A recombinant esterase mutant, gene, engineered bacteria and its application

Abstract

This invention discloses a recombinant esterase mutant, gene, engineered bacteria, and its application in the preparation of (S)-3-cyclohexene-1-carboxylic acid. The recombinant esterase mutant provided by this invention exhibits high catalytic activity, resulting in mild reaction conditions. Under the catalysis of 20 g / L of wet cells catalyzed by 100 g / L racemic methyl 3-cyclohexene-1-carboxylate, the conversion rate is >49.9% within 2 hours, reducing production costs and being environmentally friendly.

Description

(I) Technical Field

[0001] This invention belongs to the fields of biopharmaceuticals and biotransformation, specifically relating to an esterase mutant and its encoding gene, a plasmid containing the encoding gene of the mutant and recombinant bacteria, and the application of the esterase mutant in the catalytic preparation of S-3-cyclohexene-1-carboxylic acid from methyl 3-cyclohexene-1-carboxylate. (II) Background Technology

[0002] In a broad sense, esterases (EC3.1.1.X) are a general term for enzymes that catalyze the hydrolysis and synthesis of ester bonds (carboxyl ester bonds, amide bonds, thioester bonds, etc.). During ester synthesis, they catalyze the dehydration condensation of the hydroxyl group of an alcohol with the carboxyl group of an acid to obtain ester compounds. During ester hydrolysis, they produce the corresponding fatty acids and glycerol. They mainly include carboxylesterases (EC3.1.1.1) and lipases (EC3.1.1.3). Based on substrate specificity, sequence alignment, conserved domains, and enzymatic properties, esterases are classified into eight families (I-VIII), with lipases belonging to family I and carboxylesterases belonging to families II-VIII. As a class of commonly used industrial biocatalysts, carboxylesterases and lipases have significant advantages: 1) strong stereoselectivity and high specificity in the regio; 2) good tolerance and stability to organic solvents; 3) no need for coenzymes and cofactors to participate in the reaction; 4) mild reaction conditions and simple operation; 5) broad substrate spectrum; 6) reduced environmental pollution, etc.

[0003] Carboxylesterase (EC 3.1.1.3), carboxylesterase, and triacylglycerolase all belong to the α / β-sheet mono-subunit glycoprotein proteases. Their catalytic triad consists of Ser-Asp / Glu-His, and they typically share a common sequence around the serine residue at the active site (Gly-x-Ser-x-Gly). The most significant difference between carboxylesterase and lipase lies in their substrate composition: lipases primarily catalyze long-chain fatty acids (C>8), while carboxylesterases primarily catalyze short-chain esters (C<8). Furthermore, lipases exhibit interfacial activation; high activity is only observed at low substrate concentrations, while carboxylesterases follow the classic Michaelis-Menten kinetics. Structural analysis reveals that lipases possess a hydrophobic area (cap), and their interfacial activation is due to the cap blocking the active site. The cap only opens at low substrate concentrations, exposing the catalytic active site. Both enzymes have been shown to be stable and active in organic solvents. The principles of ester hydrolysis and ester synthesis are essentially the same: First, the substrate and the active serine residue of the esterase bind, forming a tetrahedral intermediate in a transition state under the action of histidine and aspartic acid residues. Through the force of serine, the ester bond breaks, forming an acyl-enzyme complex and an alcohol. Then, the covalent bond between the acyl group and serine breaks under the action of a nucleophilic reagent (water), forming the product acid and the free enzyme.

[0004] (S)-3-cyclohexene-1-carboxylic acid is an important chiral intermediate in the synthesis of edoxaban (trade name: Savaysa), a factor Xa inhibitor. Edoxaban is a small-molecule oral anticoagulant developed by Daiichi Sankyo Co., Ltd. of Japan. Compared with similar factor Xa inhibitors such as apixaban and rivaroxaban, it has significant advantages such as lower bleeding risk, less renal burden, and safer use, and has a broad market prospect. As a key intermediate in this drug, the efficient preparation technology of (S)-3-cyclohexene-1-carboxylic acid has become a research hotspot. Currently, in industry, diastereomeric resolution technology is used to prepare (S)-3-cyclohexene-1-carboxylic acid. Chiral phenylethylamine is used as a chiral resolving agent to separate the diastereomeric isomers formed by racemic 3-cyclohexene-1-carboxylic acid and phenylethylamine based on the difference in solubility of these two diastereomeric isomers in acetone. After slow cooling and recrystallization six times, the final yields of R-3-cyclohexene-1-carboxylic acid were 28.3% and (S)-3-cyclohexene-1-carboxylic acid were 28.7%, with optical purities greater than 99% for both.

