments, the volume percentage content of the solubilizer in the reaction system is ≤20%, and when the reaction system contains this amount of solubilizer, the variants of the present invention still exhibit relatively high catalytic activity and / or stereoselectivity. 【0054】 The beneficial effects of this invention will be further explained below with reference to specific examples. The substrates used in the following examples are shown as Substrates 1 to 5. 【0055】 (Example 1) 20 mg of substrate 1 was added to a 0.6 mL reaction system, along with 2 mg of esterase and 0.3 M potassium phosphate buffer at pH 7.0. After reacting at 20°C for 3 hours, 1.5 mL of anhydrous ethanol was added to the 0.6 mL reaction system, and after shaking well, an appropriate amount of anhydrous magnesium sulfate was added. The mixture was centrifuged at 12000 rpm for 3 minutes, and the supernatant was diluted 2-fold with anhydrous ethanol. The conversion rate and ee value were detected in the liquid phase. Substrates 2 and 3 were prepared using the same reaction and processing methods as described for substrate 1, and the results are shown in Table 1. 【0056】 [Table 1] 【0057】 The meaning of the numerical values ​​for the catalytic activity of wild-type esterase for different substrates is the same in each example; that is, activity represents the conversion rate. Here, we will explain using the activity value of 20 for substrate 1 as an example. Under conditions of 0.3M potassium phosphate buffer pH 7.0 and 20°C, 2 mg of wild-type enzyme, after 3 hours, resulted in a conversion rate of 20% for substrate 1. 【0058】 The ratios of decrease and increase in activity compared to the parent were as follows: --- represents a decrease of less than 5 times, -- represents a decrease of 2 to 5 times, - represents a decrease of 1 to 2 times, + represents an increase of 1 to 2 times, ++ represents an increase of 2 to 5 times, +++ represents an increase of 5 to 10 times, and ++++ represents an increase of more than 10 times. 【0059】 ee values ​​less than 0% (referring to the ee value of the product, where the substrate is racemic, meaning both S and R configurations are present, for example, each accounting for about 50%), and the enzyme selectively catalyzes the S and R configurations during the catalytic process. For example, if a large amount of substrate selectively catalyzing the R configuration is converted, the product will also have a large amount of the corresponding R configuration. In the evolutionary process, if a mutated enzyme catalyzes the R configuration, the opposite may occur as expected, meaning that the proportion of the R configuration product (e.g., 40%) may be less than the proportion of the S configuration product (e.g., 60%), and the ee value of the R configuration product (i.e., -20%) may be less than 0). These values ​​were labeled as #, ee values ​​from 0 to 50% as *, ee values ​​from 50 to 60% as **, ee values ​​from 60 to 70% as ***, ee values ​​from 70% to 80% as ****, ee values ​​from 80% to 95% as *****, and ee values ​​above 95% as ******. 【0060】 (Example 2) 20 mg of substrate 4 was added to a 0.6 mL reaction system, along with 2 mg of esterase and 0.3 M potassium phosphate buffer (pH 7.0). After reacting at 20°C for 3 hours, 1.5 mL of anhydrous ethanol was added to the 0.6 mL reaction system, and after shaking well, an appropriate amount of anhydrous magnesium sulfate was added. The mixture was centrifuged at 12000 rpm for 3 minutes, and the supernatant was diluted 2-fold with anhydrous ethanol. The conversion rate and ee value were detected in the liquid phase. For substrate 5, the same reaction and processing method as described for substrate 1 was established, and the results are shown in Table 2. 【0061】 [Table 2] 【0062】 The ratios of decrease and increase in activity compared to the parent were as follows: --- represents a decrease of less than 5 times, -- represents a decrease of 2 to 5 times, - represents a decrease of 1 to 2 times, + represents an increase of 1 to 2 times, ++ represents an increase of 2 to 5 times, +++ represents an increase of 5 to 10 times, and ++++ represents an increase of more than 10 times. 