WO2021147558A1 - 一种腈水解酶突变体及其在制备抗癫痫药物中间体中的应用 - Google Patents

一种腈水解酶突变体及其在制备抗癫痫药物中间体中的应用 Download PDF

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WO2021147558A1
WO2021147558A1 PCT/CN2020/135583 CN2020135583W WO2021147558A1 WO 2021147558 A1 WO2021147558 A1 WO 2021147558A1 CN 2020135583 W CN2020135583 W CN 2020135583W WO 2021147558 A1 WO2021147558 A1 WO 2021147558A1
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buffer
mutant
nitrilase
acn
seq
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薛亚平
熊能
吕佩锦
郑裕国
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浙江工业大学
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    • C12Y305/05—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5) in nitriles (3.5.5)
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Definitions

  • the invention relates to a mutant of CCTCC NO:M 209044 nitrilase derived from Acidovorax facilis and its application in the synthesis of 1-cyanocyclohexylacetic acid, an antiepileptic drug intermediate.
  • Gabapentin is a new type of antiepileptic drug first developed by Warner-Lambert Company of the United States. Compared with similar drugs on the market, it has the advantages of fast oral absorption, less toxic and side effects, obvious therapeutic effect and strong tolerance. It is not easy to bind to plasma proteins in the body, does not induce liver enzymes, and does not metabolize. It can pass through The blood-brain barrier of the human brain is less likely to interact with other anti-epileptic drugs, and it is particularly effective as a superimposed drug for refractory epilepsy.
  • 1-Cyanocyclohexylacetic acid is a key intermediate for the synthesis of the antiepileptic drug gabapentin, and it has broad application prospects in the market.
  • the industrial synthesis of gabapentin and the key intermediate 1-cyanocyclohexylacetic acid all adopt chemical synthesis methods, and there are problems in the production process such as harsh reaction conditions, serious environmental pollution, and high waste disposal costs.
  • Nitrilase (Nitrilase EC 3.5.5.1) is an enzyme that can directly hydrolyze nitriles (containing -CN) into corresponding carboxylic acids.
  • the catalytic reaction of nitrilase has the characteristics of high stereoselectivity, high catalytic rate, mild reaction conditions and low environmental pollution. It is an environmentally friendly green synthesis method, and has important reality for energy saving, emission reduction and building a harmonious society. significance. At present, there are many cases of nitrilase used in industry.
  • the product (R)-mandelic acid of BASF in Germany firstly reacts benzaldehyde and hydrocyanic acid to form racemic mandelonitrile, and then selects appropriate reaction conditions to hydrolyze nitrile Enzyme-catalyzed kinetic resolution can be quantitatively converted to (R)-mandelic acid.
  • Methylene glutaronitrile is first hydrolyzed to 4-cyanovaleric acid (4-CPA) ammonium salt with an immobilized nitrilase-containing microbial cell catalyst (Acidovorax facilis 72W).
  • the selectivity of the hydrolysis reaction is greater than 98%.
  • the rate is 100%, and one-half of the cyano carboxylic acid ammonium salt can be obtained, and 1 to 2% of the only reaction by-product 2-methyl glutaric acid diammonium salt can be produced.
  • the chemical-enzymatic production process can be obtained with improved yield, reduced waste, and high stereoselectivity.
  • many nitrilase enzymes have been developed and used in the synthesis of various pharmaceutical intermediates and fine chemicals.
  • thermostability of the enzyme can be achieved by molecular modification or semi-rational design of the enzyme. Since there are few reports on the crystal structure of nitrilase, there are fewer modifications on the thermostability of nitrilase. Crum, Benedik and others have studied the temperature stability of Cyanidedihydratase (CynD pum) derived from Bacillus pumilus for many years.
  • the researchers first screened out multiple forward mutant strains (K93R, D172N and E327K) using error-prone PCR, and then fused the C-terminus of Pseudomonas stutzeri Cyanidedihydratase (CynD stu ) with CynD pum, Improved its temperature stability (Frontiers in Microbiology 2016 Aug 12; 7:1264.).
  • Xu et al. used error-prone PCR to randomly mutagenize the AcN gene, and finally obtained three mutants (AcN-T201L, AcN-Q339K, AcN-Q343K) showing high thermal stability.
  • the pure enzyme was incubated at 45°C, samples were taken to measure the enzyme activity, and its half-life was calculated.
  • the substrate 1-cyanocyclohexylacetonitrile Due to the high solubility of the substrate 1-cyanocyclohexylacetonitrile under higher temperature conditions, it can promote the catalytic reaction, but the thermal stability of the catalytic enzyme is poor, and the catalytic activity is low under high temperature conditions, so the existing The nitrilase cannot meet the requirements, and it is necessary to improve the thermal stability of the nitrilase by means of molecular modification, thereby improving the catalytic efficiency and realizing industrial production.
  • the purpose of the present invention is to provide a nitrilase mutant protein with improved thermal stability and its application in the synthesis of 1-cyanocyclohexylacetic acid, including a recombinant vector containing the gene, and a recombinant genetic engineering transformed from the recombinant vector It solves the problem of poor temperature stability of nitrilase derived from Acidovorax facilis (Acidovorax facilis) CCTCC NO:M 209044.
  • the present invention provides a nitrilase mutant with improved thermal stability.
  • the mutant has one or more of the 151st, 223rd, and 250th amino acids of the amino acid sequence shown in SEQ ID No. 2. It was obtained by mutation.
  • the nitrilase mutant is preferably one of the following: (1) The threonine at position 151 of the amino acid sequence shown in SEQ ID No. 2 is mutated to valine (T151V), and the amino acid sequence is SEQ ID No. As shown in .4, the nucleotide sequence of the coding gene is shown in SEQ ID No. 3; (2) The cysteine at position 223 of the amino acid sequence shown in SEQ ID No. 2 was mutated to alanine (C223A), and the amino acid The sequence is shown in SEQ ID No. 6, and the nucleotide sequence of the coding gene is shown in SEQ ID No. 5; (3) The 250th cysteine of the amino acid sequence shown in SEQ ID No.
  • the threonine at position 151 of the amino acid sequence shown in SEQ ID No. 2 is mutated to Valine, mutated cysteine at position 223 to alanine and cysteine at position 250 to glycine, the amino acid sequence is shown in SEQ ID No. 10, and the nucleotide sequence of the coding gene is SEQ ID Shown in No.9.
  • the present invention also provides an engineered bacteria containing the gene encoding the nitrilase mutant.
  • any suitable carrier can be used.
  • suitable vectors include, but are not limited to, prokaryotic expression vectors pET28, pET20, pGEX4T1, pTrC99A, and pBV220; include, but are not limited to, eukaryotic expression vectors pPIC9K, pPICZ ⁇ , pYD1, and pYES2/GS; include, but are not limited to, cloning vectors pUC18/19 pBluscript-SK.