[0005] However, the chiral resolving agents used in this technology are expensive, require large quantities, are complex to operate, and have low yields, which does not meet the requirements of green chemistry. Therefore, the development of green and efficient (S)-3-cyclohexene-1-carboxylic acid preparation technology has become an important research area in the synthesis of edoxaban.

[0006] The asymmetric resolution of racemic methyl cyclohexene-1-carboxylate to prepare chiral 3-cyclohexene-1-carboxylic acid using esterase catalysis has been studied. Commercially available porcine liver esterase can catalyze the hydrolysis of (R,S)-3-cyclohexene-1-carboxylate to prepare chiral 3-cyclohexene-1-carboxylic acid, with a (S)-configuration carboxylic acid conversion rate of 49% and ee > 99%. Wang Jian et al. used Acinetobacter sp 192 whole-cell esterase-containing organisms as catalysts to catalyze the asymmetric resolution of 40 g / L racemic methyl cyclohexene-1-carboxylate. After 5 h of reaction, the product R-3-cyclohexene-1-carboxylic acid had an ee value of 75% and a yield of 40.7%. Wu et al. molecularly modified the carboxylesterase BioH derived from *E. coli*, adjusting its enantioselectivity by modifying the steric hindrance of the active pocket, the aromatic ring interaction between the enzyme and substrate, and hydrogen bonding, significantly improving the enzyme's stereoselectivity and increasing the ee value of the product (S)-3-cyclohexene-1-carboxylic acid to 79.9%. Ni et al. utilized an esterase derived from *Acinetobacter* to catalyze the asymmetric resolution of racemic methyl 3-cyclohexene-1-carboxylic acid. With substrate concentrations of 100-500 mM (14-70 g / L), the yield of (S)-3-cyclohexene-1-carboxylic acid was >40%, and the ee value was >99%. Therefore, the esterase-catalyzed preparation of (S)-3-cyclohexene-1-carboxylic acid demonstrates good research value and application prospects.

[0007] However, the above reports are limited to laboratory scale and suffer from problems such as the catalysts mostly being commercial enzymes, poor substrate tolerance, and insufficient product concentration or selectivity. Therefore, for cyclohexene carbamate compounds, there is an urgent need to screen for highly efficient and selective biocatalysts to meet industrial needs. (III) Summary of the Invention

[0008] The purpose of this invention is to provide a recombinant esterase mutant, its encoding gene, a recombinant vector containing the mutant gene, a recombinant genetically engineered bacterium transformed from the recombinant vector, and its application in the asymmetric resolution of methyl 3-cyclohexene-1-carboxylate to prepare (S)-3-cyclohexene-1-carboxylic acid. The recombinant esterase mutant provided by this invention exhibits high catalytic activity, resulting in milder reaction conditions, significantly improved catalytic efficiency and conversion rate, reduced production costs, and environmental friendliness.

[0009] The technical solution adopted in this invention is:

[0010] This invention provides a recombinant esterase mutant capable of asymmetricly resolving (S)-3-cyclohexene-1-carboxylic acid using methyl 3-cyclohexene-1-carboxylate as a substrate under suitable conditions. The recombinant esterase mutant is obtained by single mutation at positions 25, 26, 27, 37, 93, 118, 126, 141, 167, 171, and 224 of the amino acid sequence shown in SEQ ID NO.2.

[0011] Furthermore, the mutant (conversion rate > 49%) is preferably a mutant in which the amino acid sequence shown in SEQ ID NO. 2 is mutated to one of the following: (1) phenylalanine at position 25 is mutated to isoleucine; (2) arginine at position 141 is mutated to glycine; (3) leucine at position 167 is mutated to alanine; (4) leucine at position 167 is mutated to phenylalanine; (5) isoleucine at position 171 is mutated to methionine; (6) valine at position 224 is mutated to glutamic acid.

[0012] Due to the specificity of amino acid sequences, any fragment or variant of a peptide protein containing the amino acid sequence shown in this invention, such as its conserved variants, bioactive fragments, or derivatives, is within the scope of protection of this invention, provided that the fragment or variant shares more than 90% homology with the aforementioned amino acid sequence. Specifically, the alterations include the deletion, insertion, or substitution of amino acids in the amino acid sequence; wherein, for conserved alterations of variants, the substituted amino acid has a similar structure or chemical properties to the original amino acid, such as replacing isoleucine with leucine; variants may also have non-conserved alterations, such as replacing glycine with tryptophan.

