WO2021081868A1 - 一种温泉热碱芽孢杆菌来源的腈水合酶的突变体 - Google Patents

一种温泉热碱芽孢杆菌来源的腈水合酶的突变体 Download PDF

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WO2021081868A1
WO2021081868A1 PCT/CN2019/114605 CN2019114605W WO2021081868A1 WO 2021081868 A1 WO2021081868 A1 WO 2021081868A1 CN 2019114605 W CN2019114605 W CN 2019114605W WO 2021081868 A1 WO2021081868 A1 WO 2021081868A1
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mutant
nitrile hydratase
cell
nhase
cal
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French (fr)
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周哲敏
刘中美
李婷
陈德智
张赛兰
周丽
崔文璟
程中一
郭军玲
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江南大学
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Priority to PCT/CN2019/114605 priority Critical patent/WO2021081868A1/zh
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Priority to US17/399,423 priority patent/US20210388336A1/en

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    • C—CHEMISTRY; METALLURGY
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    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/88—Lyases (4.)
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    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • 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
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y402/00—Carbon-oxygen lyases (4.2)
    • C12Y402/01—Hydro-lyases (4.2.1)
    • C12Y402/01084—Nitrile hydratase (4.2.1.84)

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  • the invention relates to a mutant of a nitrile hydratase derived from Bacillus thermoalkali and belongs to the technical field of enzyme engineering.
  • Nitrile hydratase can be used to catalyze 3-cyanopyridine to nicotinamide with higher medicinal value.
  • Niacinamide is a vitamin and has been widely used in feed, food, pharmaceutical and other industries. Nicotinamide has a large market demand, but the current production level of niacinamide in my country is not high and the scale is small. Therefore, the use of NHase in the production of nicotinamide has great potential.
  • the reaction is an exothermic process, so the high temperature in the production process will affect the performance of the enzyme activity. The main reason is that the high temperature affects the structure of the enzyme and causes the enzyme activity to decrease, which in turn leads to a large amount of energy consumption and increases the production cost. .
  • the substrates and products of nitrile hydratase are organic matter.
  • High concentrations of organic matter will have a greater destructive effect on the structure of the enzyme, causing the enzyme activity to decrease rapidly and the catalytic activity. Therefore, in the production catalysis process, the nitrile hydration is improved. Enzyme substrate product tolerance is particularly important.
  • Rhodococcus rhodococcus (Rhodococcus rhodochrous) J1 is mainly used in industrial production to catalyze the production of nicotinamide, which adopts the method of substrate feeding in batches, but the growth cycle of Rhodococcus is long, requiring 100h, and the production efficiency is not high.
  • the highest yield is 162g/L, while the highest yield of acrylamide is 300g/L.
  • the present invention aims to provide a nitrile hydratase mutant with improved product tolerance and substrate tolerance and application thereof.
  • the first object of the present invention is to provide a nitrile hydratase mutant containing ⁇ subunits, ⁇ subunits and regulatory proteins, which contain SEQ ID NO. 2, SEQ ID NO. 3, and SEQ ID NO. 4 respectively.
  • SEQ ID NO. 2 SEQ ID NO. 3
  • SEQ ID NO. 4 SEQ ID NO. 4
  • the second object of the present invention is to provide a gene encoding the mutant.
  • the third object of the present invention is to provide a vector containing the gene.
  • the fourth object of the present invention is to provide cells expressing the nitrile hydratase mutant.
  • the cell includes Escherichia coli.
  • the cell uses Escherichia coli BL21 as a host and pET-series plasmids as a vector.
  • the plasmid is pET-24(+).
  • the fifth object of the present invention is to provide a method for improving the enzymatic activity of nitrile hydratase, which is to mutate the glycyl acid at position 20 of the alpha subunit of the nitrile hydratase shown in SEQ ID NO.5 to valine.
  • the sixth object of the present invention is to provide a composition containing the above-mentioned nitrile hydratase mutant.
  • the present invention includes, but is not limited to, a protective agent.
