WO2021169490A1 - 一种腈水解酶突变体及其在制备1-氰基环己基乙酸中的应用 - Google Patents

一种腈水解酶突变体及其在制备1-氰基环己基乙酸中的应用 Download PDF

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WO2021169490A1
WO2021169490A1 PCT/CN2020/135582 CN2020135582W WO2021169490A1 WO 2021169490 A1 WO2021169490 A1 WO 2021169490A1 CN 2020135582 W CN2020135582 W CN 2020135582W WO 2021169490 A1 WO2021169490 A1 WO 2021169490A1
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nitrilase
mutant
buffer
bacteria
final concentration
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薛亚平
熊能
李芊
郑裕国
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浙江工业大学
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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 preparing the anti-epileptic drug gabapentin.
  • Gabapentin is the first antiepileptic drug developed by Warner-Lamber in the United States, and it was first marketed in the UK in 1993. Gabapentin can prevent seizures induced by some chemicals (such as picrotoxin, bicuculine, strychnine) and non-chemical stimuli (such as sound source, electric shock), and it can prevent partial seizures and secondary generalized tonic clonus Sexual seizures are effective. Compared with similar products currently used, it has fast oral absorption, good tolerance, less toxic and side effects, and good therapeutic effect. It does not metabolize in the body, does not bind to plasma proteins, does not induce liver enzymes, and can pass through the blood of the human brain. The brain barrier is very unlikely to interact with other antiepileptic drugs, and it is particularly effective as a superimposed drug for refractory epilepsy.
  • some chemicals such as picrotoxin, bicuculine, strychnine
  • non-chemical stimuli such as sound source, electric shock
  • 1-Cyanocyclohexaneacetic acid (1-Cyanocyclohexaneacetic acid) is a key intermediate for the synthesis of a new generation of antiepileptic drug gabapentin, and the market prospect is very broad.
  • chemical synthesis technology is used to synthesize gabapentin and its key intermediate 1-cyanocyclohexyl acetic acid, and there are problems in the production process such as serious environmental pollution, serious corrosion to equipment, and high risk.
  • Nitrilase (Nitrilace EC 3.5.5.1), as an important industrial enzyme, can directly convert nitrile compounds into corresponding carboxylic acids and ammonia.
  • the nitrilase catalyzes the hydrolysis reaction of nitrile, avoiding the high temperature or strong acid and alkali conditions required in the chemical synthesis process, greatly reducing the generation of by-products and waste, reflecting high selectivity, high efficiency, and environmental economy. Meet the requirements of green chemistry.
  • the Swiss company Lonza was the first company to use nitrilase to catalyze the production of niacin.
  • 2-Cyanopyrazine is degraded into drug intermediates 5-hydroxypyridine-2-carboxylic acid and 5-hydroxypyrazine-2-carboxylic acid, respectively.
  • the reaction selectivity is strong, and the conversion rate is close to 100%, which is great compared with traditional chemical methods.
  • the advantages The Shanghai Pesticide Research Institute and Zhejiang Qianjiang Biochemical Co., Ltd.
  • a nitrilase high-activity genetically engineered strain E.coli BL21(DE3)-pETNY Nit d which can catalyze the conversion of hydroxyacetonitrile to glycolic acid, and the transformation of wild strains
  • the concentration of glycolic acid reached 11.6% in 72h, and when genetically engineered bacteria were used for transformation, the concentration of glycolic acid in 20h could reach 36%, and the catalytic efficiency was significantly improved.
  • the catalytic activity of nitrilase on the substrate can be improved.
  • Gong Jinsong et al. used the method of site-directed saturation mutagenesis to mutate the nitrilase derived from Pseudomonas putida (CGMCC3830), and screened out three types of N4OG, F50W, and Q207E that increase the catalytic activity of 3-cyanopyridine. Mutants, double mutants F50W/Q207E and triple mutants N40G/F50W/Q207E were also constructed on this basis. The catalytic activity is twice that of the wild type.
  • Liu Zhiqiang and others used the method of site-directed saturation mutagenesis to mutate the nitrilase derived from Acidovorax facilis nitrilase, and screened the best mutant F168V/T201N/S192F/M191T/F192S, and the wild-type nitrilase Compared with the best mutant F168V/T201N/S192F/M191T/F192S, the catalytic activity of the substrate iminodiacetonitrile increased by 136%.
  • the nitrilase cloned from Acidotrophic facilis has been overexpressed in Escherichia coli (E.coli BL21(DE3)), which can catalyze the production of gabapentin from 1-cyanocyclohexylacetonitrile Synthesis of intermediate 1-cyanocyclohexyl acetic acid (Catalysis Communications, 2015, 66, 121-125).
  • Escherichia coli Escherichia coli BL21(DE3)
  • gabapentin 1-cyanocyclohexylacetonitrile Synthesis of intermediate 1-cyanocyclohexyl acetic acid (Catalysis Communications, 2015, 66, 121-125).
  • Existing biocatalysts mainly exist in the form of immobilized cells and immobilized enzymes in the industrial application process.
  • Immobilized cells and immobilized enzymes have higher requirements for the activity of the starting nitrilase cells and nitrilase protein to compensate Loss of enzyme activity produced by immobilization.
  • the existing nitrilase enzymes need to be further modified to improve the catalytic efficiency and make them have higher industrial application value.