[0013] This invention provides a gene encoding the recombinant esterase mutant.

[0014] Due to the specific nature of nucleotide sequences, any variant of the polynucleotides described in this invention, provided that it shares more than 90% homology with the aforementioned polynucleotides, falls within the scope of protection of this invention. A variant of the polynucleotide refers to a polynucleotide sequence with one or more nucleotide alterations. This polynucleotide variant can be a live or non-live variant, including substitution variants, deletion variants, and insertion variants. As is known in the art, an allelic variant is a substitution of a polynucleotide, which may be a substitution, deletion, or insertion of a polynucleotide, but does not substantially alter the function of the peptide protein it encodes.

[0015] The present invention also provides a recombinant vector containing the recombinant esterase mutant encoding gene and the constructed engineered bacteria, wherein the expression vector is pET28a(+) and the engineered bacteria host is E. coli BL21(DE3).

[0016] The recombinant esterase mutant of this invention is obtained by mutating a single amino acid of the wild-type esterase EST10 to enhance its catalytic activity against racemic 3-cyclohexene-1-carboxylic acid methyl ester. First, the encoding gene of the wild-type esterase EST10 (SEQ ID NO.1) is ligated to the expression vector pET28a(+) to construct a recombinant expression plasmid. Then, the recombinant expression plasmid is transformed into *E. coli* BL21(DE3). Using the recombinant expression plasmid containing the esterase gene as a template, gene modification is performed using site-directed mutagenesis. The recombinant expression plasmid is then transformed into *E. coli* BL21(DE3) to obtain *E. coli* BL21(DE3) genetically engineered bacteria containing the esterase mutant gene. The obtained recombinant genetically engineered bacteria are induced and cultured. Cells containing the recombinant esterase mutant are isolated from the culture medium, and a crude enzyme solution of the esterase mutant is obtained from the cell cells. The catalytic activity of the mutant esterase is compared with that of the wild-type esterase, and mutants with superior catalytic performance are screened.

[0017] This invention relates to the application of the recombinant esterase mutant in the asymmetric resolution of methyl 3-cyclohexene-1-carboxylate to prepare (S)-3-cyclohexene-1-carboxylic acid. Specifically, the application involves using wet bacterial cells obtained by fermentation culture of engineered bacteria containing the recombinant esterase mutant encoding gene, or crude enzyme solution extracted by ultrasonic disruption of wet bacterial cells, or purified enzyme solution, as a catalyst. Methyl 3-cyclohexene-1-carboxylate is used as the substrate, and a potassium phosphate buffer solution with a pH of 6-9 (preferably 7) is used as the reaction medium to form a reaction system. The reaction is carried out at 20-40℃ (preferably 30℃) and 600 rpm until the reaction is complete. The reaction solution is then separated and purified to obtain S-3-cyclohexene-1-carboxylic acid. The catalyst dosage is 20-200 g / L (preferably 20 g / L) based on the weight of wet bacterial cells, and 10-30 mg / L (preferably 20 mg / L) based on the protein concentration in the crude or pure enzyme solution; the initial concentration of the substrate is 10-700 g / L (preferably 100 g / L).

[0018] The wet bacterial cells obtained by fermentation culture of the engineered bacteria containing the recombinant esterase mutant encoding gene of the present invention are prepared as follows: The engineered bacteria containing the recombinant esterase mutant encoding gene are inoculated into LB liquid medium containing a final concentration of 50 μg / mL kanamycin resistance, and cultured at 37°C and 180 rpm for 10 h. Then, the bacteria are inoculated into fresh LB liquid medium containing a final concentration of 50 μg / mL kanamycin resistance at a volume concentration of 2%, and cultured at 37°C and 180 rpm until the bacterial OD600 reaches 0.6-0.8. IPTG is added to a final concentration of 0.1 mM, and the bacteria are induced to grow at 28°C for 12 h. After centrifugation at 4°C and 8000 rpm for 10 min, the supernatant is discarded and the wet bacterial cells are collected.

[0019] Preparation of the crude enzyme solution according to this invention: Wet cells obtained by fermentation culture of engineered bacteria containing the recombinant esterase mutant encoding gene are resuspended in 9.8 mL of 100 mM, pH 7.0 potassium phosphate buffer solution at a concentration of 0.2 g wet cells. The cells are then ultrasonically disrupted under ice bath conditions (20-60 W, 2 s duration, 4 s interval, continuous disruption for 15 min) to obtain cell lysate. The ultrasonically disrupted cell lysate is centrifuged at 12000 rpm and 4℃ for 10 min, and the supernatant obtained is the desired crude enzyme solution.

