WO2022016597A1 - 环己烯甲酸酯水解酶及其突变体、编码基因、表达载体、重组菌与应用 - Google Patents

环己烯甲酸酯水解酶及其突变体、编码基因、表达载体、重组菌与应用 Download PDF

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WO2022016597A1
WO2022016597A1 PCT/CN2020/105687 CN2020105687W WO2022016597A1 WO 2022016597 A1 WO2022016597 A1 WO 2022016597A1 CN 2020105687 W CN2020105687 W CN 2020105687W WO 2022016597 A1 WO2022016597 A1 WO 2022016597A1
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cyclohexene
hydrolase
formate
residue
acid sequence
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倪晔
窦哲
许国超
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江南大学
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    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/18—Carboxylic ester hydrolases (3.1.1)
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    • C12P41/005—Processes using enzymes or microorganisms to separate optical isomers from a racemic mixture by ester formation, lactone formation or the inverse reactions by esterification of carboxylic acid groups in the enantiomers or the inverse reaction
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Definitions

  • the invention relates to a cyclohexene formate hydrolase and a mutant thereof, an encoding gene, an expression vector, a recombinant bacteria and applications, and belongs to the technical field of genetic engineering.
  • Chiral 3-cyclohexene-1-carboxylic acid is an important building block for building a variety of natural products, chiral drugs and agrochemicals, and can be used to synthesize many natural products and drugs such as the novel immunosuppressant tacrolimus, anti- Oncology drug (+)-Phyllanthocin, insect control attractant (–)-piperapine-B, neuraminidase inhibitor oseltamivir phosphate, and coagulation factor Xa inhibitor edoxaban, etc., with the chiral drug market
  • the demand for chiral cyclohexene-1-carboxylic acid is increasing rapidly.
  • the unsaturated six-membered ring of 3-cyclohexene-1-carboxylic acid has a high degree of symmetry, and the difference between the groups on the two sides of the chiral carbon is very small, and it is challenging to achieve its high enantioselective synthesis. Therefore, the establishment of a high enantioselective enzymatic synthesis of chiral 3-cyclohexene-1-carboxylic acid has important application value.
  • the synthesis methods of chiral 3-cyclohexene-1-carboxylic acid mainly include Diels-Alder reaction, chemical method of racemic acid resolution and enzymatic asymmetric hydrolysis to resolve 3-ring Hexene-1-carboxylate.
  • Diels-Alder reaction is the main method for synthesizing chiral 3-cyclohexene-1-carboxylic acid at present.
  • butadiene is a gas and the product is not easy to be separated, the reaction steps are cumbersome and the yield is low, and further improvement is still required. (Synlett, 1990, 1, 38–39.].
  • the chemical resolution process requires at least six recrystallizations in acetone to separate the optically pure enantiomers, resulting in the large use of acetone and the resolution yield. It is only 20-30%, and the atom economy is low (Drugs & Clinic, 2013, 28, 126–128). It can be seen that the chemical synthesis of chiral 3-cyclohexene-1-carboxylic acid faces complicated operations, low yields and a large amount of acetone. Biocatalytic synthesis of chiral compounds has the advantages of mild reaction conditions, high stereoselectivity, environmental friendliness, and simple operation, making it an important method to replace or expand traditional chemical synthesis in the process of sustainable development.
  • Cihangir T et al. investigated the effect of porcine liver esterase PLE, horse liver esterase HLE and porcine pancreatic lipase PPL on the hydrolysis of racemic 3-cyclohexene-1-carboxylate methyl ester, the hydrolyzates of PLE and HLE. It is (S)-3-cyclohexene-1-carboxylic acid, with ee values of >99% and 97%, respectively; the hydrolysis product of PPL is (R)-3-cyclohexene-1-carboxylic acid, with ee value of 91% (Tetrahedron Asymmetry, 2004, 15, 2057–2060).
  • a mutant Mu3 (L24A/W81A/L209A) with higher S-selectivity was obtained by combined mutagenesis to reduce steric hindrance and aromatic stacking, which hydrolyzed racemic-3-cyclohexene-1-carboxylic acid
  • the ee value of methyl ester is from 32.3% to 70.9%.
  • the reaction conditions were further optimized. In phosphate buffer (pH 8.0) containing 2.5% Tween 80 at 30°C, Mu3 could hydrolyze 40 mM substrate, and the conversion rate was 2 times that before optimization.
  • the recombinase BioH has the activity of hydrolyzing methyl 3-cyclohexene-1-carboxylate, its enantioselectivity is low.
  • the method of biocatalytic preparation of chiral cyclohexene carboxylic acid has the advantages of environmental friendliness, mild reaction conditions and simple operation. Not high, the reaction time is long and other defects, it is not suitable for industrial production.
  • the use of wild bacteria method to prepare chiral cyclohexene carboxylic acid has problems such as high enzyme loading, high catalyst cost, serious emulsification of the reaction solution, and low yield. Therefore, it is necessary to screen an enzyme with high activity, good stability, high selectivity and can obtain a higher concentration of product in a shorter reaction time to meet the needs of industrial production of chiral cyclohexenecarboxylic acid.
  • the invention provides a kind of high catalytic activity, good enantioselectivity and good substrate tolerance Cyclohexene formate hydrolase and mutant thereof, recombinant expression vector and recombinant expression transformant containing the enzyme and mutant gene, preparation method of the recombinant enzyme, and use of the cyclohexene formate hydrolase for disassembly A method for the preparation of (S)-3-cyclohexene-1-carboxylic acid.
  • the first object of the present invention is to provide a kind of cyclohexene formate hydrolase, and the described cyclohexene formate hydrolase is:
  • the amino acid sequence is a protein with 3-cyclohexene-1-carboxylate hydrolysis activity through substitution, deletion or addition of one or more amino acids from the amino acid sequence shown in SEQ ID NO.2.
  • described cyclohexene formate hydrolase replaces the 202nd alanine residue of the amino acid sequence shown in SEQ ID No.2 with a lysine residue, and the 326th glycine residue replaces simultaneously A protein with a new amino acid sequence formed after alanine residues.
