Cyclohexene formate esterase, mutant and application of cyclohexene formate esterase in production of (S)-3-cyclohexene-1-formic acid

Abstract

The invention discloses cyclohexene formate esterase, a mutant of cyclohexene formate esterase and application of cyclohexene formate esterase in production of (S)-3-cyclohexene-1-formic acid. According to the cyclohexene formate esterase and the mutant disclosed by the invention, 100-400g / L of racemic 3-cyclohexene-1-methyl formate can be split by taking wet thalli subjected to induced expression or crude enzyme liquid obtained by ultrasonically crushing the wet thalli as a catalyst, so that (S)-3-cyclohexene-1-formic acid with chemical purity and optical purity reaching 99% or above is synthesized. The enzyme activity of the cyclohexene formate esterase mutant provided by the invention is further improved, the reaction time and the catalyst dosage are obviously reduced compared with those of a substrate with the same catalysis concentration before mutation, the reaction condition is mild, the operation is relatively simple and convenient, and the cyclohexene formate esterase mutant has a very good industrial application prospect.

Description

(I) Technical field

[0001] The invention belongs to the technical field of bioengineering, and relates to a cyclohexenecarboxylate esterase with high activity and high stereoselectivity, a mutant and application thereof in asymmetric resolution of racemic 3-cyclohexene-1-carboxylate to produce (S)-3-cyclohexene-1-carboxylic acid. (II) Background technology

[0002] (S)-3-Cyclohexene-1-carboxylic acid (molecular formula: C7H 10 O2, molecular weight: 126.15, boiling point: 118℃ / 6mmHg, CAS number 5708-19-0), it is a colorless transparent liquid at room temperature and pressure, has significant acidity, and is soluble in most organic solvents. (S)-3-Cyclohexene-1-carboxylic acid is an alkyl carboxylic acid compound, mainly used as a chiral synthetic building block in the field of organic synthesis, and is mostly used for the preparation of chiral cyclohexane functional organic molecules, such as the synthesis of the new oral anticoagulant edoxaban.

[0003] The method of synthesizing (S)-cyclohexenecarboxylic acid by chemical method includes Diels-Alder reaction and racemic acid chemical resolution method, wherein Diels-Alder reaction is the main method of synthesizing (S)-3-cyclohexenecarboxylic acid at present, however, this method is difficult to operate, has harsh reaction conditions, cumbersome reaction steps, and low yield, so its application is limited. In addition, the chemical resolution method requires at least six recrystallizations in acetone to separate the optically pure enantiomers, resulting in the use of a large amount of acetone, the resolution yield is only 20-30%, and the atom economy is low.

[0004] The molecular structure of cyclohexenecarboxylic acid is highly symmetrical. The enzymatic kinetic splitting of chiral esters and hydrolysis to generate optically pure chiral acids is an important method for producing (S)-cyclohexenecarboxylic acid. The stereoselective synthesis of cyclohexenecarboxylic acid by biocatalysis mainly uses commercial enzymes, cells of wild bacteria or recombinant engineered bacteria or free enzymes for catalysis. In 2004, Cihangir et al. investigated the effect of a batch of commercial enzymes including pig liver esterase PLE and horse liver esterase HLE on the hydrolysis of 70mM racemic 3-cyclohexene-1-carboxylic acid methyl ester at 20°C. Among them, after 4h of reaction, the product of PLE was (S)-3-cyclohexene-1-carboxylic acid, with an optical purity of >99% and a yield of 43%; the product catalyzed by HLE was also (S)-3-cyclohexene-1-carboxylic acid, with an ee value of 97% and a yield of 41% (Tetrahedron Asymmetry, 2004, 15, 2057–2060). However, the commercial enzyme sequences of the above-mentioned animal sources are unknown and cannot be synthesized artificially. In 2016, Wang Zhao et al. disclosed a method for preparing chiral 3-cyclohexene-1-carboxylic acid by splitting commercial protease, using racemic 3-cyclohexene-1-carboxylic acid methyl ester as substrate and reacting in a phosphate buffer at pH 7.5 to obtain (S)-3-cyclohexenecarboxylic acid with an ee value of 99.5% and a yield of 32.8% (Chinese patent CN106119303B). In 2019, Wu et al. designed the Escherichia coli esterase BioH, and improved its enantioselectivity for (S)-3-cyclohexene-1-carboxylic acid methyl ester through rational design. The ee value of the mutant Mu3 (L24A / W81A / L209A) increased from 32.3% (wild type) to 70.9% (Biosci. Biotechnol. Biochem., 2019, 83, 1263–1269); although the recombinant enzyme BioH has the activity of hydrolyzing 3-cyclohexene-1-carboxylic acid methyl ester, its enantioselectivity is low. In 2020, Dou et al. discovered the esterase AcEst1, which can catalyze the substrate concentration of 2.0M (280g / L), and the ee value after 20h of reaction is 2.0M (280g / L). s >99%, 715mM (S)-3-cyclohexene-1-carboxylic acid methyl ester was isolated (Bioresource Technol., 2020, 317, 123984). Although there are many other reports that can catalyze the asymmetric resolution of cyclohexenecarboxylic acid methyl ester, they all have problems such as low optical activity or low substrate loading, and are not feasible for large-scale application.