  • the present invention is based on the phenomenon that nitrilase derived from Acidovorax facilis CCTCC NO:M 029044 has subunit self-assembly, and this phenomenon is related to enzyme activity.
  • the present invention provides a nitrilase with increased enzyme activity
  • the mutant protein includes the coding gene of the mutant protein, the recombinant vector containing the gene, and the recombinant genetic engineering bacteria transformed by the recombinant vector, and its application in the catalytic synthesis of 1-cyanocyclohexylacetic acid, an intermediate of gabapentin.
  • the present invention provides a nitrilase mutant, which is obtained by mutating one or more of the 180th and 205th amino acids of the amino acid sequence shown in SEQ ID No. 2.
  • the mutant is preferably one of the following: (1) The glycine at position 180 of the amino acid sequence shown in SEQ ID No. 2 is mutated to aspartic acid (G180D), and the nucleotide sequence of the coding gene is SEQ ID No. 3, the amino acid sequence is shown in SEQ ID No. 4; (2) The glycine at position 180 of the amino acid sequence shown in SEQ ID No. 2 is mutated to phenylalanine (G180F), encoding the nucleotide sequence of the gene It is shown in SEQ ID No. 5, and the amino acid sequence is shown in SEQ ID No. 6; (3) Alanine at position 205 of the amino acid sequence shown in SEQ ID No.
  • SEQ ID No. 2 is mutated to cysteine (A205C),
  • the nucleotide sequence of the coding gene is shown in SEQ ID No. 7, and the amino acid sequence is shown in SEQ ID No. 8;
  • the glycine at position 180 of the amino acid sequence shown in SEQ ID No. 2 is mutated to aspartic acid
  • the 205th alanine is mutated to cysteine (G180D/A205C)
  • the nucleotide sequence of the coding gene is shown in SEQ ID No. 9 and the amino acid sequence is shown in SEQ ID No. 10.
  • the present invention also provides a coding gene of the nitrilase mutant, a recombinant vector constructed from the coding gene, and a recombinant genetically engineered bacteria obtained by transforming a host cell with the recombinant vector.
  • Said vectors include but are not limited to prokaryotic expression vector pET28b, eukaryotic expression vector (pPIC9K, pPICZ ⁇ , pYD1 and pYES2/GS), cloning vector pUC18/19 and pBluscript-SK.
  • the host cells include, but are not limited to, various conventional host cells in the art. In the present invention, Escherichia coli E. coli BL21 (DE3) is preferred.
  • the present invention also provides an application of the nitrilase mutant in catalyzing the preparation of 1-cyanocyclohexylacetic acid from 1-cyanocyclohexylacetonitrile, and the specific application is the genetic engineering of the nitrilase mutant
  • the reaction solution is separated and purified to obtain 1-cyanocyclohexyl acetic acid.
  • the final concentration of the substrate added is 5-1000 mM based on the volume of the reaction system, preferably 200 mM, the added amount of the pure enzyme is 0.1-3 mg/mL based on the volume of the reaction system, and the specific enzyme activity is 160-170 U/g (cell wet Weight); when the catalyst is wet bacteria or immobilized cells, the amount is 10-100 g/L buffer based on the weight of the wet bacteria, preferably 50 g/L.
  • the wet bacteria are prepared as follows: the genetically engineered bacteria containing the nitrilase mutant encoding is inoculated into LB medium, cultured at 37°C for 10-12 hours, and inoculated with a volume concentration of 1% inoculum until the final
  • the LB medium with a concentration of 50 mg/L kanamycin was cultured at 37°C until the OD 600 of the medium was 0.6-0.8, and the final concentration was 0.1 mM isopropyl- ⁇ -D-thiogalactopyranoside ( IPTG), induced culture at 28°C for 10 hours, centrifuged, collected the bacterial cells, washed twice with physiological saline to obtain wet bacterial cells.
  • IPTG isopropyl- ⁇ -D-thiogalactopyranoside
  • the pure enzyme is prepared as follows: the wet cells of the genetically engineered bacteria containing the nitrilase mutant encoding genetically engineered bacteria are resuspended in a pH 7.0, 100 mM NaH 2 PO 4 -Na 2 HPO 4 buffer, and ultrasonically broken (400W , 20min, 1s break, 1s pause), centrifuge the broken product (8000rpm, 15min) and take the supernatant as the crude enzyme solution; pass the crude enzyme solution through the Ni-NTA column washed with binding buffer at a flow rate of 1 mL/min, and use The balance buffer is used to elute the weakly adsorbed impurity protein at a flow rate of 2 mL/min; the elution buffer is then used to elute and collect the target protein at a flow rate of 2 mL/min; finally, the collected target protein is collected with a concentration of 20 mM hydrogen phosphate dibasic Sodium-sodium dihydrogen phosphate buffer is the
  • the catalyst of the present invention can be a recombinant expression transformant containing the nitrilase mutant gene (ie, wet bacteria, preferably E. coli BL21 (DE3)), or an unpurified crude enzyme solution, Or the purified enzyme can be used after immobilization if necessary.
  • the nitrilase mutant gene ie, wet bacteria, preferably E. coli BL21 (DE3)
  • unpurified crude enzyme solution ie, wet bacteria, preferably E. coli BL21 (DE3)
  • the purified enzyme can be used after immobilization if necessary.
  • 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 15g/L, solvent is water, pH value is natural.
  • the beneficial effects of the present invention are mainly embodied in that the specific enzyme activity of the nitrilase double mutant AcN-G180D/A205C is increased by up to 1.6 times after semi-rational design and molecular modification of proteins.