  • the present invention also provides an application of the nitrilase mutant in the synthesis of anti-epileptic drug intermediates, specifically the nitrilase mutant catalyzes the preparation of 1-cyanocyclohexylacetonitrile from 1-cyanocyclohexylacetonitrile.
  • the final concentration of the substrate is 100-1200 mM (preferably 1000-1200 mM) based on the volume of the buffer, and the amount of the catalyst is 10-100 g/L buffer based on the weight of the wet bacteria, preferably 50 g/L.
  • wet bacterial cells are prepared as follows: inoculate the genetically engineered bacteria containing the nitrilase mutant encoding into LB medium, culture at 37°C for 10-12 hours, and inoculate the inoculum with a volume concentration of 2% until the end LB medium with a concentration of 50 mg/L kanamycin, culture at 37°C until the OD 600 of the medium is between 0.6-0.8, and add a final concentration of 0.1 mM isopropyl- ⁇ -D-thiogalactopyranoside , 28 °C induction culture for 10 hours, centrifugation, collect the bacteria, wash twice with normal saline, get wet bacteria.
  • the pure enzyme is prepared as follows: (1) Resuspend the wet bacteria in a pH 8.0, 50 mM NaH 2 PO 4 buffer containing a final concentration of 300 mM NaCl, and ultrasonically break (400W, 25min, 1s break, 1s pause) After the crushed product was centrifuged (12000rpm, 10min), the supernatant was taken as the crude enzyme solution; the ultrasonic crushing was performed at 400W for 25min, 1s crushed and 1s paused; (2) The crude enzyme solution was passed through the process at a flow rate of 1mL/min.
  • the Ni-NTA column washed with the equilibration buffer, the weakly adsorbed impurities are eluted with the elution buffer at a flow rate of 2 mL/min; then the protein elution buffer is used to elute and collect the target protein at a flow rate of 2 mL/min; finally The collected target protein was dialyzed with 50 mM disodium hydrogen phosphate-sodium dihydrogen phosphate buffer as the dialysate, and the retentate was taken as the pure enzyme; the balance buffer was pH 8.0, 50 mM NaH 2 with a final concentration of 300 mM NaCl.
  • the elution buffer is a pH 8.0, 50 mM NaH 2 PO 4 buffer containing a final concentration of 300 mM NaCl and 50 mM imidazole;
  • the protein elution buffer is a pH containing a final concentration of 300 mM NaCl and 250 mM imidazole 8.0, 50mM NaH 2 PO 4 buffer.
  • the nitrilase mutant is used as a catalyst to catalyze the synthesis of 1-cyanocyclohexylacetic acid
  • Raney nickel is used to perform chemical catalytic hydrogenation of 1-cyanocyclohexylacetic acid to synthesize gabapentin butyrolactam, and then gabapentin
  • the amide is hydrolyzed to generate gabapentin.
  • the nitrilase mutant specifically described in the present invention uses a semi-rational design method and a full-plasmid PCR technology to analyze SEQ ID No. 1 and is derived from Acidovorax facilis CCTCC NO:M 209044 nitrile.
  • the hydrolase-encoding gene strain E.coli BL21(DE3)/Pet28(+)-AcN-M was subjected to site-directed mutagenesis, and the positive mutants were screened and detected after inducing expression, and then mutants with further improved temperature stability were obtained.
  • the specific form of the nitrilase mutant described in the present invention as a catalyst may be a recombinant expression transformant containing the nitrilase mutant gene (i.e. wet bacteria, preferably E. coli BL21(DE3)), It can also be an unpurified crude enzyme, or a partially or completely purified enzyme, or it can be an immobilized enzyme prepared from the nitrilase mutant of the present invention using immobilization techniques known in the art. Or immobilized cells.
  • the nitrilase mutant gene i.e. wet bacteria, preferably E. coli BL21(DE3)
  • the nitrilase mutant gene i.e. wet bacteria, preferably E. coli BL21(DE3)
  • It can also be an unpurified crude enzyme, or a partially or completely purified enzyme, or it can be an immobilized enzyme prepared from the nitrilase mutant of the present invention using immobilization techniques known in the art. Or immobilized cells.
  • the final concentration composition of the LB liquid medium of the present invention 10g/L tryptone, 5g/L yeast extract, 10g/L sodium chloride, the solvent is water, and the pH value is natural.
  • the final concentration composition of LB solid medium 10g/L tryptone, 5g/L yeast extract, 10g/L sodium chloride, agar 20g/L, solvent is water, pH value is natural.
  • the beneficial effects of the present invention are mainly embodied in that the present invention carries out molecular modification of proteins through semi-rational design, and the thermal stability of the nitrilase mutant AcN-T151V/C223A/C250G is increased by 1.73 times, and The recombinant E. coli containing the nitrilase mutant was used to hydrolyze 1M 1-cyanocyclohexylacetonitrile at 35°C to generate 1-cyanocyclohexylacetic acid, and the final product yield reached 95%. When 1.2M 1-cyanocyclohexylacetonitrile was hydrolyzed at 35°C, the final yield reached 97%.
  • the nitrilase mutant of the present invention is used to synthesize gabapentin, and the final yield reaches 80%. Therefore, the mutant obtained by the present invention and its application lay a foundation for the high-efficiency chemical enzymatic synthesis of gabapentin.
  • FIG. 1 Electrophoresis diagram of nitrilase mutant protein after purification.
  • Lane 1 is AcN-M
  • lane 2 is AcN-T151V
  • lane 3 is AcN-C223A
  • lane 4 is AcN-C250G
  • lane 5 is AcN-T151V/C223A/C250G.
  • Figure 6 is a graph showing the comparison of the time for the transformation of 1M 1-cyanocyclohexylacetonitrile into recombinant E. coli resting cells containing the nitrilase mutant.
  • Figure 7 is a graph showing the comparison of the time of transforming 1.2M 1-cyanocyclohexylacetonitrile into recombinant E. coli resting cells containing the nitrilase mutant.
  • Figure 8 is a high performance liquid chromatogram of 1-cyanocyclohexylacetic acid.
  • the final concentration of the LB liquid medium consists of 10 g/L tryptone, 5 g/L yeast extract, 10 g/L sodium chloride, the solvent is water, and the pH value is natural.
  • the final concentration composition of LB solid medium 10g/L tryptone, 5g/L yeast extract, 10g/L sodium chloride, agar 20g/L, solvent is water, pH value is natural.
  • nitrilase gene AcN-M nucleotide sequence SEQ ID NO.1 cloned in CCTCC (A.facilis) CCTCC NO:M 209044, and the amino acid sequence of the encoded protein is shown in SEQ ID NO.2 ) PET-28b(+)-AcN-M plasmid as a template, calculate the sites that can improve thermal stability through http://kazlab.umn.edu/, and then perform site-directed mutagenesis of the whole plasmid (Table 1) PCR amplification .
  • PCR reaction system 50 ⁇ L: template 0.5-20ng, 2 ⁇ Phantamax Buffer 25 ⁇ L, 0.2mM dNTP, primers 0.2 ⁇ M each, PhantaMax Super-Fidelity DNA Polymerase 1 ⁇ L, add water to make up to 50 ⁇ L.
