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Construction of N-carbamoylase expression genes and engineering bacteria of N-carbamoylase expression genes

A hydrolase, carbamoyl technology, applied in genetic engineering, hydrolase, plant genetic improvement, etc., can solve the problems of low activity, short half-life, low conversion rate, etc., and achieve the effect of high enzyme activity

Active Publication Date: 2017-01-18
CHONGQING HONOROAD ANIMAL HEALTH
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Problems solved by technology

[0006] At present, the main problem of enzymatic splitting is that the activity of the carbamoylase contained in the microbial cells used is low, and the half-life is too short, resulting in too long hydrolysis time of intermediate products and low conversion rate

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  • Construction of N-carbamoylase expression genes and engineering bacteria of N-carbamoylase expression genes
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  • Construction of N-carbamoylase expression genes and engineering bacteria of N-carbamoylase expression genes

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[0044] The following description is disclosed to enable any person skilled in the art to make and use the invention. The preferred embodiments provided in the following description are only examples and modifications obvious to those skilled in the art, and they are not intended to limit the scope of the present invention. The general principles defined in the following description may be applied to other embodiments, alternatives, modifications, equivalent implementations and applications without departing from the spirit and scope of the invention.

[0045] According to a first preferred embodiment of the present invention, the construction of the expression vector containing the dominant gene and the engineering bacteria of the N-carbamyl hydrolase is as follows:

[0046] 1. Experimental Preparation

[0047] 1.1 Preparation of separation medium

[0048] Preparation of No. 1 Separation Medium: Dissolve 2g of chitin in 75ml of 0.4M HCl, stir at 5°C for 12 hours, adjust the ...

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Abstract

The invention provides construction of N-carbamoylase expression genes and engineering bacteria of the N-carbamoylase expression genes. A preparation method of N-carbamoylase comprises following steps: 1, screening of target bacteria is carried out; 2, extraction of bacteria total DNA is carried out; 3, amplification of the bacteria total DNA is carried out so as to obtain N-carbamoylase genes; 4, DNA segments of the N-carbamoylase genes are connected with expression vectors so as to obtain N-carbamoylase recombinant plasmids; 5, the N-carbamoylase recombinant plasmids are transferred into expression hosts, and fermental cultivation is carried out. According to the invention, amino acid sequences coded by the N-carbamoylase genes possess relatively high enzymatic activity when N-carbamoyl-D-p-hydroxyphenylglycine is taken as a substrate.

Description

technical field [0001] The invention relates to an N-carbamoyl hydrolase expression gene and construction of engineering bacteria thereof, in particular to an N-carbamoyl-D-p-hydroxyphenylglycine hydrolase expression gene and construction of engineering bacteria thereof. Background technique [0002] D-amino acid and its derivatives are important raw materials in the fields of medicine and food, and are widely used in the synthesis of semi-synthetic antibiotics, polypeptide hormones, pyrethroids, insecticides and sweeteners. Among them, D-p-hydroxyphenylglycine (D-pHPG) is the main side chain in the production of semi-synthetic penicillins and semi-synthetic cephalosporins, and is widely used in the synthesis of semi-synthetic broad-spectrum antibiotics amoxicillin, cefadroxil, and cefoperazone , Cefuroxime has cefadroxol and so on. [0003] There are two types of synthetic methods for D-p-hydroxyphenylglycine (D-p-HPG), chemical resolution and enzymatic resolution. Due to...

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Application Information

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IPC IPC(8): C12N15/55C12N9/78C12N15/70C12N15/81C12N15/75
Inventor 赵德任丽任敏李亚东金媛媛王国勤罗尚菊易丹王翠娟
Owner CHONGQING HONOROAD ANIMAL HEALTH
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