Esterase whole cell catalysts, their preparation and use

Abstract

The application discloses an esterase whole cell catalyst and preparation and application thereof, the esterase whole cell catalyst can express a carboxylic acid esterase mutant disclosed in the application, the carboxylic acid esterase mutant is subjected to the following mutations on the amino acid sequence shown in SEQ ID NO. 1: the glycine at the 236th position is replaced by valine, the arginine at the 417th position is replaced by phenylalanine, and the threonine at the 545th position is replaced by alanine. The application effectively solves the problems of low catalytic activity of a warming catalyst for resolving methyl chrysanthemate and poor product tolerance in the prior art. The esterase whole cell catalyst of the application significantly improves the activity of selectively resolving methyl chrysanthemate to prepare dextro-trans chrysanthemic acid, and the optical purity can reach more than 99%. Moreover, the carboxylic acid esterase mutant disclosed in the application can be expressed in a soluble form in cell supernatant, can be produced in a large scale through microbial fermentation, has mild reaction conditions, and is simple to operate, and is more suitable for industrial production.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, specifically to a whole-cell esterase catalyst and its preparation and application. Background Technology

[0002] Chrysanthemic acid (2,2,3,3-tetramethylcyclopropanecarboxylic acid), an important pesticide intermediate, has two chiral carbon atoms on its cyclopropane ring, resulting in two configurations: left-handed and right-handed. Including cis and trans isomers, chrysanthemic acid has four isomers. As the acidic component of pyrethroid pesticides, different isomers of chrysanthemic acid exhibit significant differences in insecticidal activity. The right-handed trans-chrysanthemic acid ((+)-trans-CA) shows higher biological activity and is an important intermediate in the synthesis of pesticides such as cypermethrin. Previous reports have indicated that chemical and enzymatic methods for resolving racemic esters or acids are important methods for synthesizing optically pure chrysanthemic acid. Currently, industrial production mainly uses the diazoacetic acid ester method (also known as the Harper and Campbell method) and the isopentenyl sulfone method (also known as the Martel method). The ethyl chrysanthemic acid (methyl ester) obtained by these methods is hydrolyzed in an alcoholic solution in the presence of alkali, followed by acidification to obtain chrysanthemic acid. The production process uses a large amount of acids, alkalis and organic reagents, causing environmental pollution and low economic value.

[0003] Current research includes studies on the biological synthesis of dextrorotatory trans-chrysanthemic acid. One example is the esterase gene from *Arthrobacter globiformis* reported by MASAKO et al., which exhibits stereoselective hydrolysis efficiency even at substrate concentrations up to 40%, removing the generated chrysanthemic acid via ultrafiltration. The resulting (+)-trans-chrysanthemic acid has an optical purity of 100%. However, some product inhibition exists, necessitating multiple ultrafiltration separations before further reaction, resulting in complex reaction conditions and high purification costs.

[0004] Therefore, there is an urgent need for a biocatalyst with high catalytic activity and high product tolerance for the resolution of racemic prochrysanthemic acid to meet the requirements of industrial production. In view of this, the present invention is proposed. Summary of the Invention

[0005] The present invention aims to overcome the problems of low catalytic activity and poor product tolerance of the heating catalysts used in the resolution of methyl chrysanthemate in the prior art. Compared with traditional enzyme catalysis, whole-cell catalysis has higher stability and reusability. Therefore, the present invention provides a whole-cell catalyst for esterase that can express a carboxylesterase mutant, as well as its preparation and application, to overcome the above-mentioned defects.

[0006] To achieve the objectives of the invention described above, the present invention is implemented through the following technical solution:

[0007] In a first aspect, this invention discloses a carboxylesterase mutant, wherein the carboxylesterase mutant undergoes the following mutations in the amino acid sequence shown in SEQ ID NO. 1: glycine at position 236 is replaced with valine, arginine at position 417 is replaced with phenylalanine, and threonine at position 545 is replaced with alanine. The amino acid sequence of the carboxylesterase mutant is shown in SEQ ID NO. 2. By selecting the above-mentioned sites for mutation, the resulting carboxylesterase product exhibits lower inhibition, which can solve the problems of low catalytic activity and poor product tolerance of the temperature-increasing catalysts used in the prior art for the resolution of methyl chrysanthemate.