  • PCR conditions (1) 95°C pre-denaturation for 3 minutes; (2) 95°C denaturation for 15s; (3) 60°C annealing for 15s; (4) 72°C extension for 5.5 minutes, steps (2) to (4) total 30 cycles; (5) Extend at 72°C for 10 minutes and store at 16°C.
  • the PCR product was verified by agarose gel electrophoresis, digested with DpnI, and then introduced into E. coli BL21 (DE3), and spread on an LB plate containing 50 ⁇ g/mL kanamycin to obtain a single clone, and then sequenced. According to the sequencing results, further reaction verification is done.
  • thermal stability-enhancing mutants were T151V, C223A, and C250G.
  • the nucleotide sequences are shown in SEQ ID NO. 3, SEQ ID NO. 5 and SEQ ID NO. 7, respectively.
  • the same method was used to construct the combined mutant T151V/C223A/C250G, the nucleotide sequence of which is shown in SEQ ID NO.9.
  • mutants were respectively introduced into E. coli BL21(DE3) to construct mutants E. coli BL21(DE3)/pET28b(+)-AcN-T151V, E.coli BL21(DE3)/pET28b(+)-AcN-C223A , E.coli BL21(DE3)/pET28b(+)-AcN-C250G, and the combined mutant E.coli BL21(DE3)/pET28b(+)-AcN-T151V/C223A/C250G, and the original strain E.coli BL21 (DE3)/pET28b(+)-AcN-M.
  • the OD 600 is between 0.6-0.8, and isopropyl- ⁇ -D-thiogalactopyranoside (IPTG) is added to a final concentration of 0.1 mM, and induced and cultured at 28°C for 10 hours.
  • IPTG isopropyl- ⁇ -D-thiogalactopyranoside
  • the obtained crude enzyme solution is passed through the Ni-NTA column at a flow rate of 1 mL/min, and the target protein is mounted on the chromatography column. After loading the sample, a large amount of unadsorbed impurity protein will not be combined with the resin and will be removed directly.
  • the collected enzyme solution uses a dialysis bag (Economical Biotech Membrane, 14KD, 34mm Width, purchased from Shenggong Bioengineering (Shanghai) Co., Ltd.), and the dialysate is sodium dihydrogen phosphate-disodium hydrogen phosphate (50mM, pH 7.0) ) Buffer, the retentate is the purified protein, and the corresponding pure enzyme solution is obtained.
  • a dialysis bag Economical Biotech Membrane, 14KD, 34mm Width, purchased from Shenggong Bioengineering (Shanghai) Co., Ltd.
  • the dialysate is sodium dihydrogen phosphate-disodium hydrogen phosphate (50mM, pH 7.0)
  • Buffer the retentate is the purified protein, and the corresponding pure enzyme solution is obtained.
  • Nitrilase activity detection reaction system (10mL): sodium dihydrogen phosphate-disodium hydrogen phosphate (200mM, pH 7.0) buffer, 200mM 1-cyanocyclohexylacetonitrile, 0.4mg pure enzyme solution. After the reaction solution was preheated at 35°C for 10 minutes, it was reacted at 180 rpm for 10 minutes.
  • Example 5 Determination of temperature stability of nitrilase mutant at 50°C
  • Example 3 The thermal stability of the purified protein in Example 3 was measured. Take a certain amount of protein in a 50mL sterile polypropylene centrifuge tube and store it in a 50°C constant temperature water bath. The protein was taken out at different times, and the activity of the protein was measured according to the method in Example 4. Taking the protein activity when it is not stored as a control, calculate the relative residual activity (Residual activity, RA for short) of the protein at each time. With time (h) as the abscissa and the natural logarithm of the relative residual activity (Ln(RA)) as the ordinate, a linear fitting was performed (see Figure 3 for the result), and the slope k was obtained. According to the formula of first-order inactivity mechanics The half-life t 1 /2 of the enzyme protein can be obtained.
  • Example 6 Determination of the viability of recombinant E. coli containing nitrilase mutants
  • Nitrilase activity detection reaction system (10mL): sodium dihydrogen phosphate-disodium hydrogen phosphate (200mM, pH 7.0) buffer, final concentration 200mM 1-cyanocyclohexylacetonitrile, recombinant E. coli wet bacteria 10g/L. After the reaction solution was preheated at 35°C for 10 minutes, it was reacted at 180 rpm for 10 minutes. Sampling 200 ⁇ L of supernatant, using liquid chromatography (Shimadzu LC-16) external standard method to determine the conversion rate of 1-cyanocyclohexylacetic acid conversion solution. The liquid phase detection conditions are as described in Example 4. After testing, the recombinant E.
  • Example 7 Determination of temperature stability of recombinant Escherichia coli containing nitrilase mutant at 50°C
  • E.coli BL21(DE3)/pET28b(+)-AcN-T151V, E.coli BL21(DE3)/pET28b(+)-AcN- containing the nitrilase mutant obtained by cultivation in Example 2 C223A, E.coli BL21(DE3)/pET28b(+)-AcN-C250G, and the combined mutant E.coli BL21(DE3)/pET28b(+)-AcN-T151V/C223A/C250G, and the original strain E.coli BL21(DE3)/pET28b(+)-AcN-M is used to prepare a 100g/L bacterial suspension with sodium dihydrogen phosphate-disodium hydrogen phosphate (200mM, pH 7.0) buffer for resting cells, stored in a 50°C constant temperature water bath In the pot.
  • the bacterial suspension was taken out at different times, and the viability of resting cells was measured according to the method in Example 6. Taking the viability of resting cells when not stored as a control, the relative residual viability of resting cells at 50°C at each time was calculated, and the results are shown in Figure 5.
  • Example 10 Transformation of 1.2M 1-cyanocyclohexylacetonitrile using recombinant E. coli containing a nitrilase mutant
  • the mutant E.coli BL21(DE3)/pET28b(+)-AcN-T151V/C223A/C250G can complete the substrate reaction within 4h, which is much better than E.coli BL21(DE3).
  • )/pET28b(+)-AcN-M the results are shown in Figure 7.
  • the immobilized cells prepared from the original strain E.coli BL21(DE3)/pET28b(+)-AcN-M reacted for 7-8 hours per batch, and the immobilized cells were prepared from E.coli BL21(DE3)/pET28b(+)-AcN.
  • Example 12 Treatment of 1-cyanocyclohexyl acetic acid by flocculation
  • Example 11 Take 1.245 kg of the conversion solution in Example 11, add 1% polyaluminum chloride for 4 hours, then add 1% diatomaceous earth to adsorb for 2 hours, filter with Buchner funnel to obtain the filtrate, add a certain amount of hydrochloric acid to adjust the pH to about 2.0 Then add an equal volume of dichloromethane and stir in a three-necked flask for 20 minutes, then transfer to a separatory funnel, let stand for about 10 minutes for liquid separation, remove the lower layer, spin-evaporate, and dry in an oven, and finally obtain a solid 1-cyano group Cyclohexyl acetic acid 158g.
