A high-throughput screening method for l-aspartate β-decarboxylase-producing bacteria

Abstract

A high-throughput screening method for an L-aspartate beta-decarboxylase producing strain is disclosed. The method includes 1) subjecting microorganisms in a sample to enrichment culture and primary separation by adopting an enrichment culture medium, 2) inoculating single colonies obtained through separation from a plane plate to a 96-micropore plate primary screening semisolid medium and performing primary screening to obtain L-aspartate beta-decarboxylase producing strains, 3) inoculating the strains obtained through primary screening to a 24-prore plate, performing liquid culturing, and performing secondary screening to obtain the L-aspartate beta-decarboxylase producing strain with high yield by combining paper chromatography, and 4) subjecting the high-yield strain obtained through secondary screening to fermentation in a shake flask, and determining enzyme activity by combining an oxidation coloration process. The method is suitable for high-throughput screening of the L-aspartate beta-decarboxylase producing strain. The strain obtained through screening is alkali-resistant, thus avoiding tedious pH value controlling steps in a cell production processes. References are provided for high-throughput screening of producing strains bio-enzymes of the same kind.

Description

technical field

[0001] The invention belongs to the field of microbial pharmacy, and relates to a high-throughput screening method for bacteria producing L-aspartic acid β-decarboxylase. Background technique

[0002] L-aspartic acid β-decarboxylase (Asd), also known as L-aspartic acid 1-decarboxylase, can catalyze the removal of the carboxyl group of L-aspartic acid to generate L-alanine (L-Ala ). L-alanine is widely used in medicine and food industry. With the increase of demand and the development of fermentation industry, the production method of L-alanine has developed from protein hydrolysis extraction and fermentation to enzymatic production. That is, L-aspartic acid is decarboxylated to L-alanine by L-aspartic acid β-decarboxylase. The production of L-Ala by Asd transformation can be divided into free cell method and immobilized cell transformation method. Japan has fixed Pseudomonas.dacunhae cells with carrageenan in 1982 for continuous production of L-Ala by L-As...

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