[0008] To address the shortcomings of existing technologies and methods and the practical needs of industrial production, this invention screened enzymes from numerous microbial sources and ultimately identified a carboxylesterase gene (EST1, GeneBank: U35855.1) from *Bacillus licheniformis* suitable for industrial production and enzyme engineering. Using this gene as the starting strain, enzyme engineering was performed to enhance its activity for catalytic synthesis, aiming to solve various problems in existing technologies. After initial mutation screening, several mutant strains with increased enzyme activity compared to the starting strain were obtained. One mutant strain exhibited 3.4 times the enzyme activity of the starting strain. Using this mutant strain to synthesize dextrorotatory trans-chrysanthemic acid (+)-trans-CA, the highest yield was 98.2 g / L after 48 h of reaction.

[0009] The carboxylesterase mutant provided by this invention, by selecting sites R417, G236, and T545 on the amino acid sequence shown in SEQ ID NO.1 as modification sites, yields the carboxylesterase mutant EST-G236V / R417F / T545A (EST-M3), which significantly improves its activity in selectively resolving methyl chrysanthemate to prepare dextrorotatory trans-chrysanthemic acid at high product concentrations, with an optical purity exceeding 99.2%. The obtained carboxylesterase mutant can be solublely expressed in cell supernatant and can be produced on a large scale through microbial fermentation. The reaction conditions are mild, the operation is simple, and it is more suitable for industrial production.

[0010] Secondly, the present invention also discloses a polynucleotide capable of encoding the above-mentioned carboxylesterase mutant, the nucleotide sequence of which is shown in SEQ ID NO.3. Herein, "polynucleotide" refers to a polymeric form of nucleotides of any length, including ribonucleotides and / or deoxyribonucleotides. Examples of polynucleotides include, but are not limited to, single-stranded, double-stranded, or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or polymers containing purine and pyrimidine bases or other naturally occurring, chemically or biochemically modified, non-natural, or derived nucleotide bases. The polynucleotide encodes the above-mentioned T7 RNA polymerase mutant, optionally encoding the sense or antisense strand. The polynucleotide can be naturally occurring, synthetic, recombinant, or any combination thereof. The terms "polynucleotide" and "nucleic acid" are used interchangeably in this specification.

[0011] Thirdly, the present invention also discloses a recombinant vector carrying the nucleotide sequence shown in SEQ ID NO. 3. The vector is known to those skilled in the art and includes, but is not limited to: plasmids, bacterial microparticles, Cos plasmids, or artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1-derived artificial chromosomes (PAC); bacteriophages such as phage or M13 phage; and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retrotranscriptoviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomaviruses. In some embodiments, the vector of the present invention contains regulatory elements commonly used in genetic engineering, such as enhancers, promoters, internal ribosome entry sites (IRES), and other expression control elements (e.g., transcription termination signals, or polyadenylation signals and poly-U sequences, etc.). In the present invention, preferably, the recombinant vector is pET28a(+), pET21a(+), or pET21b(+).

[0012] Furthermore, the recombinant vector contains a T7 promoter. The T7 promoter is a relatively weak promoter, which facilitates the screening of highly active carboxylesterase mutants and does not affect the increased activity of intracellular enzymes due to excessively high transcription levels.

[0013] Fourthly, this invention also discloses a whole-cell esterase catalyst, wherein the whole-cell esterase catalyst is a recombinant cell capable of expressing the carboxylesterase mutant described in the first aspect of this invention. The cell may refer to a single cell, a cell line, or a cell culture. In this document, "cell" includes its progeny, which may not be completely identical to the primary cell due to natural, accidental, or intentional mutations, and may differ morphologically and / or in genomic DNA from the primary cell. The cell may be a natural cell or a transformant.