  • the described cyclohexene formate hydrolase is to replace the 78th phenylalanine residue of the amino acid sequence shown in SEQ ID No.2 with a valine residue, while the 202nd position of alanine A protein with a new amino acid sequence formed after the acid residue is replaced by a lysine residue, and the glycine residue at position 326 is replaced by an alanine residue.
  • the second object of the present invention is to provide a gene encoding the cyclohexene formate hydrolase.
  • the third object of the present invention is to provide a recombinant expression vector comprising the encoding gene.
  • the recombinant expression vector is constructed by linking the nucleic acid sequence or mutant nucleic acid sequence of the cyclohexene formate hydrolase gene of the present invention to various suitable vectors by conventional methods in the art.
  • the vector is preferably a plasmid, more preferably the plasmid pET-28a(+).
  • the cyclohexene carboxylate hydrolase gene can be operably linked downstream of regulatory sequences suitable for expression to achieve constitutive or inducible expression of the cyclohexene carboxylate hydrolase.
  • the fourth object of the present invention is to provide a recombinant bacterium expressing the cyclohexene formate hydrolase.
  • the recombinant bacteria are prepared by transforming the recombinant expression vector of the present invention into a host cell.
  • the host cell can be any conventional host cell in the art, provided that the recombinant expression vector can stably replicate itself, and the cyclohexene formate hydrolase gene carried by it can be effectively expressed.
  • the present invention is preferably Escherichia coli, more preferably Escherichia coli E.coli BL21(DE3) or Escherichia coli E.coli DH5 ⁇ .
  • the fifth object of the present invention is to provide a kind of cyclohexene formate hydrolase in catalyzing 3-cyclohexene-1-carboxylate to prepare optically active (S)-3-cyclohexene-1-carboxylate application.
  • the application is to prepare (S)-3-cyclohexene-1 by catalyzing the enantioselective hydrolysis of 3-cyclohexene-1-formate in buffer by using cyclohexene formate hydrolase -methyl ester, then heated and hydrolyzed under alkaline conditions to obtain (S)-3-cyclohexene-1-carboxylic acid.
  • the buffer is a citrate buffer, a phosphate buffer or a glycine-NaOH buffer, and the pH of the buffer is 5.0-10.0.
  • the alkaline condition is 0.5-1.5M sodium hydroxide solution.
  • 3-cyclohexene-1-carboxylate is methyl 3-cyclohexene-1-carboxylate, ethyl 3-cyclohexene-1-carboxylate, 3-cyclohexene-1-carboxylate Isopropyl formate or butyl 3-cyclohexene-1-carboxylate.
  • the cyclohexene carboxylate hydrolase AcEst1 and its mutants of the present invention have efficient enantioselective resolution of 3-cyclohexene-1-carboxylate to prepare optically active (S)-3-cyclohexene-1- The function of formic acid.
  • substrate concentrations up to 2000 mM (about 280 g/L) the optical purity of the product was higher than 99%, and the substrate/catalyst was up to 3500 g/g.
  • the product prepared by the method of the present invention has high concentration, good optical purity, high catalyst efficiency, mild reaction conditions, environmental friendliness, simple operation, and easy industrial scale-up, so it has a good industrial application prospect.
  • Figure 1 shows the expression and purification results of the cyclohexene formate hydrolase AcEst1, from left to right, the standard protein marker, the crude enzyme supernatant of AcEst1 and the purified protein of AcEst1.
  • the strain Acinetobacter sp. JNU9335 was cultured in LB medium, and the total genomic DNA of high purity and large fragment was extracted by CTAB (hexadecyl trimethyl ammonium bromide) method.
  • CTAB hexadecyl trimethyl ammonium bromide
  • CTAB hexadecyl trimethyl ammonium bromide
  • the obtained recombinant plasmid pET-28a(+)-AcEst1 was transformed into E. coli BL21, and the recombinant E. coli containing cyclohexene formate hydrolase AcEst1 was constructed and inoculated into 50 ⁇ g/mL kanamycin.
  • Example 3 Obtainment of mutant A202K/G326A
  • the mutant A202K/G326A is a random mutant.
  • the random mutant library of AcEst1 was established by error-prone PCR technology, and the color change of the indicator described in Example 1 under different pH environments was used as a high-throughput screening method.
  • First design primers at both ends forward primer 5'-gtgccgcgcggcagccatatgATGGGCGTGTTGAATCAAACTT-3' (SEQ ID NO.5) reverse primer 5'-gtggtggtggtggtgctcgagTTATTTGGCATTCTTATCCCAAAAA-3' (SEQ ID NO.6), PCR system (50 ⁇ L): rTaq polymerase 0.25 ⁇ L, 10 ⁇ rTaq Buffer 5 ⁇ L, dNTP 5 ⁇ L, MgSO 4 2 ⁇ L, template plasmid about 100 ng, forward primer 2 ⁇ L, reverse primer 2 ⁇ L, MnCl 2 (10 mM) 0.5 ⁇ L, dd
  • PCR reaction procedure (1) denaturation at 98°C for 5 min; (2) denaturation at 98°C for 30s, (3) annealing at 55°C for 30s, (4) extension at 72°C for 1 min, steps (2) to (4) were carried out for a total of 30 cycles, The final extension was at 72°C for 10 min, and the PCR product was stored at -20°C.
  • the PCR fragment containing random mutation sites was double digested with NdeI and XhoI, ligated with the pET-28a(+) plasmid with the same restriction site, and then transferred to E.coli BL21(DE3) competent cells and spread evenly on LB agar plates containing 50 ⁇ g/mL kanamycin. After overnight incubation at 37°C, single clones were selected and cultured in deep-well plates to induce expression.
  • the mutant library was screened for the activity of the mutant library according to the method of pH indicator color development to obtain mutants with improved activity, which were sent to Tianlin Biotechnology Co., Ltd. for sequencing.
  • the sequencing results were compared with the cyclohexene formate hydrolase gene (AcEst1) sequence using DNAMAN software, and the 202-position alanine was mutated to lysine, and the 326-position glycine was mutated to alanine.
  • the obtained mutant was named A202K/G326A.
