Application of protein N-terminal amidase in enzymatic synthesis of dipeptides containing acidic amino acids specifically at the N-terminus

Dipeptides exhibit unique physiological functions and physical properties, e.g., L-aspartyl-L-phenylalanine-methyl. ester (Asp-Phe-OMe, aspartame) as an artificial sweetener, and functional studies of peptides have been carried out in various fields. Therefore, to establish a manufacturing process for the useful dipeptides, we investigated its enzymatic synthesis by utilizing an L-amino acid ligase (Lal), which catalyzes dipeptide synthesis in an ATP-dependent manner. Many Lals were obtained, but the Lals recognizing acidic amino acids as N-terminal substrates have not been identified. To increase the variety of dipeptides that are enzymatically synthesized, we proposed a two-step synthesis: Asn-Xaa and Gln-Xaa (Asn, L-asparagine; Gln, L-glutamine; and Xaa, arbitrary amino acids) synthesized by Lals were continuously deamidated by a novel amidase, yielding Asp-Xaa and Glu-Xaa (Asp, L-aspartic add; and Glu, L-glutamic acid). We searched for amidases that specifically deamidate the N-terminus of Asn or Gln in dipeptides since none have been previously reported. We focused on the protein N-terminal amidase from Saccharomyces cerevisiae (NTA1), and assayed its activity toward dipeptides. Our findings showed that NTA1 deamidated L-asparaginyl-L-valine (Asn-Val) and L-glutaminyl-glycine (Gln-Gly), but did not deamidate L-valyl-L-asparagine and L-alanyl-L-glutamine, suggesting that this deamidation activity is N-terminus specific. The specific activity toward Asn-Val and Gln-Gly were 190 +/- 30 nmol min(-1) mg(-1) protein and 136 +/- 6 nmol min(-1) mg(-1) protein. Additionally, we examined some characteristics of NTA1. Acidic dipeptide synthesis was examined by a combination of Lals and NTA1, resulting in the synthesis of 12 kinds of Asp-Xaa, including Asp-Phe, a precursor of aspartame, and 11 kinds of Glu-Xaa. (C) 2012, The Society for Biotechnology, Japan. All rights reserved.