[0005] Therefore, it is urgent to screen efficient and highly selective biocatalysts for cyclohexenecarboxylate compounds to meet industrial needs. (III) Summary of the invention

[0006] The invention provides a highly efficient and stereoselective cyclohexenecarboxylate esterase, a mutant and application thereof in synthesizing (S)-3-cyclohexene-1-carboxylic acid, thereby improving the enzyme activity, substrate concentration and product selectivity of the enzymatic synthesis of (S)-3-cyclohexene-1-carboxylic acid.

[0007] The technical solution adopted by the present invention is:

[0008] In a first aspect, the present invention provides a cyclohexenecarboxylate esterase with high activity and high stereoselectivity, wherein the amino acid sequence of the cyclohexenecarboxylate esterase is shown in SEQ ID NO.2.

[0009] The present invention also relates to the coding gene of the cyclohexene carboxylic acid esterase, a recombinant expression vector containing the coding gene, and a recombinant genetic engineering bacterium constructed by the recombinant expression vector. The expression vector is constructed by cloning the above-mentioned esterase gene into an expression vector by conventional methods in the art, wherein the expression vector includes various conventional vectors in the art, such as commercially available plasmids, cosmids, phages or viral vectors, etc., preferably pET-28a plasmids; the host cell is a conventional host cell in the art, as long as the expression vector can stably replicate itself and the esterase gene carried by it can be effectively expressed, for example, it can be bacteria, fungi, plant cells, animal cells, etc. The bacteria is preferably Escherichia coli, and more preferably Escherichia coli E.coli BL21 (DE3) or Escherichia coli E.coli DH5α.

[0010] In a second aspect, the present invention provides an application of the cyclohexenecarboxylate esterase in the production of (S)-3-cyclohexene-1-carboxylic acid. The application method is: using wet bacteria obtained by induction culture of recombinant genetic engineering bacteria containing the cyclohexenecarboxylate esterase encoding gene or a crude enzyme solution obtained by centrifugation after ultrasonic crushing of the wet bacteria as a catalyst, racemic 3-cyclohexene-1-carboxylate as a substrate, and a pH 6-10 buffer as a reaction medium to form a reaction system, stirring the reaction at 20-60° C. and 100-220 rpm until the reaction is completed (the substrate conversion rate is close to 50%, and the ee s The reaction solution was extracted and separated to obtain (S)-3-cyclohexene-1-carboxylic acid with a chemical purity and an optical purity of >99%.

[0011] Further, the substrate includes 3-cyclohexene-1-carboxylic acid methyl ester, 3-cyclohexene-1-carboxylic acid ethyl ester, 3-cyclohexene-1-carboxylic acid propyl ester and 3-cyclohexene-1-carboxylic acid isobutyl ester.

[0012] Furthermore, in the reaction system, the amount of substrate added is 10-400 g / L, the amount of wet bacteria added is 1-4 g / L, and the amount of crude enzyme solution added is 0.1-0.4 g / L based on protein content.

[0013] Furthermore, the reaction conditions are preferably 50° C. and 200 rpm.

[0014] In a third aspect, the present invention provides a cyclohexenecarboxylate esterase mutant, which is obtained by subjecting the 82nd, 236th and 302nd amino acid sequences of SEQ ID NO.2 to single mutation or combined mutation.

[0015] Furthermore, the mutant is a mutant in which the amino acid sequence shown in SEQ ID NO.2 is mutated into one of the following: (1) arginine at position 82 is mutated into histidine; (2) threonine at position 236 is mutated into valine; (3) leucine at position 302 is mutated into alanine; (4) arginine at position 82 is mutated into histidine, threonine at position 236 is mutated into valine, and leucine at position 302 is mutated into alanine, denoted as EstA1-R82H / T236V / L302A, the amino acid sequence is shown in SEQ ID NO.4, and the nucleotide sequence of the encoding gene is shown in SEQ ID NO.3.