  • Example 12 Take 78.3 g of 1-cyanocyclohexyl acetic acid solid prepared in Example 12 and first dissolve it in water, adjust the pH to about 10 with sodium hydroxide solution, 1M concentration, and dilute to 470 mL, and add 20% Raney nickel catalyst. React at 110°C, 2.0MPa, 450rpm and hydrogenation conditions for about 4-5h. Filter while hot to obtain 582.5 g of hydroconversion liquid. Put the hydrogenation conversion liquid in a three-necked flask, adjust the pH to about neutral with hydrochloric acid, heat at 100°C and reflux for about 4 hours, then extract with dichloromethane, rotary steam, and dry, and finally obtain gabapentin butyrolactam solid 56.3 g. The yield of this step is about 81%.

Abstract

提供了一种腈水解酶突变体及其在制备抗癫痫药物中间体中的应用，所述突变体是将SEQ ID No.2所示氨基酸序列的第151位，第223位和第250位氨基酸中的一位或多位进行突变获得的。该腈水解酶突变体AcN-T151V/C223A/C250G的热稳定性提高了1.73倍，且利用含有腈水解酶突变体的重组大肠杆菌在35℃下水解1M 1-氰基环己基乙腈生成1-氰基环己基乙酸，最终产物得率达95%。在35℃下水解1.2M 1-氰基环己基乙腈时，最终产率达97%。使用该腈水解酶突变体合成加巴喷丁，最终产率达80%。

Description

一种腈水解酶突变体及其在制备抗癫痫药物中间体中的应用 (一)技术领域
本发明涉及一种来源于敏捷食酸菌(Acidovorax facilis)CCTCC NO:M 209044腈水解酶的突变体及其在抗癫痫药物中间体1-氰基环己基乙酸合成中的应用。
(二)背景技术
加巴喷丁是由美国Warner-Lambert公司首先开发的一种新型的抗癫痫药。与目前市场上同类药品相比，其优点有口服吸收快，毒副作用小，治疗效果明显以及耐受性强，在体内不易与血浆蛋白结合，不诱导肝酶，不代谢等特点，能够穿过人体大脑的血脑屏障，与其他抗癫痫药物相互作用的可能性小，作为难治性癫痫病的叠加用药作用效果尤为突出。
1-氰基环己基乙酸是合成抗癫痫药物加巴喷丁的关键中间体，目前在市场上应用前景广阔。目前工业上合成加巴喷丁及关键中间体1-氰基环己基乙酸全部采用化学合成法，生产过程中存在反应条件苛刻，环境污染严重，废弃物处理成本高等问题。
腈水解酶(Nitrilase EC 3.5.5.1)是一类能将腈类物质(含有-CN)直接水解为相应的羧酸的酶。腈水解酶的催化反应具有高立体选择性，催化速率高，反应条件温和以及对环境污染小等特点，是一种对环境友好的绿色合成方法，对节能减排和建设和谐社会具有重要的现实意义。目前工业上应用的腈水解酶案例很多，如德国BASF公司的产品(R)-扁桃酸，首先由苯甲醛和氢氰酸反应生成外消旋扁桃腈，再选择合适的反应条件，通过腈水解酶催化的动力学拆分，可以定量地转化为(R)-扁桃酸。亚甲基戊二腈首先用固定化的含腈水解酶的微生物细胞催化剂(Acidovorax facilis 72W)水解为4-氰基戊酸(4-CPA)铵盐，水解反应的选择性大于98％，转化率为100％，能得到二分之一氰基羧酸铵盐，产生1～2％的唯一反应副产物2-甲基戊二酸二铵盐。由此可得化学-酶法生产工艺相较于传统化学法相比产量提高、减少浪费、立体选择性高。此外，还有许多腈水解酶已经被开发并用于多种药物中间体和精细化学品的合成工艺中。
但是，天然的腈水解酶普遍不耐热，这个缺点阻碍了其在工业上的应用。提高酶的热稳定性可以通过对酶进行分子改造或半理性设计等方式实现。由于腈水解酶的晶体结构报道较少，关于腈水解酶的热稳定性改造也较少。Crum和Benedik等人多年 来研究来源于短小芽胞杆菌(Bacillus pumilus)的Cyanidedihydratase(CynD pum)的温度稳定性。研究者首先利用易错PCR，筛选出了多个正向突变株(K93R,D172N和E327K)，随后将施氏假单胞菌(Pseudomonas stutzeri)Cyanidedihydratase(CynD stu)的C端与CynD pum融合，提高了其温度稳定性(Frontiers in Microbiology 2016 Aug 12；7:1264.)。Xu等人通过易错PCR对AcN基因的随机诱变，最终得到三种突变体(AcN-T201L，AcN-Q339K，AcN-Q343K)表现出较高的热稳定性。将纯酶于45℃下保温，取样测酶活，计算其半衰期，发现多重突变体AcN-T201F/Q339K/Q343K半衰期从12.5h提高到180h(Enzyme and Microbial Technology 113(2018)52-58)。从敏捷食酸菌(Acidovorax facilis)CCTCC NO:M 209044克隆的腈水解酶已经在大肠杆菌(Escherichia coli)BL21(DE3)中过量表达，通过分子改造，对底物1-氰基环己基乙酸具有较高的催化活力，能够高效催化1-氰基环己基乙腈生成1-氰基环己基乙酸(Catalysis Communications,2015，66,121-125)。由于在温度较高的条件下底物1-氰基环己基乙腈的溶解度较高，能够促进催化反应，但是催化剂酶的热稳定性较差，在高温条件下催化活力较低，因此现有的腈水解酶不能满足要求，需要通过分子改造的方法来提高腈水解酶的热稳定性，从而提高催化效率，实现工业生产。
(三)发明内容
本发明目的是提供一种热稳定性提高的腈水解酶突变体蛋白及其在1-氰基环己基乙酸合成中的应用，包括含有该基因的重组载体，以及重组载体转化得到的重组基因工程菌，解决了来源于敏捷食酸菌(Acidovorax facilis)CCTCC NO:M 209044腈水解酶温度稳定性较差的问题。