  • the conversion rate is >99%, and the recombinant E. coli containing the nitrilase mutant is used to hydrolyze 1-cyanocyclohexylacetonitrile at high temperature (50°C), and the reaction time is shortened to a quarter of the original. Therefore, the mutant obtained in the present invention has a good application prospect in the high-efficiency catalysis of 1-cyanocyclohexylacetonitrile to synthesize gabapentin intermediate 1-cyanocyclohexylacetic acid.
  • Figure 1 is a histogram of enzyme activity of different mutants.
  • Fig. 3 Comparison curve diagram of enzyme activity of nitrilase and its mutants.
  • Fig. 4 Optimal temperature curve of E.coli BL21(DE3)/pET-28b(+)-AcN-G180D/A205C double mutant of nitrilase.
  • Figure 5 The optimal pH curve of the nitrilase double mutant E. coli BL21(DE3)/pET-28b(+)-AcN-G180D/A205C.
  • the present invention uses the site-directed mutagenesis technology to perform site-directed mutation at position 168 of the nitrilase encoding gene (GenBank Accession no.: AHW42593.1) derived from A. facilis (A. facilis) CCTCC NO: M 029044, and the mutation becomes E.coli BL21(DE3)/pET-28b(+)-AcN-F168V (see Zhang X H, et al.
  • site-directed mutagenesis was carried out through whole plasmid amplification, PCR system (50 ⁇ L): template 0.5-20ng, primers G180-f and A205-f (see Table 1 for sequence) each 10-15pmol, 5 ⁇ Prime STAR Buffer (Mg 2+ plus), 0.2mM dNTP, 1.25U PrimeSTAR HS DNA Polymerase.
  • PCR conditions (1) pre-denaturation at 98°C for 3 minutes; (2) denaturation at 98°C for 10 seconds; (3) annealing at 55°C for 5 seconds; (4) extension at 72°C for 6.5 minutes; steps (2)-(4) total 30 cycles; (5) Finally, extend at 72°C for 10 minutes, and store at 4°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. After site-directed mutagenesis, 12 site-directed mutagenesis transformants were obtained at each of the two sites.
  • mutant transformant G180D nucleotide sequence SEQ ID No. 3
  • site-directed mutation was performed through whole plasmid amplification, and the PCR system was the same as single mutation.
  • 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 double mutant transformant.
  • the combined mutant E. coli BL21(DE3)/pET-28b(+)-AcN-G180D/A205C was obtained.
  • the plasmid pET-28b(+)-AcN-F168V containing Acidovorax facilis CCTCC NO:M 029044 nitrilase AcN-F168V (ie SEQ ID No. 1) was constructed.
  • the constructed pET-28b(+)-AcN-F168V expression plasmid was introduced into E. Coli BL21(DE3) to achieve overexpression.
  • the site-directed saturation mutagenesis method was used for site-directed mutagenesis and recombined into the expression vector pET-28b(+), and then the recombinant plasmid was transferred into the expression host E.Coli BL21(DE3) to construct a mutant.
  • Example 1 E.coli BL21(DE3)/pET-28b(+)-AcN-G180F, E.coli BL21(DE3)/pET-28b(+)-AcN-G180D, E.coli BL21(DE3)/pET-28b(+)-AcN-G180D, E.coli BL21(DE3)/pET-28b(+)-AcN-A205C and the combined mutant E.coli BL21(DE3)/pET-28b(+)-AcN-G180D/A205C, and the original strain E.coli BL21(DE3) /pET-28b(+)-AcN-F168V (see Zhang XH, et al.
  • the culture broth is centrifuged to collect the bacteria , Washed twice with normal saline to obtain the corresponding wet bacteria.
  • the wet bacteria are immobilized to obtain immobilized cells (see patent CN107177576A for the immobilization method).
  • the wet bacteria are purified and extracted into pure enzymes after ultrasonic disruption (see the purification process) Example 3).
  • binding buffer 50mM NaH 2 PO 4 , 300mM NaCl, pH 8.0
  • resuspend the bacteria and ultrasonically disrupt them 400W, 20min, 1s, 1s pause
  • the crushed product was centrifuged (8000 rpm, 15 min)
  • the supernatant was taken as the crude enzyme solution for separation and purification.
  • binding buffer 50 mM NaH 2 PO 4 , 300 mM NaCl, pH 8.0
  • 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 the sample is loaded, a large amount of unadsorbed contaminant protein will not be combined with the resin and will be removed directly.
  • the collected target protein is dialyzed with 20 mM disodium hydrogen phosphate-sodium dihydrogen phosphate buffer (the molecular weight of the cut-off protein is 30KDa), and the cut-off solution is the purified protein.
  • the relative enzyme activity results of each mutant are shown in Figure 3.
  • the kinetic parameters of the purified protein in Example 3 were determined, using 1-cyanocyclohexylacetonitrile as the substrate, and the pure enzyme solutions of AcN-F168V, G180D, A205C, G180F, and G180D/A205C as the catalyst.
  • the concentration of 1-cyanocyclohexylacetic acid in the reaction solution was detected by HPLC (the detection and analysis conditions were the same as in Example 4).
  • the K m and K cat values of E.coli BL21(DE3)/pET-28b(+)-AcN-G180D/A205C are shown in Table 2. It can be found that the K cat of the double mutant has a significant increase compared with AcN This shows that the activity of the modified nitrilase is indeed increased, and their Km reflects a slight decrease in the affinity of the modified enzyme to the substrate.
