WO2020147031A1 - 一种腈水合酶突变体、含该突变体的基因工程菌及其应用 - Google Patents

一种腈水合酶突变体、含该突变体的基因工程菌及其应用 Download PDF

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WO2020147031A1
WO2020147031A1 PCT/CN2019/071964 CN2019071964W WO2020147031A1 WO 2020147031 A1 WO2020147031 A1 WO 2020147031A1 CN 2019071964 W CN2019071964 W CN 2019071964W WO 2020147031 A1 WO2020147031 A1 WO 2020147031A1
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nitrile hydratase
cell
seq
mutant
substrate
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周哲敏
刘中美
郭军玲
周丽
崔文璟
蓝瑶
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江南大学
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Priority to US17/208,027 priority patent/US11332731B2/en

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    • C12Y402/01—Hydro-lyases (4.2.1)
    • C12Y402/01084—Nitrile hydratase (4.2.1.84)

Definitions

  • the invention relates to a nitrile hydratase mutant, genetic engineering bacteria containing the mutant and applications thereof, 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 is in great demand in the market, and it is estimated that it needs more than 2,000 tons per year. However, the current production level of niacinamide in my country is not high and the scale is small, requiring a large amount of imports, about 1,000 tons. Therefore, the use of NHase in the production of nicotinamide has great potential. However, the reaction is an exothermic process, so the high temperature in the production process will affect the performance of the enzyme activity.
  • 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, increase the nitrile hydration Enzyme thermal stability and substrate product tolerance are particularly important.
  • Rhodococcus rhodococcus Rhodococcus rhodochrous J1 is mainly used in industrial production to catalyze the production of nicotinamide.
  • the method of substrate feeding is used.
  • the growth cycle of Rhodococcus is long, requiring 100h, and the production efficiency is not high, only 162g/ (L ⁇ h).
  • the final product concentration is low, only 240g/L.
  • nitrile hydratase The current nitrile hydratase is widespread in nature, among which the most reported nitrile hydratase from bacteria and actinomycetes, such as nitrile hydratase from Rhodococcus, Nocardia, Bacillus, Pseudonocardia. At present, most nitrile hydratase enzymes are not high in thermal stability. Therefore, choosing a nitrile hydratase derived from Pseudonocardia thermophila to obtain a nitrile hydratase with improved stability has important applications for the industrial production of amide products value.
  • the first object of the present invention is to provide a nitrile hydratase mutant containing PtNHase- ⁇ subunit, PtNHase- ⁇ subunit and a regulatory protein PtNHase-p, the amino acid sequences of which are as SEQ ID NO.1, SEQ ID, respectively No. 2, SEQ ID No. 3.
  • the mutant is based on the nitrile hydratase shown in SEQ ID NO. 4, SEQ ID NO. 5, and SEQ ID NO. 3 as the parent enzyme with the amino acid sequences of ⁇ subunit, ⁇ subunit, and regulatory protein as the parent enzyme.
  • Leucine at position 6 of the subunit was mutated to threonine, alanine at position 19 was mutated to valine, phenylalanine at position 126 was mutated to tyrosine, and methionine at position 46 of the ⁇ subunit
  • the acid mutation was lysine, the 47th glycine was mutated to aspartic acid, the 108th glutamic acid was mutated to arginine, and the 212th serine was mutated to tyrosine.
  • the second object of the present invention is to provide a gene encoding the aforementioned nitrile hydratase mutant.
  • the third object of the present invention is to provide cells expressing the aforementioned nitrile hydratase mutant.
  • E. coli BL21 cells are included.
  • pET 24a(+) is used as an expression vector.
  • the method for constructing the above-mentioned cell is: linking the gene encoding the above-mentioned nitrile hydratase mutant with an expression vector and transferring it into E. coli.
  • the gene encoding the nitrile hydratase mutant shown in SEQ ID NO. 8 is connected to an expression vector and transferred into E. coli.
  • the gene encoding the nitrile hydratase mutant shown in SEQ ID NO. 8 is composed of a gene encoding the PtNHase- ⁇ subunit shown in SEQ ID NO.
  • the gene encoding the spacer sequence a shown in NO.6 the gene encoding the PtNHase- ⁇ subunit shown in SEQ ID NO.1
  • the gene encoding the spacer sequence b shown in SEQ ID NO.7 the regulation encoding the regulation shown in SEQ ID NO.3
  • the genes of protein PtNHase-p are connected in sequence
  • the fourth object of the present invention is to provide a composition containing the aforementioned nitrile hydratase mutant.
  • a protective agent is included but not limited to.
