Biotechnology

Biography

Biography: Ayako Tanaka

Abstract

All amino acids except for glycine can exist in either of two enantiomers, called l- or d-amino acids. l-Amino acids are predominant in nature, but d-amino acids also play an important role as physiologically active substances. d-Valine and d-Phenylalanine are components of some peptide antibiotics, and d-Aspartic acid affects the production of collagens in the skin. The increasing demand of d-amino acids by pharmaceutical and biotechnological industries and the difficulty of mass production of d-amino acids by enzymatic synthesis motivate us to establish a novel method which can produce d-amino acids on large-scale. In this study, we developed and evaluated the fermentation system for the production of N-acetyl-d-amino acids using the recombinant E. coli.

The co-expression vector of alanine racemase (Alr) from Pseudomonas putida KT2440 and d-amino-acid N-acetyltransferase (DNT) from Saccharomyces cerevisiae was constructed using a pETDuet-1 plasmid. E. coli Rosetta-Gami B (DE3) transformed with the vector was grown in LB medium, harvested and disrupted by sonication. We first checked the activity of Alr and DNT using the resulting cell lysate in vitro. The specific activity of Alr and DNT was found to be 20.2 U/mg and 3.42 U/mg, respectively. Then, we monitered the fermentation performance of the recombinant E. coli grown in LB medium containing l-Methionine (l-Met) as a substrate. After 10 hours incubation, only d-Met, but not N-acetyl-d-Met, was detected in the culture supernatant. These results suggested that Alr could convert l-Met into d-Met in the periplasmic space, and vast majority of d-Met was leaked outside the cells.