[0016] The present invention also relates to the cyclohexene carboxylic acid esterase mutant encoding gene, a recombinant expression vector containing the encoding gene, and a recombinant genetic engineering bacterium constructed by the recombinant expression vector; the expression vector is constructed by cloning the above esterase gene into the expression vector by conventional methods in the art, wherein the expression vector includes various conventional vectors in the art, such as commercially available plasmids, cosmids, phages or virus vectors, etc., preferably pET-28a plasmids; the host cell is a conventional host cell in the art, as long as the expression vector can stably replicate itself and the esterase gene carried by it can be effectively expressed, for example, it can be bacteria, fungi, plant cells, animal cells, etc. The bacteria is preferably Escherichia coli, and more preferably Escherichia coli E.coli BL21 (DE3) or Escherichia coli E.coli DH5α.

[0017] In a fourth aspect, the present invention further provides an application of the cyclohexenecarboxylate esterase mutant in the production of (S)-3-cyclohexene-1-carboxylic acid, wherein the application method comprises: using wet cells obtained by induction culture of recombinant genetically engineered bacteria containing a gene encoding the cyclohexenecarboxylate esterase mutant or a crude enzyme solution obtained by centrifugation after ultrasonic crushing of the wet cells as a catalyst, using racemic 3-cyclohexene-1-carboxylate as a substrate, and using a pH 6-10 buffer as a reaction medium to form a reaction system, stirring the reaction at 20-60° C. and 100-220 rpm until the reaction is completed (the substrate conversion rate is close to 50%, and the ee s >99%), and the reaction solution was extracted and separated to obtain (S)-3-cyclohexene-1-carboxylic acid with a chemical purity and an optical purity of >99%.

[0018] Further, the substrate includes 3-cyclohexene-1-carboxylic acid methyl ester, 3-cyclohexene-1-carboxylic acid ethyl ester, 3-cyclohexene-1-carboxylic acid propyl ester and 3-cyclohexene-1-carboxylic acid isobutyl ester.

[0019] Furthermore, in the reaction system, the amount of substrate added is 10-400 g / L, the amount of wet bacteria added is 1-4 g / L, and the amount of crude enzyme solution added is 0.1-0.4 g / L based on protein content.

[0020] Furthermore, the reaction conditions are preferably 50° C. and 200 rpm.

[0021] Further, the catalyst is prepared according to the following steps:

[0022] The recombinant genetically engineered bacteria were inoculated into LB liquid medium containing 50 mg / L kanamycin, cultured at 37°C overnight, and inoculated into LB liquid medium at a volume concentration of 1%, and cultured on a shaker at 37°C and 180 rpm. 600 When it reached 0.6, IPTG with a final concentration of 0.2 mmol / L was added as an inducer. After induction at 25°C for 12 h, the culture solution was centrifuged, the cells were collected, and washed twice with physiological saline to obtain wet bacterial resting cells;

[0023] The obtained resting cells were suspended in 100 mM sodium phosphate buffer at pH 7.0, and ultrasonically disrupted at 250 W for 10 min in an ice bath, with a 1 s disruption interval of 3 s. The supernatant was collected by centrifugation to obtain the crude enzyme solution.

[0024] The LB liquid culture medium comprises: 5-15 g / L peptone, 1-10 g / L yeast powder, 5-15 g / L NaCl, and pH 6.0-8.0.

[0025] Furthermore, the method for extracting and separating the reaction solution is as follows: after the reaction is completed, the reaction solution is first centrifuged to remove cells, the supernatant is extracted with an equal volume of ethyl acetate, and the organic solvent is removed by rotary evaporation to obtain a crude product (S)-3-cyclohexene-1-carboxylic acid methyl ester;

[0026] The crude product (S)-3-cyclohexene-1-carboxylic acid methyl ester was added with 1.0M NaOH aqueous solution, heated to reflux at 50°C with stirring for 6 hours, and then 1.0M HCl aqueous solution was added to adjust the pH to 5.0, and an equal volume of ethyl acetate was added for extraction three times. The organic layers were combined, dried over anhydrous Na2SO4, filtered, and the filtrate was rotary evaporated until the solvent was evaporated to obtain pure (S)-3-cyclohexene-1-carboxylic acid; the volume of the NaOH aqueous solution used was 2-5mL / g based on the weight of the crude product.

[0027] The mutation method of the present invention can be carried out according to conventional methods in the art, for example, directed mutagenesis, random mutagenesis, or synthesizing oligonucleotides and expressing them in host cells to obtain mutants with substitutions, insertions and / or deletions in the amino acid sequence.

[0028] The culture method and culture conditions of the engineered bacteria before and after mutation of the present invention are not particularly limited and can be appropriately selected according to factors such as host cell type and culture method according to common knowledge in the art, as long as the transformant can grow and produce esterase. Other specific operations for culturing the transformant can be performed according to conventional operations in the art.

