Method for synthesizing L-cysteine through immobilized enzyme conversion of DL-ATC (DL-2-amino-delta<2>-thiazoline-4-carboxylic acid)

Abstract

The invention discloses a method for synthesizing L-cysteine through immobilized enzyme conversion of DL-ATC (DL-2-amino-delta<2>-thiazoline-4-carboxylic acid). Fusion protein (SEQ ID NO.1) containing DL-ATC racemase and L-ATC hydrolase and nitrogen-carbamoyl-L-cysteine hydrolase (SEQ ID NO.4) are subjected to immobilization by means of polyvinyl alcohol-sodium alginate composite carrier through a crosslinking-embedding-crosslinking process, a co-immobilized enzyme system is obtained, and DL-ATC can be converted to generate L-cysteine. Sequences of genes of the fusion protein and nitrogen-carbamoyl-L-cysteine hydrolase are encoded to be shown in SEQ ID NO.2 and SEQ ID NO. 5 respectively, the genes are constructed to pET-28a, and high-purity target protein is obtained through induced expression and purification. The immobilized enzyme system conversion efficiency and the stability are high, and the method has important industrial application value in synthesis of L-cysteine.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, in particular to an immobilized enzyme converting 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...

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, its half-life is only about 100 hours), 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 process of the whole process The production capacity is greatly limited, 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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