[0020] The pure enzyme solution was prepared as follows: The crude enzyme solution was incubated with Ni2+ affinity chromatography resin equilibrated with binding buffer (50 mM, pH 7.0 sodium phosphate buffer, containing 300 mM NaCl), and then washed with rinsing buffer (50 mM, pH 7.0 sodium phosphate buffer, containing 300 mM NaCl, 50 mM imidazole) until there were basically no contaminating proteins. Then, the target protein was eluted with elution buffer (50 mM, pH 7.0 sodium phosphate buffer, containing 300 mM NaCl, 500 mM imidazole) and collected. After the purity was identified by electrophoresis (single band), the target proteins were combined and dialyzed with dialysis buffer (20 mM, pH 7.0 sodium phosphate buffer) (10 kDa molecular weight cutoff) for 24 h. The retentate was taken as the pure enzyme solution.

[0021] Compared with the prior art, the beneficial effects of the present invention are mainly reflected in:

[0022] This invention provides a series of esterase mutants with high catalytic activity and high stereoselectivity for racemic methyl 3-cyclohexene-1-carboxylate. The resulting esterase mutants, when used for the catalytic resolution to produce S-3-cyclohexene-1-carboxylic acid, offer advantages such as mild reaction conditions, high substrate concentration, high stereoselectivity, short reaction time, readily available catalysts, simplified catalyst processing steps, and environmental friendliness. Under the catalysis of 20 g / L of the esterase mutants (wet bacterial cells), the conversion rate of 100 g / L racemic methyl 3-cyclohexene-1-carboxylate is >49% within 2 hours, with an ee value reaching 99%, significantly superior to previously reported esterases. Among them, the esterase mutants EST10-F25I, EST10-R141G, and EST10-L167F achieve a conversion rate of 49.9% and an ee value of 99% within 2 hours. (iv) Description of the attached drawings

[0023] Figure 1 SDS-PAGE of esterase: Lane 3 is the protein molecular weight marker, Lane 1 is the crude enzyme solution after fermentation of E. coli BL21(DE3) / EST10, and Lane 2 is the broken-up precipitate of wet cells after fermentation of E. coli BL21(DE3) / EST10.

[0024] Figure 2 This is a schematic diagram of the asymmetric synthesis of S-3-cyclohexene-1-carboxylic acid.

[0025] Figure 3 This is a schematic diagram of GC detection of the product S / R-3-cyclohexene-1-carboxylic acid; A represents the reaction liquid without esterase for two hours, and B represents the reaction liquid with EST10-L167F added for two hours.

[0026] Figure 4 This is a schematic diagram showing the conversion rates of EST10 and its mutants. (V) Detailed Implementation

[0027] The present invention will be further described in detail below with reference to specific embodiments, but the present invention is not limited to the following embodiments. The implementation conditions used in the embodiments can be further adjusted according to different requirements of specific use, and the implementation conditions not specified are those in conventional experiments.

[0028] Example 1: Construction of wild-type esterase-engineered strain E. coli BL21(DE3) / EST10

[0029] The gene sequence of esterase EST10 (GenBank: BAA02182.1) from Geobacillus stearothermophilus in the gene bank was codon optimized, and then synthesized as a whole gene (nucleotide sequence as shown in SEQ ID NO.1, amino acid sequence as shown in SEQ ID NO.2). It was then transformed into the NcoI and XhoI sites of the vector pET28a(+) to obtain the pET28a(+)-EST10 plasmid. This plasmid was then transformed into E. coli BL21(DE3) to obtain wild-type E. coli BL21(DE3) / EST10, denoted as EST10.