  • the fifth object of the present invention is to provide the use of the aforementioned nitrile hydratase mutant or the aforementioned cell in the production of products containing nicotinamide or acrylamide.
  • the sixth object of the present invention is to provide a method for producing nicotinamide or acrylamide, using the above-mentioned nitrile hydratase mutant, or the above-mentioned cell, or the above-mentioned composition as a catalyst, and using nicotinonitrile or acrylonitrile as a substrate.
  • the substance undergoes a conversion reaction.
  • nicotinonitrile or acrylonitrile is used as a substrate, and the cell according to any one of claims 4-8 is used for fermentation, and the fermentation broth is used for whole cell transformation to produce nicotinamide or propylene Amide.
  • the conditions for the cell fermentation are: the recombinant E. coli broth cultured for 6-8 hours is inoculated into the fermentor culture medium at an inoculum of 5-8%, and cultured at 35-38°C When the OD 600 reaches 70-75, the temperature drops to 28-30°C, and the inducer is added at a constant flow rate of 0.20-0.22g/(L ⁇ h) to induce the culture for 35-40h to end the fermentation.
  • the whole cell transformation reaction conditions are as follows: adjust the temperature to 25-28°C, the mass ratio of the substrate nicotinonitrile to the wet bacteria is 0.5-2, and add the next step after the substrate reaction is completed. A batch of substrates.
  • the whole cell transformation reaction conditions are as follows: adjust the temperature to 25-28°C, the mass ratio of the substrate acrylonitrile to the wet bacteria is 1-1.5, and then add the next step after the substrate reaction is completed. A batch of substrates.
  • the nitrile hydratase mutant ⁇ L6T/A19V/F126Y- ⁇ M46K/G47N/E108R/S212Y provided by the present invention has an optimal temperature of 36°C, and a treatment at 50°C for 80 minutes still has 53% residual enzyme activity, compared to the control Enzyme mutant ⁇ L6T/A19V/F126Y- ⁇ M46K/E108R/S212Y (mutation of leucine at position 6 of the ⁇ subunit of the parent enzyme to threonine, mutation of alanine at position 19 to valine, and position 126 Phenylalanine was mutated to tyrosine, the 46th methionine of the ⁇ subunit was mutated to lysine, the 47th glycine was mutated to aspartic acid, and the 108th glutamic acid was mutated to arginine, Serine 212 was mutated to tyrosine) The remaining 37% enzyme activity was left after
  • the enzyme activity is the highest, which is defined as 100%, and the substrate tolerance of the mutant enzyme is not affected; after the mutant is treated with 2M product nicotinamide for 20 minutes, the mutation The remaining enzyme activity of the enzyme increased from 28% of the control to 52%, and the mutant also has better product tolerance; therefore, the nitrile hydratase mutant ⁇ L6T/A19V/F126Y- ⁇ M46K/G47N/ E108R/S212Y has good enzymatic properties, which is beneficial to future industrial production.
  • the present invention constructs recombinant Escherichia coli expressing the nitrile hydratase mutant ⁇ L6T/A19V/F126Y- ⁇ M46K/G47N/E108R/S212Y to obtain a nitrile hydratase strain with high enzyme activity.
  • the pure enzyme ratio of the recombinant nitrile hydratase is The enzyme activity reached 907.69U/mg.
  • FIG. 1 SDS-PAGE electrophoresis diagram of PtNHase protein expression, where M is the protein molecular weight standard (6.5-200KDa), and 1 is the cell disruption solution of E. coli BL21/pET24a- ⁇ L6T/A19V/F126Y- ⁇ M46K/E108R/S212Y control bacteria Supernatant; 2 is the supernatant of the cell disruption liquid induced by E. coli BL21/pET24a- ⁇ L6T/A19V/F126Y- ⁇ M46K/G47N/E108R/S212Y recombinant bacteria.
  • Figure 3 The relative enzyme activity changes under different 3-cyanopyridine concentrations, bacteria 1: BL21/pET24a- ⁇ L6T/A19V/F126Y- ⁇ M46K/E108R/S212Y; bacteria 2: BL21/pET24a- ⁇ L6T/A19V/F126Y- ⁇ M46K/E108R/S212Y.
  • Figure 4 Relative enzyme activity changes under different product nicotinamide concentrations, bacteria 1: BL21/pET24a- ⁇ L6T/A19V/F126Y- ⁇ M46K/E108R/S212Y; bacteria 2: BL21/pET24a- ⁇ L6T/A19V/F126Y- ⁇ M46K/ G47N/E108R/S212Y.
