WO2022073331A1 - 一种腈水解酶突变体及其在催化合成2-氯烟酸中的应用 - Google Patents

一种腈水解酶突变体及其在催化合成2-氯烟酸中的应用 Download PDF

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WO2022073331A1
WO2022073331A1 PCT/CN2021/088232 CN2021088232W WO2022073331A1 WO 2022073331 A1 WO2022073331 A1 WO 2022073331A1 CN 2021088232 W CN2021088232 W CN 2021088232W WO 2022073331 A1 WO2022073331 A1 WO 2022073331A1
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nitrilase
reaction
chloronicotinonitrile
mutant
chloronicotinic acid
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郑仁朝
戴安迪
郑裕国
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浙江工业大学
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/78—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5)
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
    • C12P17/10—Nitrogen as only ring hetero atom
    • C12P17/12—Nitrogen as only ring hetero atom containing a six-membered hetero ring
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y305/00—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5)
    • C12Y305/05—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5) in nitriles (3.5.5)
    • C12Y305/05001—Nitrilase (3.5.5.1)

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  • the invention relates to the technical field of enzyme engineering, and mainly relates to a nitrilase mutant and its application in the catalytic synthesis of 2-chloronicotinic acid.
  • 2-Chloronicotinic acid also known as 2-chloronicotinic acid
  • 2-chloronicotinic acid is an important nitrogen heterocyclic fine chemical intermediate, which is widely used in the synthesis of pesticides and pharmaceutical chemicals.
  • 2-chloronicotinic acid can be used to synthesize a series of bactericidal activities of sulfonylurea herbicide nicosulfuron, amide herbicide diflufenacil, amide fungicide boscalid and triazole thione compound.
  • cardiovascular disease treatment drugs such as the anti-AIDS drug nevirapine, the antidepressant mirtazapine, the anti-inflammatory drug pranoprofen, the anti-inflammatory and analgesic drugs niflufenac and nicotinic acid, etc.
  • the market demand for 2-chloronicotinic acid is growing due to its wide application in pesticides and medicines.
  • the production methods of 2-chloronicotinic acid are mainly chemical methods, including nicotinic acid nitrogen oxidation-chlorination-hydrolysis method, nicotinonitrile oxidation-chlorination-hydrolysis method, 3-picoline chlorination-oxidation method and alkenyl Ether and ethyl cyanoacetate ring formation, etc.
  • these methods have disadvantages such as harsh reaction conditions, serious environmental pollution, and high equipment requirements. Therefore, the development of clean and efficient production technology of 2-chloronicotinic acid has important industrial application value.
  • Biocatalytic hydrolysis and hydration of nitrile compounds has significant advantages such as high efficiency, environmental friendliness, and high chemical, regio- and stereoselectivity, and has become an important method for the industrial synthesis of carboxylic acids and amides.
  • Industrial synthesis of products are significant advantages such as high efficiency, environmental friendliness, and high chemical, regio- and stereoselectivity, and has become an important method for the industrial synthesis of carboxylic acids and amides.
  • Nitrilases can catalyze the hydrolysis of nitrile compounds to produce corresponding carboxylic acids and ammonia, but no nitrilases have been reported so far that can hydrolyze 2-chloronicotinonitrile to produce 2-chloronicotinic acid. Studies have shown that some nitrilases also have nitrile hydration activity and catalyze the formation of nitrile compounds into corresponding amides. The catalytic properties of nitrilase with both nitrile hydrolysis and nitrile hydration activities provide a new perspective for the development of new functions in bioorganic synthesis.
  • Protein molecular modification is an effective means to regulate the catalytic properties of nitrilase. Therefore, a mutant capable of specifically hydrolyzing 2-chloronicotinonitrile to synthesize 2-chloronicotinic acid was developed through molecular modification, which was used for the bioorganic 2-chloronicotinic acid. Synthesis is a problem to be solved by those skilled in the art.
  • the purpose of the present invention is to transform the nitrilase derived from Rhodococcus zopfii through protein engineering technology, and construct a mutant capable of specifically hydrolyzing 2-chloronicotinonitrile to synthesize 2-chloronicotinic acid.