[0030] SEQ ID NO.1

[0031] ATGATGAAAATCGTCCCACCAAAACCGTTCTTTTTCGAAGCCGGCGAACGTGCCGTTCTGCTGCTGCACGGCTTTACCGGTAACAGCGCAGATGTCCGCATGCTGGGTCGCTTTCTGGAAAGTAAAGGTTATACCTGTCACGCCCCGATTTATAAAGGTCATGGTGTTCCGCCGGAAGAACTGGTTCATACCGGCCCGGACGATTGGTGGCAGGATGTTATGAATGGTTATGAATTTCTGAAAAACAAAGGCTATGAGAAAATCGCAGTTGCGGGTCTGAGTCTGGGTGGTGTTTTTAGTCTGAAACTGGGTTATACCGTTCCGATTGAAGGGATTGTTACCATGTGCGCACCGATGTATATCAAAAGTGAAGAAACGATGTACGAGGGTGTTCTGGAATATGCGCGTGAATATAAAAAGCGCGAAGGTAAAAGCGAGGAACAGATTGAGCAGGAGATGGAAAAATTTAAACAGACCCCGATGAAAACCCTGAAAGCCCTGCAGGAACTGATTGCAGATGTTCGTGATCATCTGGATCTGATTTATGCACCGACATTTGTTGTGCAGGCACGTCACGATGAGATGATTAACCCGGATAGCGCAAACATTATTTATAACGAAATCGAAAGCCCGGTAAAACAGATTAAATGGTATGAGCAGAGCGGACATGTTATTACACTGGATCAGGAAAAAGATCAGCTGCATGAAGATATTTATGCATTTCTGGAGAGCCTGGATTGGCACCACCACCACCATCAC。

[0032] SEQ ID NO.2

[0033] MMKIVPPKPFFFEAGERAVLLLHGFTGNSADVRMLGRFLESKGYTCHAPIYKGHGVPPEELVHTGPDDWWQDVMNGYEFLKNKGYEKIAVAGLSLGGVFSLKLGYTVPIEGIVTMCAPMYIKSEETM YEGVLEYAREYKKREGKSEEQIEQEMEKFKQTPMKTLKALQELIADVRDHLDLIYAPTFVVQARHDEMINPDSANIIYNEIESPVKQIKWYEQSGHVITLDQEKDQLHEDIYAFLESLDWHHHHHHH.

[0034] Example 2: Site-directed mutagenesis to construct E. coli BL21(DE3) / EST10-muts

[0035] The three-dimensional structure of Est10 was constructed using Swiss-Model. After docking with the substrate, key amino acid sites near the active pocket were obtained. Energy calculations were performed using the DeepDDG online website (http: / / protein.org.cn / ddg.html). The PDB file of Est10 was uploaded, and the energy changes after saturation mutations were calculated for key amino acid sites (positions 25, 26, 27, 37, 93, 118, 126, 141, 167, 171, and 224). The mutant with the largest energy change was selected for site-directed mutagenesis to introduce a site-directed mutation into the esterase EST10. Primers were designed as follows:

[0036] F25I

[0037] Upstream primer 1: 5'-CTGCTGCTGCACGGCattACCGGTAACAGCGCA-3' Downstream primer 2: 5'-aatGCCGTGCAGCAGCAGAACGGCACGTTCG-3'

[0038] F25A

[0039] Upstream primer 3: 5'-CTGCTGCTGCACGGCgcgACCGGTAACAGCGCA-3' Downstream primer 4: 5'-cgcGCCGTGCAGCAGCAGAACGGCACGTTCG-3'

[0040] T26S

[0041] Upstream primer 5: 5'-CTGCTGCACGGCTTTagcGGTAACAGCGCAGATG-3' Downstream primer 6: 5'-gctAAAGCCGTGCAGCAGCAGAACGGCACGTTC-3'G27V

[0042] Upstream primer 7: 5'-CTGCACGGCTTTACCgtgAACAGCGCAGATGTC-3' Downstream primer 8: 5'-cacGGTAAAGCCGTGCAGCAGCAGAACGG-3'

[0043] L93A

[0044] Upstream primer 9: 5'-GAAAATCGCAGTTGCGGGTgcgAGTCTGGGTGG-3' Downstream primer 10: 5'-cgcACCCGCAACTGCGATTTTCTCATAGCCTTTG-3' P118S

[0045] Upstream primer 11: 5'-GTTACCATGTGCGCAagcATGTATATCAAAAGT-3' Downstream primer 12: 5'-gctTGCGCACATGGTAACAATCCCTTC-3'

[0046] T126A

[0047] Upstream primer 13: 5'-AAAAGTGAAGAAatgATGTACGAGGG-3'

[0048] Downstream primer 14: 5'-CCTCGTACATcatTTCTTCACTTTTGATATAC-3'

[0049] R141G

[0050] Upstream primer 15: 5'-CGTGAATATAAAAAGggcGAAGGTAAAAGCGAG-3' Downstream primer 16: 5'-gccCTTTTTATATTCACGCGCATATTCCAGAAC-3'R141E