【0063】 We labeled ee values ​​less than 0% as #, ee values ​​between 0% and 50% as *, ee values ​​between 50% and 60% as **, ee values ​​between 60% and 70% as ***, ee values ​​between 70% and 80% as ****, ee values ​​between 80% and 95% as *****, and ee values ​​above 95% as ******. 【0064】 Based on the above results, further mutations were performed to improve the ee value of the product. 【0065】 (Example 3) 20 mg of substrate 1 was added to a 0.6 mL reaction system, along with 2 mg of esterase and 0.3 M potassium phosphate buffer at pH 7.0. After reacting at 20°C for 3 hours, 1.5 mL of anhydrous ethanol was added to the 0.6 mL reaction system, and after shaking well, an appropriate amount of anhydrous magnesium sulfate was added. The mixture was centrifuged at 12000 rpm for 3 minutes, and the supernatant was diluted 2-fold with anhydrous ethanol. The conversion rate and ee value were detected in the liquid phase. Substrates 2 and 3 were prepared using the same reaction and processing methods as described for substrate 1, and the results are shown in Table 3. 【0066】 [Table 3] 【0067】 The ratios of decrease and increase in activity compared to the parent were as follows: --- represents a decrease of less than 5 times, -- represents a decrease of 2 to 5 times, - represents a decrease of 1 to 2 times, + represents an increase of 1 to 2 times, ++ represents an increase of 2 to 5 times, +++ represents an increase of 5 to 10 times, and ++++ represents an increase of more than 10 times. 【0068】 We labeled ee values ​​less than 0% as #, ee values ​​between 0% and 50% as *, ee values ​​between 50% and 60% as **, ee values ​​between 60% and 70% as ***, ee values ​​between 70% and 80% as ****, ee values ​​between 80% and 95% as *****, and ee values ​​above 95% as ******. 【0069】 (Example 4) 20 mg of substrate 4 was added to a 0.6 mL reaction system, along with 2 mg of esterase and 0.3 M potassium phosphate buffer at pH 7.0. After reacting at 20°C for 3 hours, 1.5 mL of anhydrous ethanol was added to the 0.6 mL reaction system, and after shaking well, an appropriate amount of anhydrous magnesium sulfate was added. The mixture was centrifuged at 12000 rpm for 3 minutes, and the supernatant was diluted 2-fold with anhydrous ethanol. The conversion rate and ee value were detected in the liquid phase. For substrate 5, the same reaction and processing method as described for substrate 1 was established, and the results are shown in Table 4. 【0070】 [Table 4] 【0071】 The ratios of decrease and increase in activity compared to the parent were as follows: --- represents a decrease of less than 5 times, -- represents a decrease of 2 to 5 times, - represents a decrease of 1 to 2 times, + represents an increase of 1 to 2 times, ++ represents an increase of 2 to 5 times, +++ represents an increase of 5 to 10 times, and ++++ represents an increase of more than 10 times. 【0072】 We labeled ee values ​​less than 0% as #, ee values ​​between 0% and 50% as *, ee values ​​between 50% and 60% as **, ee values ​​between 60% and 70% as ***, ee values ​​between 70% and 80% as ****, ee values ​​between 80% and 95% as *****, and ee values ​​above 95% as ******. 【0073】 The following examples further combined beneficial mutation sites to improve the ee value of the product. 【0074】 (Example 5) 20 mg of substrate 1 was added to a 0.6 mL reaction system, along with 1 mg of esterase and 0.3 M potassium phosphate buffer at pH 6.5. After reacting at 20°C for 3 hours, 1.5 mL of anhydrous ethanol was added to the 0.6 mL reaction system, and after shaking well, an appropriate amount of anhydrous magnesium sulfate was added. The mixture was centrifuged at 12000 rpm for 3 minutes, and the supernatant was diluted 2-fold with anhydrous ethanol. The conversion rate and ee value were detected in the liquid phase. Substrates 2 and 3 were prepared using the same reaction and processing methods as described for substrate 1, and the results are shown in Table 5. 