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Heat-resistant recombinant nitrile hydratase gene, encoded enzyme, engineering bacterium and application of gene engineering bacterium

A technology of genetically engineered bacteria and nitrile hydratase, which is applied in the field of heat-resistant recombinant nitrile hydratase gene, encoded enzyme, engineering bacteria and its application, can solve the problems of low substrate and product tolerance, poor stability of nitrile hydratase, and Target product purity and actual yield and other issues, to achieve the effect of high activity and high expression

Inactive Publication Date: 2015-04-29
HANGZHOU NORMAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the amidase gene is often contained in the gene cluster of nitrile hydratase, which can convert acrylamide and nicotinamide into acrylic acid and nicotinic acid, which seriously affects the purity and actual yield of the target product
In addition, reported nitrile hydratases have problems such as poor stability and low tolerance to substrates and products.

Method used

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  • Heat-resistant recombinant nitrile hydratase gene, encoded enzyme, engineering bacterium and application of gene engineering bacterium
  • Heat-resistant recombinant nitrile hydratase gene, encoded enzyme, engineering bacterium and application of gene engineering bacterium
  • Heat-resistant recombinant nitrile hydratase gene, encoded enzyme, engineering bacterium and application of gene engineering bacterium

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Example 1: Cloning of full-length nitrile hydratase gene

[0029] Bacterial Genome Extraction Kit (AxyPrep, Corning Life Sciences Co., Ltd.) was used to extract the genome of Bradyrhizobium japonicum USDA 110 (American Agricultural Culture Collection, NRRL B-4361), and nucleic acid electrophoresis was detected as follows: figure 1 shown.

[0030] Design primers NH05-Up and NH05-Down according to the nucleotide sequence (such as SEQ ID NO.1) of putative nitrile hydratase in the genome of brady-growing type soybean rhizobia USDA 110, with the genome of brady-growing type soybean rhizobia USDA 110 as template for PCR amplification of the full-length nitrile hydratase gene, such as figure 2 shown. The PCR product is recovered using a DNA gel recovery and purification kit, and the obtained gene fragment is shown in the α subunit shown in SEQ ID NO.2 (the encoded amino acid sequence is shown in SEQ ID NO.5), shown in SEQ ID NO.3 The β subunit (the encoded amino acid seque...

Embodiment 2

[0041] Example 2: Design of a full-length nitrile hydratase gene with E. coli SD sequence and spacer sequence

[0042] In order to realize the soluble functional expression of the nitrile hydratase gene in Escherichia coli BL21 (DE3) cells, the SD sequence (as shown in SEQ ID NO.8) and the spacer sequence ( As shown in SEQ ID NO.9). Design primer Link-Up and Link-Down according to nitrile hydratase gene sequence (by SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO.4 sequential connection composition), with the PCR product among the embodiment 1 as template, The nitrile hydratase gene containing Escherichia coli SD sequence and spacer sequence was amplified by overlapping PCR. PCR products were recovered using a DNA gel recovery and purification kit.

[0043] The SD sequence (shown in SEQ ID NO.8) of Escherichia coli is as follows:

[0044] 5'-aaggag-3'

[0045] The spacer sequence is as follows (shown in SEQ ID NO.9):

[0046] 5'-atatacc-3'

[0047] Link-Up (SD sequence and spacer...

Embodiment 3

[0066] Example 3: Construction and identification of genetically engineered strain E.coli pET28a(+)-NH05

[0067] The plasmid vector pET28a(+) and the nitrile hydratase gene (prepared in Example 2) containing the Escherichia coli SD sequence and the spacer sequence were double digested with NeI I and Hid III, mixed in equal amounts, and ligated with T4 DNA ligase at 16°C Overnight, obtain recombinant plasmid pET28a(+)-NH05 (schematic diagram as figure 2 shown). Then, the recombinant plasmid was transformed into competent E.coli BL21(DE3) cells. Then spread the transformed bacterial liquid on the LB plate containing the final concentration of 100μg / ml Kanamycin (Kanamycin, Kan), culture it statically at 37°C, pick a single colony, and determine the gene sequence by Shanghai Sangong , thus verifying the correctness of the recombinant plasmid pET28a(+)-NH05 and the recombinant strain.

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Abstract

The invention discloses a heat-resistant recombinant nitrile hydratase gene, an encoded enzyme, a vector, an engineering bacterium and an application of the gene engineering bacterium to catalysis of a nitrile compound to produce amide and a biodegradation herbicide, that is, dichlobenil. The functional expression of the tentative nitrile hydratase gene from bradyrhizobium japonicum USDA 110 is realized, and the crucial role of an activation element of the nitrile hydratase in the expression process is proved. Meanwhile, the invention provides a method for constructing the nitrile hydratase gene engineering bacterium in Escherichia coli, and the nitrile hydratase with the high expression quantity and the high activity is obtained from the nitrile hydratase gene with the participation of the activation element.

Description

(1) Technical field [0001] The invention relates to a heat-resistant recombinant nitrile hydratase gene from rhizobia, an encoded enzyme, an engineering bacterium, and the application of the genetically engineered bacterium in preparing amide compounds and degrading herbicide--dichonil. (2) Background technology [0002] Nitrile compounds are a class of organic compounds containing a cyano group (-CN). In the field of chemical industry, nitrile compounds can be used as important chemical raw materials, widely used in the organic synthesis of chemical amides and carboxylic acids and their derivatives. However, chemical conversion of nitrile compounds usually requires conditions such as strong acid, strong base and high pressure, which will inevitably cause serious pollution to the environment. Compared with the chemical hydrolysis method, the reaction conditions of the biocatalytic method are mild (normal temperature, normal pressure and neutral pH value), and at the same ti...

Claims

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Application Information

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IPC IPC(8): C12N15/60C12N9/88C12N15/70C12N1/21C12P17/12A62D3/02A62D101/04A62D101/26
Inventor 裴晓林王秋岩杨立荣吴坚平
Owner HANGZHOU NORMAL UNIVERSITY
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