本发明采用的技术方案是：
本发明提供一种热稳定性提高的腈水解酶突变体，所述突变体是将SEQ ID No.2所示氨基酸序列的第151位，第223位和第250位氨基酸中的一位或多位进行突变获得的。
进一步，优选所述腈水解酶突变体为下列之一：(1)将SEQ ID No.2所示氨基酸序列的第151位苏氨酸突变为缬氨酸(T151V)，氨基酸序列为SEQ ID No.4所示，编码基因核苷酸序列为SEQ ID No.3所示；(2)将SEQ ID No.2所示氨基酸序列第223位半胱氨酸突变为丙氨酸(C223A)，氨基酸序列为SEQ ID No.6所示，编码基因核苷酸序列为SEQ ID No.5所示；(3)将SEQ ID No.2所示氨基酸序列第250位半胱氨酸突变为甘氨酸(C250G)，氨基酸序列为SEQ ID No.8所示，编码基因核苷酸序列 为SEQ ID No.7所示；(4)将SEQ ID No.2所示氨基酸序列第151位的苏氨酸突变为缬氨酸，同时将第223位半胱氨酸突变为丙氨酸和第250位半胱氨酸突变为甘氨酸，氨基酸序列为SEQ ID No.10所示，编码基因核苷酸序列为SEQ ID No.9所示。
本发明又提供了一种包含所述腈水解酶突变体编码基因的工程菌。
在本发明腈水解酶突变体的制备方法中，可以采用任何适当的载体。例如，适当的载体包括但不限于原核表达载体pET28、pET20、pGEX4T1、pTrC99A和pBV220；包括但不限于真核表达载体pPIC9K、pPICZα、pYD1和pYES2/GS；包括但不限于克隆载体pUC18/19和pBluscript-SK。
本发明又提供一种所述腈水解酶突变体在合成抗癫痫药物中间体中的应用，具体为一种所述腈水解酶突变体在催化1-氰基环己基乙腈制备1-氰基环己基乙酸中的应用，所述的应用以含腈水解酶突变体编码基因工程菌经发酵培养获得的湿菌体、湿菌体固定化细胞或者湿菌体超声破碎后提取的纯酶为催化剂，以1-氰基环己基乙腈为底物，以pH＝7.0、200mM磷酸氢二钠-磷酸二氢钠缓冲液为反应介质构成反应体系，在25-50℃(优选35℃)恒温水浴中反应，反应完全后，将反应液分离纯化，获得1-氰基环己基乙酸。
所述底物终浓度以缓冲液体积计为100～1200mM(优选1000-1200mM)，所述催化剂用量以湿菌体重量计为10～100g/L缓冲液，优选50g/L。
进一步，所述湿菌体按如下方法制备：将含腈水解酶突变体编码基因工程菌接种到LB培养基中，37℃培养10-12小时，按体积浓度2％的接种量接种至含终浓度50mg/L卡那霉素的LB培养基，37℃培养至培养液的OD 600为0.6-0.8之间，加入终浓度为0.1mM异丙基-β-D-硫代吡喃半乳糖苷，28℃诱导培养10小时，离心，收集菌体，用生理盐水清洗2次，得到湿菌体。
进一步，所述纯酶按如下方法制备：(1)将湿菌体用含终浓度300mM NaCl的pH 8.0、50mM NaH 2PO 4缓冲液重悬，超声波破碎(400W，25min，1s破碎1s暂停)，破碎产物离心(12000rpm，10min)后，取上清液作为粗酶液；所述超声波破碎是在400W下25min，1s破碎1s暂停；(2)将粗酶液以1mL/min的流速通过经平衡缓冲液冲洗的Ni-NTA柱，用洗脱缓冲液洗脱弱吸附的杂蛋白，流速为2mL/min；再用蛋白洗脱缓冲液洗脱并收集目的蛋白，流速为2mL/min；最后将收集的目的蛋白以50mM磷酸氢二钠-磷酸二氢钠缓冲液为透析液进行透析，取截留液即为纯酶；所述平衡缓冲液为含终浓度300mM NaCl的pH 8.0、50mM NaH 2PO 4缓冲液；所述洗脱缓冲液为含终 浓度300mM NaCl和50mM咪唑的pH 8.0、50mM NaH 2PO 4缓冲液；所述蛋白洗脱缓冲液为含终浓度300mM NaCl和250mM咪唑的pH 8.0、50mM NaH 2PO 4缓冲液。
进一步，湿菌体固定化细胞按如下方法制备：将湿菌体悬浮于pH＝7.0、200mM磷酸氢二钠-磷酸二氢钠缓冲液体系中，加入终浓度6mg/mL硅藻土，室温搅拌1h，随后加入质量浓度5％聚乙烯亚胺水溶液，室温搅拌1h；最后加入质量浓度25％戊二醛水溶液，搅拌0.5h，真空抽滤，获得固定化细胞；所述聚乙烯亚胺水溶液体积加入量以缓冲液体积计为3％，所述戊二醛水溶液体积加入量以缓冲液体积计为1％。
本发明使用所述腈水解酶突变体作为催化剂进行催化合成1-氰基环己基乙酸，使用雷尼镍对1-氰基环己基乙酸进行化学催化加氢，合成加巴喷丁内酰胺，然后将加巴喷丁内酰胺水解，生成加巴喷丁。
本发明具体所述的腈水解酶突变体为利用半理性设计方法及全质粒PCR技术，通过分子手段对SEQ ID No.1所示来源于敏捷食酸菌(Acidovorax facilis)CCTCC NO:M 209044腈水解酶编码基因菌株E.coli BL21(DE3)/Pet28(+)-AcN-M进行定点突变，诱导表达后筛选检出正突变子，然后得到温度稳定性进一步提高的突变体，是能在更耐热环境下催化双腈化合物区域选择性水解合成单氰基羧酸化合物的突变体蛋白。
本发明具体所述的腈水解酶突变体作为催化剂的形式可以是含有所述腈水解酶突变体基因的重组表达转化体(即湿菌体，优选为大肠杆菌E.coli BL21(DE3))，也可以是未纯化的粗酶，也可以是经过经部分纯化的或完全纯化后的酶，也可以是利用本领域已知的固定化技术将本发明的腈水解酶突变体制成的固定化酶或者固定化细胞。
本发明所述LB液体培养基终浓度组成：10g/L胰蛋白胨，5g/L酵母提取物，10g/L氯化钠，溶剂为水，pH值自然。LB固体培养基终浓度组成：10g/L胰蛋白胨，5g/L酵母提取物，10g/L氯化钠，琼脂20g/L，溶剂为水，pH值自然。
与现有技术相比，本发明的有益效果主要体现在：本发明经过半理性设计对蛋白质进行分子改造，腈水解酶突变体AcN-T151V/C223A/C250G的热稳定性提高了1.73倍，且利用含有腈水解酶突变体的重组大肠杆菌在35℃下水解1M 1-氰基环己基乙腈生成1-氰基环己基乙酸，最终产物得率达95％。在35℃下水解1.2M 1-氰基环己基乙腈时，最终产率达97％。使用本发明腈水解酶突变体合成加巴喷丁，最终产率达80％。因此，本发明所获得的突变体及应用为加巴喷丁的高效化学酶法合成奠定了基础。
(四)附图说明
图1腈水解酶突变体蛋白纯化后的电泳图。泳道1为AcN-M，泳道2为AcN-T151V，泳道3为AcN-C223A，泳道4为AcN-C250G，泳道5为AcN-T151V/C223A/C250G。