  • Example 4 Plasmids of mutant A202K/G326A and preparation of recombinant bacteria and recombinase
  • the plasmid pET-28a(+)-A202K/G326A obtained in Example 3 was extracted, transformed into E. coli BL21, and inoculated into LB medium containing 50 ⁇ g/mL kanamycin (peptone 10 g /L, yeast extract 5g/L, NaCl 10g/L, pH 7.0), shake culture at 37°C overnight, insert into a 2L Erlenmeyer flask with 600mL LB medium at an inoculum of 1% (v/v), Set at 37°C, shake culture at 180rpm, when the OD 600 of the culture medium reaches 0.6 to 2.0, add IPTG with a final concentration of 0.2mmol/L as an inducer, and after induction at 16°C for 16h, centrifuge the culture medium to collect cells.
  • kanamycin peptone 10 g /L
  • yeast extract 5g/L yeast extract 5g/L, NaCl 10g/L, pH 7.0
  • the obtained resting cells were suspended in Tris-HCl buffer (20 mM, pH 8.0), crushed by a high-pressure homogenizer, and freeze-dried to obtain A202K/G326A recombinase.
  • Site-directed mutagenesis of A202K/G326A was performed using the protocol described in the Site-Directed Mutagenesis Kit (Stratagene, Catalog #200522).
  • N represents the mixture of four bases of A, T, C, and G
  • D represents the mixture of three bases of A, G, and T
  • H represents the mixture of three bases of A, C, and T.
  • PCR reaction system 50 ⁇ L: KOD plus Neo 0.25 ⁇ L, template 0.5 ⁇ 20ng, 5 ⁇ L 10 ⁇ KOD plus Neo buffer, 5 ⁇ L dNTP (2.0mM each), 2 ⁇ L MgSO 4 (25mM), 2 ⁇ L forward primer, 2 ⁇ L reverse primer , ddH 2 O was made up to 50 ⁇ L.
  • the template described therein is the cyclohexene formate hydrolase mutant plasmid pET-28a(+)-A202K/G326A obtained in Example 3.
  • PCR reaction procedure (1) denaturation at 98 °C for 5 min; (2) denaturation at 98 °C for 30 s, (3) annealing at 55 °C for 30 s, (4) extension at 68 °C for 3.5 min, and repeat steps (2) to (4) for 20 cycles , the final extension at 68°C for 10min, and the PCR product was stored at -20°C.
  • the amplified PCR product was digested with endonuclease DpnI for 2 h at 37°C, transformed into E. coli BL21 competent cells, and spread evenly on LB agar plates containing 50 ⁇ g/mL kanamycin. After overnight culture at 37°C, 200 monoclonal strains were selected and cultured and induced to express in deep-well plates.
  • the activity of the mutant library was screened according to the method of pH indicator color development, and the obtained mutants with improved activity were sent to Tianlin Biotechnology Co., Ltd. for sequencing.
  • the sequencing results were compared with the sequence of the cyclohexene formate hydrolase gene (AcEst1) using DNAMAN software.
  • the 78 position was mutated to valine, the 202 position was mutated to lysine, and the 326 position was mutated to alanine.
  • the resulting mutant was named F78V/A202K/G326A.
  • the mutant protein F78V/A202K/G326A obtained by mutating the glycine residue at the position of the glycine residue to an alanine residue has a 6-fold increase in the hydrolysis activity of methyl 3-cyclohexene-1-carboxylate.
  • the mutant F78V/A202K/G326A can achieve a conversion rate similar to that of WT in only 1 h, and the reaction time is shortened by 6 times.
  • Example 6 Plasmids of mutant F78V/A202K/G326A and preparation of recombinant bacteria and recombinase
  • the plasmid pET-28a(+)-F78V/A202K/G326A obtained as in Example 5 was extracted, transformed into E. coli BL21, and inoculated into LB medium containing 50 ⁇ g/mL kanamycin ( Peptone 10g/L, yeast extract 5g/L, NaCl 10g/L, pH 7.0), shake and culture at 37°C overnight, and insert into a 2L conical flask with 600mL LB medium at an inoculum of 1% (v/v) medium, placed at 37 °C, shaken at 180 rpm for culture, when the OD 600 of the culture medium reached 0.6 to 2.0, IPTG with a final concentration of 0.2 mmol/L was added as an inducer, and after induction at 16 °C for 16 h, the culture medium was centrifuged and collected.
  • kanamycin Peptone 10g/L, yeast extract 5g/L, NaCl 10g/L, pH 7.0
  • the cells were washed twice with normal saline to obtain resting cells.
  • the obtained resting cells were suspended in Tris-HCl buffer (20 mM, pH 8.0), crushed by a high-pressure homogenizer, and freeze-dried to obtain F78V/A202K/G326A recombinant enzyme.
  • Example 7 Catalytic properties of the recombinase AcESt1
  • the buffer system used is: sodium citrate buffer (pH 5.0-6.0); sodium phosphate buffer (pH 6.0-8.0); Tris-HCl buffer (pH 8.0-9.0) and glycine-NaOH buffer (pH 9.0) ⁇ 10.0).
  • the results are shown in Table 3.
  • the optimum pH of AcEst1 is Tris-HCl buffer at pH 9.0.
  • Example 8 Recombinase AcESt1 catalyzes the hydrolysis and resolution of different 3-cyclohexene-1-carboxylate esters
  • Recombinant AcEst1 can catalyze the enantioselective hydrolysis of various 3-cyclohexene-1-carboxylate esters to generate (S)-3-cyclohexene-1-carboxylate in high optical purity.
  • AcEst1 was investigated for methyl 3-cyclohexene-1-carboxylate, ethyl 3-cyclohexene-1-carboxylate, isopropyl 3-cyclohexene-1-carboxylate and 3-cyclohexene-1-carboxylate, respectively.
  • Typical conditions for the enzymatic 3-cyclohexene-1-carboxylate hydrolysis and resolution reaction are as follows: 16 mg of recombinant AcEst1 lyophilized enzyme powder was dissolved in 200 mL of Tris-HCl buffer (200 mM, pH 9.0), and the substrate was added The final concentration of methyl 3-cyclohexene-1-carboxylate is 200-2000 mM (28-280 g/L), and the corresponding S/C is 350-3500 g/g, respectively. The reaction is carried out at 30 °C, 200rpm mechanical stirring, by added 1.0M Na 2 CO 3 pH was controlled at 9.0 until the substrate ee> 99%.