  • the seventh object of the present invention is to provide the application of the above-mentioned nitrile hydratase mutant or the above-mentioned cell in the production of products containing nicotinamide or acrylamide.
  • the above-mentioned nitrile hydratase mutant, or the above-mentioned cell, or the above-mentioned composition is used as a catalyst, and nicotinonitrile or acrylonitrile is used as a substrate for the conversion reaction.
  • nicotinonitrile or acrylonitrile is used as a substrate, the above-mentioned cells are used for fermentation, and the fermentation broth is used for whole cell transformation to produce nicotinamide or acrylamide.
  • the OD 600 of the fermentation broth is 5-10, and the concentration of the substrate is 0.2-1 mol/L.
  • the whole cell transformation reaction conditions are: the temperature is 25-28° C., and the mass ratio of the substrate nicotinonitrile to the wet bacteria is 0.5-2.
  • the whole cell transformation reaction conditions are: the temperature is 25-28° C., and the mass ratio of the substrate acrylonitrile to the wet bacteria is 1-1.5.
  • the eighth object of the present invention is to provide a method for recombinantly expressing the nitrile hydratase mutant.
  • the cells expressing the nitrile hydratase mutant are inoculated in LB medium and cultured at 35-37°C until the OD 600 is At 0.6-0.8, add inducer IPTG and induce 12-18h at 20-30°C.
  • the method is to inoculate the cells in an LB expression medium containing kanamycin, culture with shaking at 37° C. and 200 r/min until the OD 600 is 0.6-0.8, and then add Inducer IPTG to 0.1mM, Co 2+ to 0.1mg/L, 25°C for 12-18h.
  • the method further includes collecting the bacterial cells of the cell, crushing the bacterial cells and collecting the supernatant, filtering the supernatant membrane, and separating it with a Strep Trap HP column to obtain the nitrile hydratase mutation body.
  • the invention also provides the application of the cell in the field of fermentation.
  • the nitrile hydratase mutant Cal.t Nhase-A20V provided by the present invention has a half-life of about 10 minutes at 70°C, and the thermal stability of the mutant Cal.t Nhase-A20V does not change much compared with the wild enzyme.
  • the specific enzyme activity is 650U/mg, which is 128% of the wild enzyme.
  • the mutant also has better substrate tolerance and product tolerance, and the final yield of nicotinamide catalyzed by whole cells reaches 598 g/L; therefore, the nitrile hydratase mutant Cal.t Nhase- A20V has good enzymatic properties, which is conducive to future industrial production.
  • Figure 1 The thermal stability of wild enzyme and mutant enzyme Cal.t Nhase-A20V at 70°C for 10 min.
  • FIG. 2 Substrate tolerance of wild enzyme and mutant enzyme Cal.t Nhase-A20V at different concentrations of substrate 3-cyanopyridine.
  • Figure 3 Product tolerance of wild enzyme and mutant enzyme Cal.t Nhase-A20V at different concentrations of product nicotinamide.
  • LB medium peptone 10g/L, yeast extract 5g/L, NaCl 10g/L.
  • Cal.t NHase-T104A (mutate the threonine at position 104 of the ⁇ subunit shown in SEQ ID NO. 2 to alanine)
  • Cal.t NHase-S152K (change the amino acid sequence as SEQ ID The Serine at position 152 of the ⁇ subunit shown in NO.2 is mutated to lysine)
  • Cal.t NHase-K185A (the amino acid sequence is shown as the lysine at position 185 of the ⁇ subunit shown in SEQ ID NO.2 Mutation to alanine).
  • PCR was performed with the primers shown in Table 1 under the conditions shown in Table 2, and the PCR product was transformed into E.coli JM109 and sequenced by Suzhou Jinweizhi, and the plasmid with the correct sequencing result was retrieved to carry the coding mutant Gene recombinant plasmid pET24a-NHase-A20V, pET24a-Cal.t Nhase-H150S, pET24a-Cal.t Nhase-T104A, pET24a-Cal.t Nhase-S152K, pET24a-Cal.tNhase-K185A, transform the recombinant plasmid into E .coli BL21 strain was expressed to obtain a recombinant strain.
