Study On Enzymatic Synthesis Of L-Tyrosine

L-tyrosine is an essential amino acid, which plays important roles in the metabolism of people and animal. In the field of food, medicine and cosmetics, manufacturers use L-tyrosine as raw materials for the production of pharmaceutical and chemical products, such as L-dopa, p-coumaric acid and hydroxy styrene. With the development of gene engineering and enzyme engineering, biosynthesis of L-tyrosine will have broader application prospect. The paper is based on the enzymatic biosynthesis of L-tyrosine by pyruvate fermenting liquer, mixed amino acids and phenol, which has great theoretical and practical significance.The TPL gene (Tyrosine phenol-lyase, EC 4.1.99.2, TPL) encoding L-tyrosine, from Citrobacter freundii, was obtained by nucleotide synthesis after rare codon optimization. The synthetic gene was digested with Nco I and EcoR I, and the isolated DNA fragments were recovered and inserted into pETDuet-1 vector. The recombinant plasmid was transformed into E.coli BL21 (DE3). The reaction was optimal at pH 8.5 and 45℃. Triton-X 100 (1 mM), Ammonium chloride (350 mM) and Pyridoxine phosphate (2 mM) could promote TPL activity. L-tyrosine conversion rate reached 78.6% with a final concentration of 16.17 g L-tyrosine/1.With the development of biotechnology, multienzyme reaction system has been put on increasing emphasis on study. In this paper, an enzymatic process for L-tyrosine production was established through coupled SdaA (Serine Deaminase, EC 4.3.1.17, SdaA) and TPL system. The reaction property of the coupled enzymatic system of SdaA and TPL was studied, and optimum reaction condition was obtained: the ratio between SdaA and TPL 0.6:1 (ratio of cell quantity), pH 8.5, reaction temperature 45℃. Triton-X 100 (1 mM), Ammonium chloride (250 mM) and Pyridoxine phosphate (2 mM) could promote the reaction. L-tyrosine conversion rate reached 83.3%. By dual-enzyme coupled reaction system, the cost of L-tyrosine production from mixed amino acid would be reduced.Gene tandem technology connects small molecule peptide series in the fore and aft direction, then increase gene copy number and the molecular weight. To implement co-expression of SdaA and TPL in prokaryotic expression system, a tandem gene fragment encoding SdaA has been synthesized chemically. The fragment was inserted into vector pETDuet-1-TPL and expressed in E.coli BL21 (DE3). L-tyrosine conversion rate reached 61.7%,14.7% higher than single-enzyme system (TPL).The tandem gene bacteria could catalytic three sources of L-serine (Serine enzyme liquid, mixed amino acid and electrodialysis mixure of amino acids) for the production of L-tyrosine, which will enrich the L-tyrosine synthesis pathway and improve resource utilization efficiency of L-serine.