  • the wild-type AcEst1 obtained in Example 3, the mutant A202K/G326A obtained in Example 4, and the mutant F78V/A202K/G326A obtained in Example 5 respectively catalyzed the hydrolysis reaction of methyl 3-cyclohexene-1-carboxylate.
  • the reaction was carried out in a 1L three-necked flask, and 200 mL of Tris-HCl buffer at pH 9.0 was added, 16 mg of F78V/A202K/G326A crude enzyme powder prepared as in Example 5 and 58.7 g of racemic 3-cyclohexene-1-methyl formate , at 30 ° C, 200 rpm mechanical stirring under the reaction, stream added 1M Na 2 CO 3 to control the pH of the reaction solution maintained at 9.0. After the reaction for 12 h, the conversion rate reached 61.1%, and the optical purity of (S)-methyl 3-cyclohexene-1-carboxylate was >99% at this time.

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Abstract

一种环己烯甲酸酯水解酶及其突变体、编码基因、表达载体、重组菌与应用。环己烯甲酸酯水解酶AcEst1及其突变体具有高效对映选择性拆分3-环己烯-1-甲酸酯，制备光学活性(S)-3-环己烯-1-甲酸的功能。在底物浓度高达2000mM(约280g/L)时，产物的光学纯度高于99％，底物/催化剂高达3500g/g。相对于其它制备方法，使用本方法制备所得的产物浓度高，光学纯度好，催化剂效率高，反应条件温和，对环境友好，操作简便，易于工业放大，因此具有很好的工业应用前景。

Description

环己烯甲酸酯水解酶及其突变体、编码基因、表达载体、重组菌与应用 技术领域
本发明涉及一种环己烯甲酸酯水解酶及其突变体、编码基因、表达载体、重组菌与应用，属于基因工程技术领域。
背景技术
(S)-3-环己烯-1-甲酸[(S)-3-cyclohexene-1-carboxylic acid，(S)-CHCA]，分子式：C 7H 10O 2，分子量：126.15，沸点：118℃/6mmHg(lit.)，CAS号为5708-19-0。
手性3-环己烯-1-甲酸是构建多种天然产物、手性药物和农用化学品的重要砌块单元，可用于合成许多天然产物和药物如新型免疫抑制剂他克莫司，抗肿瘤药(+)-Phyllanthocin，昆虫防治引诱剂(–)-哌拉平-B，神经氨酸酶抑制剂磷酸奥司他韦，和凝血因子Xa抑制剂依度沙班等，随着手性药物市场的不断扩大，手性环己烯-1-甲酸的需求迅速增加。3-环己烯-1-甲酸的不饱和六元环具有高度的对称性，手性碳两侧基团的差异非常小，实现其高对映选择性合成充满挑战。因此，建立高对映选择性的酶法合成手性3-环己烯-1-甲酸的工艺具有重要的应用价值。
目前手性3-环己烯-1-甲酸的合成方法主要有狄尔斯-阿尔德(Diels-Alder)反应，化学法外消旋酸拆分法和酶法不对称水解拆分3-环己烯-1-甲酸酯。其中Diels-Alder反应是目前合成手性3-环己烯-1-甲酸的主要方法，然而由于丁二烯为气体且产物为不易于分离，反应步骤繁琐，收率较低，仍然需要进一步改进(Synlett,1990,1,38–39.]。化学拆分过程需要在丙酮中进行至少六次重结晶才可分离出光学纯的对映异构体，导致丙酮的大量使用，拆分收率仅20～30％，原子经济性低(Drugs&Clinic,2013,28,126–128)。由此可见，化学法合成手 性3-环己烯-1-甲酸面临操作繁琐、收率较低和丙酮的大量使用等诸多问题。生物催化法合成手性化合物具有反应条件温和、立体选择性高、环境友好、操作简便等优势，使其成为可持续发展过程中替代或拓展传统化学合成的重要方法。
2004年，Cihangir T等考察了猪肝酯酶PLE，马肝酯酶HLE和猪胰脂肪酶PPL水解外消旋3-环己烯-1-羧酸甲酯的效果，PLE和HLE的水解产物为(S)-3-环己烯-1-甲酸，ee值分别为>99％和97％；PPL的水解产物为(R)-3-环己烯-1-甲酸，ee值为91％(Tetrahedron Asymmetry,2004,15,2057–2060)。然而，上述酶为动物来源的商品酶，其实际应用的过程中存在：价格昂贵、同工酶干扰、批次间差异较大和引入病毒的风险等。2019年，Xiafen Wu等对大肠杆菌来源的羧酯酶BioH进行分子改造以提高其对(S)-3-环己烯-1-甲酸甲酯的对映选择性(Biosci.Biotechnol.Biochem.,2019,83,1263–1269)。通过降低空间位阻和芳香堆积作用的组合突变，获得了具有较高S-选择性的突变体Mu3(L24A/W81A/L209A)，其水解外消旋-3-环己烯-1-羧酸甲酯的ee值由32.3％至70.9％。进一步优化反应条件，在30℃和含有2.5％吐温80的磷酸盐缓冲液(pH 8.0)中，Mu3可水解40mM底物，转化率是优化前的2倍。重组酶BioH虽然具有水解3-环己烯-1-甲酸甲酯的活性，但对映选择性较低。2019年，窦哲等以3-环己烯-1-甲酸甲酯为底物进行野生菌的筛选，发现Acinetobacter sp.JNU9335能够选择性地水解底物生成(S)-3-环己烯-1-甲酸甲酯，通过在碱性条件下水解得到(S)-3-环己烯-1-甲酸。其中在50mL反应中添加了3.50g(R,S)-3-环己烯-1-甲酸甲酯，反应12h后，最终产物的收率为40％，ee s为99％(中国发明专利，申请公开号CN 110272839 A)。