  • the efficient and green production is of great significance.
  • the present invention adopts the following technical solutions:
  • the present invention utilizes error-prone PCR technology to randomly mutate the nitrilase encoding gene (SEQ ID NO. 1) derived from R. zopfii, specifically, firstly uses T7 primer to carry out PCR amplification, and randomly introduces mutations to obtain nitrilase
  • the nucleotide sequence of the mutant was connected to the expression vector pET-28b(+) and then introduced into the E. coli host. After induction and expression, the mutant with reduced nitrile hydration activity and increased nitrile hydrolysis activity was obtained through high-throughput screening.
  • the present invention provides a nitrilase mutant whose amino acid sequence is shown in SEQ ID NO.4, that is, the 167th tryptophan W mutation of the parent nitrilase whose amino acid sequence is shown in SEQ ID NO.2 Glycine G.
  • the present invention also provides a gene encoding the nitrilase mutant, the nucleotide sequence of which is shown in SEQ ID NO.3.
  • the present invention also provides a recombinant plasmid comprising the gene.
  • the original vector is pET-28b(+).
  • the present invention also provides a recombinant engineering bacterium comprising the recombinant plasmid.
  • the above-mentioned recombinant plasmid transforms host cells to obtain recombinant genetically engineered bacteria, and the host cells can be various conventional host cells in the art.
  • the host cells are Escherichia coli BL21 (DE3).
  • Another object of the present invention is to provide the application of the nitrilase mutant in catalyzing the hydrolysis of 2-chloronicotinonitrile to synthesize 2-chloronicotinic acid.
  • the application includes: using the wet cell body, the wet cell body immobilized cell or the pure enzyme extracted after the wet cell body is ultrasonically broken, obtained after the recombinant engineering bacteria containing the nitrilase mutant encoding gene is induced and expressed, as a catalyst, and using 2 -Chloronicotinonitrile is the substrate, and the NaH 2 PO 4 -Na 2 HPO 4 buffer solution with pH value of 6-8 is used as the reaction medium to form a reaction system, and the reaction is carried out at 25-45 ° C. After the reaction, separation and purification are performed to obtain 2- Chloronic acid.
  • the dosage of the catalyst is 0.2-3 g/L based on the dry weight of the bacterial cells, and the substrate concentration is 50-500 mM.
  • the thalline collected after the induction culture is used as a catalyst, and a 200 mM NaH 2 PO 4 -Na 2 HPO 4 buffer with a pH value of 7 is used as a reaction medium.
  • the concentration of the substrate 2-chloronicotinonitrile is 300mM, bacterial cells 2g/L (dry weight), react at 30°C for 30-40h.
  • the wet cell preparation method is as follows: the engineered bacteria containing the nitrilase mutant gene are inoculated into a liquid LB medium containing 50 ⁇ g/mL kanamycin, cultured at 37° C. at 200 rpm overnight, and then incubated at 2% (volume). Concentration) of the inoculum was transferred to a fresh liquid LB medium containing 50 ⁇ g/mL kanamycin, and cultured at 37°C at 180 rpm until the bacterial concentration OD 600 was 0.4-0.8, and then added to the medium with a final concentration of 0.1-1mM IPTG, 28°C, 180rpm for 12h induction. The fermentation broth was centrifuged at 8000 rpm for 10 min at 4°C, and the cells were collected. The composition of LB liquid medium was (g/L): peptone 10, yeast extract 5, NaCl 10, pH 7.0.
  • the nitrilase mutant W167G provided by the invention can eliminate the hydration activity of the parent nitrilase to 2-chloronicotinonitrile, no by-product 2-chloronicotinamide is produced in the catalytic process, and the nitrile hydrolysis activity is greatly improved, and can be specifically catalyzed
  • the hydrolysis of 2-chloronicotinonitrile to synthesize 2-chloronicotinic acid has important potential in the enzymatic industrial synthesis of 2-chloronicotinic acid.
  • Figure 1 shows the HPLC analysis chromatograms of the reaction products of 2-chloronicotinonitrile catalyzed by the parent nitrilase (A) and the mutant W167G (B).