  • Figure 5 Schematic diagram of whole-cell catalytic production of nicotinamide.
  • Figure 6 Schematic diagram of whole-cell catalytic production of acrylamide.
  • the reaction solution was filtered through a 0.22 ⁇ m microporous membrane and loaded onto a C18 chromatographic column for HPLC analysis.
  • Cell density: UV-1800PC UV-Vis spectrophotometer measures OD 600 and converts it according to the relationship between absorbance and OD. Conversion relationship: 1g/L 0.3683 OD 600 .
  • LB medium peptone 10g/L, yeast extract 5g/L, NaCl 10g/L.
  • Fermenter culture medium (g/L): glucose 12.0, potassium dihydrogen phosphate 13.5, diammonium hydrogen phosphate 4.0, citric acid 1.7, magnesium sulfate 1.68, and trace elements 10 mL.
  • Feed medium g/L: glucose 500.0, magnesium sulfate 7.33, yeast extract 4.0, tryptone 4.0.
  • Inducer (g/100mL): lactose 10.0, CoCl 2 ⁇ 6H 2 O 0.8.
  • Trace elements (g/100mL): ferrous sulfate heptahydrate 1.0, zinc sulfate heptahydrate 0.525, copper sulfate pentahydrate 0.3, manganese sulfate tetrahydrate 0.05, borax 0.023, calcium chloride 0.2, ammonium molybdate 0.01.
  • HPLC detection of nitrile hydratase content Agilent 1260 was used for HPLC detection, the mobile phase was water acetonitrile buffer; the detection wavelength was 210nm, the flow rate was 0.6mL/min; the chromatographic column was a C18 column.
  • the nitrile hydratase mutant ⁇ L6T/A19V/F126Y- ⁇ M46K/E108R/S212Y gene was synthesized by chemical synthesis, and the gene was cloned at the NdeI and Bpu10I restriction sites of pET24a plasmid, completed by General Biological Systems (Anhui) Co., Ltd. , The recombinant plasmid pET24a- ⁇ L6T/A19V/F126Y- ⁇ M46K/E108R/S212Y was obtained. Using pET24a- ⁇ L6T/A19V/F126Y- ⁇ M46K/E108R/S212Y as a template, PCR was performed under the conditions shown in Table 1.
  • the upstream primer sequence information used is shown in SEQ ID NO. 9, and the downstream primer sequence information used is shown in SEQ ID NO. Shown in .10.
  • the PCR product was transformed into E. coli JM109 to obtain the recombinant plasmid pET24a-- ⁇ L6T/A19V/F126Y- ⁇ M46K/G47N/E108R/S212Y carrying the gene encoding the mutant.
  • the recombinant plasmid pET24a- ⁇ L6T/A19V/F126Y- ⁇ M46K/G47N/E108R/S212Y was transformed into E. coli BL21 strain to obtain the recombinant strain BL21/pET24a- ⁇ L6T/A19V/F126Y- ⁇ M46K/G47N/E108R/S212Y.
  • a control plasmid and a control strain expressing the nitrile hydratase mutant ⁇ L6T/A19V/F126Y- ⁇ M46K/E108R/S212Y were constructed in a similar way.
  • 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.
  • the above-mentioned overnight culture was inoculated into 100 mL LB expression medium (peptone 10g/L, yeast extract 5g/L, NaCl 10g/L) containing kanamycin at a concentration of 100 ⁇ g/mL at an inoculum of 1% (v/v).
  • LB expression medium peptone 10g/L, yeast extract 5g/L, NaCl 10g/L
  • kanamycin 100 ⁇ g/mL at an inoculum of 1% (v/v).
  • L at 37°C and 200r/min shaking culture until the OD 600 reaches 0.6-0.8 at a wavelength of 600nm, add the inducer IPTG to 0.1mM, induce at 20°C for 12-18h to obtain the bacteria, and centrifuge at 5000g to harvest the bacteria.
  • the BL21/pET24a- ⁇ L6T/A19V/F126Y- ⁇ M46K/G47N/E108R/S212Y recombinant E. coli was inoculated into 5 mL of LB medium with a kanamycin concentration of 100 ⁇ g/mL, and cultured overnight at 37° C. with 200 r/min shaking.
  • Example 2 Add 10 ⁇ L of the mutant enzyme purified in Example 1 with 0.5 mg/ml in a 500 ⁇ L buffer reaction system (20 mmol/L Na 2 HPO 4 , 280 mmol/L NaCl and 6 mmol/L KCl), and store it in a metal bath at 50°C. Take a sample for 20 minutes to determine the residual enzyme activity.