图2腈水解酶突变体的活力比较。
图3腈水解酶突变体在50℃下的热稳定性。
图4含有腈水解酶突变体的重组大肠杆菌静息细胞的活力比较。
图5含有腈水解酶突变体的重组大肠杆菌静息细胞50℃下温度稳定性。
图6含有腈水解酶突变体的重组大肠杆菌静息细胞转化1M 1-氰基环己基乙腈时间比较曲线图。
图7含有腈水解酶突变体的重组大肠杆菌静息细胞转化1.2M 1-氰基环己基乙腈时间比较曲线图。
图8为1-氰基环己基乙酸高效液相色谱图。
(五)具体实施方式
下面结合具体实施例对本发明进行进一步描述，但本发明的保护范围并不仅限于此：
所述LB液体培养基终浓度组成：10g/L胰蛋白胨，5g/L酵母提取物，10g/L氯化钠，溶剂为水，pH值自然。
LB固体培养基终浓度组成：10g/L胰蛋白胨，5g/L酵母提取物，10g/L氯化钠，琼脂20g/L，溶剂为水，pH值自然。
实施例1：半理性设计及定点突变
以含有克隆于敏捷食酸菌(A.facilis)CCTCC NO:M 209044腈水解酶基因AcN-M(核苷酸序列SEQ ID NO.1所示，编码蛋白氨基酸序列为SEQ ID NO.2所示)的pET-28b(+)-AcN-M质粒为模版，通过http://kazlab.umn.edu/计算能够提高热稳定性的位点，再进行全质粒定点突变(表1)PCR扩增。PCR反应体系(50μL)：模版0.5～20ng，2×Phanta max Buffer 25μL，0.2mM dNTP，引物各0.2μM，Phanta Max Super-Fidelity DNA Polymerase 1μL，加水补至50μL。PCR条件：(1)95℃预变性3min；(2)95℃变性15s；(3)60℃退火15s；(4)72℃延伸5.5min，步骤(2)～(4)共30个循环；(5)最后72℃延伸10min，16℃保存。PCR产物经过琼脂糖凝胶电泳验证，利用DpnI消化后导入E.coli BL21(DE3)，涂布至含有50μg/mL卡那霉素的LB平板，得到单克隆，然后进行测序。根据测序结果，进一步做反应验证。
表1引物设计表
Figure PCTCN2020135583-appb-000001
利用液相色谱筛选验证，最终确定热稳定性提高突变体为T151V，C223A，C250G核苷酸序列分别为SEQ ID NO.3，SEQ ID NO.5和SEQ ID NO.7所示。并且，利用同样的方法，构建组合突变体T151V/C223A/C250G，其核苷酸序列如SEQ ID NO.9所示。
将上述突变体分别导入E.coli BL21(DE3)，构建突变体E.coli BL21(DE3)/pET28b(+)-AcN-T151V，E.coli BL21(DE3)/pET28b(+)-AcN-C223A，E.coli BL21(DE3)/pET28b(+)-AcN-C250G，和组合突变体E.coli BL21(DE3)/pET28b(+)-AcN-T151V/C223A/C250G，以及原始菌株E.coli BL21(DE3)/pET28b(+)-AcN-M。
实施例2：腈水解酶突变体的表达
实施例1中得到的突变体E.coli BL21(DE3)/pET28b(+)-AcN-T151V，E.coli BL21(DE3)/pET28b(+)-AcN-C223A，E.coli BL21(DE3)/pET28b(+)-AcN-C250G，和组合突变体E.coli BL21(DE3)/pET28b(+)-AcN-T151V/C223A/C250G，以及原始菌株E.coli BL21(DE3)/pET28b(+)-AcN-M接种到LB培养基中，37℃培养10-12小时，按体积接种量2％接种至含有卡那霉素(终浓度50mg/L)的LB培养基，37℃扩大培养至培养液的OD 600为0.6-0.8之间，加入异丙基-β-D-硫代吡喃半乳糖苷(IPTG)至终浓度为0.1mM，28℃诱导培养10小时。培养液离心收集菌体，用生理盐水清洗2次，得到相应的湿菌体。
实施例3：腈水解酶突变体蛋白的纯化
(1)向实施例2中收集到的湿菌体中加入平衡缓冲液(50mM NaH 2PO 4，300mM NaCl缓冲液，pH 8.0)重悬菌体后，超声波破碎(400W，25min，1s破碎1s暂停)。破碎产物离心(12000×g，10min)，取上清液作为粗酶液，准备分离纯化。
(2)预装好20mL Ni-NTA亲和层析柱后，使用平衡缓冲液(50mM NaH 2PO 4，300mM NaCl，pH 8.0)进行平衡，流速为2mL/min。
(3)清洗8-10个柱体积后将所得到的粗酶液以1mL/min的流速通过Ni-NTA柱，目的蛋白则挂载在层析柱上。上样后大量未吸附的杂蛋白不与树脂结合，将直接被除去。
(4)使用洗脱缓冲液(50mM NaH 2PO 4，300mM NaCl，50mM咪唑，pH 8.0)洗脱弱吸附的杂蛋白，流速为2mL/min。
(5)使用蛋白洗脱缓冲液(50mM NaH 2PO 4，300mM NaCl，250mM咪唑，pH 8.0)洗脱并收集目的蛋白，流速为2mL/min。
(6)收集的酶液使用透析袋(Economical Biotech Membrane，14KD，34mm Width，购自生工生物工程(上海)股份有限公司)，透析液为磷酸二氢钠-磷酸氢二钠(50mM，pH 7.0)缓冲液，取截留液即为纯化后的蛋白，获得相应的纯酶液。
(7)通过SDS-PAGE分析纯化后的蛋白，蛋白电泳结果如图1所示。
实施例4：腈水解酶活力的测定
对实施例3中纯化后的蛋白进行酶活的测定。腈水解酶活力检测反应体系(10mL)：磷酸二氢钠-磷酸氢二钠(200mM、pH 7.0)缓冲液，200mM 1-氰基环己基乙腈，0.4mg纯酶液。反应液于35℃预热10min后，180rpm反应10min。取样200μL上清，加入4μL 6M的HCl终止反应后，利用液相色谱(岛津LC-16)外标法测定转化液1-氰基环己基乙酸转化率，1-氰基环己基乙酸高效液相色谱图见图8所示。
色谱柱为
Figure PCTCN2020135583-appb-000002
column(250mm×4.6mm，5μm)，流动相为缓冲液(0.58g/L磷酸氢二铵，1.83g/L高氯酸钠，高氯酸调节pH为1.8，溶剂为去离子水)∶乙腈＝76∶24(v/v)，流速为1mL/min，紫外检测波长215nm，柱温40℃。