[0029] Compared with the prior art, the beneficial effects of the present invention are mainly reflected in:

[0030] (1) The cyclohexenecarboxylate esterase screened by the present invention has an optimum temperature of 50°C and an optimum pH of 8.0, and has a high optimum reaction temperature, a fast reaction rate, and a high catalytic substrate concentration.

[0031] (2) The cyclohexenecarboxylate esterase mutant screened by the present invention has significantly improved hydrolysis activity and stereoselectivity, and can also catalyze high-concentration substrate reactions.

[0032] (3) The cyclohexenecarboxylic acid esterase and mutants of the present invention can use wet cells after induced expression or crude enzyme solution of wet cells ultrasonically crushed as catalyst to split 100-400 g / L of racemic 3-cyclohexene-1-carboxylic acid methyl ester to synthesize (S)-3-cyclohexene-1-carboxylic acid with a chemical purity and optical purity of more than 99%.

[0033] (3) The cyclohexenecarboxylate esterase mutant provided by the present invention has further improved enzyme activity. Compared with the mutant, the reaction time and the amount of catalyst used for catalyzing the same concentration of substrate are significantly reduced. In addition, the reaction conditions are mild and the operation is relatively simple, which has a good industrial application prospect. (IV) Description of the drawings

[0034] Figure 1 , relative activity curve of cyclohexenecarboxylate esterase at different temperatures.

[0035] Figure 2 , relative activity curve of cyclohexenecarboxylate esterase at different pH.

[0036] Figure 3 , gas chromatogram; A represents (R)-3-cyclohexene-1-carboxylic acid methyl ester and (S)-3-cyclohexene-1-carboxylic acid methyl ester standards; B represents (S)-3-cyclohexene-1-carboxylic acid and (R)-3-cyclohexene-1-carboxylic acid standards.

[0037] Figure 4, Gas chromatogram of the wet bacterial reaction liquid of mutant EstA1-R82H / T236V / L302A in Example 9. (V) Specific implementation methods

[0038] The present invention is further described below in conjunction with specific embodiments, but the protection scope of the present invention is not limited thereto:

[0039] In the examples of the present invention, if there is no other special explanation, % means wt%, i.e. weight percentage. The reagents or instruments used without indicating the manufacturer are all conventional reagent products that can be obtained through commercial purchase.

[0040] LB liquid medium: peptone 10 g / L, yeast extract 5 g / L, NaCl 10 g / L, pH 7.0.

[0041] LB solid medium is prepared by adding 18 g / L agar to LB liquid medium.

[0042] Example 1: Cloning of cyclohexenecarboxylate esterase gene EstA1

[0043] 1. Screening of cyclohexenecarboxylate esterase ancestor enzyme

[0044] The ancestral sequence reconstruction (ASR) method was used to select the amino acid sequence of cyclohexene carboxylate esterase from Acinetobacter sp. WCHAc010052 as the probe.

[0045] (Genebank accession number: AXY61874.1), the cyclohexenecarboxylate esterase ancestral enzyme (amino acid sequence as shown in SEQ ID NO.2) was screened by the FireProt-ASR online tool (https: / / loschmidt.chemi.muni.cz / fireprotasr / ), its coding gene (nucleotide sequence as shown in SEQ ID NO.1) was obtained by codon optimization, and the full-length sequence was artificially synthesized by a gene synthesis company.

[0046] 2. Obtaining the gene fragment of the ancestral enzyme of cyclohexenecarboxylate esterase

[0047] PCR primers were designed according to the artificially synthesized SEQ ID NO.1 in step 1. PCR amplification was performed using the artificially synthesized cyclohexene carboxylic acid esterase ancestral enzyme DNA as a template. The PCR product was purified by agarose gel electrophoresis, and the PCR product was recovered using an agarose gel DNA recovery kit. A complete esterase full-length gene sequence of 1047 bp was obtained by DNA sequencing, which was named cyclohexene carboxylic acid esterase EstA1, and the nucleotide sequence was shown in SEQ ID NO.1.

[0048] Upstream primer:

[0049] 5'-gtgccgcgcggcagc catatg ATGCCTAGTGCCGCAATGG-3'

[0050] Downstream primer:

[0051] 5'-acggagctcgaattc ggatcc TGCTGCAGCCGGTGCAGC-3'

[0052] Among them, the underlined part of the upstream primer is the NdeI restriction site, and the underlined part of the downstream primer is the BamHI restriction site.

[0053] The PCR system was: 2×Taq PCR MasterMix 10 μL, upstream primer and downstream primer 1 μL each (0.3 μmol / L), DNA template 1 μL (0.1 μg) and ddH2O 7 μL.