【0075】 [Table 5] 【0076】 The ratios of decrease and increase in activity compared to the parent were as follows: --- represents a decrease of less than 5 times, -- represents a decrease of 2 to 5 times, - represents a decrease of 1 to 2 times, + represents an increase of 1 to 2 times, ++ represents an increase of 2 to 5 times, +++ represents an increase of 5 to 10 times, and ++++ represents an increase of more than 10 times. 【0077】 We labeled ee values ​​less than 0% as #, ee values ​​between 0% and 50% as *, ee values ​​between 50% and 60% as **, ee values ​​between 60% and 70% as ***, ee values ​​between 70% and 80% as ****, ee values ​​between 80% and 95% as *****, and ee values ​​above 95% as ******. 【0078】 (Example 6) 20 mg of substrate 4 was added to a 0.6 mL reaction system, along with 1 mg of esterase and 0.3 M potassium phosphate buffer at pH 6.5. After reacting at 20°C for 3 hours, 1.5 mL of anhydrous ethanol was added to the 0.6 mL reaction system, and after shaking well, an appropriate amount of anhydrous magnesium sulfate was added. The mixture was centrifuged at 12000 rpm for 3 minutes, and the supernatant was diluted 2-fold with anhydrous ethanol. The conversion rate and ee value were detected in the liquid phase. For substrate 5, the same reaction and processing method as described for substrate 1 was established, and the results are shown in Table 6. 【0079】 [Table 6] 【0080】 The ratios of decrease and increase in activity compared to the parent were as follows: --- represents a decrease of less than 5 times, -- represents a decrease of 2 to 5 times, - represents a decrease of 1 to 2 times, + represents an increase of 1 to 2 times, ++ represents an increase of 2 to 5 times, +++ represents an increase of 5 to 10 times, and ++++ represents an increase of more than 10 times. 【0081】 We labeled ee values ​​less than 0% as #, ee values ​​between 0% and 50% as *, ee values ​​between 50% and 60% as **, ee values ​​between 60% and 70% as ***, ee values ​​between 70% and 80% as ****, ee values ​​between 80% and 95% as *****, and ee values ​​above 95% as ******. 【0082】 Based on the reaction conditions in the above examples, the following examples further optimized the reaction system. 【0083】 (Example 7) The reaction system consisted of 0.6 mL of reaction system with 20 mg of substrate 1, 1 mg of esterase (G19S+H86A+M113I+S218F+F219Y+M137L), and 0.3 M potassium phosphate buffer pH 6.5. Based on the reaction conditions, the reaction system was optimized using solubilizers with different solubility (2-MeTHF (0%~20%), DCM (0%~20%), DMSO (0%~20%)), buffers of different concentrations (0.1 M~1 M potassium phosphate buffer pH 6.5), buffers of different pH (0.3 M potassium phosphate buffer pH 6.0~7.5), and reaction temperatures (20°C~40°C). After reacting for 3 hours, 1.5 mL of anhydrous ethanol was added to 0.6 mL of the reaction system, and after shaking well, an appropriate amount of anhydrous magnesium sulfate was added. The mixture was centrifuged at 12000 rpm for 3 minutes, and the supernatant was diluted 2-fold with anhydrous ethanol. The conversion rate and ee value were detected in the liquid phase, and the results are shown in Tables 7-10. 【0084】 [Table 7] Note: The reaction conditions in Table 7 were as follows: different solubilizers were included, the buffer solution was 0.3 M potassium phosphate buffer pH 6.5, and the reaction temperature was 20°C. 【0085】 [Table 8] Note: The reaction conditions in Table 8 were as follows: No solubilizer was used, and the reaction temperature was 20°C. 【0086】 [Table 9] Note: The reaction conditions in Table 9 were as follows: No solubilizer was used, the buffer solution was 0.3 M potassium phosphate buffer, and the reaction temperature was 20°C. 【0087】 [Table 10] Note: The reaction conditions in Table 10 were as follows: No solubilizers were used, the buffer solution was 0.3 M potassium phosphate buffer, and the pH was 6.5. 【0088】 The ratios of decrease and increase in activity compared to the parent were as follows: --- represents a decrease of less than 5 times, -- represents a decrease of 2 to 5 times, - represents a decrease of 1 to 2 times, + represents an increase of 1 to 2 times, ++ represents an increase of 2 to 5 times, +++ represents an increase of 5 to 10 times, and ++++ represents an increase of more than 10 times. 