  • the PCR amplification reaction conditions are:
  • PCR products were identified by agarose gel electrophoresis. Then the PCR product was purified, digested and transferred into E. coli BL21 competent cells.
  • E. coli BL21/pET24a-Cal.t NHase-A20V, BL21/pET24a-Cal.t Nhase-H150S, BL21/pET24a-Cal.t Nhase-T104A, BL21/pET24a- Cal.t Nhase-S152K, BL21/pET24a-Cal.t Nhase-K185A were inoculated in 5 mL of LB medium with a kanamycin concentration of 50 ⁇ g/mL (peptone 10g/L, yeast extract 5g/L, NaCl 10g/L ), cultivate overnight at 37°C, shaking at 200r/min.
  • the above-mentioned overnight culture was inoculated into 100mL LB expression medium (peptone 10g/L, yeast extract 5g/L, NaCl 10g/L) containing kanamycin at a concentration of 50 ⁇ g/mL at an inoculum of 1% (v/v).
  • LB expression medium peptone 10g/L, yeast extract 5g/L, NaCl 10g/L
  • kanamycin at a concentration of 50 ⁇ g/mL at an inoculum of 1% (v/v).
  • L at 37°C, 200r/min shaking culture to OD 600 to 0.6-0.8, add the inducer IPTG to 0.1mM, Co 2+ to 0.1mg/L, 25°Cto induce 12-18h to obtain the bacteria, 12000rpm Centrifuge at speed to collect bacteria.
  • Example 2 Add 10 ⁇ L of the mutant enzyme purified in Example 1 with 0.5 mg/mL to 500 ⁇ L buffer reaction system, and treat them in a metal bath at 70°C for 0 min, 10 min, 20 min, and 30 min, respectively, and determine the residual enzyme activity. Among them, the enzyme activity is determined for 0 min. Is 100%.
  • the enzyme activity without product treatment is defined as 100%.
  • the remaining enzyme activity of the mutant enzyme Cal.t Nhase-A20V is 40% of that of the wild enzyme.
  • the remaining enzyme activity of the other mutant enzymes Cal.t NHase-H150S, Cal.t NHase-T104A, Cal.t NHase-S152K, Cal.t NHase-K185A, compared with the wild enzymes, all appeared Various degrees of decline.
  • the product tolerance of the mutant enzyme Cal.t Nhase-A20V has been significantly improved, and the Cal.t Nhase-A20V mutant enzyme was selected for follow-up research.
  • the enzyme activity during 0M substrate treatment was defined as 100%. It was found that after the mutant was treated with 1M substrate 3-cyanopyridine at 30°C for 30 minutes, the residual enzyme activity of the mutant was determined by the wild enzyme. From 52% to 72%, the substrate tolerance of the mutants was significantly improved.