[0054] The PCR amplification procedure was as follows: (1) 95°C, pre-denaturation for 3 min; (2) 94°C, denaturation for 30 s; (3) 55°C, annealing for 30 s; (4) 72°C, extension for 2 min; steps (2) to (4) were repeated for 30 cycles; (5) 72°C, extension for 10 min, and cooling to 4°C.

[0055] Example 2: Construction of Cyclohexenecarboxylate Esterase Expression Vector and Engineering Bacteria

[0056] 1. Expression vector

[0057] The cyclohexene carboxylate esterase EstA1 gene DNA fragment obtained in Example 1 and the pET-28a empty plasmid were double-digested with restriction endonucleases NdeI and BamHI at 37°C for 2h, purified by agarose gel electrophoresis, and the target fragment was recovered using an agarose gel DNA recovery kit. The target fragment was ligated overnight at 4°C under the action of T4 DNA ligase to obtain the expression plasmid pET28a-EstA1.

[0058] 2. Engineered bacteria

[0059] The expression plasmid pET28a-EstA1 was heat-shock transformed into Escherichia coli DH5α competent cells, spread on resistant LB plates containing 50 mg / L kanamycin, and cultured at 37°C for 12 hours. Single clones were picked, colony PCR was performed for verification, and positive clones were screened.

[0060] The positive clones were inoculated into LB solid medium containing 50 mg / L kanamycin and cultured at 37°C for 12 h. The plasmids were extracted using a plasmid mini-extraction kit and re-transformed into Escherichia coli BL21 (DE3) competent cells by heat shock. The transformation solution was spread onto LB plates containing 50 mg / L kanamycin and cultured inverted at 37°C overnight to obtain positive transformants, recombinant Escherichia coli BL21 (DE3) / pET28a-EstA1. The positive clones were verified by colony PCR and gene sequencing.

[0061] Example 3: Inducible expression of esterase EstA1

[0062] 1. Wet bacteria

[0063] The recombinant E. coli obtained in Example 2 was inoculated into LB liquid medium containing 50 mg / L kanamycin, and cultured at 37°C overnight with shaking. The inoculum was inoculated into a 500 mL Erlenmeyer flask containing 100 mL LB medium at a rate of 1% (v / v), and cultured on a shaking table at 37°C and 180 rpm. When the OD of the culture solution reached 600 When it reached 0.6, IPTG with a final concentration of 0.2 mmol / L was added as an inducer. After induction at 25°C for 12 h, the culture solution was centrifuged, the cells were collected, and washed twice with physiological saline to obtain wet bacterial resting cells.

[0064] 2. Cryogenic Stem Cells

[0065] The resting cells were freeze-dried at -40°C for 24 h to obtain freeze-dried cells, which were collected and stored at 4°C.

[0066] 3. Crude enzyme solution

[0067] 1 g of the obtained resting cells was suspended in 10 mL of 100 mM sodium phosphate buffer at pH 7.0, and ultrasonically disrupted at 250 W for 10 min in an ice bath, with an interval of 3 s after disruption for 1 s. The supernatant was collected by centrifugation to obtain the crude enzyme solution of esterase EstA1.

[0068] Example 4: Determination of esterase EstA1 activity

[0069] Esterase activity assay reaction system: 1 mmol / L cyclohexenecarboxylic acid p-nitrophenolate was added to 200 μL of pH 8.0, 100 mmol / L sodium phosphate buffer, and the mixture was incubated at 30°C for 2 min. Then, 10 μL of the crude enzyme solution prepared in Example 3 was added and mixed quickly. The change in absorbance at 405 nm was detected by a microplate reader. The concentration of cyclohexenecarboxylic acid p-nitrophenolate consumed was calculated using the standard curve of the concentration of cyclohexenecarboxylic acid p-nitrophenolate and the absorbance value, and then the enzyme activity was calculated.

[0070] The enzyme activity (U) was defined as the amount of enzyme required to catalyze 1 μmol of cyclohexenecarboxylic acid p-nitrophenolate per minute under the above conditions.

[0071] Example 5: Effect of reaction temperature and pH on cyclohexenecarboxylate esterase activity

[0072] The activity changes of the crude enzyme solution of esterase EstA1 prepared in Example 3 at different reaction temperatures and pH values ​​were investigated, and the relative enzyme activities under other conditions were calculated with the highest activity being 100%.