  • Figure 2 shows the progress of the hydrolysis reaction of mutant W167G catalyzed by 300 mM 2-chloronicotinonitrile.
  • primers T7 F and T7 R were used to carry out PCR amplification and random introduction of mutations.
  • PCR reaction system 50 ⁇ L: template pET-RZ 0.5-20 ng, 1 ⁇ Taq Buffer (without Mg 2+ ), 0.2 mM dNTP, 0.3 mM MnCl 2 , 2 mM MgCl 2 , 0.2 each of upstream and downstream primers T7 F and T7 R ⁇ M, Taq DNA polymerase 5U.
  • PCR conditions (1) pre-denaturation at 95°C for 5 min; (2) denaturation at 95°C for 15s; (3) annealing at 60°C for 5s; (4) extension at 72°C for 30s, steps (2) to (4) for a total of 30 cycles; ( 5) The final extension at 72°C for 3 min, and storage at 4°C.
  • the PCR products were analyzed by agarose gel electrophoresis and recovered by cutting the gel.
  • the above-mentioned gel recovery product was used as a primer to amplify to obtain a complete plasmid.
  • PCR system 50 ⁇ L: 2 ⁇ Phanta Max buffer, 0.2 mM dNTPs, 2.5 U Phanta Max high-fidelity polymerase, 50 ng of gel recovery product, 20 ng of pET-RZ plasmid.
  • PCR conditions (1) pre-denaturation at 95 °C for 5 min; (2) denaturation at 95 °C for 15 s, annealing at 60 °C for 5 s, and extension at 72 °C for 3.5 min, step (2) for a total of 35 cycles; (3) final extension at 72 °C for 5 min, 4 Store at °C.
  • the amplified PCR product was digested with endonuclease DpnI at 37°C for 3h, inactivated at 65°C for 10min, transformed into E.coli BL21 (DE3), spread on LB plates containing kanamycin (50 ⁇ g/mL), 37 Cultivate overnight.
  • Example 2 Pick a single colony in Example 1 and culture it in a 96 deep-well plate, add 1 mL of LB medium (containing a final concentration of 50 ⁇ g/mL kanamycin) to each well plate, cultivate at 37°C for 12 h, and take 200 ⁇ L of bacterial liquid for transfer. to 800 ⁇ L of fresh LB medium (containing a final concentration of 50 ⁇ g/mL kanamycin, 0.1 mM IPTG), and cultured at 28° C. for 18 h.
  • LB medium containing a final concentration of 50 ⁇ g/mL kanamycin
  • the cells of the 96 deep-well plate were centrifuged for 30 min (3000 rpm, 4° C.), the supernatant was discarded, washed with NaH 2 PO 4 -Na 2 HPO 4 buffer (200 mM, pH 7.0), and the cells were resuspended in 600 ⁇ L.
  • Substrate 2-chloronicotinonitrile (final concentration 100mM) was added to each well, and the reaction was carried out at 30°C for 12h.
  • reaction solution of the 96 deep-well plate was centrifuged for 30 min (3000 rpm, 4°C), and 20 ⁇ L of the supernatant was transferred to a 96-well micro-reaction plate containing 150 ⁇ L of a mixture of o-phthalaldehyde and mercaptoethanol in each well. Incubate at 37°C for 30min. The fluorescence intensity was then measured using a microplate reader (excitation wavelength 412 nm, emission wavelength 467 nm). Stronger fluorescence indicates higher nitrile hydrolysis activity and more NH3 and 2-chloronicotinic acid produced. After verification by liquid chromatography, sequencing analysis was performed.
  • the mutant W167A (Table 2) was obtained by screening, that is, the codon at position 167 was mutated from TGG to GCC.
  • PCR system 50 ⁇ L: template pET-W167A 0.5-20 ng, primers W167 F and W167 R 10-15 pmol each, 2 ⁇ Phanta Max buffer, 0.2 mM dNTP, 2.5 U Phanta Max high-fidelity polymerase.
