A method for enzymatic conversion of dl-2-amino-△2-thiazoline-4-carboxylic acid to synthesize l-cysteine

Abstract

The invention discloses a method for synthesizing L-cysteine by transforming DL-2-amino-delta<2>-thiazolinyl-4-carboxylic acid (DL-ATC) by an enzyme process. A gene engineering bacterium subjected to membrane permeation treatment is immobilized by a calcium alginate embedding process to obtain immobilized cells, and the immobilized cells can transform the DL-ATC to generate L-cysteine. The gene engineering bacterium is Escherichia coli in which cysteine desulfhydrase gene is knocked out, which can heterologously co-express DL-ATC racemase / L-ATC hydrolase fusion protein and nitro-carbamyl-L-cysteine hydrolase. Sequences disclosed as SEQ ID NO.2 and 5 are respectively constructed onto an expression vector, and the expression vector is transformed into tnaA / malY-gene-knock-out Escherichia coli to obtain the engineering bacterium. The transformation efficiency of the immobilized cells from DL-ATC to L-cysteine is up to 93%. The method has the advantages of high transformation rate, low production cost and the like, and has important application value in synthesis of L-cysteine.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, in particular to an enzymatic conversion of DL-2-amino-△ 2 - A method for synthesizing L-cysteine ​​from thiazoline-4-carboxylic acid (DL-ATC). Background technique [0002] L-cysteine ​​is the only amino acid with a reducing group sulfhydryl (-SH) among more than 20 kinds of amino acids that make up proteins, and has important physiological functions. It has been widely used in medicine, food additives and cosmetics. my country is a major producer of L-cysteine, accounting for 80% of the world's total output. The products not only occupy the domestic market, but also are exported in large quantities to all over the world. At present, the domestic production of L-cysteine ​​mainly relies on the keratin in human or animal hair to extract L-cystine through acid hydrolysis, and then obtains L-cysteine ​​through electrolytic reduction. The method has low yield, high energy consumption, a la...

AI Technical Summary

Problems solved by technology

[0003] Although the technology of microbial enzymatic conversion of DL-ATC to synthesize L-cysteine ​​has been relatively mature in the world, the service life of the bacteria is not long (due to the thermal stability of L-ATC hydrolase at physiological temperature Not high, variable, and its half-life is very short), the enzymes in the enzymatic reaction pathway are all induced enzymes, the response time to the substrate is long, the enzyme amount in the wild strain is insufficient, and the activity is relatively low, which makes the continuous production capacity of the whole process It is greatly restricted, and the production cost remains high. Generally, this method is only used for the production of medical-grade high-purity L-cysteine

In particular, there is a catabolic pathway of cysteine ​​in wild bacteria, and the L-cysteine ​​accumulated in the transformation process is decomposed to release a large amount of H 2 S gas, which seriously affects the yield

At present, due to the lack of genome information on strains and some unknown reasons, the genetic engineering of wild Pseudomonas is very difficult and has been stagnant.

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