  • the optimal temperature of the purified protein in Example 3 was determined, using 1-cyanocyclohexylacetonitrile as the substrate, and the nitrilase AcN-F168V (specific enzyme activity 104U/g cell wet weight) or nitrile hydrolysis
  • the pure enzyme solution of the enzyme combination mutant G180D/A205C (specific enzyme activity of 165U/g cell wet weight) is the catalyst.
  • Example 7 Determination of the optimum pH of nitrilase and its mutants
  • the optimal temperature of the purified protein in Example 3 was determined, using 1-cyanocyclohexylacetonitrile as the substrate, and the nitrilase AcN-F168V (specific enzyme activity 104U/g cell wet weight) or nitrile hydrolysis
  • the pure enzyme solution of the enzyme combination mutant G180D/A205C (specific enzyme activity of 165U/g wet cell weight) is the catalyst.
  • the concentration of 1-cyanocyclohexylacetic acid in the reaction solution is detected by HPLC.
  • the optimal pH of the double mutant is 8.5.
  • the result is shown in Figure 5. Shown. The optimum pH for AcN-F168V is 7 under the same conditions.
  • Example 8 Nitrilase and its mutants transform 200mM cyanocyclohexylacetonitrile
  • the reaction process of the purified nitrilase and its mutant protein in Example 3 was measured, using 1-cyanocyclohexylacetonitrile as the substrate and the nitrilase AcN-F168V (specific enzyme activity of 104U/g cell Wet weight) or the pure enzyme solution of the nitrilase combination mutant G180D/A205C (specific enzyme activity of 165U/g cell wet weight) is the catalyst.
  • the concentration of 1-cyanocyclohexylacetic acid in the reaction solution was detected by HPLC.

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Abstract

提供一种腈水解酶突变体及其在制备1-氰基环己基乙酸中的应用，所述突变体是将SEQ ID No.2所示氨基酸序列的第180位，第205位氨基酸中的一位或多位进行突变获得的。经过半理性设计，对蛋白质进行分子改造，腈水解酶双突变体AcN-G180D/A205C的比酶活最高提高了1.6倍，转化率>99％，且利用含有腈水解酶突变体的重组大肠杆菌在高温下(50℃)水解1-氰基环己基乙腈，反应时间缩短为原来的四分之一。因此，所获得的突变体在高效催化1-氰基环己基乙腈合成加巴喷丁中间体1-氰基环己基乙酸中具有良好的应用前景。

Description

一种腈水解酶突变体及其在制备1-氰基环己基乙酸中的应用 (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制备抗癫痫药物加巴喷丁中的应用。
(二)背景技术
加巴喷丁是美国Warner-Lamber公司首先开发的抗癫痫药，于1993年首次在英国上市。加巴喷丁能防止由部分化学品(如印防己毒素、荷包牡丹碱、士的宁)和非化学品刺激(如音源、电击休克)诱发的惊厥，对部分性癫痫发作和继发全身性强直阵挛性癫痫发作有效果。与目前使用的同类产品相比，具有口服吸收快，耐受性好，毒副作用小，治疗效果好，在体内不代谢，不与血浆蛋白结合，不诱导肝酶，能够穿过人体大脑的血脑屏障，与其他抗癫痫病药物相互作用的可能性很低，作为难治性癫痫病的叠加用药作用尤为突出。
1-氰基环己基乙酸(1-Cyanocyclohexaneacetic acid)是合成新一代抗癫痫药物加巴喷丁的关键中间体，市场前景非常广阔。目前，用于合成加巴喷丁及其关键中间体1-氰基环己基乙酸采用化学合成技术，生产过程中存在环境污染严重，对设备腐蚀严重，危险性大等问题。
