Application of protein N-terminal amidase in enzymatic synthesis of dipeptides containing acidic amino acids specifically at the N-terminus(ENZYMOLOGY, PROTEIN ENGINEERING, AND ENZYME TECHNOLOGY)
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概要
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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 acid; 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.
- 2013-04-00
著者
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Noguchi Atsushi
Department Of Applied Chemistry Faculty Of Science And Engineering Waseda University
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Kino Kuniki
Department Of Applied Chemistry Faculty Of Science And Engineering Waseda University
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Arai Toshinobu
Department Of Applied Chemistry Faculty Of Science And Engineering Waseda University
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Takano Eriko
Department of Applied Chemistry, Faculty of Science and Engineering, Waseda University
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