与传统化学合成法相比，生物催化制备手性环己烯甲酸的方法具有环境友好、反应条件温和、操作简单等优点，但是目前以上方法仅限于实验室规模，且存在酶自身活力低，产物浓度不高，反应时间较长等缺陷，不适合工业化生产。采用野生菌法制备手性环己烯甲酸存在酶上载量过高，导致催化剂成本高、反应液乳化严重、收率较低等问题。因此，需要筛选活力高、稳定性好、选择 性高且能在较短反应时间获得较高浓度产物的酶，以满足工业化生产手性环己烯甲酸的需求。
发明内容
本发明针对酶法拆分制备(S)-3-环己烯-1-甲酸中现有技术上的不足，提供了一种催化活力高、对映选择性好、底物耐受性好的环己烯甲酸酯水解酶及其突变体，含有该酶及突变体基因的重组表达载体及重组表达转化体，该重组酶的制备方法，以及该环己烯甲酸酯水解酶用于拆分制备(S)-3-环己烯-1-甲酸的方法。
本发明的第一个目的是提供一种环己烯甲酸酯水解酶，所述的环己烯甲酸酯水解酶为：
(a)氨基酸序列如SEQ ID NO.2所示的蛋白质；
(b)氨基酸序列由SEQ ID NO.2所示的氨基酸序列经过替换、缺失或添加一个或多个氨基酸，且具有3-环己烯-1-甲酸酯水解活性的蛋白质。
进一步地，所述的环己烯甲酸酯水解酶是将SEQ ID No.2所示氨基酸序列的第202位丙氨酸残基替换为赖氨酸残基，同时第326位甘氨酸残基替换为丙氨酸残基后形成的新氨基酸序列的蛋白质。
进一步地，所述的环己烯甲酸酯水解酶是将SEQ ID No.2所示氨基酸序列的第78位苯丙氨酸残基替换为缬氨酸残基，同时第202位的丙氨酸残基替换为赖氨酸残基，同时第326位甘氨酸残基替换为丙氨酸残基后形成的新氨基酸序列的蛋白质。
本发明的第二个目的是提供一种所述的环己烯甲酸酯水解酶的编码基因。
本发明的第三个目的是提供一种重组表达载体，包含所述的编码基因。
进一步地，所述的重组表达载体通过本领域常规方法将本发明的环己烯甲酸酯水解酶基因的核酸序列或突变体核酸序列连接于各种合适载体上构建而 成。所述载体优选质粒，更优选质粒pET-28a(+)。所述环己烯甲酸酯水解酶基因可以操作性地连接于适合表达的调控序列的下游，以实现所述环己烯甲酸酯水解酶的组成型或诱导型表达。
本发明的第四个目的是提供一种表达所述的环己烯甲酸酯水解酶的重组菌。
进一步地，所述的重组菌是通过将本发明的重组表达载体转化至宿主细胞中来制得所述重组菌。所述宿主细胞可以是本领域的各种常规宿主细胞，前提是能使所述重组表达载体稳定地自行复制，且其所携带的环己烯甲酸酯水解酶基因可被有效表达。本发明优选大肠杆菌，更优选大肠杆菌E.coli BL21(DE3)或大肠杆菌E.coli DH5α。
本发明的第五个目的是提供一种环己烯甲酸酯水解酶在催化3-环己烯-1-甲酸酯制备光学活性(S)-3-环己烯-1-甲酸中的应用。
进一步地，所述的应用是采用环己烯甲酸酯水解酶在缓冲液中催化3-环己烯-1-甲酸酯对映选择性水解制备(S)-3-环己烯-1-甲酯，然后在碱性条件下加热水解得到(S)-3-环己烯-1-甲酸。
进一步地，所述的缓冲液为柠檬酸盐缓冲液、磷酸盐缓冲液或甘氨酸-NaOH缓冲液，所述缓冲液的pH为5.0～10.0。
进一步地，所述的碱性条件为0.5～1.5M氢氧化钠溶液。
进一步地，所述的3-环己烯-1-甲酸酯为3-环己烯-1-甲酸甲酯、3-环己烯-1-甲酸乙酯、3-环己烯-1-甲酸异丙酯或3-环己烯-1-甲酸丁酯。
本发明的有益效果：
本发明环己烯甲酸酯水解酶AcEst1及其突变体具有高效对映选择性拆分3-环己烯-1-甲酸酯，制备光学活性(S)-3-环己烯-1-甲酸的功能。在底物浓度高达2000mM(约280g/L)时，产物的光学纯度高于99％，底物/催化剂高达3500g/g。相对于其它制备方法，使用本发明方法制备所得的产物浓度高，光学纯度好， 催化剂效率高，反应条件温和，对环境友好，操作简便，易于工业放大，因此具有很好的工业应用前景。
附图说明
图1为环己烯甲酸酯水解酶AcEst1的表达及纯化结果，从左到右依次为，标准蛋白marker，AcEst1粗酶上清液和AcEst1纯化后的蛋白。
具体实施方式
下面结合附图和具体实施例对本发明作进一步说明，以使本领域的技术人员可以更好地理解本发明并能予以实施，但所举实施例不作为对本发明的限定。
实施例1：环己烯甲酸酯水解酶AcEst1基因的克隆
将菌株Acinetobacter sp.JNU9335在LB培养基中进行培养，采用CTAB(十六烷基三甲基溴化铵)法提取高纯度、大片段的基因组总DNA。将适量Acinetobacter sp.JNU9335菌体加入液氮冷冻，研磨成粉，加入适量2×CTAB提取缓冲液(100mmol/L Tris-HCl，pH 8.0，20mmol/L EDTA，1.4mol/L NaCl，2％(w/v)CTAB，40mmol/L巯基乙醇)，65℃保温10min，间歇摇动。随后加入等体积的氯仿/异戊醇，轻缓颠倒离心管混匀，室温下，8000rpm离心10min，将上清液转入另一离心管中，加入等体积的氯仿/异戊醇，颠倒离心管混匀，室温下8000rpm离心10min。将上层水相转入新的离心管中，加入等体积异丙醇混匀，室温放置30min。4000rpm离心10min，移去上清液，用70％乙醇洗两次，烘干后加入20μL的TE缓冲液(100mM Tris-HCl，10mM EDTA pH 8.0，)溶解DNA，-20℃保存备用。采用Sau3AI对总DNA进行部分酶切，酶切后的DNA片段通过电泳进行纯化，采用胶回收纯化试剂盒回收大约2～6kb的片段，回收的DNA溶解于ddH 2O中，置于-20℃保藏。