  • a 500 ⁇ L buffer reaction system (20 mmol/L Na 2 HPO 4 , 280 mmol/L NaCl and 6 mmol/L KCl
  • the enzyme activity is defined as 100%, and the substrate tolerance is not significantly affected after mutation.
  • the enzyme activity without product treatment was defined as 100%. It was found that after the mutant was treated with 2M product nicotinamide for 20 minutes, the remaining enzyme activity of the mutant enzyme increased from 28% of the control to 52%. The tolerance of the mutants to the product nicotinamide was significantly improved.
  • Recombinant Escherichia coli BL21/pET24a- ⁇ L6T/A19V/F126Y- ⁇ M46K/G47N/E108R/S212Y were inoculated into 5 mL of LB medium with a kanamycin concentration of 100 ⁇ g/mL, and cultured overnight at 37° C. with 200 r/min shaking.
  • the above-mentioned overnight culture was inoculated into an LB medium containing a kanamycin concentration of 100 ⁇ g/mL at an inoculum amount of 1%, and cultured with shaking at 37° C. and 200 r/min for 6-8 hours.
  • the above-mentioned culture was inoculated into a 2L fermenter fermentation medium with a kanamycin concentration of 100 ⁇ g/mL at a 6% inoculum, and fed at 37°C.
  • the OD 600 reached 60
  • the temperature dropped to 30°C.
  • Add 140-150 mL of inducer at a constant flow rate of 0.20-0.22g/(L ⁇ h) and induce the culture for 36 hours to end the fermentation.
  • the enzyme activity reached 24763.48U/mL.

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Abstract

本发明公开了一种腈水合酶突变体、含该突变体的基因工程菌及其应用，属于酶工程技术领域。本发明是将腈水合酶突变体αL6T/A19V/F126Y-βM46K/E108R/S212Y(公开于发明专利CN102216455A)的第47位甘氨酸突变为天冬酰胺，获得的新突变酶具有更好的温度耐受性和产物耐受性，有利于以后的工业生产。将含该腈水合酶突变体的重组菌株高密度发酵，以烟腈为底物，进行全细胞催化反应制备烟酰胺。此法与化学生产法相比，生产工艺安全清洁，无环境污染，与酶法相比，底物价格便宜，催化效率高，终产物烟酰胺产率95％以上，浓度达到680g/L，简化了产物的分离纯化步骤。

Description

一种腈水合酶突变体、含该突变体的基因工程菌及其应用 技术领域
本发明涉及一种腈水合酶突变体、含该突变体的基因工程菌及其应用，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，已经被广泛的用于饲料、食品、制药等行业。烟酰胺市场需求量很大，估计每年需要2000多吨，但目前我国烟酰胺的生产水平不高，规模不大，需要大量的进口，约1000吨。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，提高腈水合酶的热稳定性和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Rhodococcus rhodochrous 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，只有162g/(L·h)。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。