酶活定义(U)：在35℃、pH 7.0，200mM磷酸二氢钠-磷酸氢二钠缓冲液条件下，每分钟催化生成1μmol 1-氰基环己基乙酸所需要的酶量定义为1U。经检测，突变体AcN-T151V和突变体AcN-C223A的相对活力分别是原始腈水解酶AcN-M的1.17和1.31倍，突变体AcN-C250G和组合突变体AcN-T151V/C223A/C250G的初始活力与原始腈水解酶AcN-M相比仅有90.38％和84.71％，结果见图2所示。
实施例5：腈水解酶突变体50℃下温度稳定性的测定
对实施例3中纯化后的蛋白进行热稳定性的测定。取一定量的蛋白于50mL无菌聚丙烯离心管中，保存于50℃恒温水浴锅中。于不同时间取出蛋白，按照实施例4的方法，测定蛋白的活力。以未保存时，蛋白的活力为对照，计算各时间下，蛋白的相对残余活力(Residual activity，简称RA)。以时间(h)为横坐标，相对残余活力的自然对数(Ln(RA))为纵坐标，进行线性拟合(结果见图3)，得出斜率k。根据一级失活动力学的公式
Figure PCTCN2020135583-appb-000003
可以得到酶蛋白的半衰期t 1/2。
经测定，原始腈水解酶AcN-M的半衰期为13.6h，突变体AcN-T151V的半衰期为14h，突变体AcN-C223A的半衰期为14.2h，突变体AcN-C250G的半衰期为19.9h，组合突变体AcN-T151V/C223A/C250G的半衰期为23.6h，结果见表2所示。
表2腈水解酶突变体在50℃下的半衰期
Figure PCTCN2020135583-appb-000004
实施例6：含有腈水解酶突变体的重组大肠杆菌活力的测定
将实施例2中培养得到的含有腈水解酶突变体的重组大肠杆菌E.coli BL21(DE3)/pET28b(+)-AcN-T151V，E.coli BL21(DE3)/pET28b(+)-AcN-C223A，E.coli BL21(DE3)/pET28b(+)-AcN-C250G，和组合突变体E.coli BL21(DE3)/pET28b(+)-AcN-T151V/C223A/C250G，以及原始菌株E.coli BL21(DE3)/pET28b(+)-AcN-M进行活力的测定。腈水解酶活力检测反应体系(10mL)：磷酸二氢钠-磷酸氢二钠(200mM、pH 7.0)缓冲液，终浓度200mM 1-氰基环己基乙腈，重组大肠杆菌湿菌体10g/L。反应液于35℃预热10min后，180rpm反应10min。取样200μL上清，利用液相色谱(岛津LC-16)外标法测定转化液1-氰基环己基乙酸转化率。液相检测条件如实施例4中所述，经检测，含有腈水解酶突变体的重组大肠杆菌E.coli BL21(DE3)/pET28b(+)-AcN-T151V、E.coli BL21(DE3)/pET28b(+)-AcN-C223A和E.coli BL21(DE3)/pET28b(+)-AcN-T151V/C223A/C250G的相对活力分别是原始菌株E.coli  BL21(DE3)/pET28b(+)-AcN-M的1.02、1.32和1.54倍，而E.coli BL21(DE3)/pET28b(+)-AcN-C250G突变体的初始活力与原始菌株E.coli BL21(DE3)/pET28b(+)-AcN-M相比仅有86.9％，结果见图4所示。
实施例7：含有腈水解酶突变体的重组大肠杆菌50℃下温度稳定性的测定
将实施例2中培养得到的含有腈水解酶突变体的重组大肠杆菌E.coli BL21(DE3)/pET28b(+)-AcN-T151V，E.coli BL21(DE3)/pET28b(+)-AcN-C223A，E.coli BL21(DE3)/pET28b(+)-AcN-C250G，和组合突变体E.coli BL21(DE3)/pET28b(+)-AcN-T151V/C223A/C250G，以及原始菌株E.coli BL21(DE3)/pET28b(+)-AcN-M静息细胞用磷酸二氢钠-磷酸氢二钠(200mM、pH 7.0)缓冲液配置成100g/L的菌悬液，保存于50℃恒温水浴锅中。于不同时间取出菌悬液，按照实施例6的方法，测定静息细胞的活力。以未保存时，静息细胞的活力为对照，计算各时间下，静息细胞在50℃下的相对残余活力，结果见图5所示。
实施例9：使用含腈水解酶突变体的重组大肠杆菌转化1M 1-氰基环己基乙腈
分别称取0.5g实施例2方法获得的组合突变体E.coli BL21(DE3)/pET28b(+)-AcN-T151V/C223A/C250G，以及原始菌株E.coli BL21(DE3)/pET28b(+)-AcN-M的湿菌体悬浮于10mL磷酸氢二钠-磷酸二氢钠缓冲液体系中(200mM，pH＝7.0)，加入1.48g 1-氰基环己基乙腈(终浓度1M)，35℃恒温水浴反应。于不同时间取样，12000rpm离心，弃去沉淀，取上清用高效液相色谱分析产物浓度。高效液相色谱分析条件如实施例4中所述。
经测定，如图6所示，突变体E.coli BL21(DE3)/pET28b(+)-AcN-AcN-T151V/C223A/C250G在2h内几乎完全转化，转化速率远快于E.coli BL21(DE3)/pET28b(+)-AcN-M。
实施例10：使用含腈水解酶突变体的重组大肠杆菌转化1.2M 1-氰基环己基乙腈
分别称取0.5g实施例2方法获得的组合突变体E.coli BL21(DE3)/pET28b(+)-AcN-T151V/C223A/C250G，以及原始菌株E.coli BL21(DE3)/pET28b(+)-AcN-M的湿菌体悬浮于10mL磷酸氢二钠-磷酸二氢钠缓冲液体系中(200mM，pH＝7.0)，加入1.78g 1-氰基环己基乙腈(终浓度1.2M)，35℃恒温水浴反应。于不同时间取样，12000rpm离心，弃去沉淀，上清液用高效液相色谱分析产物浓度。高效液相色谱分析条件如实施例4中所述。
经测定，如表3所示，突变体E.coli  BL21(DE3)/pET28b(+)-AcN-T151V/C223A/C250G可在4h内将底物反应完全，远优于E.coli BL21(DE3)/pET28b(+)-AcN-M，结果见图7所示。
表3含有腈水解酶突变体的重组大肠杆菌静息细胞转化1.2M 1-氰基环己基乙腈
Figure PCTCN2020135583-appb-000005
实施例11：使用固定化细胞转化1M 1-氰基环己基乙腈