[0014] Furthermore, the recombinant cells carry a nucleotide sequence as shown in SEQ ID NO.2, or carry the recombinant vector described in the third aspect of the present invention and the above-mentioned recombinant vector containing the T7 promoter.

[0015] Fifthly, the present invention also discloses a method for constructing the above-mentioned recombinant cells, comprising: transforming the above-mentioned recombinant vector into a host bacterium to obtain recombinant cells. The host bacterium is preferably *Escherichia coli*, such as *E. coli* W3110, *E. coli* W3110, *E. coli* BL21, *E. coli* BW25113, and other model strains commonly used as genetic engineering hosts in the art, as long as the genes to be overexpressed or heterologously overexpressed can be expressed in it. However, the host bacterium is not limited to *Escherichia coli*, and other suitable host bacterium species, such as *Bacillus subtilis* or *Saccharomyces cerevisiae*, can be selected according to specific circumstances.

[0016] Sixthly, this invention also discloses the application of a whole-cell esterase catalyst in the preparation of dextrorotatory trans-chrysanthemic acid. The carboxylesterase mutant disclosed in this invention catalyzes the one-step conversion of the substrate methyl chrysanthemate to dextrorotatory trans-chrysanthemic acid, as shown in the example... Figure 2 As shown.

[0017] In a seventh aspect, the present invention also discloses a method for preparing dextrorotatory trans-chrysanthemic acid, comprising the following steps:

[0018] (1) Mix the whole-cell esterase catalyst, buffer solution and racemic methyl chrysanthemate disclosed in this invention evenly and carry out the catalytic reaction;

[0019] (2) The reaction product was purified to obtain dextrorotatory trans-chrysanthemic acid.

[0020] In some specific embodiments, the temperature of the catalytic reaction is 40~55℃, preferably 45~50℃;

[0021] The catalytic reaction takes 1-6 hours, preferably 3-6 hours.

[0022] The pH of the catalytic reaction is 6-9, preferably 6-8;

[0023] Preferably, the mass concentration of the racemic methyl chrysanthemate is 50 g / L to 140 g / L, and more preferably 100 g / L to 140 g / L;

[0024] Preferably, the amount of the carboxylesterase mutant is 1 g / L to 20 g / L, more preferably 5 g / L to 10 g / L;

[0025] Preferably, the buffer solution is selected from phosphate buffer, Tris-HCl or NaOH-glycine solution;

[0026] Preferably, the buffer solution is a phosphate buffer solution;

[0027] Preferably, the molar concentration of phosphate in the phosphate buffer solution is 30 mM to 100 mM.

[0028] Therefore, the present invention has the following beneficial effects:

[0029] (1) The present invention obtains a carboxylesterase mutant by selecting a mutation site on the amino acid sequence shown in SEQ ID NO.1 as the site for modification, which effectively solves the problems of low catalytic activity and poor product tolerance of the heating catalyst used to resolve methyl chrysanthemate in the prior art.

[0030] (2) The carboxylesterase mutant of the present invention significantly improves its activity in selectively resolving methyl chrysanthemate to prepare dextrorotatory trans-chrysanthemic acid, and the optical purity can reach more than 99%.

[0031] (3) The carboxylesterase mutant of the present invention can be expressed solublely in cell supernatant and can be produced on a large scale by microbial fermentation. The reaction conditions are mild and the operation process is simple, making it more suitable for industrial production. Attached Figure Description

[0032] Figure 1 This is a schematic diagram of the plasmid vector pET28a(+)-EST1 containing the carboxylesterase gene.

[0033] Figure 2 This diagram illustrates the one-step catalytic reaction of engineered bacteria containing carboxylesterase and its mutant genes to convert methyl chrysanthemate to dextrorotatory trans-chrysanthemic acid.