目前的腈水合酶在自然界中广泛存在，其中有关来自细菌和放线菌的腈水合酶报道最多例如来自红球菌，诺卡氏菌，芽孢杆菌，假诺卡氏菌的腈水合酶。目前大部分腈水合酶热稳定性都不高，因此，选择来源于嗜热假诺卡氏菌(Pseudonocardia thermophila)获得一种稳定性提高的腈水合酶对于酰胺类产品的工业化生产具有重要的应用价值。
发明内容
本发明的第一个目的是提供一种腈水合酶突变体，含有PtNHase-α亚基、PtNHase-β亚基以及一个调控蛋白PtNHase-p，其氨基酸序列分别如SEQ ID NO.1、SEQ ID NO.2、SEQ ID NO.3所示。
所述突变体是以α亚基、β亚基、调控蛋白的氨基酸序列分别如SEQ ID NO.4、SEQ ID NO.5、SEQ ID NO.3所示的腈水合酶为亲本酶，将α亚基的第6位亮氨酸突变为苏氨酸、第19位丙氨酸突变为缬氨酸、第126位苯丙氨酸突变为酪氨酸，β亚基的第46位甲硫氨酸突变为赖氨酸、第47位甘氨酸突变为天冬氨酸、第108位谷氨酸突变为精氨酸、第212位丝氨酸突变为酪氨酸。
。
本发明的第二个目的是提供编码上述腈水合酶突变体的基因。
本发明的第三个目的是提供表达上述腈水合酶突变体的细胞。
在本发明的一种实施方式中，包括大肠杆菌BL21细胞。
在本发明的一种实施方式中，以pET 24a(+)为表达载体。
在本发明的一种实施方式中，上述细胞的构建方法是：将编码上述腈水合酶突变体的基因与表达载体连接，转入大肠杆菌中。
在本发明的一种实施方式中，是将如SEQ ID NO.8所示的编码腈水合酶突变体的基因与表达载体连接，转入大肠杆菌中。
在本发明的一种实施方式中，所述如SEQ ID NO.8所示的编码腈水合酶突变体的基因是由编码SEQ ID NO.2所示PtNHase-β亚基的基因、编码SEQ ID NO.6所示间隔序列a的基因、编码SEQ ID NO.1所示PtNHase-α亚基的基因、编码SEQ ID NO.7所示间隔序列b的基因、编码SEQ ID NO.3所示调控蛋白PtNHase-p的基因依次连接
本发明的第四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。
本发明的第五个目的是提供上述的腈水合酶突变体或上述的细胞在生产含烟酰胺或丙烯酰胺的产品中的应用。
本发明的第六个目的是提供一种生产烟酰胺或丙烯酰胺的方法，以上述的腈水合酶突变体，或上述的细胞，或上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。
在本发明的一种实施方式中，以烟腈或丙烯腈作为底物，利用权利要求4-8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。
在本发明的一种实施方式中，所述细胞发酵的条件为：将培养6-8h的重组大肠杆菌菌液按5-8％的接种量接种于发酵罐培养基中，35-38℃培养，当OD 600达到70-75时，温度降至28-30℃，以0.20-0.22g/(L·h)的恒速流加诱导剂，诱导培养35-40h结束发酵。
在本发明的一种实施方式中，全细胞转化反应条件为：调整温度为25-28℃，底物烟腈与湿菌体的质量比为0.5-2，当底物反应完毕后再加入下一批底物。
在本发明的一种实施方式中，全细胞转化反应条件为：调整温度为25-28℃，底物丙烯腈与湿菌体的质量比为1-1.5，当底物反应完毕后再加入下一批底物。
本发明的有益效果：
第一，本发明提供的腈水合酶突变体αL6T/A19V/F126Y-βM46K/G47N/E108R/S212Y的最 适温度为36℃，50℃处理80分钟仍有53％残留的酶活性，相比对照酶突变体αL6T/A19V/F126Y-βM46K/E108R/S212Y(将亲本酶的α亚基的第6位亮氨酸突变为苏氨酸、第19位丙氨酸突变为缬氨酸、第126位苯丙氨酸突变为酪氨酸，β亚基的第46位甲硫氨酸突变为赖氨酸、第47位甘氨酸突变为天冬氨酸、第108位谷氨酸突变为精氨酸、第212位丝氨酸突变为酪氨酸)50℃处理80分钟剩余37％酶活，提高了43％，突变体热稳定性有明显提高。在当底物3-氰基吡啶浓度为0.2M时，酶活最高，定义为100％，突变酶的底物耐受性未受到影响；突变体在2M的产物烟酰胺下处理20min后，突变酶的剩余酶活由对照的28％提高到52％同时该突变体也具备了更好的产物耐受性；因此，本发明提供的腈水合酶突变体αL6T/A19V/F126Y-βM46K/G47N/E108R/S212Y具有很好的酶学性质，有利于以后的工业生产。
第二，本发明通过构建表达腈水合酶突变体αL6T/A19V/F126Y-βM46K/G47N/E108R/S212Y的重组大肠杆菌，获得具有高酶活的腈水合酶菌株，重组腈水合酶的纯酶比酶活达907.69U/mg。将重组菌株高密度发酵，以烟腈和丙烯腈作为底物，进行全细胞催化反应制备烟酰胺和丙烯酰胺，烟酰胺的产量达680g/L，丙烯酰胺的产量达514.8g/L。此法与玫瑰色红球菌Rhodococcus rhodochrous J1催化生成烟酰胺相比，终产物烟酰胺和丙烯酰胺的产率达95％以上，且简化了产物的分离纯化步骤，发酵周期短，生产效率高。
附图说明
图1：PtNHase蛋白表达的SDS-PAGE电泳图，其中M为蛋白分子量标准(6.5-200KDa)，1为大肠杆菌BL21/pET24a-αL6T/A19V/F126Y-βM46K/E108R/S212Y对照菌的细胞破碎液上清；2为大肠杆菌BL21/pET24a-αL6T/A19V/F126Y-βM46K/G47N/E108R/S212Y重组菌诱导后的细胞破碎液上清。