  • PCR conditions (1) pre-denaturation at 95 °C for 5 min; (2) denaturation at 95 °C for 15 s, annealing at 60 °C for 5 s, and extension at 72 °C for 3.5 min, step (2) for a total of 35 cycles; (3) final extension at 72 °C for 5 min, 4 Store at °C.
  • the amplified PCR product was digested with endonuclease DpnI at 37°C for 3h, inactivated at 65°C for 10min, transformed into E.coli BL21 (DE3), spread on LB plates containing kanamycin (50 ⁇ g/mL), 37 Cultivate overnight.
  • a total of 200-300 single clones were generated by the saturation mutation at position 167, which, after sequencing, contained all 20 natural amino acids.
  • the mutants with no nitrile hydration activity and improved nitrile hydrolysis activity were determined to be W167S (that is, the codon at position 167 was mutated from TGG to TCC), W167C (that is, the codon at position 167 was mutated from TGG to TGC), and W167G (that is, the codon at position 167 was mutated from TGG to TGC).
  • the codons were mutated from TGG to GGC), as shown in Table 2.
  • the parent nitrilase has hydratase activity when hydrolyzing 2-chloronicotinonitrile, producing a large amount of by-product 2-chloronicotinamide (Fig. 1A).
  • the transformed mutant W167G only has hydrolase activity and no by-product 2-chloronicotinamide Nicotinamide was produced ( Figure IB).
  • the preferred mutant W167G in Example 3 adopts the method of Example 4 to induce and cultivate the collected wet cells as a catalyst, and is added to the reaction system (total system 10 mL, substrate 2-chloronicotinonitrile 300 mM, pH 7 200 mM NaH 2 PO 4 -Na 2 HPO 4 buffer solution, 0.02 g of bacterial cells (dry weight)), react at 30°C. 100 ⁇ L of the reaction solution was sampled at certain time intervals, and 10 ⁇ L of 6M HCl was added to terminate the reaction, and the content of the product was detected by HPLC.
  • the mutant W167G can completely convert 300 mM substrate 2-chloronicotinonitrile to 2-chloronicotinic acid.