[0034] Figure 3 This is a schematic diagram illustrating the protein structure and substrate docking of carboxylesterase.

[0035] Figure 4 This is a schematic diagram of the amino acid composition of the substrate active pocket.

[0036] Figure 5 The reaction process diagram shows the synthesis of dextrorotatory trans-chrysanthemic acid using wild-type strains EST1 and EST-M3 under optimal conditions with 200 g / L substrate. Detailed Implementation

[0037] The present invention will be further described below with reference to specific embodiments. Those skilled in the art will be able to implement the present invention based on these descriptions. Furthermore, the embodiments of the present invention described below are generally only some, not all, of the embodiments of the present invention. Therefore, all other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort should fall within the scope of protection of the present invention.

[0038] Example 1:

[0039] Construction of the recombinant vector pET28a(+)-EST1, as follows: Figure 1 As shown in the figure. In this embodiment, the expression vector pET28a(+) was purchased from Hangzhou Qingke Biotechnology Co., Ltd., and the carboxylesterase gene sequence having the amino acid sequence shown in SEQ ID NO.1 was synthesized by Hangzhou Qingke Biotechnology Co., Ltd.

[0040] The cloning vector was transformed into Escherichia coli BL21(DE3) competent cells. Recombinants were screened on LB agar plates containing kanamycin resistance, and single clones were selected for colony PCR verification. The nucleotide sequences of the identification primers used are shown in SEQ ID NO.4 and SEQ ID NO.5. Positive clones were sent to Qingke Biotechnology Co., Ltd. for sequencing.

[0041] Example 2:

[0042] Culture of carboxylesterase-expressing cells. The recombinant bacteria from Example 1 were cultured, the recombinant vector was amplified and extracted, and transformed into Escherichia coli E. coli BL21(DE3) competent cells. The cells were then plated on LB agar plates containing kanamycin and incubated overnight at 37°C inverted, thus forming carboxylesterase-expressing cells.

[0043] The obtained carboxylesterase-expressing cells were streaked onto LB agar plates containing kanamycin. Single colonies were picked and inoculated into 5 mL of LB medium containing kanamycin (10 g / L peptone, 5 g / L yeast extract, 10 g / L NaCl, pH 7.0) and cultured overnight at 37°C and 180 rpm. The inoculum was then transferred at a 1:100 ratio to 100 mL of seed culture medium in a 500 mL Erlenmeyer flask and cultured at 37°C with shaking at 180 rpm until the OD reached approximately 0.6–0.8.

[0044] IPTG was added to a final concentration of 0.1 mM, and the induction temperature was adjusted to 28°C to begin culturing. The culture medium was collected after 12 hours. Cells were collected by centrifugation and washed three times with physiological saline. The cells were resuspended in phosphate buffer (pH 7.0) and disrupted using an ultrasonic homogenizer, maintaining a cell dry weight concentration of 40 g / L. The ultrasonic homogenizer power was 250 W. The supernatant was collected by centrifugation, which is the crude carboxylesterase enzyme solution. Next, the crude enzyme solution can be purified using gravity nickel column affinity chromatography to obtain the purified carboxylesterase enzyme solution. Considering the simplicity of the reaction, the subsequent catalytic reaction was carried out using the crude carboxylesterase enzyme solution.

[0045] Example 3:

[0046] Three-dimensional modeling of carboxylesterases and modification of substrate channel amino acid residues. For example... Figure 3 As shown, carboxylesterase was homology-modeled using the online website Swiss-model (https: / / swissmodel.expasy.org / ) to obtain a simulated three-dimensional protein structure. The EST1 protein structure was then docked with methyl chrysanthemate via MOE, and the docking results were analyzed and simulated. The simulation ultimately yielded a substrate pocket consisting of 19 amino acids, as shown below. Figure 4 As shown. Site-directed saturation mutagenesis was performed on these 19 amino acids. The amino acid residues contained in the substrate pocket are as follows: I235, G236, S382, P411, G414, S415, R417, A418, A421, Y426, G429, S442, A447, V448, L451, T545, V546, H568.