[0051] Upstream primer 17: 5'-CGTGAATATAAAAAGgaaGAAGGTAAAAGCGAG-3' Downstream primer 18: 5'-ttcCTTTTTATATTCACGCGCATATTCCAGAAC-3'

[0052] L167A

[0053] Upstream primer 19: 5'-AAAACCCTGAAAGCCgcgCAGGAACTGATTGC-3' Downstream primer 20: 5'-cgcGGCTTTCAGGGTTTTCATCGGGGTCTG-3'

[0054] L167F

[0055] Upstream primer 21: 5'-AAAACCCTGAAAGCCtttCAGGAACTGATTGC-3'

[0056] Downstream primer 22: 5'-aaaGGCTTTCAGGGTTTTCATCGGGGTCTG-3'

[0057] I171M

[0058] Upstream primer 23: 5'-GCCCTGCAGGAACTGatgGCAGATGTTCGTGAT-3'

[0059] Downstream primer 24: 5'-catCAGTTCCTGCAGGGCTTTCAGGGTTTTCAT-3'

[0060] I171V

[0061] Upstream primer 25: 5'-GCCCTGCAGGAACTGgtgGCAGATGTTCGTGAT-3'

[0062] Downstream primer 26: 5'-cacCAGTTCCTGCAGGGCTTTCAGGGTTTTCAT-3'

[0063] V224E

[0064] Upstream primer 27: 5'-GAGCAGAGCGGACATgaaATTACACTGGATCAG-3'

[0065] Downstream primer 28: 5'-ttcATGTCCGCTCTGCTCATACCATTTAATCTG-3'

[0066] The site-directed mutagenesis primers were as described above, with lowercase letters indicating the mutation sites. Similarly, using plasmid DNA containing the EST10 gene as a template, the mutation was introduced via PCR. The PCR reaction program was as follows: 95℃ for 5 min; 95℃ for 30 s, 55℃ for 10 s, 72℃ for 4 min 30 s, repeated for 35 cycles; extension at 72℃ for 10 min. The PCR product was treated with DpnI at 37℃ for 3 h, inactivated at 80℃ for 10 min, and then transformed into E. coli BL21(DE3) recipient bacteria. The transformed product was plated on LB agar plates containing a final concentration of 50 mg / L kanamycin and incubated at 37℃ for 12 h. Randomly selected single colonies were sequenced and analyzed to obtain the EST10 mutants -- E. coli BL21(DE3) / EST10-muts, namely: EST10-F25I, EST10-F25A, EST10-T26S, EST10-G27V, EST10-L93A, EST10-P118S, EST10-T126A, EST10-R141G, EST10-R141E, EST10-L167A, EST10-L167F, EST10-I171M, EST10-I171V, and EST10-V224E.

[0067] Example 3: Preparation of wet cells of recombinant esterase mutant

[0068] Recombinant *E. coli* BL21(DE3) / EST10-muts containing the recombinant esterase mutant gene obtained in Example 2 were inoculated into LB liquid medium containing a final concentration of 50 μg / mL kanamycin resistance and cultured at 37°C and 180 rpm for 10 h. Then, 2% (v / v) inoculation was added to fresh LB liquid medium containing a final concentration of 50 μg / mL kanamycin resistance and cultured at 37°C and 180 rpm until the bacterial OD600 reached 0.6-0.8. IPTG was added to a final concentration of 0.1 mM, and the culture was induced at 28°C for 12 h. After centrifugation at 4°C and 8000 rpm for 15 min, the supernatant was discarded, and the precipitate was collected to obtain recombinant *E. coli* wet cells containing the recombinant esterase mutant gene. These wet cells can be used directly as a biocatalyst or for protein purification. Recombinant *E. coli* BL21(DE3) / EST10 wet cells containing the recombinant esterase gene were prepared using the same method.

[0069] Example 4: Isolation and purification of esterase mutants

[0070] Preparation of the crude esterase solution according to the present invention: The recombinant esterase-producing engineered bacteria prepared by the method in Example 3 were resuspended at a concentration of 0.2 g wet cells in 9.8 mL of 100 mM, pH 7.0 potassium phosphate buffer solution. The cells were then subjected to ultrasonic disruption under ice bath conditions (20 W, 40% power, 2 s duration, 4 s interval, continuous disruption for 15 min) to obtain cell lysate. The cell lysate obtained after ultrasonic disruption was centrifuged at 12000 rpm and 4℃ for 10 min, and the supernatant obtained was the crude enzyme solution. Gel electrophoresis images of the crude enzyme solution of E. coli BL21(DE3) / EST10 and the cell lysate are shown below. Figure 1 As shown.