腈水解酶(Nitrilace EC 3.5.5.1)作为一种重要的工业酶，能够直接将腈化物转化为相应的羧酸和氨。腈水解酶催化腈的水解反应，避免了化学合成过程中所需要的高温或强酸强碱等条件，极大减少了副产物和废物的产生，体现出高选择性、高效率以及环境经济性，符合绿色化学的要求。目前腈水解酶应用于合成医药中间体的例子很多，瑞士Lonza公司最早采用腈水解酶催化生产烟酸，还利用腈水解酶和烟酰胺脱氢酶共同作用，将底物2-氰基吡啶与2-氰基吡嗪分别降解为药物中间体5-羟基吡啶-2-甲酸与5-羟基吡嗪-2-甲酸，反应选择性强，转化率接近100％，相比传统化学法有极大的优势。上海市农药研究所与浙江钱江生物化学股份有限公司合作构建了一株腈水解酶高活性基因工程菌E.coli BL21(DE3)-pETNY Nit d能够催化羟基乙腈转化为乙醇酸，野生菌株转化72h乙醇酸浓度达到11.6％,而采用基因工程菌进行转化,20h乙醇酸浓度即可达到36％,催化效率显著提升。Banerjee等将恶臭假单胞菌(P.putida)MTCC 5110腈水解酶基因在大肠杆菌中进行了重组表达,并对产酶条件进行了系统优 化,重组酶对扁桃腈表现出较高的腈水解酶活力,最终转化结果表明(R)-扁桃酸的收率及ee值分别达到87％和99.99％。Chauhan等扩增获得一株敏捷食酸菌(Acidovorax facilis)72W的腈水解酶编码基因,并在大肠杆菌中进行了过量表达,重组酶对脂肪族二腈具有较强的立体选择性,能将2-甲基戊二腈转化为4-氰基戊酸,底物转化率达到100％,产物中无酰胺化合物生成,2-甲基戊二酸是唯一的副产物且含量低于2％。此外，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。
通过分子改造，可以提高腈水解酶对底物的催化活性，目前关于通过分子改造来提高腈水解酶活性的研究很多。龚劲松等人利用定点饱和突变的方法对来源于恶臭假单胞菌(Pseudomonas putida CGMCC3830)的腈水解酶进行突变，筛选出N4OG、F50W、Q207E三种对3-氰基砒啶催化活性提高的突变体，在此基础上还构建了双突变体F50W/Q207E和三突变体N40G/F50W/Q207E，催化活性是野生型的两倍。柳志强等人利用定点饱和突变的方法对来源于敏捷食酸菌(Acidovorax facilis nitrilase)的腈水解酶进行突变，筛选出最佳突变体F168V/T201N/S192F/M191T/F192S，与野生型腈水解酶相比，最佳突变体F168V/T201N/S192F/M191T/F192S对底物亚氨基二乙腈的催化活性提高了136％。
从敏捷食酸菌(A.facilis CCTCC NO:M 029044)中克隆的腈水解酶已经在大肠杆菌(E.coli BL21(DE3))中过量表达，其能够催化1-氰基环己基乙腈生成加巴喷丁合成中间体1-氰基环己基乙酸(Catalysis Communications,2015,66,121-125)。现有的生物催化剂在工业应用过程中主要以固定化细胞和固定化酶的形式存在，固定化细胞和固定化酶对出发的腈水解酶细胞和腈水解酶蛋白的活性要求较高，以弥补固定化产生的酶活损失。现有的腈水解酶还需要进一步的改造，提高催化效率，使其具有更高的工业应用价值。
(三)发明内容
本发明基于来源于敏捷食酸菌(Acidovorax facilis)CCTCC NO:M 029044腈水解酶具有亚基自组装的现象，并且该现象与酶活有关，本发明提供了一种酶活提高的腈水解酶突变体蛋白，包括该突变体蛋白的编码基因，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，及其在催化合成加巴喷丁中间体1-氰基环己基乙酸中的应用。
本发明采用的技术方案是：
本发明提供一种腈水解酶突变体，所述突变体是将SEQ ID No.2所示氨基酸序列 的第180位，第205位氨基酸中的一位或多位进行突变获得的。
进一步，优选所述突变体为下列之一：(1)将SEQ ID No.2所示氨基酸序列的第180位甘氨酸突变为天冬氨酸(G180D)，编码基因核苷酸序列为SEQ ID No.3所示，氨基酸序列为SEQ ID No.4所示；(2)将SEQ ID No.2所示氨基酸序列的第180位甘氨酸突变为苯丙氨酸(G180F)，编码基因核苷酸序列为SEQ ID No.5所示，氨基酸序列为SEQ ID No.6所示；(3)将SEQ ID No.2所示氨基酸序列的第205位丙氨酸突变为半胱氨酸(A205C)，编码基因核苷酸序列为SEQ ID No.7所示，氨基酸序列为SEQ ID No.8所示；(4)将SEQ ID No.2所示氨基酸序列的第180位甘氨酸突变为天冬氨酸，同时将第205位丙氨酸突变为半胱氨酸(G180D/A205C)，编码基因核苷酸序列为SEQ ID No.9所示，氨基酸序列为SEQ ID No.10所示。
本发明又提供了一种一种所述腈水解酶突变体的编码基因、由所述编码基因构建的重组载体以及由所述重组载体转化宿主细胞获得的重组基因工程菌。所述的载体包括但不限于原核表达载体pET28b、真核表达载体(pPIC9K、pPICZα、pYD1和pYES2/GS)、克隆载体pUC18/19和pBluscript-SK。所述的宿主细胞包括但不限于本领域的各种常规宿主细胞，本发明优选大肠杆菌E.Coli BL21(DE3)。
本发明又提供了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，具体所述的应用以含腈水解酶突变体的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为4.0-10.5、200M磷酸缓冲液为反应介质构成反应体系，在20-60℃、600rpm恒温水浴条件下反应，反应完全后，将反应液分离纯化，获得1-氰基环己基乙酸。所述底物加入终浓度以反应体系体积计为5-1000mM，优选200mM，所述纯酶加入量以反应体系体积计为0.1-3mg/mL，比酶活为160～170U/g(细胞湿重)；所述催化剂为湿菌体或固定化细胞时，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1％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(IPTG)，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：将含腈水解酶突变体编码基因工程菌湿菌体 用含pH 7.0、100mM的NaH 2PO 4-Na 2HPO 4缓冲液重悬，超声波破碎(400W，20min，1s破碎1s暂停)，破碎产物离心(8000rpm，15min)后取上清液作为粗酶液；将粗酶液以1mL/min的流速通过经结合缓冲液冲洗的Ni-NTA柱，用平衡缓冲液洗脱弱吸附的杂蛋白，流速为2mL/min；再用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浓度为20mM的磷酸氢二钠-磷酸二氢钠缓冲液为透析液进行透析(透析袋截留分子量为30KDa)，取截留液即为纯酶液；所述结合缓冲液为含终浓度300mM NaCl的pH 8.0、50mM NaH 2PO 4缓冲液，所述平衡缓冲液为含终浓度300mM NaCl和50mM咪唑的pH 8.0、50mM NaH 2PO 4缓冲液，所述洗脱缓冲液为含终浓度300mM NaCl和500mM咪唑的pH 8.0、50mM NaH 2PO 4缓冲液。
