Enzymatic Synthesis Of L-arginine Chiral Metabolic Products Research

Chiral is one of the basic properties of nature. Most molecules of constituting living organism are asymmetric chiral molecules, which play important physiological functions in vivo.The pharmacologic action, toxicity and therapeutic effect is different between enantiomer in vivo. The chiral research has been attracted more and more attention of the word. As common chiral molecules, amino acids with various functions and applications in many areas, including drug manufacturing, and the health care and food industry.In this paper the target genes were synthesized after rare codon optimization and expressed in E. coli BL21 (DE3)via pETDuet-1. Use the reconbinant enzymes to biotransformation L-arginine to L-ornithine and L-citrulline.Specifically the following:1. Recombinant expression of arginase. The gene (ARG1) encoding arginase, from bovine Liver, was obtained by nucleotide synthesis after rare codon optimization. Thirty-four rare codons of ARG1 gene were replaced with E. coli favored codons. The synthetic gene was digested with NcoⅠ and HindⅢ, and the isolated DNA fragments were recovered and inserted into pETDuet vector. The recombinant plasmids, pETDuet-ARG1, was transformed into E. coli BL21 (DE3). The reaction was optimal at pH 9.5 and 37℃. Manganese (10-5 M) and Emulsifier OP-10 (0.033%(v/v)) could promote arginase activity. L-arginine conversion rate reached 98% with a final concentration of 111.52 g L-ornithine/1 in 10ml transfer solution.2. Recombinant expression of arginine deiminase. The gene (ADI) encoding arginine deiminase, from enterococcus faecalis, was obtained by nucleotide synthesis after rare codon optimization. Eight rare codons of ADI gene were replaced with E. coli favored codons. The synthetic gene was digested with NcoⅠ and HindⅢ, and the isolated DNA fragments were recovered and inserted into pETDuet vector. The recombinant plasmids, pETDuet-ADI, was transformed into E. coli BL21 (DE3).The reaction was optimal at pH 6.5 and 49℃. Triton X-100 (0.049%(v/v)) could promote arginine deiminase activity. L-arginine conversion rate reached 98.5% with a final concentration of 198.12 g L-citrulline/1 in 10ml transfer solution.Although the biological synthesis of chiral compounds is still very limited range of applications, only to provide intermediate for the chemical reaction or alternative steps, however, with the development of genetic engineering and enzyme engineering technology, biological synthesis of chiral drug intermediates will accomplish much. The paper based on the biological synthesis of chiral drug intermediates, combined with genetic engineering recombinant enzyme, increased the efficiency of enzymatic conversion, prepared L-ornithine and L-citrulline, has important theoretical significance and potential applications.