[0073] 1. Temperature

[0074] Reaction system: Add 1 mmol / L cyclohexenecarboxylic acid p-nitrophenolate to 200 μL of pH 7.0, 100 mmol / L sodium phosphate buffer, keep at 30°C for 2 min, add 10 μL of the crude enzyme solution prepared in Example 3, mix quickly, and detect the enzyme activity at 20°C, 30°C, 40°C, 50°C and 60°C using the method described in Example 4. Results are shown in Figure 1 The reaction has higher activity between 40-60°C, and has the highest activity when the reaction temperature is 50°C.

[0075] 2. pH

[0076] The pH of the buffer solution in the reaction system of step 1 was changed to pH 6.0, 7.0, 8.0, 9.0 and 10.0, the enzyme activity assay temperature was fixed at 50°C, and other operations were the same. The results are shown in Figure 2 , with the highest enzyme activity at pH 8.0.

[0077] Therefore, the optimal reaction temperature of cyclohexenecarboxylate esterase is 50°C and the optimal reaction pH is 8.0.

[0078] Example 6: Determination of the hydrolysis reaction of racemic 3-cyclohexene-1-methyl carboxylate catalyzed by cyclohexenecarboxylate ester

[0079] The crude enzyme solution obtained in Example 3 was subjected to the hydrolysis reaction assay of racemic 3-cyclohexene-1-methyl carboxylate.

[0080] Reaction system 1mL: final concentration 10g / L racemic 3-cyclohexene-1-carboxylic acid methyl ester, 0.1g / L (based on protein content) crude enzyme solution, 100mM potassium phosphate buffer solution (pH 8.0) supplemented to 1mL. React at 50℃, 200rpm for 5min. After the reaction, take 100μL of the reaction solution and add 10μL 2M NaOH solution to terminate the reaction. After centrifugation at 12000rpm for 1min, take the supernatant and pass it through a 0.22μm membrane. Detect the characteristic peak area by gas chromatography (GC), calculate the content of substrate and product according to the standard curve of standard concentration and peak area, and then calculate the substrate conversion rate and product enantioselectivity ee. p , the enantioselective ee of the substrate s .

[0081]

[0082] Wherein S1 represents (S)-3-cyclohexene-1-carboxylic acid methyl ester, R1 represents (R)-3-cyclohexene-1-carboxylic acid methyl ester, S2 represents (S)-3-cyclohexene-1-carboxylic acid, and R2 represents (R)-3-cyclohexene-1-carboxylic acid.

[0083] GC detection conditions: instrument Agilent 8860 (Agilent Technologies, USA), chiral column BD-M (Merck, USA), 1 μL injection volume, 1 mL / min flow rate, 100 °C for 5 min, heating to 200 °C at a rate of 5 °C / min. The GC detection results of substrate and product standards are shown in Figure 3 As shown in A and B.

[0084] Example 7. Preparation of cyclohexenecarboxylate esterase mutants

[0085] 1. Screening of mutation sites

[0086] The structural model of cyclohexenecarboxylate esterase EstA1 (amino acid sequence as shown in SEQ ID NO.2) was constructed using AlphaFold 2, and the substrate was docked into the active center using AutoDock to analyze the interaction between cyclohexenecarboxylate esterase EstA1 and the substrate, and positions 82, 236, and 302, which are close to the substrate, were selected for mutation.

[0087] 2. Single-site mutation

[0088] According to the mutation sites screened in step 1, mutation primers were designed as shown in Table 1. Using the plasmid pET28a-EstA1 of E.coli BL21(DE3) / pET28a-EstA1 as a template, PCR site-directed saturation mutagenesis was performed. The PCR product was treated with DpnI at 37°C for 3h, then transformed into the E.coli BL21(DE3) host bacteria, spread on an LB solid plate containing a final concentration of 50mg / L kanamycin resistance, and cultured at 37°C for 12h. Single colonies were randomly picked for sequencing analysis to obtain a mutant library. The crude enzyme solution was prepared using the method in Example 3, and the substrate conversion rate and the enantioselectivity of the product, ee, were detected using the method in Example 6. p , the enantioselective ee of the substrate s .

[0089] The results of some mutants are shown in Table 2. The dominant mutants EstA1-R82H (R at position 82 mutated to H), EstA1-T236V (T at position 236 mutated to V), and EstA1-L302A (L at position 302 mutated to A) were obtained. p There is a significant improvement, and the conversion rate is closer to 50%.

[0090] PCR system: 30 ng of recombinant plasmid pET28a-EstA1 as template, 1.0 μL of rTaq DNA polymerase, 10× PCR buffer (Mg 2+ Plus) 5.0 μL, dNTP Mixture 4.0 μL, MnCl2 with a final concentration of 50 μM, 1.5 μL each of upstream and downstream primers (10 μM), and then make up to 50 μL with sterile water.