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Abstract

一种温泉热碱芽孢杆菌来源的腈水合酶的突变体，属于酶工程技术领域。本发明提供的腈水合酶突变体Cal.t Nhase-A20V在70℃的半衰期大约是10min，相比野生酶热稳定性并未有太大变化，突变体Cal.t Nhase-A20V的比酶活是野生酶的128％。同时该突变体也具备了更好的底物耐受性和产物耐受性，全细胞催化生成烟酰胺最终产量达到598g/L。所述腈水合酶突变体Cal.t Nhase-A20V，具有很好的酶学性质，有利于以后的工业生产。

Description

一种温泉热碱芽孢杆菌来源的腈水合酶的突变体 技术领域
本发明涉及一种温泉热碱芽孢杆菌来源的腈水合酶的突变体，属于酶工程技术领域。
背景技术
腈水合酶(NHase)可用于催化3-氰基吡啶为药用价值更高的烟酰胺，烟酰胺是一种维生素，已经被广泛地用于饲料、食品、制药等行业。烟酰胺市场需求量很大，但目前我国烟酰胺的生产水平不高，规模不大。因此，将NHase用于烟酰胺的生产有很大潜力。但是，该反应是一个放热的过程，所以生产过程中高温会影响酶活的发挥，主要是温度高，影响酶的结构，导致酶活下降，进而导致了大量的能耗，提高了生产成本。同时腈水合酶的底物和产物都是有机物，高浓度的有机物对酶的结构会产生较大的破坏作用，使酶活急速降低，催化活性下降，所以，在生产催化过程中，提高腈水合酶底物产物耐受性尤为重要。
目前工业生产中主要用玫瑰色红球菌(Rhodococcusrhodochrous)J1催化生成烟酰胺，采用的是底物分批补料的方式，但是红球菌生长周期较长，需要100h，并且生产效率不高，烟酰胺产量最高为162g/L，而丙烯酰胺产量最高为300g/L。目前也有通过重组菌生产烟酰胺的报道，但终产物浓度较低，只有240g/L。因此，获得一株高效且有机溶剂耐受性较好的腈水合酶对于烟酰胺工业化生产具有重要的应用价值。
发明概述
技术问题
本发明旨在提供一种产物耐受性和底物耐受性都提高的腈水合酶突变体及其应用。
问题的解决方案
技术解决方案
本发明的第一个目的是提供一种腈水合酶突变体，含有β亚基、α亚基以及调控 蛋白，其分别含SEQ ID NO.2、SEQ ID NO.3、SEQ ID NO.4所示的氨基酸序列。。
本发明的第二个目的是提供编码所述突变体的基因。
本发明的第三个目的是提供含有所述基因的载体。
本发明的第四个目的是提供表达所述腈水合酶突变体的细胞。
在本发明的一种实施方式中，所述细胞包括大肠杆菌。
在本发明的一种实施方式中，所述细胞是以以大肠杆菌BL21为宿主，以pET-系列质粒为载体。
在本发明的一种实施方式中，所述质粒为pET-24(+)。
本发明的第五个目的是提供一种提高腈水合酶酶活力的方法，是将氨基酸序列如SEQ ID NO.5所示的腈水合酶α亚基第20位甘丙基酸突变为缬氨酸。
本发明的第六个目的是提供含有上述腈水合酶突变体的组合物。
在本发明的一种实施方式中，包括但不限于保护剂。
本发明的第七个目的是提供上述的腈水合酶突变体或上述细胞在生产含烟酰胺或丙烯酰胺的产品中的应用。
在本发明的一种实施方式中，以上述腈水合酶突变体，或上述细胞，或上述组合物为催化剂，以烟腈或丙烯腈作为底物进行转化反应。
在本发明的一种实施方式中，以烟腈或丙烯腈作为底物，利用上述细胞进行发酵，发酵后的菌液用于全细胞转化生产烟酰胺或丙烯酰胺。
在本发明的一种实施方式中，所述发酵后的菌液的OD 600为5-10，所述底物的浓度为0.2-1mol/L。
在本发明的一种实施方式中，全细胞转化反应条件为：温度为25-28℃，底物烟腈与湿菌体的质量比为0.5-2。
在本发明的一种实施方式中，全细胞转化反应条件为：温度为25-28℃，底物丙烯腈与湿菌体的质量比为1-1.5。
本发明的第八个目的是提供一种重组表达所述腈水合酶突变体的方法，将表达所述腈水合酶突变体的细胞接种于LB培养基中，35-37℃培养至OD 600为0.6-0.8时，加入诱导剂IPTG于20-30℃诱导12-18h。