本发明所述催化剂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液，或是经过纯化后的纯酶，如果需要可以对其进行固定化后进行使用。
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15g/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，腈水解酶双突变体AcN-G180D/A205C的比酶活最高提高了1.6倍，转化率>99％，且利用含有腈水解酶突变体的重组大肠杆菌在高温下(50℃)水解1-氰基环己基乙腈，反应时间缩短为原来的四分之一。因此，本发明所获得的突变体在高效催化1-氰基环己基乙腈合成加巴喷丁中间体1-氰基环己基乙酸中具有良好的应用前景。
(四)附图说明
图1为不同突变体酶活柱形图。
图2腈水解酶E.coli BL21(DE3)/pET-28b(+)-AcN及其突变转化子E.coli BL21(DE3)/pET-28b(+)-AcN-G180D，E.coli BL21(DE3)/pET-28b(+)-AcN-G180F，E.coli BL21(DE3)/pET-28b(+)-AcN-A205C以及双突变体E.coli BL21(DE3)/pET-28b(+)-AcN-G180D/A205C的SDS-PAGE图，泳道1为AcN粗酶液，泳道2为G180D/A205C粗酶液，泳道3为AcN纯酶液，泳道4为G180D/A205C纯酶液，泳道5为G180D粗酶液，泳道6为G180F粗酶液，泳道7为A205C粗酶液，泳道8为G180D纯酶液，泳道9为G180F纯酶液，泳道10为A205C纯酶液。
图3腈水解酶及其突变体的酶活比较曲线图。
图4腈水解酶双突变体E.coli BL21(DE3)/pET-28b(+)-AcN-G180D/A205C的最适温度曲线图。
图5腈水解酶双突变体E.coli BL21(DE3)/pET-28b(+)-AcN-G180D/A205C的最适pH曲线图。
图6腈水解酶AcN-F168V及其双突变体的反应进程。
(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：
实施例1：定点突变及筛选
1、突变位点选择
本发明利用定点突变技术对来源于敏捷食酸菌(A.facilis)CCTCC NO:M 029044的腈水解酶编码基因(GenBank Accession no.:AHW42593.1)进行168位点的定点突变，突变成E.coli BL21(DE3)/pET-28b(+)-AcN-F168V(参见Zhang X H,et al.Activity improvement of a regioselective nitrilase from Acidovorax facilis and its application in the production of 1-(cyanocyclohexyl)acetic acid[J].Process Biochemistry,2014.)，以此为基础，主要针对的是“A surface”上的氨基酸位点为突变位点，利用全质粒PCR定点突变成功后，将目的基因所在的表达载体转入大肠杆菌宿主，诱导表达后通过酶活检测方法筛选出正突变子，进行酶活的重复检测，确定酶活提高的突变体，从而获得有自组装倾向并能够高效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。
2、单突变
以含有克隆于敏捷食酸菌(A.facilis)CCTCC NO:M 029044腈水解酶基因AcN-F168V(核苷酸序列SEQ ID No.1，氨基酸序列为SEQ ID No.2)的pET-28b(+)-AcN-F168V质粒为模板，通过全质粒扩增进行定点突变，PCR体系(50μL)为：模板0.5-20ng，引物G180-f及A205-f(序列见表1)各10-15pmol，5×Prime STAR Buffer(Mg 2+plus)，0.2mM dNTP，1.25U PrimeSTAR HS DNA Polymerase。PCR条件:(1)98℃预变性3min；(2)98℃变性10s；(3)55℃退火5s；(4)72℃延伸6.5min；步骤(2)-(4)共30个循环；(5)最后72℃延伸10min，4℃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。经过定点突变，得到两个位点各12种定点突变转化子，对获得的共23种单突变体进行酶活的检测，活力测定方法同实施例4，测定的酶活结果见图1，最终筛选获得酶活提高的突变体转化子E.coli BL21(DE3)/pET-28b(+)-AcN-G180F(记为G180F)，E.coli BL21(DE3)/pET-28b(+)-AcN-G180D(记为G180D)，E.coli BL21(DE3)/pET-28b(+)-AcN-A205C(记为A205C)。
表1突变位点的引物设计
Figure PCTCN2020135582-appb-000001
3、组合突变
以突变体转化子G180D(核苷酸序列SEQ ID No.3)的pET-28b(+)-AcN-G180D质粒为模板，通过全质粒扩增进行定点突变，PCR体系同单突变。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，得到双突变转化子。即获得组合突变体E.coli BL21(DE3)/pET-28b(+)-AcN-G180D/A205C(记为G180D/A205C)。