[0047] PCR reaction system (50 µL): 2 µL forward primer (10 μM), 2 µL reverse primer (10 μM), 25 µL 2×Phanta buffer, 1 µL dNTP mixture (10 mM each), 1 µL plasmid template, 1 µL DNA polymerase, and 18 µL ultrapure water. The PCR program set according to the Phanta Super-Fidelity DNA polymerase instructions was as follows: 95℃ pre-denaturation for 5 min, followed by 29 cycles (95℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 5 min), final extension at 72℃ for 5 min, and incubation at 16℃. The resulting recombinant plasmid was transformed into E. coli BL21(DE3) competent cells via heat shock at 42℃, and the plated cells were incubated at 37℃ for 12 h.

[0048] Example 4:

[0049] Screening of carboxylesterase mutants. The mutants from Example 3 were expanded and transferred to test tubes containing 10 mL LB liquid medium (50 µg / mL kanamycin). After incubation at 37°C and 180 rpm for 10 h, the cultures were sent to Hangzhou Qingke Biotechnology Co., Ltd. for sequencing. One mL of the successfully sequenced mutant culture was transferred to a shake flask containing 100 mL LB liquid medium (50 µg / mL kanamycin) and cultured until an OD600 value of 0.8 was reached. After induction at 28°C for 12 h, the cells were collected by centrifugation. The obtained mutants were screened, and the original strain of *E. coli* BL21(DE3) / pET28a(+)-EST1 was simultaneously cultured in shake flasks as a positive control. A 1 mL transformation system (cell concentration 10 g WCW / L) was prepared by adding 50 mM pH 7.5 phosphate buffer to a final concentration of 10 g / L methyl chrysanthemate. The reaction was carried out at 45 °C and 800 rpm for 10 min. The reaction was terminated by adding 20 µL 6 M HCl to each 1 mL of the solution. After mixing with 500 μL ethyl acetate, the solution was centrifuged at 12000 rpm for 3 min and the supernatant was collected. This process was repeated three times. The solution was dried overnight in a fume hood and then dissolved in 1 mL of n-hexane before liquid chromatography analysis.

[0050] Example 5:

[0051] A liquid chromatography method for the synthesis of chrysanthemic acid catalyzed by carboxylesterase was developed. The dissolved sample was filtered through a 0.22 µm nylon membrane for liquid chromatography analysis. A CHIRALPAKQN-AX chiral column (quinine alkaloids) was used to separate the enantiomers of chrysanthemic acid. The mobile phase ratio was acetonitrile:water:acetic acid = 90:10:0.5 (v / v), the flow rate was 0.8 mL / min, the temperature was 15 °C, the detection wavelength was 230 nm, the column temperature was 15 °C, and the injection volume was 20 μL. The solvent retention time was 1.93 min, and the peak elution time of dextrorotatory trans-chrysanthemic acid was 2.85 min. HPLC results showed that the increased carboxylesterase activity was observed at EST-G236V, EST-S382W, EST-R417F, EST-Y426E, and EST-T545A, with enzyme activities of each mutant being 113.2%, 124.1%, 161.5%, 119%, and 143.8% of the wild-type carboxylesterase activity, respectively.

[0052] Example 6:

[0053] Determination of specific enzyme activity of wild-type carboxylesterase and its mutants. Enzyme activity unit definition: The amount of cells required to generate 1 micromole of dextrorotatory trans-chrysanthemic acid per minute at 45℃ and pH 8 is defined as one enzyme activity unit, U. Specific enzyme activity is defined as the number of activity units per gram of cells, U / g. Enzyme activity detection standard conditions: In a 1000 µL reaction system, add 20 g / L methyl chrysanthemic acid, 10 g / L resting cells, and use 100 mM phosphate buffer at 45℃ and pH 8 as the reaction medium. React at 800 rpm for 10 min, process the samples, and perform HPLC analysis.

