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Heat-resistant nitrile hydratase, engineering bacteria and application thereof in production of amide by catalyzing hydration reaction of nitrile compounds

A technology of nitrile hydratase and engineering bacteria, applied in the field of genetic engineering, can solve the problems of poor thermal stability and achieve high thermal stability

Inactive Publication Date: 2018-04-06
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Poor thermal stability is also one of the factors limiting the large-scale application of nitrile hydratase

Method used

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  • Heat-resistant nitrile hydratase, engineering bacteria and application thereof in production of amide by catalyzing hydration reaction of nitrile compounds
  • Heat-resistant nitrile hydratase, engineering bacteria and application thereof in production of amide by catalyzing hydration reaction of nitrile compounds
  • Heat-resistant nitrile hydratase, engineering bacteria and application thereof in production of amide by catalyzing hydration reaction of nitrile compounds

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Cloning of embodiment 1 heat-resistant nitrile hydratase 1229 gene

[0043] The genome of Auromonas manganese oxidans ATCC BAA-1229 was extracted using a bacterial genome extraction kit. Then design primers Ama_Alpha F and Ama_Act R according to the nucleotide sequence (such as SEQ ID NO.7) of putative nitrile hydratase in the genome of Auromonas manganese oxidans ATCC BAA-1229, with Auromonas manganese oxidans ATCC BAA-1229 Using the genome as a template, the full-length nitrile hydratase gene was amplified by PCR. Restriction sites BamHI and HindIII (underlined) were added to the upstream and downstream primers, respectively.

[0044]

[0045] The PCR reaction system and reaction conditions are as follows:

[0046] PCR amplification system:

[0047]

[0048] PCR amplification conditions:

[0049] 1) Pre-denaturation: 95°C for 2 minutes;

[0050] 2) Denaturation: 95°C for 10s; Annealing: 58°C for 15s; Extension: 72°C for 15s; a total of 30 cycles;

[0051] ...

Embodiment 2

[0055] Embodiment 2 Construction of recombinant heat-resistant nitrile hydratase expression plasmid and genetically engineered bacteria

[0056] In order to realize the high-efficiency functional expression of thermostable nitrile hydratase 1229 gene in Escherichia coli BL21(DE3) cells, the optimized pET28a(+) containing T7 promoter was selected as the expression vector. The vector pET28a(+) and the full-length nitrile hydratase gene were double digested with BamHI and HindIII, and the digested product was recovered using a DNA gel recovery kit.

[0057] Double enzyme digestion system and reaction conditions:

[0058] Gene or plasmid 20μl;

[0059] BamHI enzyme 2μl;

[0060] HindIII enzyme 2μl;

[0061] Add double distilled water to 40 μl;

[0062] Incubate at 37°C for 3h.

[0063] 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...

Embodiment 3

[0064] Example 3 Expression and purification of nitrile hydratase 1229 of genetically engineered E.coli BL21(DE3) / pENHase-1229

[0065] The genetically engineered bacteria E.coli BL21(DE3) / pENHase-1229 glycerol-preserved strain was inoculated in 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. Take 2 mL of the culture solution and transfer it to 100 mL of fresh LB liquid medium containing 50 μg / ml Kan, culture at 37°C with shaking at 200 rpm until the cell density (OD600) reaches 0.8, add IPTG to the final concentration of 0.1 mM, 18-37 Induce at ℃ 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 ...

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Abstract

The invention discloses heat-resistant nitrile hydratase, engineering bacteria and application thereof in production of amide by catalyzing hydration reaction of nitrile compounds. The heat-resistantnitrile hydratase contains alpha-subunit coding genes with a base sequence shown as SEQ ID NO.4, beta-subunit coding genes with a base sequence shown as SEQ ID NO.5, and active element coding genes with a base sequence shown as SEQ ID NO.6. The nitrile hydratase genes are cloned from aurantimonas manganoxydans SI859A and successfully subjected to heterologous expression in E. coli BL21 (DE3), so as to obtain nitrile hydratase with high expression quantity and high activity and proteins with specific activities reaching 404.5U / mg. The nitrile hydratase has high catalytic activity on aromatic nitrile compounds and aliphatic nitrile compounds, and is a kind of nitrile hydratase with wide substrate spectrum, high thermal stability and excellent industrial application potential.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to a heat-resistant nitrile hydratase, engineering bacteria and the application thereof in catalyzing the hydration reaction of nitrile compounds to generate amides. Background technique [0002] Nitriles are an important class of precursor compounds, which are used to synthesize amides and their derivatives through hydration reactions, and play an important role in chemical synthesis. The hydration reaction of traditional cyano groups requires harsh reaction conditions such as strong acid, strong alkali and high temperature, which will inevitably produce a large amount of waste liquid and cause serious pollution to the environment. [0003] Compared with the traditional chemical hydrolysis method, the biotransformation method has the advantages of mild conditions, high reaction efficiency, environmental friendliness, and the ability to achieve chemical, regio and enantioselectivi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/88C12N15/60C12N15/63C12P13/02
CPCC12N9/88C12N15/63C12P13/02C12Y402/01084
Inventor 杨立荣周海胜吴坚平徐刚
Owner ZHEJIANG UNIV
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