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Abstract

提供了一种腈水解酶突变体及其在催化合成2-氯烟酸中的应用，所述腈水解酶突变体的氨基酸序列如SEQ ID NO.4所示，是将亲本腈水解酶的第167位色氨酸（Trp）突变为甘氨酸（Gly）获得的，所述腈水解突变体消除了亲本腈水解酶对2-氯烟腈的水合活力，催化过程无副产物2-氯烟酰胺产生，且腈水解活力提高，可专一性催化2-氯烟腈水解合成2-氯烟酸。

Description

一种腈水解酶突变体及其在催化合成2-氯烟酸中的应用 技术领域
本发明涉及酶工程技术领域，主要涉及一种腈水解酶突变体及其在催化合成2-氯烟酸中的应用。
背景技术
2-氯烟酸，又名2-氯尼古丁酸，是一种重要的氮杂环类精细化工中间体，广泛用于农药、医药化学品的合成。在农药领域，2-氯烟酸可用于合成磺酰脲类除草剂烟嘧磺隆、酰胺类除草剂吡氟草胺、酰胺类杀菌剂啶酰菌胺以及三唑硫酮类的系列杀菌活性化合物。在医药方面，可用于合成抗生素、心血管疾病治疗药物如抗艾滋病药奈韦拉平、抗抑郁药米氮平、消炎药普拉洛芬、抗炎镇痛药尼氟灭酸和烟甲灭酸等。由于2-氯烟酸在农药和医药中的广泛应用，其市场需求日益增长。
目前，2-氯烟酸的生产方法主要为化学法，包括烟酸氮氧化-氯化-水解法、烟腈氧化-氯化-水解法、3-甲基吡啶氯化-氧化法和烯基醚与氰乙酸乙酯成环法等。然而，这些方法存在反应条件严苛、环境污染严重、设备要求较高等缺陷。因此，开发清洁、高效的2-氯烟酸生产技术具有重要的工业应用价值。
近年来，生物技术领域的突破性进展使生物催化在化学工业中发挥越来越重要的作用，逐渐成为工业可持续发展最有希望的技术之一。生物催化腈化合物水解与水合具有过程高效、环境友好以及高度化学、区域和立体选择性等显著优势，成为羧酸、酰胺工业合成的重要方法，成功实现了丙烯酰胺、烟酰胺等大宗和精细化学品的工业合成。
腈水解酶能够催化腈化合物水解生成相应的羧酸和氨，但迄今未报道能够水解2-氯烟腈生成2-氯烟酸的腈水解酶。研究表明，一些腈水解酶同时具有腈水合活性，催化腈化合物生成相应的酰胺。腈水解酶兼具腈水解与腈水合活性的催化特性为其生物有机合成新功能的开发提供了新视角。
蛋白质分子改造是调控腈水解酶催化特性的有效手段，因此，通过分 子改造开发能够专一性水解2-氯烟腈合成2-氯烟酸的突变体，用于2-氯烟酸的生物有机合成，是本领域技术人员需要解决的问题。
发明内容
本发明的目的是通过蛋白质工程技术对Rhodococcus zopfii来源的腈水解酶进行改造，构建一种能够专一性水解2-氯烟腈合成2-氯烟酸的突变体，对于实现2-氯烟酸的高效、绿色生产具有重要意义。
为实现上述目的，本发明采用如下技术方案：
本发明利用易错PCR技术对来源于R.zopfii的腈水解酶编码基因(SEQ ID NO.1)进行随机突变，具体地，首先利用T7引物进行PCR扩增，随机引入突变，得到腈水解酶突变体核苷酸序列，连接到表达载体pET-28b(+)后导入大肠杆菌宿主，诱导表达后通过高通量筛选获得腈水合活力降低、腈水解活力提高的突变体，测序分析发现SEQ ID NO.2所示的R.zopfii腈水解酶氨基酸序列的第167位色氨酸发生突变。然后结合定点突变方法，对第167位定点突变，得到腈水合活力消除且腈水解活力进一步提高的突变体，从而获得能够高效催化2-氯烟腈合成2-氯烟酸的腈水解酶突变体W167G。
本发明提供了一种腈水解酶的突变体，其氨基酸序列如SEQ ID NO.4所示，即氨基酸序列如SEQ ID NO.2所示的亲本腈水解酶的第167位色氨酸W突变为甘氨酸G。
本发明还提供了编码所述腈水解酶突变体的基因，其核苷酸序列如SEQ ID NO.3所示。
本发明还提供了一种包含所述基因的重组质粒。作为优选，原始载体为pET-28b(+)。
本发明还提供了一种包含所述重组质粒的重组工程菌。上述重组质粒转化宿主细胞获得重组基因工程菌，所述宿主细胞可以为本领域的各种常规宿主细胞，作为优选，宿主细胞为大肠杆菌Escherichia coli BL21(DE3)。
本发明的另一个目的是提供所述的腈水解酶突变体在催化2-氯烟腈水解合成2-氯烟酸中的应用。