[0091] PCR amplification procedure: (1) 95°C, pre-denaturation for 3 min; (2) 94°C, denaturation for 30 s; (3) 55°C annealing for 30 s; (4) 72°C extension for 2 min; steps (2) to (4) were repeated for 30 cycles; (5) 72°C extension for 10 min, and then cooled to 4°C.

[0092] Table 1. Site-directed saturation mutagenesis primers

[0093]

[0094] In Table 1, N represents A / C / G / T, M represents A / C, and K represents G / T.

[0095] Table 2. Single mutant reaction results

[0096]

[0097] 3. Combination mutation

[0098] Based on the superior single mutants in step 2, combined mutations were performed using the primers in Table 3, and the PCR amplification system and procedure and screening method in step 2 were used. The results are shown in Table 4, and the superior mutants were screened.

[0099] EstA1-R82H / T236V / L302A, the amino acid sequence is shown in SEQ ID NO.4, the gene sequence is shown in SEQ ID NO.3, and the wet bacteria and crude enzyme solution of the mutant were prepared according to the method described in Example 3.

[0100] Table 3. Combined mutation primers

[0101]

[0102] Table 4. Combined mutation reaction results

[0103]

[0104] Example 8: EstA1 and its mutants catalyze the hydrolysis of racemic 3-cyclohexene-1-carboxylic acid methyl ester at different concentrations

[0105] Reaction system: In 10 mL of pH 8.0, 100 mmol / L sodium phosphate buffer, add 100-400 g / L racemic 3-cyclohexene-1-carboxylic acid methyl ester, 1-4 g / L EstA1 wet cells prepared in Example 3 and mutant EstA1-R82H / T236V / L302A wet cells prepared by the method of Example 6, mix quickly, react at 50°C, 200 rpm for 2-8 h, and take samples at regular intervals to monitor the reaction progress until ee s >99%, the contents of substrate and product were detected by gas chromatography as described in Example 5, and the substrate conversion rate was calculated. The results are shown in Table 5.

[0106] When the substrate concentration was 100 g / L, the same amount of catalyst EstA1 and EstA1-R82H / T236V / L302A were added. s When the reaction rate was >99%, the substrate conversion rate of EstA1 was 55.5% after 4 h of reaction, while the substrate conversion rate of EstA1-R82H / T236V / L302A was 51.1% after 2 h of reaction, which significantly shortened the reaction time. Therefore, the mutant had better substrate conversion ability.

[0107] In addition, as the substrate concentration increases, the reaction catalyzed by EstA1 and EstA1-R82H / T236V / L302A takes longer to reach the ee value for the same substrate concentration. s >99%, but EstA1-R82H / T236V / L302A achieved ee at the same substrate concentration sThe amount of enzyme and time required for >99% were significantly less than those of EstA1, and it has certain potential for industrial application.

[0108] Table 5 Effects of different substrate concentrations on the hydrolysis reaction of cyclohexene carboxylate esterase mutants

[0109]

[0110]

[0111] Example 9: Preparation of (S)-3-cyclohexene-1-carboxylic acid catalyzed by cyclohexenecarboxylate esterase mutant

[0112] Reaction system: In 100 mL of pH 8.0, 100 mmol / L sodium phosphate buffer, add 400 g / L racemic 3-cyclohexene-1-carboxylic acid methyl ester and 4 g / L EstA1 wet cells prepared in Example 3 and mutant EstA1-R82H / T236V / L302A wet cells prepared by the method of Example 6, mix quickly, react at 50°C, 200 rpm, take samples every 1 h during the reaction, detect by the method of Example 5, and react for 8 h until the remaining substrate ee s >99%, i.e., the (R)-configuration substrate was completely converted. The gas chromatogram of the wet bacterial reaction liquid of the mutant EstA1-R82H / T236V / L302A is shown in Figure 4 shown.

[0113] After the reaction, the reaction solution was first centrifuged to remove cells, and then the supernatant was extracted with an equal volume of ethyl acetate, and the organic solvent was removed by rotary evaporation to obtain a crude product (S)-3-cyclohexene-1-carboxylic acid methyl ester. For EstA1 and EstA1-R82H / T236V / L302A, 16 g and 18 g of the product (S)-3-cyclohexene-1-carboxylic acid methyl ester were obtained, respectively, and the product yields were 40% and 45%, respectively.

