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Amidase, coding gene and applications thereof

An amidase and gene technology, which is applied to amidase and its encoding gene and application fields, can solve the problems of low amidase yield, reduced product purity, inability of amidase to effectively hydrolyze macromolecular aromatic amide compounds, etc. The effect of stereoselectivity and high catalytic activity

Active Publication Date: 2015-03-11
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] However, the currently studied amidases still have the problem that the substrate spectrum is not wide enough, especially many amidases cannot effectively hydrolyze macromolecular aromatic amides.
Furthermore, the wild strain of microorganisms is used to directly carry out the catalytic reaction of the substrate, and there are still problems such as the low yield of amidase and the generation of a large amount of by-products so that the purity of the product is reduced.

Method used

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  • Amidase, coding gene and applications thereof
  • Amidase, coding gene and applications thereof
  • Amidase, coding gene and applications thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] 1. Cloning the AmiH gene from the genome of Klebsiella oxytoca KCTC 1686 (Klebsiella oxytoca KCTC 1686).

[0039] Primers AmiH-F and AmiH-R were designed according to the genomic DNA sequence of Klebsiella oxytoca KCTC 1686 (GenBank accession number: CP003218.1).

[0040] AmiH-F sequence: 5'-CCG GAATTCATGGCTATTCAACGTCCCACTG-3'

[0041] AmiH-R sequence: 5'-TTCCC AAGCTT GTTAAAACGTCCGCCAGTCAC-3'

[0042] 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 AmiH-F and AmiH-R were used as primers for PCR amplification. The PCR reaction system and reaction conditions were as follows:

[0043] PCR amplification system:

[0044]

[0045] PCR amplification conditions:

[0046] 1) Pre-denaturation: 95°C for 5 minutes;

[0047] 2) Denaturation: 98°C for 10s; Annealing: 55°C for 15s; Extension: 72°C for 60s; a total of 30 cycles;

[0048] 3)...

Embodiment 2

[0058] Embodiment 2 amidase hydrolysis butanamide

[0059] Get 50mL of the engineering bacteria E.coli BL21(DE3) / pET-30a(+)-AmiH fermentation broth in Example 1, centrifuge at 10000 rpm to collect the thalline, then resuspend with 50mM Tris-HCl (pH 7.5) buffer Bacteria cells, that is, the resting cell suspension of engineering bacteria E.coliBL21(DE3) / pET-30a(+)-AmiH. The control cell load was 3g stem cells / L. Butanamide was added to the resuspension to a final concentration of 20 mM, and the catalytic reaction was carried out at 35° C. for 2 hours. Then detect the content of butanamide and butyric acid in the reaction system by gas chromatography. As a result, it was found that there was no butanamide in the reaction system, and all of them were converted into butyric acid.

Embodiment 3

[0060] Embodiment 3 amidase hydrolysis caproamide

[0061] Get 50mL of the engineering bacteria E.coli BL21(DE3) / pET-30a(+)-AmiH fermentation broth in Example 1, centrifuge at 10000rpm to collect the thalline, then resuspend the bacteria with a buffer of 50mM Tris-HCl (pH 7.5) Somatic cells, that is, the resting cell suspension of engineering bacteria E.coli BL21(DE3) / pET-30a(+)-AmiH. The control cell load was 3g stem cells / L. Hexanamide was added to the resuspension to a final concentration of 30 mM, and the catalytic reaction was carried out at 30° C. for 2 hours. Then gas chromatography was used to detect the contents of caproamide and caproic acid in the reaction system. As a result, it was found that there was no caproamide remaining in the reaction system, and all of them were converted into caproic acid.

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Abstract

The invention discloses amidase, a coding gene and applications thereof. The amino acid sequence of the amidase is represented by the SEQ ID No.2. The amidase gene is cloned from acid-producing Klebsiella oxytoca KCTC 1686 genome. After the expression of the amidase gene, amidase can be successfully obtained, and the amidase has the advantages of high catalytic activity, good stereo-selectivity, high expression amount, and wind substrate spectrum. The obtained amidase can be used to prepare optically-pure chiral compounds especially an important medicine intermediate 2-(4-chlorophenyl)-3-methylbutyric acid. The recombinant amidase can be used to catalyze a racemic substrate 2-(4-chlorophenyl)-3-methylbutyramide, and when the substrate conversion rate is near 50%, the optical purity of the (S)-2-(4-chlorophenyl)-3-methylbutyric acid is 98.5%.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to amidase, its coding gene and application. Background technique [0002] Amidases are a class of hydrolases that catalyze the hydrolysis of amides to the corresponding carboxylic acids and ammonia. Amidase has a wide range of substrates and can hydrolyze various natural and synthetic aliphatic and aromatic amides. Some amidases usually have stricter chemical, regio, and stereoselectivity, especially for substrates with chiral centers at the α-position. It has great potential in the preparation of optically pure amides, carboxylic acids and their corresponding derivatives, and is increasingly valued by the industry. [0003] Optically pure amides, carboxylic acids and their derivatives are important chemical products and chiral drug intermediates. Due to the increasing demand for chiral drugs and intermediates, the use of biological or enzymatic synthesis of chiral compounds h...

Claims

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Application Information

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IPC IPC(8): C12N9/10C12N15/66C12N15/63C12P7/40
CPCC12N9/80C12P7/40C12Y305/01004
Inventor 杨立荣郭法谋吴坚平徐刚
Owner ZHEJIANG UNIV
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