所述应用包括：以含腈水解酶突变体编码基因的重组工程菌经诱导表达后获得的湿菌体、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2-氯烟腈为底物，以pH值6～8的NaH 2PO 4-Na 2HPO 4缓冲液为反应介质构成反应体系，在25～45℃下反应，反应结束后，分离纯化获得2-氯烟酸。
作为优选，反应体系中，催化剂的用量以菌体干重计为0.2～3g/L，底物浓度为50～500mM。
更为优选，以诱导培养后收集的菌体为催化剂，pH值为7的200mM NaH 2PO 4-Na 2HPO 4缓冲液为反应介质，反应体系中，底物2-氯烟腈的浓度为300mM，菌体2g/L(干重)，30℃下反应30～40h。
所述湿菌体制备方法如下：将含腈水解酶突变体基因的工程菌接种到含有50μg/mL卡那霉素的液体LB培养基中，37℃，200rpm培养过夜，随后以2％(体积浓度)的接种量转接到新鲜的含有50μg/mL卡那霉素的液体LB培养基中，37℃，180rpm培养至菌体浓度OD 600为0.4～0.8，随后向培养基中加入终浓度为0.1～1mM的IPTG，28℃，180rpm诱导培养12h。将发酵液于4℃下8000rpm离心10min，收集菌体。LB液体培养基组成为(g/L)：蛋白胨10，酵母提取物5，NaCl 10，pH 7.0。
本发明具备的有益效果：
本发明提供的腈水解酶突变体W167G，消除亲本腈水解酶对2-氯烟腈的水合活力，催化过程无副产物2-氯烟酰胺产生，且腈水解活力大幅提高，可专一性催化2-氯烟腈水解合成2-氯烟酸，在酶法工业化合成2-氯烟酸中具有重要潜力。
附图说明
图1为亲本腈水解酶(A)及突变体W167G(B)催化2-氯烟腈反应产物的HPLC分析谱图。
图2为突变体W167G催化300mM 2-氯烟腈水解反应进程。
具体实施方式
下面结合具体实施例对本发明作进一步描述，但本发明的保护范围并不仅限于此。
实施例1 腈水解酶突变文库的构建
以含有来源于Rhodococcus zopfii的腈水解酶基因(核苷酸序列SEQ ID NO.1、氨基酸序列SEQ ID NO.2)的pET-RZ质粒为模板，利用引物T7 F和T7 R(表1)进行PCR扩增，随机引入突变。
PCR反应体系(50μL)：模板pET-RZ 0.5～20ng，1×Taq Buffer(不含Mg 2+)，0.2mM dNTP，0.3mM MnCl 2，2mM MgCl 2,上下游引物T7 F和T7 R各0.2μM，Taq DNA聚合酶5U。
PCR条件：(1)95℃预变性5min；(2)95℃变性15s；(3)60℃退火5s；(4)72℃延伸30s，步骤(2)～(4)共30个循环；(5)最后72℃延伸3min，4℃保藏。
PCR产物经过琼脂糖凝胶电泳分析后切胶回收。
随后以上述胶回收产物为引物，扩增得到完整的质粒。
PCR体系(50μL)：2×Phanta Max buffer，0.2mM dNTPs，2.5U Phanta Max高保真聚合酶，胶回收产物50ng，pET-RZ质粒20ng。
PCR条件：(1)95℃预变性5min；(2)95℃变性15s，60℃退火5s，72℃延伸3.5min，步骤(2)共35个循环；(3)最后72℃延伸5min，4℃保存。
扩增得到的PCR产物经内切酶DpnI 37℃消化3h，65℃灭活10min，转化导入E.coli BL21(DE3)，涂布于含卡那霉素(50μg/mL)的LB平板，37℃培养过夜。
表1 引物设计表
Figure PCTCN2021088232-appb-000001
实施例2 突变体的高通量筛选
挑取实施例1中的单菌落至96深孔板中培养，每个孔板加入1mL LB培养基(含有终浓度50μg/mL卡那霉素)，37℃培养12h，取200μL菌液 转接到800μL新鲜LB培养基(含终浓度50μg/mL卡那霉素，0.1mM IPTG)，28℃培养18h。将96深孔板的菌体离心30min(3000rpm，4℃)，弃去上清后，用NaH 2PO 4-Na 2HPO 4缓冲液(200mM，pH 7.0)洗涤并用600μL重悬菌体。各孔中加入底物2-氯烟腈(终浓度100mM)，30℃反应12h。反应结束后，将96深孔板的反应液离心30min(3000rpm，4℃)，取20μL上清液转移每个孔中含有150μL邻苯二甲醛与巯基乙醇混合液的96孔微量反应板中，放置于37℃保温30min。随后利用酶标仪(激发波长412nm，发射波长467nm)测定荧光强度。荧光越强表明腈水解活力越高，产生的NH 3和2-氯烟酸越多。利用液相色谱复筛验证后，进行测序分析。液相色谱流动相为乙腈：水：磷酸＝250:750:1，流速为1mL/min，检测波长为210nm。筛选获得突变体W167A(表2)，即167位的密码子由TGG突变为GCC。
