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A strain of Escherichia coli recombinant strain heterologously expressing heat-resistant nitrile hydratase and its application

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

Active Publication Date: 2020-12-29
JIANGNAN UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

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
Moreover, the existing nitrile hydratase is difficult to maintain higher activity at high temperature, so it is unfavorable for its industrial production application

Method used

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  • A strain of Escherichia coli recombinant strain heterologously expressing heat-resistant nitrile hydratase and its application
  • A strain of Escherichia coli recombinant strain heterologously expressing heat-resistant nitrile hydratase and its application
  • A strain of Escherichia coli recombinant strain heterologously expressing heat-resistant nitrile hydratase and its application

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Experimental program
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Effect test

Embodiment 1

[0035] Embodiment 1 Construction of recombinant Escherichia coli BL21 / pET24a-Cal.tNHase

[0036] The NHase gene sequence derived from Caldalkalibacillus thermarum TA2.A1 was retrieved through NCBI, optimized through the codon online optimization website Graphical Codon Usage Analyzer, and a spacer sequence was added between the genes encoding each subunit, specifically: the encoded amino acid sequence The gene of SEQ ID NO.1, the spacer sequence shown in SEQ ID NO.4, the gene encoding the amino acid sequence SEQ ID NO.2, the spacer sequence shown in SEQ ID NO.4 and the gene encoding the amino acid sequence SEQ ID NO.3 The genes are connected sequentially to obtain the gene NHase of nitrile hydratase, the nucleotide sequence is shown in SEQID NO.5, and the electrophoresis result is shown in figure 1 Shown, synthesized by Jinweizhi Biotechnology Company.

[0037] The optimized NHase sequence does not contain restriction endonuclease sites. Specific primers P1 and P2 are designe...

Embodiment 2

[0042] The expression of embodiment 2 nitrile hydratase

[0043] Inoculate recombinant Escherichia coli BL21 / pET24-Cal.t NHase in 5 mL of LB medium with a kanamycin concentration of 100 μg / mL, and culture 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, and 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 397.35...

Embodiment 3

[0044] Embodiment 3 Cal.t NHase thermal stability analysis

[0045] The pure enzyme with a concentration of 0.5 mg / mL was treated at 60°C, and samples were taken to determine the residual enzyme activity. The enzyme activity at time 0 is defined as 100%. Such as Figure 4 As shown, 60% of the enzyme activity can still be maintained at 60°C for 6 hours. The most commonly used nitrile hydratase in industry is H-NHase derived from Rhodococcus rhodochrous J1, which has a half-life of 1 hour at 60°C.

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Abstract

The invention discloses a Escherichia coli recombinant strain heterologously expressing heat-resistant nitrile hydratase and application of the Escherichia coli recombinant strain, and belongs to thefield of bioengineering. According to the invention, the Escherichia coli recombinant strain having a high nitrile hydratase activity is obtained by constructing of recombinant Escherichia coli, and the recombinant nitrile hydratase obtained by fermentation has better thermal stability. In addition, the Escherichia coli recombinant strain can be used as a catalyst, and nicotinonitrile or acrylonitrile can be used as a substrate to carry out a whole-cell catalytic reaction to prepare nicotinamide or acrylamide. Compared with a chemical production method, the production process is safe and clean, and has no environmental pollution. Compared with an enzymatic method, the production process employs a cheap substrate, simplifies separation and purification steps of products, has high catalyticefficiency, and has a yield of the final product nicotinamide or acrylamide of 95% or above.

Description

technical field [0001] The invention relates to an Escherichia coli recombinant bacterium strain heterologously expressing heat-resistant nitrile hydratase 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 acrylamid...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/21C12N15/70C12N9/88C12N15/60C12P17/12C12P13/02C12R1/19
CPCC12N9/88C12N15/70C12P13/02C12P17/12C12Y402/01084
Inventor 周哲敏刘中美马东崔文璟周丽郭军玲张赛兰
Owner JIANGNAN UNIV
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