按如下反应体系与载体pUC118进行连接：
表1 连接反应体系
Figure PCTCN2020105687-appb-000001
16℃温育12h，取10μL酶连接产物转化至200μL E.coli DH5α感受态细胞，得到的重组子诱导表达后，加入底物3-环己烯-1-甲酸甲酯反应，若有产物3-环己烯-1-甲酸生成，根据溴百里酚蓝和酚红双指示剂的在不同pH下的颜色变化进行高通量筛选。将具有明显颜色变化，即由棕绿色变为黄色的菌落进行二次筛选，将产物进行HPLC检测，将有明显产物峰的重组子委托天霖生物科技有限公司进行序列测定，获得如SEQ ID NO.1所示的核苷酸序列，根据该核苷酸序列所推导的氨基酸序列为SEQ ID NO.2，将该序列表达的环己烯甲酸酯水解酶命名为AcEst1。
实施例2：重组AcEst1的质粒以及重组菌、重组酶的制备
以正向引物5’-gtgccgcgcggcagccatatgATGGGCGTGTTGAATCAAACTT-3’(SEQ ID NO.3)，反向引物5’-gtggtggtggtggtgctcgagTTA-TTTGGCATTCTTATCCCAAAA-3’(SEQ ID NO.4)，利用聚合酶链式反应技术对实施例1中获得的AcEst1的核苷酸序列进行扩增，将获得的含有AcEst1序列的DNA片段分别用NdeI和XhoI双酶切，随后与同样经过NdeI和XhoI双酶切的质粒pET-28a(+)进行连接，获得重组质粒pET-28a(+)-AcEst1。
将获得的重组质粒pET-28a(+)-AcEst1转化到大肠杆菌E.coli BL21中，构建含环己烯甲酸酯水解酶AcEst1的重组大肠杆菌并接种至含50μg/mL卡那霉素的LB培养基(蛋白胨10g/L，酵母膏5g/L，NaCl 10g/L，pH 7.0)中，37℃振荡培养过夜，按1％(v/v)的接种量接入装有600mL LB培养基的2L三角瓶中，置37℃，180rpm摇床振荡培养，当培养液的OD 600达到0.6～2.0时，加入 终浓度为0.2mmol/L的IPTG作为诱导剂，16℃诱导16h后，将培养液离心，收集细胞，并用生理盐水洗涤两次，得静息细胞。将所得的静息细胞悬浮于Tris-HCl缓冲液(20mM，pH 8.0)中，高压匀浆机破碎，冷冻干燥即得AcEst1重组酶。
实施例3：突变体A202K/G326A的获得
突变体A202K/G326A为随机突变体，通过易错PCR技术建立AcEst1的随机突变体库，利用实施例1所述指示剂在不同pH环境下颜色的变化作为高通量筛选的手段。首先设计两端引物：正向引物5’-gtgccgcgcggcagccatatgATGGGCGTGTTGAATCAAACTT-3’(SEQ ID NO.5)反向引物5’-gtggtggtggtggtgctcgagTTATTTGGCATTCTTATCCCAAAA-3’(SEQ ID NO.6)，PCR体系(50μL)：rTaq polymerase 0.25μL，10×rTaq Buffer 5μL，dNTP 5μL，MgSO 4 2μL，模板质粒约100ng，正向引物2μL，反向引物2μL，MnCl 2(10mM)0.5μL，ddH 2O补足至50μL。
PCR反应程序：(1)98℃变性5min；(2)98℃变性30s，(3)55℃退火30s，(4)72℃延伸1min，步骤(2)～(4)共进行30个循环，最后72℃延伸10min，-20℃保藏PCR产物。
含有随机突变位点的PCR片段经NdeI和XhoI双酶切后与具有相同酶切位点的pET-28a(+)质粒连接后转入E.coli BL21(DE3)感受态细胞，并均匀涂布于含有50μg/mL卡那霉素的LB琼脂平板。37℃过夜培养后，挑选单克隆至深孔板进行培养及诱导表达。根据pH指示剂显色的方法进行突变库的活力筛选得到活性提高的突变体，送天霖生物科技有限公司进行测序。测序结果用DNAMAN软件与环己烯甲酸酯水解酶基因(AcEst1)序列进行比对，202位丙氨酸突变为赖氨酸，326位甘氨酸突变为丙氨酸。获得的突变体命名为A202K/G326A。
将SEQ ID No.2所示氨基酸序列的第202位的丙氨酸残基替换为赖氨酸残基，同时第326位的甘氨酸残基替换为丙氨酸残基获得的突变体蛋白A202K/G326A对3-环己烯-1-甲酸甲酯的水解活性提高了3倍。催化相同的底物浓度，突变体A202K/G326A仅需反应2h就可达到与WT相近的转化率，反 应时间缩短了3倍。
实施例4：突变体A202K/G326A的质粒以及重组菌、重组酶的制备
提取如实施例3中所获得的质粒pET-28a(+)-A202K/G326A，将其转化到大肠杆菌E.coli BL21中，接种至含50μg/mL卡那霉素的LB培养基(蛋白胨10g/L，酵母膏5g/L，NaCl 10g/L，pH 7.0)中，37℃振荡培养过夜，按1％(v/v)的接种量接入装有600mL LB培养基的2L三角瓶中，置37℃，180rpm摇床振荡培养，当培养液的OD 600达到0.6～2.0时，加入终浓度为0.2mmol/L的IPTG作为诱导剂，16℃诱导16h后，将培养液离心，收集细胞，并用生理盐水洗涤两次，得静息细胞。将所得的静息细胞悬浮于Tris-HCl缓冲液(20mM，pH 8.0)中，高压匀浆机破碎，冷冻干燥即得A202K/G326A重组酶。