[0071] Pure enzyme solution: The crude enzyme solution was incubated with Ni2+ affinity chromatography resin equilibrated with binding buffer (50 mM, pH 7.0, sodium phosphate buffer containing 300 mM NaCl), followed by washing with rinsing buffer (50 mM, pH 7.0, sodium phosphate buffer containing 300 mM NaCl and 50 mM imidazole) until virtually free of contaminating proteins. The target protein was then eluted with elution buffer (50 mM, pH 7.0, sodium phosphate buffer containing 300 mM NaCl and 500 mM imidazole) and collected. After electrophoresis to determine purity (single band), the target proteins were combined and dialyzed with dialysis buffer (20 mM, pH 7.0, sodium phosphate buffer) (10 kDa molecular weight cutoff) for 24 h. The retentate was used to determine the protein content using a BCA kit and stored at -80°C to obtain the pure enzyme solution.

[0072] Example 5: Esterase Activity Assay

[0073] The purified wild-type EST10 enzyme and recombinant esterase mutant enzymes EST10-F25I, EST10-F25A, EST10-T26S, EST10-G27V, EST10-L93A, EST10-P118S, EST10-T126A, EST10-R141G, EST10-R141E, EST10-L167A, EST10-L167F, EST10-I171M, EST10-I171V, and EST10-V224E obtained by the method in Example 4 were used to detect enzyme activity of the catalytic substrate (racemic 3-cyclohexene-1-carboxylate).

[0074] Table 1. Esterase and mutant enzyme activities

[0075]

[0076]

[0077] The enzyme catalytic system composition and catalytic conditions are as follows: 0.02 g of pure enzyme solution (based on protein content) was diluted with phosphate buffer (100 mM, pH 7.0), and racemic methyl 3-cyclohexene-1-carboxylate with a final concentration of 20 g / L was added. A 1 mL reaction system was prepared with phosphate buffer (100 mM, pH 7.0). The reaction was carried out at 30 °C and 600 rpm for 10 minutes. 200 μL of the sample was taken and 200 μL of 2 M HCl was added to terminate the reaction. The sample was extracted with 800 μL of ethyl acetate. The aqueous phase and organic phase were separated by centrifugation. A certain amount of anhydrous sodium sulfate was added to the organic phase for drying. After centrifugation, 200 μL of the sample was taken and the contents of methyl 3-cyclohexene-1-carboxylate and S-3-cyclohexene-1-carboxylic acid were detected by high performance gas chromatography (GC). The conversion rate and ee value were calculated.

[0078] The GC detection method is as follows: Gas chromatograph: Agilent 6890N; Chiral gas column: B-DM (0.25mm×30m×0.12mm), injection port temperature: 250℃; FID detector temperature: 250℃; air flow rate: 300mL / min, make-up gas flow rate: 25mL / min; split ratio: 50; constant flow rate: 1mL / min. Column oven temperature program: initial temperature 80℃, hold for 0.5min, increase to 120℃ at 8℃ / min, hold for 0.5min, increase to 140℃ at 2℃ / min, hold for 2min.

[0079] Enzyme activity unit (U) is defined as the amount of enzyme required to consume 1 mM methyl 3-cyclohexene-1-carboxylate in 1 min at 30°C and pH 7.0.

[0080] The protein content in each enzyme solution was determined using the BCA method. This experiment utilized the KGI BCA protein content assay kit, and the procedure was performed according to the kit's instructions.

[0081] Specific activity is the number of enzyme activity units per milligram of protein, usually expressed as U / mg protein.

[0082] Example 6: Application of recombinant esterase in the preparation of S-3-cyclohexene-1-carboxylic acid

[0083] Using the recombinant Escherichia coli BL21(DE3) / EST10 wet cells and E. coli BL21(DE3) / EST10-muts wet cells containing the recombinant plasmid obtained in Example 3 as biocatalysts, and methyl 3-cyclohexene-1-carboxylate as substrate, S-3-cyclohexene-1-carboxylic acid was prepared by catalytic reaction.