在本发明的一种实施方式中，所述方法是将所述细胞接种于含卡那霉素的LB表达培养基中，37℃、200r/min振荡培养至OD 600为0.6-0.8时，加入诱导剂IPTG至0.1mM，Co 2+至0.1mg/L，25℃诱导12-18h。
在本发明的一种实施方式中，所述方法还包括收集所述细胞的菌体，将菌体破碎后收集上清，将上清膜过滤后，用Strep Trap HP柱分离得到腈水合酶突变体。
本发明还提供所述细胞在发酵领域中的应用。
发明的有益效果
有益效果
有益效果：本发明提供的腈水合酶突变体Cal.t Nhase-A20V在70℃的半衰期大约是10min，相比野生酶热稳定性并未有太大变化，突变体Cal.t Nhase-A20V的比酶活为650U/mg，是野生酶的128％。同时该突变体也具备了更好的底物耐受性和产物耐受性，全细胞催化生成烟酰胺最终产量达到598g/L；因此，本发明提供的腈水合酶突变体Cal.t Nhase-A20V，具有很好的酶学性质，有利于以后的工业生产。
对附图的简要说明
附图说明
图1：野生酶和突变酶Cal.t Nhase-A20V 70℃下处理10min的热稳定性。
图2：野生酶和突变酶Cal.t Nhase-A20V在不同浓度底物3-氰基吡啶浓度下的底物耐受性。
图3：野生酶和突变酶Cal.t Nhase-A20V在不同浓度产物烟酰胺浓度下的产物耐受性。
发明实施例
本发明的实施方式
酶活的定义(U)：每分钟转化3-氰基吡啶生成1μmol/L烟酰胺所需的酶量定义为1U。
比酶活(U/mg)：每毫克NHase的酶活。
相对酶活的定义：突变酶在pH＝7.4，温度为30℃反应10 min测得的酶活定义为100％。
LB培养基：蛋白胨10g/L，酵母浸膏5g/L，NaCl 10g/L。
腈水合酶反应体系：底物为490μL 200mM的3-氰基吡啶，加入浓度为0.5mg/mL的纯酶溶液10μL或OD 600＝8的菌液10μL在30℃的温度下反应10min后用500μL乙腈终止反应，并离心去除沉淀，取上清过0.22μm的膜后作为液相测定的样品。
腈水合酶的检测：用HPLC检测，流动相为水∶乙腈＝1∶2；检测波长210nm，流速为0.6mL/min；色谱柱为C18柱。
温度稳定性的确定：将野生酶和突变体在pH＝7.4的KPB缓冲液中，70℃分别处理10min，30min后测定残留酶活，未处理的酶的酶活定义为100％，得到热稳定性结果。
底物耐受性的确定：将野生酶和突变体稀释在pH＝7.4的KPB缓冲液中成为OD 600＝8的菌液，30℃分别在0M、1M的3-氰基吡啶的浓度下30℃孵育30分钟后测定残留酶活，得到底物耐受性结果。
产物耐受性的确定：将野生酶和突变体稀释到pH＝7.4的KPB缓冲液中成为OD 600＝8的菌液，30℃分别在0M、1M、2M的烟酰胺的浓度下孵育30分钟后测定残留酶活，得到产物耐受性结果。
实施例1
将来源于嗜热假诺卡氏菌(Pseudonocardiathermophila)来源的腈水合酶(PtNHase)与来源于温泉热碱芽孢杆菌(Caldalkalibacillusthermarum)Cal.t NHase进行动力学模拟发现，某些氨基酸RMSF值较大，推测这些氨基酸可能影响其热稳定性。因此，构建了以下几个突变体：Cal.t NHase-A20V，Cal.t NHase-H150S(将氨基酸序列如SEQ ID NO.2所示的β亚基第150位的组氨酸突变为丝氨酸)，Cal.t NHase-T104A(将氨基酸序列如SEQ ID NO.2所示的β亚基第104位的苏氨酸突变为丙氨酸)，Cal.t NHase-S152K(将氨基酸序列如SEQ ID NO.2所示的β亚基第152位的丝氨酸突变为赖氨酸)，Cal.t NHase-K185A(将氨基酸序列如SEQ ID NO.2所示的β亚基第185位的赖氨酸突变为丙氨酸)。
(1)突变体的构建：