实施例5：突变体F78V/A202K/G326A的获得
对A202K/G326A进行定点突变，采用Site-Directed Mutagenesis Kit(Stratagene，Catalog#200522)所述方案进行操作。
首先设计含有突变点的简并引物：F：5’-TCGCGTAAATTGNDTGATCATCAAATT-3’(SEQ ID NO.7)，R：5’-AATTTGATGATCAHNCAATTTACGCGA-3’(SEQ ID NO.8)。
其中N代表A、T、C、G四种碱基的混合；D代表A、G、T三种碱基的混合，H代表A、C、T三种碱基的混合。
PCR反应体系(50μL)：KOD plus Neo 0.25μL，模板0.5～20ng，5μL 10×KOD plus Neo buffer，5μL dNTP(各2.0mM)，2μL MgSO 4(25mM)，正向引物2μL，反向引物2μL，ddH 2O补足至50μL。
其中所述的模板为实施例3获得环己烯甲酸酯水解酶突变体质粒pET-28a(+)-A202K/G326A。
PCR反应程序：(1)98℃变性5min；(2)98℃变性30s，(3)55℃退火30s，(4)68℃延伸3.5min，步骤(2)～(4)重复进行20个循环，最后68℃延伸10min，-20℃保藏PCR产物。
扩增得到的PCR产物在37℃经内切酶DpnI消化2h后转化E.coli BL21 感受态细胞，并均匀涂布于含有50μg/mL卡那霉素的LB琼脂平板。37℃过夜培养后，挑选单克隆200株至深孔板进行培养及诱导表达。根据pH指示剂显色的方法进行突变库的活力筛选，得到的活力提高的突变体，送天霖生物科技有限公司进行测序。测序结果用DNAMAN软件与环己烯甲酸酯水解酶基因(AcEst1)序列进行比对，78位突变为缬氨酸，202位突变为赖氨酸，326位突变为丙氨酸。得到的突变体命名为F78V/A202K/G326A。
将SEQ ID No.2所示氨基酸序列的第78位的苯丙氨酸残基替换为缬氨酸残基，同时第202位的丙氨酸残基替换为赖氨酸残基，同时第326位的甘氨酸残基突变为丙氨酸残基获得的突变体蛋白F78V/A202K/G326A对3-环己烯-1-甲酸甲酯的水解活性提高了6倍。催化相同的底物浓度，突变体F78V/A202K/G326A仅需反应1h就可达到与WT相近的转化率，反应时间缩短了6倍。
实施例6：突变体F78V/A202K/G326A的质粒以及重组菌、重组酶的制备
提取如实施例5中所获得的质粒pET-28a(+)-F78V/A202K/G326A，将其转化到大肠杆菌E.coli BL21中，接种至含50μg/mL卡那霉素的LB培养基(蛋白胨10g/L，酵母膏5g/L，NaCl 10g/L，pH 7.0)中，37℃振荡培养过夜，按1％(v/v)的接种量接入装有600mL LB培养基的2L三角瓶中，置37℃，180rpm摇床振荡培养，当培养液的OD 600达到0.6～2.0时，加入终浓度为0.2mmol/L的IPTG作为诱导剂，16℃诱导16h后，将培养液离心，收集细胞，并用生理盐水洗涤两次，得静息细胞。将所得的静息细胞悬浮于Tris-HCl缓冲液(20mM，pH 8.0)中，高压匀浆机破碎，冷冻干燥即得F78V/A202K/G326A重组酶。
实施例7：重组酶AcESt1的催化特性
将本发明的环己烯甲酸酯水解酶应用于外消旋3-环己烯-1-甲酸酯的酶法水解以制备光学活性的(S)-3-环己烯-1-甲酸。
在不同温度(20～65℃)下，以磷酸钠(100mM，pH 7.0)为缓冲液，1mM对硝基苯酚环己烯甲酸酯为测活底物，根据分光光度计405nm处吸光值的变化考察了环己烯甲酸酯水解酶AcEst1的活性。结果如表2所示，AcEst1在40℃时催化活性最高。当温度继续升高后，酶活性开始下降。
表2 环己烯甲酸酯水解酶AcEst1在不同温度下的活性
Figure PCTCN2020105687-appb-000002
反应温度为30℃时，以1mM对硝基苯酚环己烯甲酸酯为测活底物，根据分光光度计405nm处吸光值的变化，考察了AcEst1在不同pH值缓冲液中的活性。所用的缓冲液体系为：柠檬酸钠缓冲液(pH 5.0～6.0)；磷酸钠缓冲液(pH 6.0～8.0)；Tris-HCl缓冲液(pH 8.0～9.0)和甘氨酸-NaOH缓冲液(pH 9.0～10.0)。结果如表3所示，AcEst1的最适pH在pH 9.0的Tris-HCl缓冲液。
表3 环己烯甲酸酯水解酶AcEst1在不同pH缓冲液中的活性
Figure PCTCN2020105687-appb-000003
实施例8：重组酶AcESt1催化不同3-环己烯-1-甲酸酯水解拆分
重组AcEst1可以催化多种3-环己烯-1-甲酸酯的对映选择性水解，生成高光 学纯度的(S)-3-环己烯-1-甲酸。分别考察了AcEst1对3-环己烯-1-甲酸甲酯、3-环己烯-1-甲酸乙酯、3-环己烯-1-甲酸异丙酯和3-环己烯-1-甲酸丁酯的活性以及水解产物的光学纯度，利用实施例3获得的野生型AcEst1催化3-环己烯-1-甲酸酯水解反应。称取5mg重组AcEst1粗酶粉，溶解于10mL Tris-HCl缓冲液(200mM，pH 9.0)中，加入底物3-环己烯-1-甲酸酯至终浓度为50mM，反应在30℃下进行。结果见表4。
表4 AcEst1对不同3-环己烯-1-甲酸酯的活性和产物光学纯度
Figure PCTCN2020105687-appb-000004