【0089】 We labeled ee values ​​less than 0% as #, ee values ​​between 0% and 50% as *, ee values ​​between 50% and 60% as **, ee values ​​between 60% and 70% as ***, ee values ​​between 70% and 80% as ****, ee values ​​between 80% and 95% as *****, and ee values ​​above 95% as ******. 【0090】 An amplification reaction was performed using 10g of substrate. 【0091】 (Example 8) Based on the optimized reaction system, the amplification reaction was carried out, 10 g of substrate 1 was added, and 100 mL of the reaction system was prepared. The esterase (G19S+H86A+M113I+S218F+F219Y+M137L) was 100 mg, and the solution was 0.3 M potassium phosphate buffer at pH 6.5. The reaction was carried out at 20°C, and the reaction time was tracked, samples were taken for detection, and the pH was adjusted to approximately 6.4. After 3 hours of reaction, the conversion rate was 49% and the ee value was 99%. The reaction sample was post-processed, 100 mL of methylene chloride was added, and after extraction and thorough shaking, the organic layer was separated, an appropriate amount of anhydrous sodium sulfate was added, and the mixture was further filtered. Finally, the organic layer was subjected to rotary evaporation to obtain 4.8 g of sample with a purity of 98% and an ee value of 98%. Nuclear magnetic analysis was performed, and the yield was 45%. 【0092】 (Example 9) Based on the optimized reaction system, the amplification reaction was carried out, 10 g of substrate 4 was added, and 100 mL of the reaction system was prepared. The esterase (G19S+H86A+M113I+S218F+F219Y+M137L) was 100 mg, and the solution was 0.3 M potassium phosphate buffer at pH 6.5. The reaction was carried out at 20°C, and the reaction time was tracked, samples were taken for detection, and the pH was adjusted to approximately 6.4. After 6 hours of reaction, the conversion rate was 49% and the ee value was 99%. The reaction sample was post-processed, 100 mL of methylene chloride was added, and after extraction and thorough shaking, the organic layer was separated, an appropriate amount of anhydrous sodium sulfate was added, and the mixture was further filtered. Finally, the organic layer was subjected to rotary evaporation to obtain 4.6 g of sample with a purity of 98% and an ee value of 98%. Nuclear magnetic analysis was performed, and the yield was 44%. 【0093】 As is clear from the above description, the above embodiment of the present invention achieves the following technical effects. The esterase mutant obtained by rational design based on the amino acid sequence shown in Sequence ID No. 1 and through several enzyme evolution screenings has a modified protein structure and function compared to the parent esterase. In actual application, the catalytic activity and / or stereoselectivity of the esterase mutant is greatly improved, and it still maintains relatively stable catalytic activity and / or stereoselectivity when a specific organic solubilizer is included in the system. Furthermore, the improved catalytic activity and / or stereoselectivity of the esterase mutant reduces the amount of enzyme used to some extent and the difficulty of post-processing, making it suitable for industrial production. 【0094】 The above description is merely a preferred embodiment of the present invention and does not limit it, and various modifications and variations are possible for those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made without departing from the spirit and principles of the present invention should also be included within the scope of protection of the present invention.

Claims

[Claim 1] An esterase mutant, wherein the amino acid sequence of the esterase mutant is based on the amino acid sequence shown in SEQ ID NO:

1. G19S、G19S+H86S、G19S+H86A、G19S+H86Q、G19S+H86M、G19S+H86T、G19S+H86C、G19S+H86N、G19S+F88N、G19S+F88R、G19S+F88Y、G19S+F88K、G19S+S111T、G19S+S111V、G19S+M113I、G19S+M113L、G19S+M113V、G19S+F125Y、G19S+F125S、G19S+Y128F、G19S+L157V、G19S+M166L、G19S+L187V、G19S+L187I、G19S+S218Y、G19S+S218H、G19S+S218F、G19S+S218N、G19S+H86S+S111T、G19S+H86S+S111V、G19S+H86S+M113A、G19S+H86S+M113G、G19S+H86S+M113I、G19S+H86S+M113L、G19S+H86S+M113V、G19S+H86S+M113I+L157A、G19S+H86S+M113I+L157G、G19S+H86S+M113I+L157V、G19S+H86A+S111T、G19S+H86A+S111V、G19S+H86A+M113A、G19S+H86A+M113G、G19S+H86A+M113I、G19S+H86A+M113L、G19S+H86A+M113V、G19S+H86A+M113I+S218E、G19S+H86A+M113I+S218F、G19S+H86A+M113I+S218I、G19S+H86A+M113I+S218L、G19S+H86A+M113I+S218M、G19S+H86A+M113I+S218T、G19S+H86A+M113I+S218V、G19S+H86A+M113I+S218Y、G19S+M113I+S218F+H86C、G19S+M113I+S218F+H86M、G19S+M113I+S218F+H86N、G19S+M113I+S218F+H86Q、G19S+M113I+S218F+H86S、G19S+H86A+M113I+S218F+M137F、G19S+H86A+M113I+S218F+M137L、G19S+H86A+M113I+S218F+M137Y、G19S+H86A+M113I+S218F+M137E、G19S+H86A+M113I+S218F+M137W, G19S+H86A+M113I+S218F+F219Y, G19S+H86A+M113I+S218F+F219 L, G19S+H86A+M113I+S218F+F219T, G19S+H86A+M113I+S218F+F219Q, G19S+H86A+M113I+S218F+F21 These are amino acid mutations in which the following types occur: 9Y+M137F, G19S+H86A+M113I+S218F+F219Y+M137L, G19S+H86A+M113I+S218F+F219Y+M137W, G19S+H86A+M113I+S218F+F219Y+M137Y, or G19S+H86A+M113I+S218F+F219Y+M137S. The amino acid sequence of Sequence ID No. 1 is as follows: Esterase mutants characterized by: [Claim 2] The esterase variant is derived from Rauvolfia serpentina. The esterase mutant according to claim 1. [Claim 3] Encoding the esterase variant described in claim 1 or 2, A DNA molecule characterized by the following features. [Claim 4] The DNA molecule described in claim 3 is linked, A recombinant plasmid characterized by the following features. [Claim 5] The recombinant plasmids include 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-21 a(+), pET-23a(+), pET-23b(+), pET-24a(+), pET-25b(+), pET-26b(+), pET-27b(+), p ET-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, pQE 80, one selected from the group consisting of 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 feature 4. [Claim 6] A recombinant plasmid as described in claim 4 or 5 A non-plant host cell characterized by the following features. [Claim 7] A prokaryotic cell or a eukaryotic cell, wherein the eukaryotic cell is a yeast cell. The host cell according to feature 6. [Claim 8] The host cells are competent cells. The host cell according to feature 7. [Claim 9] The competent cells are E. coli BL21 cells or E. coli W3110 cells. The host cell according to feature 8. [Claim 10] This includes catalyzing the hydrolysis of an ester compound represented by formula I to an acid compound represented by formula II and an alcohol compound represented by formula III, using the esterase variant described in claim 1 or 2. A method for preparing chiral compounds characterized by the following: 【Chemistry 1】 (wherein n = 1, 2, 3 or 4, X = -CH ２ -, -O- or -S-, R １ = -CH ３ -, -CH ２ CH ３ -, -CH ２ CH ２ CH ３ or -CH(CH ３ )2, and R ２ =-H, -F, -Cl, -Br, -CH ３ or -CH ２ CH ３ (That is the case.) [Claim 11] The aforementioned ester compound is 【Chemistry 2】 or 【Transformation 3】 It is one of the following: The preparation method according to feature 10. [Claim 12] The esterase variant catalyzes the hydrolysis reaction of ester compounds represented by formula I at temperatures of 20°C to 40°C. The preparation method according to feature 10. [Claim 13] The ester compound and the esterase mutant are dissolved in potassium phosphate buffer to form a catalytic reaction system, where the concentration of potassium phosphate buffer is 0.1 M to 1 M and the pH is 6.0 to 7.

5. The preparation method according to feature 10. [Claim 14] The mass ratio of the esterase variant to the ester compound is 0.2 to 2:

20. The preparation method according to feature 10. [Claim 15] The mass ratio of the esterase variant to the ester compound is 0.1 to 0.5:

10. The preparation method according to feature 10. [Claim 16] The catalytic reaction system further includes a solubilizing agent selected from the group consisting of DMSO, DCM, and 2-MeTHF. The preparation method according to feature 13. [Claim 17] The volume percentage content of the solubilizer in the catalytic reaction system is ≤20%. The preparation method according to feature 16.

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