合成Cal.t-NHase基因(如SEQ ID NO.1所示)，并将该基因克隆于pET24a质粒的NdeI和EcoRI酶切位点处，由苏州金唯智完成，获得pET24a-Cal.tNHase重组质粒。以pET24a-Cal.t NHase为模版，用表1所示引物在表2所示条件下PCR，PCR产物转化E.coli JM109后苏州金唯智测序，将测序结果正确的质粒重新获得携带编码突变体基因的重组质粒pET24a-NHase-A20V，pET24a-Cal.t Nhase-H150S，pET24a-Cal.t Nhase-T104A，pET24a-Cal.t Nhase-S152K，pET24a-Cal.tNhase-K185A，将重组质粒转化E.coli BL21菌株进行表达，获得重组菌株。
表1引物
[Table 1]
Figure PCTCN2019114605-appb-000001
表2全质粒PCR扩增反应体系
[Table 2]
试剂 用量
ddH2O 32μL
5×PS Buffer(Mg2+plus) 10μL
dNTPMixture(2mmol/L) 4μL
P1(10mmol/L) 1μL
P2(10mmol/L) 1μL
pET24a-Cal.tNHase 1μL
Primer STAR HS DNA聚合酶 1μL
共计 50μL
PCR扩增反应条件为：
95℃预变性5min
Figure PCTCN2019114605-appb-000002
95℃ 变性 1min
58℃ 退火 30s 30个循环
72℃ 延伸 2min
72℃ 延伸10min
PCR产物用琼脂糖凝胶电泳方法鉴定。然后将PCR产物纯化、消化后转入大肠杆菌BL21感受态细胞。
(2)将步骤(1)得到的重组大肠杆菌BL21/pET24a-Cal.t NHase-A20V，BL21/pET24a-Cal.t Nhase-H150S，BL21/pET24a-Cal.t Nhase-T104A，BL21/pET24a-Cal.t Nhase-S152K，BL21/pET24a-Cal.t Nhase-K185A接种于5mL卡那霉素浓度为50μg/mL的LB培养基(蛋白胨10g/L、酵母提取物5g/L、NaCl 10g/L)，37℃、200r/min振荡过夜培养。
将上述过夜培养物按1％(v/v)的接种量接种于含卡那霉素浓度为50μg/mL的100mL LB表达培养基(蛋白胨10g/L、酵母提取物5g/L、NaCl 10g/L)中，37℃、200r/min振荡培养至OD 600至0.6-0.8时，加入诱导剂IPTG至0.1mM，Co 2+至0.1mg/L，25℃诱导12-18h得到菌体，12000rpm的转速离心收菌。
(3)将重组菌体用结合缓冲溶液(20mmol/L Na 2HPO 4、280mmol/L NaCl、6mmol/L KCl)浓缩5倍，超声破碎，12000rpm离心40min，上清用0.22μm滤膜过滤。用10倍柱体积的结合缓冲溶液平衡1mL的strep Trap HP柱，用15倍柱体积的结合缓冲溶液洗去非特异性吸附的蛋白，用8倍柱体积的20mM Na 2HPO 4，280mM NaCl，6mM KCl，2.5mM脱硫生物素缓冲液洗脱蛋白，收集样品并用SDS-PAGE分析鉴定。
实施例2
在500μL缓冲反应体系中加入0.5mg/mL实施例1纯化后的突变酶10μL，于70℃金属浴中分别处理0min、10min、20min、30min，测定残留酶活，其中，处理0min的酶活定为100％。
如图1所示，发现突变体在70℃下处理10min，突变酶Cal.t NHase-H150S的酶活急剧下降，其他突变酶相较于野生型并未有太大变化，在后续研究中不研究Cal.t NHase-H150S突变酶的性质。
实施例3
配制不同浓度的产物烟酰胺溶液：0M、2M，将OD 600＝8野生酶和突变体菌液分别在不同底物浓度的溶液中30℃处理30min后用KPB重悬清洗两次细胞，取10μL测定残余酶活，用0M处理的酶活定义为100％。
