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Escherichia coli recombinant strain for heterologous expression of nitrile hydratase and application thereof

A technology of Escherichia coli and nitrile hydratase, applied in the field of bioengineering, can solve the problems of low production efficiency, long growth cycle, difficult product purification, etc., and achieve the effects of high production efficiency, short fermentation cycle, and simplified separation and purification steps

Inactive Publication Date: 2019-01-22
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The technology for producing acrylamide was the first to use sulfuric acid hydration method, but the process is complicated, the product purification is difficult, and it pollutes the environment. Therefore, the development of new production methods has always been the key direction of research in related fields.
[0003] At present, in industrial production, Rhodococcus rhodochrous J1 is mainly used to catalyze the formation of nicotinamide and acrylamide, and the substrate feeding method is adopted. However, the growth cycle of Rhodococcus is long, requiring 100 hours, and the production efficiency is not high. Niacinamide yields up to 162g / L, while acrylamide yields up to 300g / L
At present, there are also reports on the production of nicotinamide by recombinant bacteria, but the concentration of the final product is low, only 240g / L

Method used

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  • Escherichia coli recombinant strain for heterologous expression of nitrile hydratase and application thereof
  • Escherichia coli recombinant strain for heterologous expression of nitrile hydratase and application thereof
  • Escherichia coli recombinant strain for heterologous expression of nitrile hydratase and application thereof

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Embodiment 1

[0044] Embodiment 1 Construction of recombinant Escherichia coli BL21 / pET24a-PtNHase

[0045] The NHase gene sequence derived from Pseudonocardia thermophila was retrieved through NCBI, optimized through the codon online optimization website Graphical Codon Usage Analyzer, and a spacer sequence was added between the nhhB and nhhA genes, nhhA and nhhP genes, specifically: the encoded amino acid sequence The nhhB gene of SEQ ID NO.1, the spacer sequence a shown in SEQ ID NO.4, the nhhA gene of the encoded amino acid sequence SEQ ID NO.2, the spacer sequence b shown in SEQ ID NO.5, the encoded amino acid sequence SEQ The nhhP gene of ID NO.3 is sequentially connected to obtain the gene NHase of nitrile hydratase, the nucleotide sequence is shown in SEQ ID NO.6, and the electrophoresis result is shown in figure 1 Shown, synthesized by General Biosystems (Anhui) Co., Ltd.

[0046] The optimized NHase sequence did not contain any restriction endonuclease sites. Specific primers P1 ...

Embodiment 2

[0050] The expression of embodiment 2 nitrile hydratase

[0051] Recombinant Escherichia coli BL21 / pET24-PtNHase was inoculated in 5 mL of LB medium with a kanamycin concentration of 100 μg / mL, and cultured overnight at 37°C with shaking at 200 r / min. Inoculate the above-mentioned overnight culture into LB medium containing 100 μg / mL of kanamycin at an inoculum size of 1%, and culture it with shaking at 37°C and 200 r / min until the bacterial liquid OD 600 To 0.6-0.8, add IPTG to a final concentration of 0.4mmol / L, induce culture at 20°C for 16-20h, collect the bacteria and ultrasonically disrupt them, analyze and identify the expression level of nitrile hydratase recombinant protein by Tris-tricine SDS-PAGE method, the results are as follows image 3 shown. After sonication and centrifugation at 12000rpm for 60min, the protein was purified with affinity chromatography column Strep Trap FF, and the specific enzyme activity of the pure recombinant nitrile hydratase was 907.69U / ...

Embodiment 3

[0052] Embodiment 3 Recombinant escherichia coli high-density fermentation

[0053] Recombinant Escherichia coli BL21 / pET24-PtNHase was inoculated in 5 mL of LB medium with a kanamycin concentration of 100 μg / mL, and cultured overnight at 37°C with shaking at 200 r / min. The above-mentioned overnight culture was inoculated into LB medium containing 100 μg / mL of kanamycin 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 2L fermenter fermentation medium containing kanamycin concentration of 100 μg / mL according to the inoculum size of 6%, fed culture at 37 ° C, when OD 600 When it reaches 60°C, the temperature is lowered to 30°C, and 140-150mL of inducer is added at a constant rate of 0.20-0.22g / (L·h), and the induction culture is 36h to end the fermentation. After the fermentation, the enzyme activity reached 24763.48U / mL.

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Abstract

The invention discloses an escherichia coli recombinant strain for heterologous expression of nitrile hydratase and application thereof, and belongs to the field of bioengineering. The escherichia coli recombinant strain with high nitrile hydrase activity is obtained by constructing recombinant escherichia coli. The recombinant escherichia coli is subjected to high-density fermentation. Nicotinamide or acrylamide is adopted as a substrate to conduct whole-cell catalytic reaction to prepare nicotinamide or acrylonitrile. Compared with a chemical production method, the method is high in catalytic efficiency, and yield of the nicotinamide or acrylamide is over 95%.

Description

technical field [0001] The invention relates to an Escherichia coli recombinant bacterium expressing nitrile hydratase heterologously and its application, belonging to the technical field of bioengineering. Background technique [0002] Niacinamide is a vitamin that has been widely used in feed, food, pharmaceutical and other industries. The market demand for nicotinamide is huge, and it is estimated that more than 2,000 tons are needed per year. However, the current production level of nicotinamide in my country is not high, and the scale is small, requiring a large amount of imports, about 1,000 tons. Acrylamide is one of the important products of amide compounds produced by the microbial method of nitrile hydratase. It is also a kind of fine chemical raw material with a wide range of uses. It is widely used in petroleum, papermaking, mining, textile, food and other industries. It has huge potential Economic Value. As for the production of acrylamide, sulfuric acid hydra...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/70C12N9/88C12N15/60C12P13/02C12P17/12C12R1/19
CPCC12N9/88C12N15/70C12P13/02C12P17/12C12Y402/01084
Inventor 周哲敏刘中美周丽蓝瑶崔文璟郭军玲
Owner JIANGNAN UNIV
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