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

Paper ID Volume ID Publish Year Pages File Format Full-Text
20511 43178 2013 6 PDF Available
Title
Application of protein N-terminal amidase in enzymatic synthesis of dipeptides containing acidic amino acids specifically at the N-terminus
Abstract

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.

Keywords
Protein N-terminal amidase; l-Amino acid ligase; Peptide synthesis; Functional peptide; Aspartame
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Application of protein N-terminal amidase in enzymatic synthesis of dipeptides containing acidic amino acids specifically at the N-terminus
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Publisher
Database: Elsevier - ScienceDirect
Journal: Journal of Bioscience and Bioengineering - Volume 115, Issue 4, April 2013, Pages 382–387
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
Get Full-Text Now
Don't Miss Today's Special Offer
Price was $35.95
You save - $31
Price after discount Only $4.95
100% Money Back Guarantee
Full-text PDF Download
Online Support
Any Questions? feel free to contact us