[0054] Table 1. Relative and specific enzyme activities of carboxylesterases and their mutants

[0055]

[0056] Example 7:

[0057] (+)-trans-CA was synthesized from 10 g / L wild-type carboxylesterase-expressing cells in an aqueous system at 35 °C using 20 g / L methyl chrysanthemate as a substrate. A 20 mL transformation system (cell concentration 10 g WCW / L) was prepared by adding methyl chrysanthemate ((+)-trans:(+)-cis:(-)-trans:(-)-cis = 48.8:48.1:1.4:1.7) to a final concentration of 10 mM pH 8 phosphate buffer. The reaction was carried out at 35 °C and 800 rpm for 12 h. After the reaction, 1 mL of the reaction solution was taken and 20 µL of 6 M HCl was added to terminate the reaction. After extraction with ethyl acetate and drying, the solution was filtered through a 1 mL n-hexane membrane and analyzed by HPLC, yielding a (+)-trans-CA concentration of 4.80 g / L.

[0058] Example 8:

[0059] In an aqueous system at 45℃, 10 g / L wild-type carboxylesterase-expressing cells were used to catalyze the synthesis of (+)-trans-CA using 20 g / L methyl chrysanthemate as a substrate. A 20 mL transformation system (cell concentration 10 g WCW / L) was prepared by adding 20 g / L methyl chrysanthemate to a final concentration of 100 mM pH 8 phosphate buffer. The reaction was carried out at 35℃ and 800 rpm for 6 h. After the reaction, 1 mL of the reaction solution was taken and 20 µL of 6 M HCl was added to terminate the reaction. After extraction with ethyl acetate and drying, the solution was filtered through a 1 mL n-hexane membrane and analyzed by HPLC, yielding a (+)-trans-CA concentration of 9.40 g / L. Compared with the reaction at 35℃, it can be seen that the appropriate reaction temperature can significantly increase the catalytic synthesis efficiency of (+)-trans-CA, thereby increasing the yield.

[0060] Example 9:

[0061] In an aqueous system at 45℃, EST-G236V / R417F / T545A carboxylesterase mutant cells (10 g / L) were used to catalyze the synthesis of (+)-trans-CA using methyl chrysanthemate as a substrate. A transformation system of 20 mL (cell concentration 10 g WCW / L) was prepared by adding methyl chrysanthemate to a final concentration of 20 g / L in 100 mM pH 8 phosphate buffer. The reaction was carried out at 45℃ and 800 rpm for 6 h. After the reaction, 1 mL of the reaction solution was taken and 20 µL of 6 M HCl was added to terminate the reaction. After extraction with ethyl acetate and drying, the solution was filtered through a 1 mL n-hexane membrane and analyzed by HPLC, yielding a (+)-trans-CA concentration of 9.60 g / L.

[0062] Example 10:

[0063] In an aqueous system at 45℃, wild-type and mutant EST-G236V / R417F / T545A (M3) cells expressing carboxylesterase (10 g / L) were used to catalyze the synthesis of (+)-trans-CA using 400 g / L methyl chrysanthemate as a substrate. A 100 mL transformation system (wild-type cell concentration 10 g WCW / L) was prepared by adding methyl chrysanthemate to a final concentration of 400 g / L using 100 mM pH 8 phosphate buffer as a buffer. The reaction was carried out at 45℃ and 800 rpm for 3 h. After the reaction, 500 μL of the reaction solution was taken and 10 µL of 6M HCl was added to terminate the reaction. The solution was extracted with ethyl acetate, dried, filtered through a 500 μL n-hexane filter, and the concentration of (+)-trans-CA was determined by HPLC to be 38%. The reaction was continued for 6 hours. At 9 hours, the concentrations of (+)-trans-CA were measured to be 48.5 g / L and 51.75 g / L, respectively. After 10 hours, the product no longer accumulated, and the reaction rate stagnated, which was suspected to be due to product inhibition. The reaction solution was filtered through a hollow fiber ultrafiltration membrane to remove the generated chrysanthemic acid and the reaction was continued. The above steps were repeated 4 times until the reaction was terminated after 50 hours. All aqueous phases were extracted in batches in a separatory funnel. The extracts were collected in a beaker, dried, and then hexane was added to 100 ml to fully dissolve the product. After filtration, the concentration of (+)-trans-CA was detected by HPLC to be 172 g / L.