图2：在50℃下保存后酶的热稳定性曲线，酶1：αL6T/A19V/F126Y-βM46K/E108R/S212Y酶2：αL6T/A19V/F126Y-βM46K/G47N/E108R/S212Y。
图3：在不同3-氰基吡啶浓度下的相对酶活变化，菌1：BL21/pET24a-αL6T/A19V/F126Y-βM46K/E108R/S212Y；菌2：BL21/pET24a-αL6T/A19V/F126Y-βM46K/E108R/S212Y。
图4：在不同产物烟酰胺浓度下的相对酶活变化，菌1：BL21/pET24a-αL6T/A19V/F126Y-βM46K/E108R/S212Y；菌2：BL21/pET24a-αL6T/A19V/F126Y-βM46K/G47N/E108R/S212Y。
图5：全细胞催化生产烟酰胺示意图。
图6：全细胞催化生产丙烯酰胺示意图。
具体实施方式
(一)酶活、比酶活和相对酶活的定义及酶活的测定方法
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αL6T/A19V/F126Y-βM46K/G47N/E108R/S212Y在pH＝8.4，温度为36℃反应10分钟测得的酶活定义为100％。
测发酵液酶活的方法：将100μL，OD 600＝1.0的菌体(磷酸缓冲液溶解)加入400μL、125mmol/L烟腈溶液中，25℃反应10min，500μL乙腈终止反应，加入终止液后立即在4℃，12000r/min条件下离心1min，吸取上清。反应液经0.22μm微孔滤膜过滤，载入C18色谱柱进行HPLC分析，流动相为乙腈水(乙腈：水＝1:2)的混合溶液。烟腈与烟酰胺测定方法：流动相为乙腈水(乙腈：水＝1:2)的混合溶液，流速0.6mL/min，吸光值215nm，采集时间12min。细胞密度：UV-1800PC型紫外可见分光光度计测量OD 600，根据吸光值和OD的关系换算，换算关系：1g/L＝0.3683OD 600。
测腈水合酶酶活的方法：底物为490μL 200mM的烟腈，加入浓度为0.5μg/μL的纯酶溶液10μL或OD＝10的菌液10μL在36℃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。
(二)培养基
LB培养基：蛋白胨10g/L，酵母浸膏5g/L，NaCl 10g/L。
发酵罐培养基(g/L)：葡萄糖12.0，磷酸二氢钾13.5，磷酸氢二铵4.0，柠檬酸1.7，硫酸镁1.68，微量元素10mL。
补料培养基(g/L)：葡萄糖500.0，硫酸镁7.33，酵母提取物4.0，胰蛋白胨4.0。
诱导剂(g/100mL)：乳糖10.0，CoCl 2·6H 2O 0.8。
微量元素(g/100mL)：七水硫酸亚铁1.0，七水硫酸锌0.525，五水硫酸铜0.3，四水硫酸锰0.05，硼砂0.023，氯化钙0.2，钼酸铵0.01。
(三)HPLC检测腈水合酶、烟酰胺和丙烯酰胺含量的方法
腈水合酶反应体系：底物为490μL 200mM的3-氰基吡啶，加入浓度为0.5μg/μL的纯酶溶液10μL或在600nm波长下，OD＝10的菌液10μL在36℃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。
HPLC检测腈水合酶含量：采用安捷伦1260进行HPLC检测，流动相为水乙腈缓冲液；检测波长210nm，流速为0.6mL/min；色谱柱为C18柱。
HPLC检测烟酰胺或丙烯酰胺的含量：采用安捷伦1260进行HPLC检测，流动相为水乙腈缓冲液；检测波长210nm，流速为0.6mL/min；色谱柱为C18柱。
(四)温度稳定性的确定
以酶突变体αL6T/A19V/F126Y-βM46K/E108R/S212Y(将同一亲本酶的α亚基的第6位亮氨酸突变为苏氨酸、第19位丙氨酸突变为缬氨酸、第126位苯丙氨酸突变为酪氨酸，β亚基的第46位甲硫氨酸突变为赖氨酸、第47位甘氨酸突变为天冬氨酸、第108位谷氨酸突变为精氨酸、第212位丝氨酸突变为酪氨酸；公开于发明专利CN102216455A中)；公开于发明专利CN102216455A中)为对照。
将对照和突变体在pH＝8.4的KPB缓冲液中，50℃分别保温20分钟、40分钟、60分钟、80分钟、100分钟后测定残留酶活，得到温度稳定性结果。
(五)底物耐受性的确定
将对照和突变体稀释在pH＝8.4的KPB缓冲液中成为在600nm波长下OD＝10的菌液，36℃分别在200mM、400mM、600mM、800mM、1000mM的3-氰基吡啶的浓度下保温20分钟后测定残留酶活，得到底物耐受性结果。
(六)产物耐受性的确定
将对照和突变体稀释到pH＝8.4的KPB缓冲液中成为在600nm波长下OD＝10的菌液，36℃分别在0.5M、1M、1.5M、2M的烟酰胺的浓度下保温20分钟后测定残留酶活，得到产耐受性结果。
实施例1重组大肠杆菌的构建
(1)突变体αL6T/A19V/F126Y-βM46K/G47N/E108R/S212Y的构建：