实施例9：重组酶AcEst1催化3-环己烯-1-甲酸甲酯水解拆分
典型的酶促3-环己烯-1-甲酸酯水解拆分反应的条件如下：16mg重组AcEst1冻干酶粉，溶解于200mL Tris-HCl缓冲液(200mM，pH 9.0)中，加入底物3-环己烯-1-甲酸甲酯至终浓度为200-2000mM(28-280g/L)，对应的S/C分别为350-3500g/g。反应在30℃下进行，200rpm机械搅拌，通过补充1.0M Na 2CO 3将pH控制在9.0，直至底物e.e.>99％。反应结束后用2M NaOH调pH至12，然后用二氯甲烷萃取三次，合并萃取液，加无水硫酸钠干燥过夜。旋转蒸发除去溶剂即可得到(S)-3-环己烯-1-甲酸甲酯。用气相色谱(手性毛细管柱B-DM)分析测定转化率和水解产物的ee值。具体分析条件为：以N 2为载气，进样口温度280℃，检测器温度280℃，初始柱温50℃，2℃/min至100℃保持10min。结果见表5。然后将(S)-3-环己烯-1-甲酸甲酯加入1M NaOH水溶液50℃加热回流搅拌反应6h，再加入1M HCl水溶液调节pH至5.0，加入等体积二氯甲烷萃取3次，合并有机层，用无水Na 2SO 4干燥，过滤，旋转蒸发得到(S)-3-环己烯-1-甲酸，所得产物为具有特殊气味的液体，分离后总得率为38％，光学 纯度为99％e.e.。
表5 重组酶AcEst1催化3-环己烯-1-甲酸甲酯不对称拆分的结果
Figure PCTCN2020105687-appb-000005
实施例10：AcEst1及其突变体催化3-环己烯-1-甲酸酯水解拆分
实施例3获得的野生型AcEst1、实施例4获得的突变体A202K/G326A与实施例5获得的突变体F78V/A202K/G326A分别催化3-环己烯-1-甲酸甲酯水解反应。称取5mg重组AcEst1或突变体粗酶粉，溶解于10mL Tris-HCl缓冲液(200mM，pH 9.0)中，加入底物3-环己烯-1-甲酸甲酯至终浓度为50mM，反应在30℃下进行。从反应情况看(如表6)，突变体转化率达到50％左右所需的时间明显减少。
表6 重组酶AcEst1及其突变体催化3-环己烯-1-甲酸甲酯不对称拆分的结果
Figure PCTCN2020105687-appb-000006
实施例11：(S)-3-环己烯-1-甲酸的制备
反应在1L三口烧瓶中进行，加入200mL pH 9.0的Tris-HCl缓冲液，16mg如实施例5制备的F78V/A202K/G326A粗酶粉以及58.7g消旋3-环己烯-1-甲酸甲酯，在30℃，200rpm机械搅拌下反应，流加1M Na 2CO 3控制反应液pH维持在9.0。反应12h后，转化率达到61.1％，此时(S)-3-环己烯-1-甲酸甲酯的光学纯度>99％。反应结束后用2M NaOH调pH至12，然后用二氯甲烷萃取三次，合并萃取液，加无水Na 2SO 4干燥过夜。旋转蒸发除去溶剂即可得到(S)-3-环己 烯-1-甲酸甲酯。然后将(S)-3-环己烯-1-甲酸甲酯加入1M NaOH水溶液50℃加热回流搅拌反应6h，再加入1M HCl水溶液调节pH至5.0，加入等体积二氯甲烷萃取3次，合并有机层，用无水Na 2SO 4干燥，过滤，旋转蒸发得到(S)-3-环己烯-1-甲酸22.3g，收率为38.0％，GC纯度为99.0％，光学纯度为99.5％e.e.。
以上所述实施例仅是为充分说明本发明而所举的较佳的实施例，本发明的保护范围不限于此。本技术领域的技术人员在本发明基础上所作的等同替代或变换，均在本发明的保护范围之内。本发明的保护范围以权利要求书为准。

Claims (10)

  1. 一种环己烯甲酸酯水解酶，其特征在于，所述的环己烯甲酸酯水解酶为：
    (a)氨基酸序列如SEQ ID NO.2所示的蛋白质；或，
    (b)氨基酸序列由SEQ ID NO.2所示的氨基酸序列经过替换、缺失或添加一个或多个氨基酸，且具有3-环己烯-1-甲酸酯水解活性的蛋白质。
  2. 根据权利要求1所述的环己烯甲酸酯水解酶，其特征在于，所述的环己烯甲酸酯水解酶是将SEQ ID No.2所示氨基酸序列的第202位丙氨酸残基替换为赖氨酸残基，同时第326位甘氨酸残基替换为丙氨酸残基后形成的新氨基酸序列的蛋白质。
  3. 根据权利要求1所述的环己烯甲酸酯水解酶，其特征在于，所述的环己烯甲酸酯水解酶是将SEQ ID No.2所示氨基酸序列的第78位苯丙氨酸残基替换为缬氨酸残基，同时第202位的丙氨酸残基替换为赖氨酸残基，同时第326位甘氨酸残基替换为丙氨酸残基后形成的新氨基酸序列的蛋白质。
  4. 一种权利要求1-3任一项所述的环己烯甲酸酯水解酶的编码基因。
  5. 一种重组表达载体，其特征在于，包含权利要求4所述的编码基因。
  6. 一种表达权利要求1-3任一项所述的环己烯甲酸酯水解酶的重组菌。
  7. 一种权利要求1-3任一项所述的环己烯甲酸酯水解酶在催化3-环己烯-1-甲酸酯制备光学活性(S)-3-环己烯-1-甲酸中的应用。
  8. 根据权利要求7所述的应用，其特征在于，所述的应用是采用环己烯甲酸酯水解酶在缓冲液中催化3-环己烯-1-甲酸酯对映选择性水解制备(S)-3-环己烯-1-甲酯，然后在碱性条件下加热水解得到(S)-3-环己烯-1-甲酸。
  9. 根据权利要求8所述的应用，其特征在于，所述的缓冲液为柠檬酸盐缓冲液、磷酸盐缓冲液或甘氨酸-NaOH缓冲液，所述缓冲液的pH为5.0～10.0。
  10. 根据权利要求8所述的应用，其特征在于，所述的碱性条件为0.5～1.5M氢氧化钠溶液。
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