[0084] The catalytic reaction system and catalytic conditions are as follows: the final concentration of wet bacterial cells was 20 g / L, the final concentration of racemic methyl 3-cyclohexene-1-carboxylate was 100 g / L, and 10 mL of sodium phosphate buffer solution (pH 7.0) was used as the reaction medium. The reaction was carried out in a 30°C water bath with magnetic stirring at 600 rpm. The pH was controlled at 7.0 by automatically adding 2M NaOH solution. The reaction was carried out for 2 hours, and samples were taken for GC analysis as described in Example 5. The chromatogram is shown below. Figure 3 The substrate conversion rate and ee value results are shown in Table 2 and . Figure 4 .

[0085] Table 2. Esterase catalytic effect

[0086] EST10 37.3 76.0 EST10-F25I 49.91 99.2 EST10-F25A 45.3 83.2 EST10-T26S 46.6 71.7 EST10-G27V 47.5 63.3 EST10-L93A 44.0 93.7 EST10-P118S 16.9 45.7 EST10-T126A 45.6 76.8 EST10-R141G 49.96 99.5 EST10-R141E 46.3 83.7 EST10-167A 48.9 99.1 EST10-L167F 49.99 99.3 EST10-I171M 49.3 98.4 EST10-I171V 44.1 85.4 EST10-V224E 49.6 99.7

Claims

1. A recombinant esterase mutant, characterized in that, The mutant was obtained by mutating valine at position 224 of the amino acid sequence shown in SEQ ID NO.2 to glutamic acid.

2. The encoding gene of the recombinant esterase mutant of claim 1.

3. A recombinant genetically engineered bacterium constructed from the encoding gene of claim 2.

4. The use of the recombinant esterase mutant of claim 1 in the asymmetric resolution of methyl 3-cyclohexene-1-carboxylate to prepare S-3-cyclohexene-1-carboxylic acid.

5. The application as described in claim 4, characterized in that, The application involves using engineered bacteria containing a recombinant esterase mutant encoding gene, obtained through fermentation culture, as a catalyst. The reaction system is constructed using methyl 3-cyclohexene-1-carboxylate as a substrate and a potassium phosphate buffer solution with a pH of 6-9 as the reaction medium. The reaction is carried out at 20-40℃ and 600 rpm until complete. The reaction solution is then separated and purified to obtain S-3-cyclohexene-1-carboxylic acid.

6. The application as described in claim 5, characterized in that, The catalyst dosage, based on the wet cell weight, is 20-200 g / L buffer solution; the initial concentration of the substrate is 10-700 g / L buffer solution.

7. The application as described in claim 5, characterized in that, The wet bacterial cells were prepared as follows: Engineered bacteria containing the recombinant esterase mutant encoding gene were inoculated into LB liquid medium containing a final concentration of 50 μg / mL kanamycin resistance and cultured at 37°C and 180 rpm for 10 h. Then, at a volume concentration of 2%, the inoculum was transferred to fresh LB liquid medium containing a final concentration of 50 μg / mL kanamycin resistance and cultured at 37°C and 180 rpm until the bacterial cell OD reached a certain level. 600 When the concentration reaches 0.6-0.8, add IPTG to a final concentration of 0.1mM, induce culture at 28℃ for 12h, centrifuge at 4℃ and 8000rpm for 15min, discard the supernatant, and collect the wet cells.

8. The application as described in claim 5, characterized in that, The preparation method of the crude enzyme solution is as follows: wet cells obtained by fermentation culture of engineered bacteria containing the recombinant esterase mutant encoding gene are resuspended in 9.6 mL of 100 mM, pH 7.0 potassium phosphate buffer solution at 0.4 g wet cells. The cells are then ultrasonically disrupted at 20-60 W under ice bath conditions for 2 seconds followed by 4 seconds of interval, for a total of 15 min to obtain cell lysate. The cell lysate is then centrifuged at 8000 rpm and 4℃ for 10 min, and the supernatant obtained is the crude enzyme solution.

9. The application as described in claim 5, characterized in that, The pure enzyme solution was prepared as follows: The crude enzyme solution was incubated with Ni2+ affinity chromatography resin equilibrated with binding buffer, then washed with rinsing buffer until virtually free of contaminating proteins. The target protein was then eluted with elution buffer and collected. The solution was dialyzed with dialysis buffer for 24 hours, and the retentate was collected as the pure enzyme solution. The binding buffer was a 50mM sodium phosphate buffer containing 300mM NaCl at pH 7.

0. The rinsing buffer was a 50mM sodium phosphate buffer containing 300mM NaCl and 50mM imidazole at pH 7.

0. The elution buffer was a 50mM sodium phosphate buffer containing 300mM NaCl and 500mM imidazole at pH 7.0.

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