化学合成法合成腈水合酶突变体αL6T/A19V/F126Y-βM46K/E108R/S212Y基因，并将该基因克隆于pET24a质粒的NdeI和Bpu10I酶切位点处，由通用生物系统(安徽)有限公司完成，获得pET24a-αL6T/A19V/F126Y-βM46K/E108R/S212Y重组质粒。以pET24a-αL6T/A19V/F126Y-βM46K/E108R/S212Y为模版，在表1所示条件下进行PCR，所用上游引物序列信息如SEQ ID NO.9所示，所用下游引物序列信息如SEQ ID NO.10所示。PCR产物转入E.coli JM109后获得携带编码突变体基因的重组质粒pET24a--αL6T/A19V/F126Y-βM46K/G47N/E108R/S212Y。将重组质粒pET24a--αL6T/A19V/F126Y-βM46K/G47N/E108R/S212Y转化E.coli BL21菌株，获得重组菌株 BL21/pET24a-αL6T/A19V/F126Y-βM46K/G47N/E108R/S212Y。
以类似的方法构建表达腈水合酶突变体αL6T/A19V/F126Y-βM46K/E108R/S212Y的对照质粒和对照菌株。
表1全质粒PCR扩增反应体系
Figure PCTCN2019071964-appb-000001
PCR扩增反应条件为：
Figure PCTCN2019071964-appb-000002
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BL21/pET24a-αL6T/A19V/F126Y-βM46K/G47N/E108R/S212Y重组大肠杆菌接种于4mL卡那霉素浓度为100μg/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100μg/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振荡培养至在600nm波长下OD 600至0.6-0.8时，加入诱导剂IPTG至0.1mM，20℃诱导12-18h得到菌体，5000g的转速离心收菌。
(3)将重组菌体溶于20mL结合缓冲溶液(20mmol/LNa 2HPO 4、280mmol/L NaCl、6mmol/L KCl)，超声破碎，13000g离心25min，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20mMNa 2HPO 4、280mMNaCl、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腈水合酶的表达
将BL21/pET24a-αL6T/A19V/F126Y-βM46K/G47N/E108R/S212Y重组大肠杆菌接种于5mL卡那霉素浓度为100μg/mL的LB培养基，37℃、200r/min振荡过夜培养。将上述过夜培养物按1％的接种量接种于含卡那霉素浓度为100μg/mL的LB培养基，37℃、200r/min振荡培养至菌液OD 600至0.6-0.8，加入IPTG至终浓度0.4mmol/L，20℃诱导培养16-20h，收集菌体超声破碎，通过Tris-tricine SDS-PAGE方法分析鉴定腈水合酶重组蛋白表达水平，结果如图1所示。通过超声破碎，12000rpm离心60min，用亲和层析柱Strep Trap FF纯化蛋白，重组腈水合酶的纯酶的比酶活为907.69U/mg。
实施例3热稳定性测定
在500μL缓冲反应体系(20mmol/L Na 2HPO 4、280mmol/L NaCl和6mmol/L KCl)中加入0.5mg/ml实施例1纯化后的突变酶10μL，保存于50℃金属浴中，每隔20分钟取样，测定残留酶活。
如图2所示，发现突变体在50℃下处理80min后，突变酶的剩余酶活由对照的37％(剩余酶活333U/mg)提高到53％(剩余酶活为424U/mg)；在50℃处理100min后，突变酶的相对酶活由对照的24％(剩余酶活216U/mg)提高到45％(剩余酶活360U/mg)。突变体热稳定性有明显提高。
实施例4底物耐受性测定
配制0.2M、0.4M、0.6M、0.8M、1M不同浓度的底物溶液，将在600nm波长下，OD＝10的对照和突变体菌液分别在不同底物浓度的溶液中36℃处理20min后用KPB重悬清洗两次细胞，取10μL测定残余酶活。
如图3所示，在当底物3-氰基吡啶浓度为0.2M时，酶活定义为100％，经过突变以后其底物耐受性并没有受到明显影响。
实施例5产物耐受性测定
配制0.5M、1M、1.5M、2M不同浓度的产物烟酰胺溶液，将在600nm波长下，OD 600＝10的对照和突变体菌液分别在不同底物浓度的溶液中36℃处理20min后用KPB重悬清洗两次 细胞，取10μL测定残余酶活。
如图4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，突变酶的剩余酶活由对照的28％提高到52％。突变体对产物烟酰胺的耐受性有明显提高。
实施例6重组大肠杆菌高密度发酵