实施例2：腈水解酶突变体的表达
构建含敏捷食酸菌(Acidovorax facilis)CCTCC NO:M 029044腈水解酶AcN-F168V(即SEQ ID No.1)的质粒pET-28b(+)-AcN-F168V。将构建获得的pET-28b(+)-AcN-F168V表达质粒导入大肠杆菌E.Coli BL21(DE3)中实现过表达。采用定点饱和突变方法进行定点突变，并重组至表达载体pET-28b(+)，再将重组质粒转入表达宿主E.Coli BL21(DE3)中，构建突变体。得到实施例1的突变体转化子E.coli BL21(DE3)/pET-28b(+)-AcN-G180F，E.coli BL21(DE3)/pET-28b(+)-AcN-G180D，E.coli BL21(DE3)/pET-28b(+)-AcN-A205C和组合突变体E.coli BL21(DE3)/pET-28b(+)-AcN-G180D/A205C，以及原始菌株E.coli BL21(DE3)/pET-28b(+)-AcN-F168V(参见Zhang X H,et al.Activity improvement of a regioselective nitrilase from Acidovorax facilis and its application in the production of 1-(cyanocyclohexyl)acetic acid[J].Process Biochemistry,2014.)接种到LB培养基中，37℃培养10-12小时，按体积接种量1％接种至含有卡那霉素(终浓度50mg/L)的LB培养基，37℃、150rpm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，湿菌体进行固定化获得固定化细胞(固定化方法见专利CN107177576A)，湿菌体经过超声破碎后纯化提取为纯酶(纯化过程见实施例3)。
实施例3：腈水解酶及其突变体蛋白的纯化
(1)向实施例2中收集到的湿菌体中加入结合缓冲液(50mM NaH 2PO 4，300mM NaCl，pH 8.0)，重悬菌体后超声波破碎(400W，20min，1s破碎1s暂停)。破碎产物离心(8000rpm，15min)后，取上清液作为粗酶液，准备分离纯化。
(2)预装好10mL Ni-NTA亲和层析柱后，使用结合缓冲液(50mM NaH 2PO 4，300mM NaCl，pH 8.0)进行冲洗，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)使用平衡缓冲液(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，500mM咪唑，pH 8.0)洗脱并收集目的蛋白，流速为2mL/min。
(6)收集的目的蛋白以浓度为20mM的磷酸氢二钠-磷酸二氢钠缓冲液为透析液进行透析(截留蛋白分子量为30KDa)，取截留液即为纯化后的蛋白。
(7)通过SDS-PAGE分析纯化后的蛋白，蛋白电泳结果如图2所示。
实施例4腈水解酶活性的测定
对实施例3中纯化后的蛋白进行酶活的测定。腈水解酶活性检测反应体系(10mL):100mM、pH＝7.0磷酸氢二钠-磷酸二氢钠缓冲液，200mM 1-氰基环己基乙腈，30mg纯酶。反应液于45℃预热10min后，150rpm反应10min。取样500μL上清，加入500μL 2M的HCl终止反应后，利用液相色谱(Agilent)外标法测定转化液1-氰基环己基乙酸转化率。色谱柱为J&K Scientific C18-H柱(4.6×250mm,5μm,
Figure PCTCN2020135582-appb-000002
)，流动相为缓冲液(0.58g/L磷酸氢二铵，1.8375g/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℃。各个突变体的相对酶活结果见图3。
酶活定义(U):在45℃，pH 7.0、100mM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。
实施例5：腈水解酶及其突变体的动力学参数测定
对实施例3中纯化后的蛋白进行动力学参数测定，以1-氰基环己基乙腈为底物，分别以AcN-F168V、G180D、A205C、G180F、G180D/A205C的纯酶液为催化剂。
反应体系为10mL：纯酶液(165U/g)用pH 7.0、20mM磷酸盐缓冲液稀释10倍后加入反应容器，使其加入终浓度为0.2mg/mL，分别加入终浓度6.75-40.49mM(6.75、13.50、20.24、26.99、33.74、40.49mM)底物，用200mM的pH＝7的磷酸缓冲液做为反应介质补足到10mL，45℃、600rpm反应5min，取样500μL，并用500μL 2M的盐酸终止反应，用HPLC(检测分析条件同实施例4)对反应液中1-氰基环己基乙酸的浓度进行检测。
通过Origin对所获得的数据进行非线性拟合得出的腈水解酶E.coli BL21(DE3)/pET-28b(+)-AcN-F168V和腈水解酶组合突变体E.coli BL21(DE3)/pET-28b(+)-AcN-G180D、E.coli BL21(DE3)/pET-28b(+)-AcN-G180F、E.coli BL21(DE3)/pET-28b(+)-AcN-A205C、E.coli BL21(DE3)/pET-28b(+)-AcN-G180D/A205C的K m和K cat的值见表2所示，可以发现双突变体的K cat与AcN相比有明显的提高，这说明改造后的腈水解酶活性确实提高了，而它们的Km反映出改造后的酶对底物的亲和力有微弱的下降。
表2腈水解酶突变体的动力学参数
Enzyme K m[mM] V max[mmolmg -1min -1] K cat[s -1] K cat/K m[mM -1h -1]
AcN-F168V 16.25±5.37 1.53±0.19 5573s -1 342.95
G180D 3.21±1.41 1.98±0.13 6624s -1 2063.55
G180F 5.88±1.58 2.35±0.14 7612s -1 1294.56