实施例3 定点突变
以质粒pET-W167A为模板，通过全质粒扩增进行定点突变。
PCR体系(50μL)：模板pET-W167A 0.5～20ng，引物W167 F和W167 R各10～15pmol，2×Phanta Max buffer，0.2mM dNTP，2.5U Phanta Max高保真聚合酶。
PCR条件：(1)95℃预变性5min；(2)95℃变性15s，60℃退火5s，72℃延伸3.5min，步骤(2)共35个循环；(3)最后72℃延伸5min，4℃保存。
扩增得到的PCR产物经内切酶DpnI 37℃消化3h，65℃灭活10min，转化导入E.coli BL21(DE3)，涂布于含卡那霉素(50μg/mL)的LB平板，37℃培养过夜。
167位点的饱和突变共产生了200～300个单克隆，经过测序，包含了所有20种天然氨基酸。
通过实施例2的高通量筛选方法初筛后，利用液相色谱复筛验证。确定无腈水合活力且腈水解活力提高的突变体为W167S(即167位的密码子由TGG突变为TCC)，W167C(即167位的密码子由TGG突变为TGC)，W167G(即167位的密码子由TGG突变为GGC)，如表2所示。
亲本腈水解酶在水解2-氯烟腈时具有水合酶活力，产生了大量副产物2-氯烟酰胺(图1A)，改造以后的突变体W167G只具有水解酶活力，无副 产物2-氯烟酰胺产生(图1B)。
表2 腈水解酶相对活力比较
Figure PCTCN2021088232-appb-000002
实施例4 腈水解酶的诱导及表达
取10μL甘油管保藏的菌液接种至10mL液体LB培养基(含有50μg/mL的卡那霉素)，37℃，200rpm培养过夜，以2％接种量转接至100mL新鲜LB培养基中(含有50μg/mL的卡那霉素)，继续培养至OD 600为0.4～0.8，加入终浓度为0.1mM的IPTG，于28℃下诱导培养12h。培养结束后，4℃下8 000rpm离心10min收集菌体，用0.9％的生理盐水洗涤2次，得到湿菌体。
实施例5 利用重组腈水解酶突变体制备2-氯烟酸
实施例3中的优选突变体W167G采用实施例4的方法诱导培养收集到的湿菌体作为催化剂，添加入反应体系中(总体系10mL，底物2-氯烟腈300mM，pH 7的200mM NaH 2PO 4-Na 2HPO 4缓冲液，0.02g菌体(干重))，30℃反应。间隔一定时间取样100μL反应液加入10μL 6M HCl终止反应，HPLC检测产物含量。
由图2可知，突变体W167G可将300mM底物2-氯烟腈完全转化为2-氯烟酸。

Claims (10)

  1. 一种腈水解酶突变体，其特征在于，其氨基酸序列如SEQ ID NO.4所示。
  2. 一种编码如权利要求1所述的腈水解酶突变体的基因，其特征在于，其核苷酸序列如SEQ ID NO.3所示。
  3. 一种包含如权利要求2所述的基因的重组质粒。
  4. 如权利要求3所述的重组质粒，其特征在于，原始载体为pET-28b(+)。
  5. 一种包含如权利要求3或4所述的重组质粒的重组工程菌。
  6. 如权利要求5所述的重组工程菌，其特征在于，宿主细胞为大肠杆菌Escherichia coli BL21。
  7. 如权利要求1所述的腈水解酶突变体在催化2-氯烟腈水解合成2-氯烟酸中的应用。
  8. 如权利要求7所述的应用，其特征在于，包括：以含腈水解酶突变体编码基因的重组工程菌经诱导表达后获得的湿菌体、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2-氯烟腈为底物，以pH值6～8的NaH 2PO 4-Na 2HPO 4缓冲液为反应介质构成反应体系，在25～45℃下反应，反应结束后，分离纯化获得2-氯烟酸。
  9. 如权利要求8所述的应用，其特征在于，反应体系中，催化剂的用量以菌体干重计为0.2～3g/L，底物浓度为50～500mM。
  10. 如权利要求9所述的应用，其特征在于，以诱导培养后收集的菌体为催化剂，pH值为7的200mM NaH 2PO 4-Na 2HPO 4缓冲液为反应介质，反应体系中，底物2-氯烟腈的浓度为300mM，菌体以干重计为2g/L，30℃下反应30～40h。
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