将重组大肠杆菌BL21/pET24a-αL6T/A19V/F126Y-βM46K/G47N/E108R/S212Y接种于5mL卡那霉素浓度为100μg/mL的LB培养基，37℃、200r/min振荡过夜培养。将上述过夜培养物按1％的接种量接种于含卡那霉素浓度为100μg/mL的LB培养基，37℃、200r/min振荡培养6-8h。将上述培养物按6％的接种量接种于含卡那霉素浓度为100μg/mL的2L发酵罐发酵培养基中，37℃补料培养，当OD 600达到60时，温度降至30℃，以0.20-0.22g/(L·h)的恒速流加140-150mL的诱导剂，诱导培养36h结束发酵。发酵结束后，酶活达到24763.48U/mL。
实施例7全细胞催化法生产烟酰胺
将高密度发酵后的菌液离心收集，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＝150-160.0的发酵液中，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680g/L，如图5所示。
实施例8全细胞催化法生产丙烯酰胺
将高密度发酵后的菌液离心收集，水洗后再次离心收集。调整温度为25-28℃，丙烯腈以64g/L的终浓度加入到OD 600＝150-160.0的发酵液中，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，计算得到丙烯酰胺的浓度为514.8g/L，如图6所示。
虽然本发明已以较佳实施例公开如上，但其并非用以限定本发明，任何熟悉此技术的人，在不脱离本发明的精神和范围内，都可做各种的改动与修饰，因此本发明的保护范围应该以权利要求书所界定的为准。
Figure PCTCN2019071964-appb-000003
Figure PCTCN2019071964-appb-000004
Figure PCTCN2019071964-appb-000005
Figure PCTCN2019071964-appb-000006
Figure PCTCN2019071964-appb-000007
Figure PCTCN2019071964-appb-000008
Figure PCTCN2019071964-appb-000009
Figure PCTCN2019071964-appb-000010

Claims (16)

  1. 一种腈水合酶突变体，其特征在于，含有PtNHase-α亚基、PtNHase-β亚基以及一个调控蛋白PtNHase-p，其氨基酸序列分别如SEQ ID NO.1、SEQ ID NO.2、SEQ ID NO.3所示。
  2. 编码权利要求1所述腈水合酶突变体的基因。
  3. 表达权利要求1所述腈水合酶突变体的细胞。
  4. 如权利要求3所述的细胞，其特征在于，包括大肠杆菌BL21细胞。
  5. 如权利要求4所述的细胞，其特征在于，以pET 24a(+)为表达载体。
  6. 如权利要求3所述的细胞，其特征在于，其构建方法是：将编码权利要求1所述腈水合酶突变体的基因与表达载体连接，转入大肠杆菌中。
  7. 如权利要求6所述的细胞，其特征在于，是将如SEQ ID NO.8所示的编码腈水合酶突变体的基因与表达载体连接，转入大肠杆菌中。
  8. 如权利要求7所述的细胞，其特征在于，所述如SEQ ID NO.8所示的编码腈水合酶突变体的基因是由编码SEQ ID NO.2所示PtNHase-β亚基的基因、编码SEQ ID NO.6所示间隔序列a的基因、编码SEQ ID NO.1所示PtNHase-α亚基的基因、编码SEQ ID NO.7所示间隔序列b的基因、编码SEQ ID NO.3所示调控蛋白PtNHase-p的基因依次连接
  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9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3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3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4-8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，所述细胞发酵的条件为：将培养6-8h的重组大肠杆菌菌液按5-8％的接种量接种于发酵罐培养基中，35-38℃培养，当OD 600达到70-75时，温度降至28-30℃，以0.20-0.22g/(L·h)的恒速流加诱导剂，诱导培养35-40h结束发酵。
  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：调整温度为25-28℃，底物烟腈与湿菌体的质量比为0.5-2，当底物反应完毕后再加入下一批底物。
  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：调整温度为25-28℃，底物丙烯腈与湿菌体的质量比为1-1.5，当底物反应完毕后再加入下一批底物。
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CN113462677B (zh) * 2021-07-29 2023-02-10 浙江大学杭州国际科创中心 一种α亚基突变的腈水合酶突变体及其应用
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