Nitrile hydratase and application thereof

A technology for nitrile hydratase and nitrile compounds, which is applied in the field of genetic engineering and can solve the problems of accumulation of carboxylic acid compounds, intermittency, and unstable product quality.

Active Publication Date: 2015-04-08
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Although the process of using wild strains to industrially produce amide compounds has been widely used, the above-mentioned processes generally have poor enzyme activity stability of wild strains of nitrile hydratase, low tolerance to substrates and products, and intermittent Production, product quality is not stable enough and other issues
In addition, in addition to nitrile hydratase, there are amidase and nitrilase in wild strains, which can cause the accumulation of by-product carboxylic acid compounds, thereby reducing the yield and purity of amides and increasing the separation and purification. difficulty and production cost

Method used

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  • Nitrile hydratase and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] 1. Cloning of nitrile hydratase gene and activator gene from Bordetella petrii DSM 12804 genome

[0050] Primers NP-F (upstream primer) and NP-R (downstream primer) were designed according to the Bordetella DSM 12804 genomic DNA sequence (GenBank accession number: AM902716.1).

[0051] NP-F sequence: 5'-CCG GAATTC ATGCTCGAAGTTCTTTGCATGG-3'

[0052] NP-R sequence: 5'-TTCCC AAGCTT GTTAAGCCATTGCGGCAACG-3'

[0053] Restriction sites EcoRI and HindIII (underlined) were added to the upstream and downstream primers, respectively. Using Bordetella DSM 12804 genomic DNA as template, NP-F and NP-R as primers for PCR amplification, the PCR reaction system and reaction conditions are as follows:

[0054] PCR amplification system:

[0055]

[0056]

[0057] PCR amplification conditions:

[0058] 1) Pre-denaturation: 95°C for 5 minutes;

[0059] 2) Denaturation: 98°C for 10s; Annealing: 56°C for 15s; Extension: 72°C for 100s; a total of 30 cycles;

[0060] 3) Extensio...

Embodiment 2

[0071] Example 2 Genetically engineered bacteria catalyze acrylonitrile to generate acrylamide

[0072] 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.

[0073] Get the fermented liquid of the engineered bacterium E.coli BL21(DE3) / pET-30a(+)-NHaseP that 25ml embodiment 1 constructs, 10000rpm, 10min centrifugal collection thalline, then use the buffering of 250ml 50mM Tris-HCl (pH 8.5) The collected bacterial cells were resuspended in the liquid, and the enzyme activity of the resuspended enzyme solution was 1700U / ml. Add 1.0ml of acrylonitrile to the resuspension, and carry out hydration reaction at 20°C for 120min. Then the contents of acrylonitrile, acrylamide and acrylic acid in the reaction system were detected by gas chromatography. The conversion rate of the substrate is greater than 99%, the yield is greater than 92%, and no acrylic acid is found ...

Embodiment 3

[0074] Example 3 Genetically engineered bacteria catalyze 3-cyanopyridine to generate nicotinamide

[0075] Get the fermented liquid of the engineered bacterium E.coli BL21(DE3) / pET-30a(+)-NHaseP that 25ml embodiment 1 constructs, 10000rpm, 10min centrifugal collection thalline, then use the buffering of 250ml 50mM Tris-HCl (pH 6.0) The collected bacteria were resuspended. Add 5.0 g of 3-cyanopyridine to the resuspension, and carry out hydration reaction at 37° C. for 120 min. Then use high performance liquid chromatography to detect the content of 3-cyanopyridine and nicotinamide in the reaction system. The substrate conversion rate is greater than 99%, and the yield is greater than 89%.

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Abstract

The invention discloses a nitrile hydratase and an application thereof. The nitrile hydratase is composed of an alpha subunit and a beta subunit, wherein an amino acid sequence of the alpha subunit is as shown in SEQ ID NO.6; the amino acid sequence of the beta subunit is as shown in SEQ ID NO.7. The nitrile hydratase is cloned to a nitrile hydratase gene from Burdett bacteria DSM 12804 (Bordetella petrii DSM 12804); the nitrile hydratase which is high in expression quantity, high in activity, wide in substrate spectrum and good in chiral selectivity is successfully obtained after gene expression. An existing nitrile hydratase is relatively high in activity on aliphatic acrylic compounds in general, and is low in activity on aromatic nitrile compounds in general. The nitrile hydratase disclosed by the invention has relatively high catalytic activity on the aromatic nitrile compounds, especially 2-isopropyl-4-chlorophenyl acetonitrile.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to a nitrile hydratase and its application. Background technique [0002] Nitrile is an important compound that exists widely in nature. The methods of nitrile hydrolysis mainly include chemical hydrolysis and biotransformation, wherein the biotransformation mainly involves nitrile hydratase, amidase and nitrilase. Nitrile hydratase is a class of enzymes that can catalyze nitrile hydrolysis with sulfur atoms and cysteine ​​sulfinic acid residues as the absolute center. It can catalyze the hydration of nitriles to generate amides, and amidase further catalyzes the hydrolysis of amides into carboxylic acid compounds, while Nitrilase can directly catalyze nitriles to carboxylic acid compounds. Through the enzymatic reaction of these enzymes, nitrile substances can be converted into some compounds with higher value and wider application range, such as amides, acids, amino compounds,...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/88C12N15/70C12N1/21C12P13/02
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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