Nitrile hydratase gene, encoded enzyme, vector, engineering bacterium as well as application of engineering bacterium to preparation of amide compound

A nitrile hydratase and gene-encoded technology, applied in genetic engineering, using vectors to introduce foreign genetic material, applications, etc., can solve problems such as low activity and limited applications, and achieve high-efficiency transformation effects

Active 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

At the same time, nitrile hydratase generally has strong substrate specificity, and often shows very low activity to larger aromatic nitriles, which limits its application in the transformation of some pharmaceutical intermediates

Method used

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  • Nitrile hydratase gene, encoded enzyme, vector, engineering bacterium as well as application of engineering bacterium to preparation of amide compound
  • Nitrile hydratase gene, encoded enzyme, vector, engineering bacterium as well as application of engineering bacterium to preparation of amide compound
  • Nitrile hydratase gene, encoded enzyme, vector, engineering bacterium as well as application of engineering bacterium to preparation of amide compound

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Example 1: Cloning of full-length nitrile hydratase gene

[0025] The genome of Sulphitobacter sp. E-36ATCC BAA-1142 was extracted using a bacterial genome extraction kit. Design primers NH_E36-Up and NH_E36-Down according to the nucleotide sequence (such as SEQ ID NO.1) of hypothetical nitrile hydratase in the Bacillus bisulfite E-36 genome again, take Bacillus bisulfite E-36 genome as template, PCR amplifies Amplify the full-length nitrile hydratase gene, such as figure 1 shown. The PCR product is recovered using a DNA gel recovery and purification kit, and the obtained full-length nitrile hydratase gene is composed of the alpha subunit shown in SEQ ID NO.2 (the encoded amino acid sequence is shown in SEQ ID NO.5), SEQ ID NO The β subunit shown in .3 (the encoded amino acid sequence is shown in SEQ ID NO.6) and the activation element shown in SEQ ID NO.4 (the encoded amino acid sequence is shown in SEQ ID NO.7) consists of SEQ ID NO . The α subunit shown in 2, the ...

Embodiment 2

[0036] Embodiment 2: Construction and identification of genetic engineering strain E.coli pET28a(+)-NH_E36

[0037] In order to achieve efficient functional expression of the nitrile hydratase gene derived from the Bacillus bisulfite E-36 genome in Escherichia coli BL21(DE3) cells, pET28a(+) containing T7 promoter was selected as the expression vector. The vector pET28a(+) and the full-length nitrile hydratase gene were double-digested with Nell and Xho I, and the digested product was recovered using a DNA gel recovery kit.

[0038] Double enzyme digestion system and reaction conditions:

[0039]

[0040] Incubate at 37°C for 5h.

[0041] Nucleic acid electrophoresis was used to preliminarily determine the concentration of the two, and the gene / plasmid (mol / mol, 2:1) was mixed, and T4 DNA ligase was added to connect overnight at 16°C to obtain the recombinant plasmid pET28a(+)-NH_E36 (schematic diagram as shown in figure 2 shown). Then, the recombinant plasmid was trans...

Embodiment 3

[0042] Example 3: Expression and purification of nitrile hydratase of genetic engineering strain E.coli pET28a(+)-NH_E36

[0043] The genetically engineered bacteria E.coli pET28a(+)-NH_E36 glycerol-preserved strain was inoculated into 5 mL of LB liquid medium containing 50 μg / ml Kan, and cultured at 37° C. with shaking at 200 rpm for 10 to 14 hours. Transfer 2 mL of the culture solution to 100 mL of fresh LB liquid medium containing 50 μg / ml Kan, and culture at 37 °C with shaking at 200 rpm until the cell density (OD 600 ) reaches about 0.8, add IPTG to a final concentration of 0.1 mM, and induce at 18-37°C for 12-18 hours. Cells were collected by centrifugation at 5000-10000×g for 5-10 min, washed twice with phosphate buffer (50-200 mM, pH 5.0-8.0), and resuspended in phosphate buffer. In an ice bath, use an ultrasonic breaker to break the cells, centrifuge at 5000-10000×g for 5-30 minutes, collect the supernatant of the broken cells, and pass the supernatant of the broken ...

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Abstract

The invention discloses a nitrile hydratase gene, an encoded enzyme, a vector, an engineering bacterium as well as an application of the engineering bacterium to preparation of an amide compound. The recombinant nitrile hydratase gene is formed by sequentially connecting an alpha subunit shown in SEQ ID NO.2, a beta subunit shown in SEQ ID NO.3 and an activation element shown in SEQ ID NO.4. The functional expression of the tentative nitrile hydratase gene from sulfitobacter sp E-36 in Escherichia coil cells is realized, the gene engineering bacterium for expressing the nitrile hydratase efficiently is provided, and aromatic nitrile can be converted into the corresponding amide compound efficiently.

Description

(1) Technical field [0001] The invention relates to the construction of a nitrile hydratase gene for efficiently hydrating aromatic nitriles, a genetically engineered bacterium for efficiently expressing the recombinase, and the application of the genetically engineered bacterium in preparing amide compounds. (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 and intermediates, and are 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 p...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/60C12N9/88C12N15/70C12N1/21C12P17/12C12P13/02
Inventor 裴晓林王秋岩杨立荣吴坚平
Owner HANGZHOU NORMAL UNIVERSITY
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