[0064] Using 100 mM pH 8 phosphate buffer as the buffer system, 100 mL of substrate methyl chrysanthemate (concentration of mutant EST-M3 cells 10 g WCW / L) was added to form a transformation system. The reaction was carried out at 45℃ and 800 rpm for 3 h. After the reaction, 500 μL of the reaction solution was added to 10 µL of 6 M phosphate buffer. The reaction was terminated with HCl. After extraction with ethyl acetate and drying, 500 μL of n-hexane was added and filtered through a membrane. The concentration of (+)-trans-CA was then detected by HPLC, yielding a concentration of 47.75 g / L. The reaction was continued for 6 h. At 9 h, the concentration of (+)-trans-CA was measured to be 114.75 g / L and 120.25 g / L. At 10 h, the product concentration was 121.75 g / L. The reaction solution was then filtered through a hollow fiber ultrafiltration membrane to remove the generated chrysanthemic acid, and the reaction was continued. The same steps were repeated for sampling and testing. The reaction was terminated after 50 h, and the concentration of (+)-trans-CA was measured to be 194.75 g / L.

[0065] like Figure 5 As shown, the wild-type carboxylesterase requires four ultrafiltrations to achieve a high concentration of product (+)-trans-CA during catalysis. The carboxylesterase mutant prepared in this invention exhibits high tolerance to (+)-trans-CA, successfully overcoming product inhibition. Only one ultrafiltration is needed to achieve a product concentration of 194.75 g / L with an optical purity of 99%. It can be produced on a large scale through bio-fermentation, which is simple to operate and has high selectivity and catalytic activity for methyl chrysanthemate, making it more suitable for industrial production.

[0066] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A carboxylesterase mutant, characterized in that, The carboxylesterase mutant has the following mutations in the amino acid sequence shown in SEQ ID NO.1: glycine at position 236 is replaced with valine, arginine at position 417 is replaced with phenylalanine, and threonine at position 545 is replaced with alanine. The amino acid sequence of the carboxylesterase mutant is shown in SEQ ID NO.

2.

2. A polynucleotide encoding a carboxylesterase mutant as described in claim 1, characterized in that: The nucleotide sequence of the polynucleotide is shown in SEQ ID NO.

3.

3. A recombinant vector carrying the nucleotide sequence as described in claim 2.

4. The recombinant vector according to claim 3, characterized in that: The recombinant vector contains the T7 promoter.

5. A whole-cell catalyst for an esterase, characterized in that: The whole-cell esterase catalyst is a recombinant cell capable of expressing the carboxylate esterase mutant as described in claim 1.

6. The whole-cell catalyst for esterase according to claim 5, characterized in that: The recombinant cells carry a nucleotide sequence as shown in SEQ ID NO.3, or carry a recombinant vector as described in claim 3 or 4.

7. A method for constructing recombinant cells, characterized in that: The recombinant vector as described in claim 3 or 4 is transformed into a host bacterium to obtain recombinant cells.

8. The application of the whole-cell esterase catalyst as described in claim 5 in the preparation of dextrorotatory trans-chrysanthemic acid.

9. A method for preparing dextrorotatory trans-chrysanthemic acid, characterized in that, Includes the following steps: (1) Mix the whole-cell esterase catalyst, buffer solution, and racemic methyl chrysanthemate as described in claim 5 or 6 evenly and carry out the catalytic reaction; (2) The reaction product was purified to obtain dextrorotatory trans-chrysanthemic acid.

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