如图2所示，不用产物处理时的酶活定义为100％，发现突变体在2M的产物烟酰胺下处理20min后，突变酶Cal.t Nhase-A20V的剩余酶活由野生酶的40％提高到69％，而其余突变酶Cal.t NHase-H150S，Cal.t NHase-T104A，Cal.t NHase-S152K，Cal.t NHase-K185A的剩余酶活，相较于野生酶，均出现了不同程度的下降。突变酶Cal.t Nhase-A20V产物耐受性有明显提高，选定Cal.t Nhase-A20V突变酶做后续研究。
实施例4
配制不同浓度的底物溶液：0M、1M，将OD 600＝8野生酶和突变体菌液分别在不同底物浓度的溶液中30℃处理30min后用KPB重悬清洗两次细胞，取10μL测定残余酶活，用0M处理的酶活定义为100％。
如图3所示，0M的底物处理时的酶活定义为100％，发现突变体在1M的底物3-氰基吡啶下30℃处理30min后，突变体的残余酶活由野生酶的52％提高到72％，突变体的底物耐受性有明显的提高。
实施例5
将实施例1步骤(2)中得到的BL21/pET24a-Cal.t NHase-A20V菌液离心收集，水洗后再次离心收集。调整温度为25-28℃，烟腈以0.4mol/L的终浓度加入到OD 600＝8的菌液中，并不断搅拌，当批底物反应完毕后再加入下一批底物，用HPLC检测反应液中各成分的含量，计算得到烟酰胺的浓度为598g/L，如图4所示。

Claims (18)

  1. 一种腈水合酶突变体，其特征在于，含有β亚基、α亚基以及调控蛋白，其分别含SEQ ID NO.2、SEQ ID NO.3、SEQ ID NO.4所示的氨基酸序列。
  2. 编码权利要求1所述腈水合酶突变体的基因。
  3. 含有权利要求2所述基因的载体。
  4. 表达权利要求1所述腈水合酶的细胞。
  5. 如权利要求4所述的细胞，其特征在于，所述细胞包括大肠杆菌。
  6. 如权利要求4或5所述的细胞，其特征在于，所述细胞是以以大肠杆菌BL21为宿主，以pET-系列质粒为载体。
  7. 如权利要求6所述的细胞，其特征在于，所述质粒为pET-24(+)。
  8. 一种提高腈水合酶酶活力的方法，其特征在于，将氨基酸序列如SEQ ID NO.5所示的腈水合酶α亚基第20位甘丙基酸突变为缬氨酸。
  9. 含有权利要求1所述腈水合酶突变体的组合物。
  10. 如权利要求8所述的组合物，其特征在于，包括但不限于保护剂。
  11. 权利要求1所述的腈水合酶突变体或权利要求4所述的细胞在生产含烟酰胺或丙烯酰胺的产品中的应用。
  12. 一种生产烟酰胺或丙烯酰胺的方法，其特征在于，以权利要求1所述的腈水合酶突变体，或权利要求4-7任一所述的细胞，或权利要求9所述的组合物为催化剂，以烟腈或丙烯腈作为底物进行转化反应。
  13. 根据权利要求12所述的方法，其特征在于，以烟腈或丙烯腈作为底物，利用权利要求5-7任一所述的细胞进行发酵，发酵后的菌液用于全细胞转化生产烟酰胺或丙烯酰胺。
  14. 如权利要求13所述的方法，其特征在于，所述发酵后的菌液的OD 600为5-10，所述底物的浓度为0.2-1mol/L。
  15. 如权利要求13所述的方法，其特征在于，全细胞转化反应条件为：温度为25-28℃，底物烟腈与湿菌体的质量比为0.5-2。
  16. 如权利要求13所述的方法，其特征在于，全细胞转化反应条件为：温度为25-28℃，底物丙烯腈与湿菌体的质量比为1-1.5。
  17. 一种生产腈水合酶的方法，其特征在于，将权利要求4-7任一所述的细胞接种于LB培养基中，于35-37℃培养至OD 600为0.6-0.8，加入诱导剂IPTG于20-30℃诱导12-18h。
  18. 权利要求4-7任一所述细胞在发酵领域的应用。
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