[0114] 16 g and 18 g of the product (S)-3-cyclohexene-1-carboxylic acid methyl ester obtained above were added to 50 mL of 1.0 M NaOH aqueous solution, heated to reflux at 50° C. with stirring for 6 h, and then 1.0 M HCl aqueous solution was added to adjust the pH to 5.0. An equal volume of ethyl acetate was added for extraction three times. The organic layers were combined, dried over anhydrous Na2SO4, filtered, and the filtrate was rotary evaporated until the solvent was evaporated to obtain pure (S)-3-cyclohexene-1-carboxylic acid. For EstA1 and EstA1-R82H / T236V / L302A, 14.4 g and 16.2 g of pure (S)-3-cyclohexene-1-carboxylic acid were obtained, respectively. The obtained product was a liquid with a special odor. The total yields after separation were 36% and 40.5%, respectively, and the optical purity reached 99% ee.

[0115] The above is only the preferred embodiment of the present application, and does not limit the present application in other forms. Any technician familiar with the profession may use the above disclosed technical content to change or modify it into an equivalent embodiment with equivalent changes. However, any simple modification, equivalent change and modification made to the above embodiment based on the technical essence of the present application without departing from the technical solution of the present application still falls within the protection scope of the technical solution of the present application.

Claims

1. A highly active and stereoselective cyclohexenecarboxylate esterase, characterized in that: The amino acid sequence of the cyclohexene carboxylate esterase is shown in SEQ ID NO.

2.

2. Use of the cyclohexenecarboxylic acid esterase according to claim 1 in the production of (S)-3-cyclohexene-1-carboxylic acid.

3. The use according to claim 2, characterized in that The application method comprises the following steps: using wet bacteria obtained by induction culture of recombinant genetic engineering bacteria containing a cyclohexene carboxylate esterase encoding gene or a crude enzyme solution obtained by centrifugation after ultrasonic crushing of the wet bacteria as a catalyst, using racemic 3-cyclohexene-1-carboxylate as a substrate, and using a buffer solution with a pH of 6-10 as a reaction medium to form a reaction system; stirring the reaction at 20-60° C. and 100-220 rpm until the reaction is completed; and extracting and separating the reaction solution to obtain (S)-3-cyclohexene-1-carboxylic acid.

4. The method according to claim 3, characterized in that In the reaction system, the amount of substrate added is 10-400 g / L, the amount of wet bacteria added is 1-4 g / L, and the amount of crude enzyme solution added is 0.1-0.4 g / L based on protein content.

5. A mutant of cyclohexenecarboxylate esterase according to claim 1, characterized in that: The mutant is obtained by performing single mutation or combined mutation on the 82nd, 236th and 302nd positions of the amino acid sequence shown in SEQ ID NO.

2.

6. The mutant according to claim 5, characterized in that The mutant is a mutant in which the amino acid sequence shown in SEQ ID NO.2 is mutated into one of the following: (1) the arginine at position 82 is mutated into histidine; (2) the threonine at position 236 is mutated into valine; (3) the leucine at position 302 is mutated into alanine; (4) the arginine at position 82 is mutated into histidine, the threonine at position 236 is mutated into valine, and the leucine at position 302 is mutated into alanine.

7. Use of the cyclohexenecarboxylate esterase mutant according to claim 5 in producing (S)-3-cyclohexene-1-carboxylic acid.

8. The use according to claim 7, characterized in that The application method comprises the following steps: using wet bacteria obtained by induction culture of recombinant genetic engineering bacteria containing a gene encoding a cyclohexene carboxylate esterase mutant or a crude enzyme solution obtained by centrifugation after ultrasonic crushing of the wet bacteria as a catalyst, using racemic 3-cyclohexene-1-carboxylate as a substrate, and using a buffer solution with a pH of 6-10 as a reaction medium to form a reaction system; stirring the reaction at 20-60° C. and 100-220 rpm until the reaction is completed; and extracting and separating the reaction solution to obtain (S)-3-cyclohexene-1-carboxylic acid.

9. The use according to claim 8, characterized in that In the reaction system, the amount of substrate added is 10-400 g / L, the amount of wet bacteria added is 1-4 g / L, and the amount of crude enzyme solution added is 0.1-0.4 g / L based on protein content.

10. The use according to claim 8, characterized in that The catalyst is prepared according to the following steps: The recombinant genetically engineered bacteria were inoculated into LB liquid medium containing 50 mg / L kanamycin, cultured at 37°C overnight, and inoculated into LB liquid medium at a volume concentration of 1%, and cultured on a shaker at 37°C and 180 rpm. 600 When it reaches 0.6, add IPTG with a final concentration of 0.2mmol / L as an inducer. After induction at 25℃ for 12h, centrifuge the culture medium, collect the cells, and wash twice with physiological saline to obtain wet bacterial resting cells; the obtained resting cells are suspended in 100mM, pH 7.0 sodium phosphate buffer, and ultrasonically disrupted at 250W for 10min in an ice bath, with an interval of 3s for 1s, and the supernatant is collected by centrifugation to obtain the crude enzyme solution.

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