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Nitrile hydratase and its coding gene and application

A technology of nitrile hydratase and coding gene, which is applied in the field of genetic engineering, can solve the problems of poor substrate and product tolerance, low enzyme expression level, low thermal stability, etc., and achieve high expression effect

Active Publication Date: 2017-10-03
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the research on recombinant nitrile hydratase has attracted widespread attention. After years of hard work by many scientific researchers, considerable achievements have been made, but there are still many problems, such as low enzyme expression level and low thermal stability. , poor tolerance to substrates and products, which seriously restricts the industrial application of nitrile hydratase

Method used

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  • Nitrile hydratase and its coding gene and application
  • Nitrile hydratase and its coding gene and application
  • Nitrile hydratase and its coding gene and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] 1. Cloning of nitrile hydratase and its activator gene from the genome of Klebsiella oxytoca KCTC 1686 (Klebsiella oxytoca KCTC 1686)

[0042] Primers NH-F and NH-R were designed according to the genomic DNA sequence of Klebsiella oxytoca KCTC 1686 (GenBank accession number: CP003218.1).

[0043] NH-F sequence: 5'-CCG GAATTC ATGAGCCATAAACACGACCACG-3'

[0044]NH-R sequence: 5'-TTCCC AAGCTT GTTATGGTGTAACTCCATTATCG-3

[0045] Restriction sites EcoRI and HindIII (underlined) were added to the upstream and downstream primers, respectively. Klebsiella oxytoca KCTC 1686 genomic DNA was used as a template, and NH-F and NH-R were used as primers for PCR amplification. The PCR reaction system and reaction conditions were as follows:

[0046] PCR amplification system:

[0047]

[0048] PCR amplification conditions:

[0049] 1) Pre-denaturation: 95°C for 5 minutes;

[0050] 2) Denaturation: 98°C for 10s; Annealing: 57°C for 15s; Extension: 72°C for 60s; a total of 30 cy...

Embodiment 2

[0061] Example 2 Genetically engineered bacteria catalyze acrylonitrile to generate acrylamide

[0062] The enzyme activity unit is defined as: under the reaction conditions, the amount of enzyme that catalyzes the substrate reaction to produce 1 μmol of product per minute.

[0063] Take 25ml of the fermentation broth of the engineering bacteria E.coli BL21(DE3) / pET-30a(+)-NHaseK in Example 1, centrifuge at 10000rpm for 10min to collect the bacterial cells, and then resuspend with 250ml of 50mM Tris-HCl (pH 7.5) buffer Bacteria cells, that is, the resting cell suspension of engineering bacteria E.coli BL21(DE3) / pET-30a(+)-NHaseK. 1.5 ml of acrylonitrile was added to the resuspension, and the hydration reaction was carried out at 35°C for 2 hours. Then the contents of acrylonitrile and acrylamide in the reaction system were detected by gas chromatography. As a result, it was found that no acrylonitrile remained in the reaction system, and all of them were converted into acryl...

Embodiment 3

[0064] Example 3 Genetically engineered bacteria catalyze butyronitrile to generate butanamide

[0065] Take 25ml of the fermentation broth of the engineering bacteria E.coli BL21(DE3) / pET-30a(+)-NHaseK in Example 1, centrifuge at 10000rpm for 10min to collect the bacteria, and then resuspend with 250ml of 50mM Tris-HCl (pH 7.0) buffer Bacteria cells, that is, the resting cell suspension of engineering bacteria E.coli BL21(DE3) / pET-30a(+)-NHaseK. Add 7.0 g of butyronitrile to the resuspension liquid, carry out hydration reaction at 35° C., and react for 2 hours. Then detect the content of butyronitrile and butyramide in the reaction system with gas chromatography. As a result, it was found that there was no residue of butyronitrile in the reaction system, and all of them were converted into butanamide.

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Abstract

The invention discloses a nitrile hydratase as well as an encoding gene and application thereof. The nitrile hydratase is prepared from an alpha subunit and a beta subunit, wherein an amino acid sequence of the alpha subunit is shown as SEQ ID NO.4, and an amino acid sequence of the beta subunit is shown as SEQ ID NO.5. According to the nitrile hydratase as well as the encoding gene and the application thereof, a nitrile hydratase gene is cloned from acid-producing Klebsiella spp KCTC 1686, and the nitrile hydratase with high expression level, high activity, wide substrate spectrum and chiral selectivity is successfully obtained after the gene is expressed.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to a nitrile hydratase and its coding gene and application. Background technique [0002] Nitrile is an important class of compounds. Its hydrolysis reaction is widely used in the synthesis of amino acids, amides, carboxylic acids and their derivatives, and occupies an important position in organic synthesis. Nitrile hydrolysis methods mainly include chemical hydrolysis and biotransformation. Compared with chemical hydrolysis, biotransformation has the advantages of mild conditions, less environmental pollution, and the ability to achieve chemical, regio and enantioselectivity. Biotransformation methods mainly involve nitrile hydratase, amidase and nitrilase. Among them, nitrile hydratase can catalyze the hydration of nitrile to generate amide, amidase can further catalyze the hydrolysis of amide to generate carboxylic acid compound, and nitrilase can directly catalyze nitrile to...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/88C07K14/00C12N15/60C12N15/63C12P13/02
CPCC12N9/88C12P13/02C12Y402/01084
Inventor 杨立荣郭法谋王丽燕吴坚平徐刚
Owner ZHEJIANG UNIV
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