A205C 3.40±0.78 1.63±0.059 8317s -1 2446.18
G180D/A205C 19.65±7.40 4.78±0.73 24139s -1 1228.45
实施例6：腈水解酶及其突变体最适温度的测定
对实施例3中纯化后的蛋白进行最适温度的测定，以1-氰基环己基乙腈为底物，以腈水解酶AcN-F168V(比酶活为104U/g细胞湿重)或腈水解酶组合突变体G180D/A205C的纯酶液(比酶活为165U/g细胞湿重)为催化剂。
反应体系为10mL：收集的纯酶液(165U/g)用pH 7.0、20mM磷酸盐缓冲液稀释10倍后加入反应容器，使其加入终浓度为0.2mg/mL，再加入终浓度200mM底物，用100mM的pH＝7的磷酸缓冲液做为反应介质补足到10mL，温度为20-60℃(20、25、30、35、40、45、50、55、60℃)、600rpm反应10min，取样500μL，并用500μL 2M的盐酸终止反应，用HPLC对反应液中1-氰基环己基乙酸的浓度进行检测，双突变体的最适温度为50℃，结果见图4所示。同样条件下检测腈水解酶AcN-F168V最适温度为45℃。
实施例7：腈水解酶及其突变体最适pH的测定
对实施例3中纯化后的蛋白进行最适温度的测定，以1-氰基环己基乙腈为底物，以腈水解酶AcN-F168V(比酶活为104U/g细胞湿重)或腈水解酶组合突变体G180D/A205C(比酶活为165U/g细胞湿重)的纯酶液为催化剂。
反应体系为10mL：收集G180D/A205C的纯酶液(165U/g)用pH 7.0、20mM磷 酸盐缓冲液稀释10倍加入反应容器中，使其加入终浓度为0.2mg/mL，加入终浓度200mM底物，分别用100mM的pH＝(4、4.5、5、5.5、6、6.5、7、7.5、8、8.5、9、9.5、10、10.5)的磷酸缓冲液做为反应介质补足到10mL，45℃、600rpm反应10min，取样500μL，并用500μL 2M的盐酸终止反应，用HPLC对反应液中1-氰基环己基乙酸的浓度进行检测，双突变体的最适pH为8.5，结果见图5所示。同样条件下检测AcN-F168V最适pH为7。
实施例8：腈水解酶及其突变体转化200mM氰基环己基乙腈
对实施例3中纯化后的腈水解酶及其突变体蛋白进行反应进程的测定，以1-氰基环己基乙腈为底物，以腈水解酶AcN-F168V(比酶活为104U/g细胞湿重)或腈水解酶组合突变体G180D/A205C的纯酶液(比酶活为165U/g细胞湿重)为催化剂。
反应体系为10mL：收集的纯酶液用pH 7.0、20mM磷酸盐缓冲液稀释10倍后加入反应容器，使其加入终浓度为0.2mg/mL，再加入终浓度200mM底物，用100mM的pH＝7的磷酸缓冲液做为反应介质补足到10mL，600rpm、45℃恒温水浴反应，于不同时间取样500μL，并用500μL 2M的盐酸终止反应，12000rpm离心，弃去沉淀。用HPLC对反应液中1-氰基环己基乙酸的浓度进行检测，腈水解酶AcN-F168V及其双突变体的反应进程结果见图6所示,转化率大于99％。经测定，如图6所述，双突变体G180D/A205C可在60min内将底物反应完全，反应时间较腈水解酶AcN-F168V相比有所缩短。

Claims (9)

  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180位，第205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180位甘氨酸突变为天冬氨酸；(2)将SEQ ID No.2所示氨基酸序列的第180位甘氨酸突变为苯丙氨酸；(3)将SEQ ID No.2所示氨基酸序列的第205位丙氨酸突变为半胱氨酸；(4)将SEQ ID No.2所示氨基酸序列的第180位甘氨酸突变为天冬氨酸，同时将第205位丙氨酸突变为半胱氨酸。
  3. 一种权利要求1所述腈水解酶突变体的编码基因。
  4. 一种由权利要求3所述腈水解酶突变体的编码基因构建的重组基因工程菌。
  5. 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。
  6. 如权利要求5所述的应用，其特征在于所述的应用以含腈水解酶突变体的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为4.0-10.5、200M磷酸缓冲液为反应介质构成反应体系，在20-60℃、600rpm恒温水浴条件下反应，反应完全后，将反应液分离纯化，获得1-氰基环己基乙酸。
  7. 如权利要求5所述的应用，其特征在于所述底物加入终浓度以反应体系体积计为5-1000mM；所述纯酶比酶活为160～170U/g，加入量以反应体系体积计为0.1-3mg/mL；所述催化剂为湿菌体或固定化细胞时，用量以湿菌体重量计为10-100g/L缓冲液。
  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1％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体按如下方法制备：将含腈水解酶突变体编码基因工程菌湿菌体用含pH 7.0、100mM的NaH 2PO 4-Na 2HPO 4缓冲液重悬，超声波破碎，破碎产物离心后取上清液作为粗酶液；所述超声波破碎条件为：400W，20min，1s破碎1s暂停；将粗酶液以1mL/min的流速通过经结合缓冲液冲洗的Ni-NTA柱，用平衡缓冲液洗脱弱吸附的杂蛋白，流速为2mL/min；再用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浓度为20mM的磷酸氢二钠-磷酸二氢钠缓冲液为透析液进行透析，取截留液即为纯酶液；所述结合缓冲液为含终浓度300mM NaCl的pH 8.0、50mM NaH 2PO 4缓冲液，所述平衡缓冲液为含终浓度300mM NaCl和50mM咪唑的pH 8.0、50mM NaH 2PO 4缓冲液，所述洗脱缓冲液为含终浓度300mM NaCl和500mM咪唑的pH 8.0、50mM NaH 2PO 4缓冲液。
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