分别称取2g实施例2方法获得的组合突变体E.coli BL21(DE3)/pET28b(+)-AcN-T151V/C223A/C250G，以及原始菌株E.coli BL21(DE3)/pET28b(+)-AcN-M湿菌体悬浮于20mL磷酸氢二钠-磷酸二氢钠缓冲液体系中(200mM，pH＝7.0)，加入终浓度0.006g/mL硅藻土，室温搅拌1h。随后加入质量浓度5％聚乙烯亚胺水溶液，室温搅拌1h。最后加入质量浓度25％戊二醛水溶液，搅拌0.5h，真空抽滤，获得固定化细胞；其中聚乙烯亚胺水溶液体积加入量以缓冲液体积计为3％，戊二醛水溶液体积加入量以缓冲液体积计为1％。
取0.5g湿菌体所对应的固定化细胞悬浮于10mL磷酸氢二钠-磷酸二氢钠缓冲液体系中(200mM，pH＝7.0)，加入1.48g 1-氰基环己基乙腈(终浓度1M)，25℃恒温水浴反应。其中由原始菌株E.coli BL21(DE3)/pET28b(+)-AcN-M制得的固定化细胞每批次反应7-8小时，由E.coli BL21(DE3)/pET28b(+)-AcN-T151V/C223A/C250G制得的固定化细胞，每批反应时间4-6小时。每批反应结束后，进行真空抽滤固液分离，反应液用高效液相色谱分析产物浓度(见实施例4)，固定化细胞则投入下一批次反应。结果如表4所示。
表4使用固定化细胞转化1M 1-氰基环己基乙腈
Figure PCTCN2020135583-appb-000006
实施例12：用絮凝法处理1-氰基环己基乙酸
取实施例11中的转化液1.245kg加1％聚氯化铝絮凝4h，再加1％硅藻土吸附2h， 用布氏漏斗抽滤获得滤液，加入一定量的盐酸将pH调节到2.0左右，后加入等体积的二氯甲烷在三口烧瓶中搅拌20min，然后移入分液漏斗中，静置10min左右进行分液，取下层旋蒸，并在烘箱中烘干，最后得到固体1-氰基环己基乙酸158g。
实施例13：用化学法将1-氰基环己基乙酸合成加巴喷丁
取实施例12制备的1-氰基环己基乙酸固体78.3g先溶解在水里，用氢氧化钠溶液调节pH到10左右，1M浓度，定容至470mL，加入20％的雷尼镍催化剂。在110℃、2.0MPa、450rpm，加氢条件下反应约4-5h。趁热过滤，得到加氢转化液582.5g。将加氢转化液置于三口烧瓶中，经盐酸调节pH到中性左右，在100℃下加热高温回流4h左右，再用二氯甲烷萃取、旋蒸、烘干，最后得到加巴喷丁内酰胺固体56.3g。该步收率在81％左右。
将15.3g加巴喷丁内酰胺溶于50ml的HCl溶液，150rpm下加热回流4h，自然冷却至室温，并用二氯甲烷萃取未反应完全的加巴喷丁内酰胺，水相在0-4℃条件下冷却1h，之后过滤获得白色晶体，在40℃下烘干获得加巴喷丁盐酸盐。母液回收循环利用。36.4g加巴喷丁在40℃下溶于50ml水，之后加入12.5ml甲苯，并用200g/L的碳酸钠调节pH至7.0-7.5，搅拌30min，之后用甲醇或异丙醇重结晶获得加巴喷丁纯品，母液循环用于下一次结晶提纯，最终加巴喷丁产率达80％。

Claims (10)

  1. 一种腈水解酶突变体，其特征在于所述突变体是将SEQ ID No.2所示氨基酸序列的第151位，第223位和第250位氨基酸中的一位或多位进行突变获得的。
  2. 如权利要求1所述腈水解酶突变体，其特征在于所述突变体为下列之一：(1)将SEQ ID No.2所示氨基酸序列的第151位苏氨酸突变为缬氨酸；(2)将SEQ ID No.2所示氨基酸序列第223位半胱氨酸突变为丙氨酸；(3)将SEQ ID No.2所示氨基酸序列第250位半胱氨酸突变为甘氨酸；(4)将SEQ ID No.2所示氨基酸序列第151位的苏氨酸突变为缬氨酸，同时将第223位半胱氨酸突变为丙氨酸和第250位半胱氨酸突变为甘氨酸。
  3. 一种权利要求1所述腈水解酶突变体的编码基因。
  4. 一种权利要求1所述腈水解酶突变体在催化1-氰基环己基乙腈制备1-氰基环己基乙酸中的应用。
  5. 如权利要求4所述的应用，其特征在于所述的应用以含腈水解酶突变体编码基因工程菌经发酵培养获得的湿菌体、湿菌体固定化细胞或者湿菌体超声破碎后提取的纯酶为催化剂，以1-氰基环己基乙腈为底物，以pH＝7.0、200mM磷酸氢二钠-磷酸二氢钠缓冲液为反应介质构成反应体系，在25-50℃恒温水浴中反应，反应完全后，将反应液分离纯化，获得1-氰基环己基乙酸。
  6. 如权利要求5所述的应用，其特征在于所述底物终浓度以缓冲液体积计为100～1200mM，所述催化剂用量以湿菌体重量计为10～100g/L缓冲液。
  7. 如权利要求5所述的应用，其特征在于所述反应温度为35℃。
  8. 如权利要求5所述的应用，其特征在于所述湿菌体按如下方法制备：将含腈水解酶突变体编码基因工程菌接种到LB培养基中，37℃培养10-12小时，按体积浓度2％的接种量接种至含终浓度50mg/L卡那霉素的LB培养基，37℃培养至培养液的OD 600为0.6-0.8之间，加入终浓度为0.1mM异丙基-β-D-硫代吡喃半乳糖苷，28℃诱导培养10小时，离心，收集菌体，用生理盐水清洗2次，得到湿菌体。
  9. 如权利要求8所述的应用，其特征在于所述纯酶按如下方法制备：(1)将湿菌体用含终浓度300mM NaCl的pH 8.0、50mM NaH 2PO 4缓冲液重悬，超声波破碎(400W，25min，1s破碎1s暂停)，破碎产物离心(12000rpm，10min)后，取上清液作为粗酶液；所述超声波破碎是在400W下25min，1s破碎1s暂停；(2)将粗酶液以1mL/min的流速通过经平衡缓冲液冲洗的Ni-NTA柱，用洗脱缓冲液洗脱弱吸附的杂蛋白，流速为2mL/min；再用蛋白洗脱缓冲液洗脱并收集目的蛋白，流速为2mL/min；最后将收集的目的蛋白以50mM 磷酸氢二钠-磷酸二氢钠缓冲液为透析液进行透析，取截留液即为纯酶；所述平衡缓冲液为含终浓度300mM NaCl的pH 8.0、50mM NaH 2PO 4缓冲液；所述洗脱缓冲液为含终浓度300mM NaCl和50mM咪唑的pH 8.0、50mM NaH 2PO 4缓冲液；所述蛋白洗脱缓冲液为含终浓度300mM NaCl和250mM咪唑的pH 8.0、50mM NaH 2PO 4缓冲液。
  10. 如权利要求8所述的应用，其特征在于湿菌体固定化细胞按如下方法制备：将湿菌体悬浮于pH＝7.0、200mM磷酸氢二钠-磷酸二氢钠缓冲液体系中，加入终浓度6mg/mL硅藻土，室温搅拌1h，随后加入质量浓度5％聚乙烯亚胺水溶液，室温搅拌1h；最后加入质量浓度25％戊二醛水溶液，搅拌0.5h，真空抽滤，获得固定化细胞；所述聚乙烯亚胺水溶液体积加入量以缓冲液体积计为3％，所述戊二醛水溶液体积加入量以缓冲液体积计为1％。
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