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Purification, characterization, and primary structure of a novel N-acyl-d-amino acid amidohydrolase from Microbacterium natoriense TNJL143-2

Paper ID Volume ID Publish Year Pages File Format Full-Text
20909 43197 2012 7 PDF Available
Title
Purification, characterization, and primary structure of a novel N-acyl-d-amino acid amidohydrolase from Microbacterium natoriense TNJL143-2
Abstract

A novel N-acyl-d-amino acid amidohydrolase (DAA) was purified from the cells of a novel species of the genus Microbacterium. The purified enzyme, termed AcyM, was a monomeric protein with an apparent molecular weight of 56,000. It acted on N-acylated hydrophobic d-amino acids with the highest preference for N-acetyl-d-phenylalanine (NADF). Optimum temperature and pH for the hydrolysis of NADF were 45°C and pH 8.5, respectively. The kcat and Km values for NADF were 41 s−1 and 2.5 mM at 37°C and pH 8.0, although the enzyme activity was inhibited by high concentrations of NADF. Although many known DAAs are inhibited by 1 mM EDTA, AcyM displayed a 65% level of its full activity even in the presence of 20 mM EDTA. Based on partial amino acid sequences of the purified enzyme, the full-length AcyM gene was cloned and sequenced. It encoded a protein of 495 amino acids with a relatively low sequence similarity to a DAA from Alcaligenes faecalis DA1 (termed AFD), a binuclear zinc enzyme of the α/β-barrel amidohydrolase superfamily. The unique cysteine residue that serves as a ligand to the active-site zinc ions in AFD and other DAAs was not conserved in AcyM and was replaced by alanine. AcyM was the most closely related to a DAA of Gluconobacter oxydans (termed Gox1177) and phylogenetically distant from AFD and all other DAAs that have been biochemically characterized thus far. AcyM, along with Gox1177, appears to represent a new phylogenetic subcluster of DAAs.

Keywords
N-Acyl-d-amino acid amidohydrolase; d-Amino acids; d-Aminoacylase; α/β-Barrel amidohydrolase superfamily; d-Phenylalanine; Microbacterium natoriense; Optical resolution
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Purification, characterization, and primary structure of a novel N-acyl-d-amino acid amidohydrolase from Microbacterium natoriense TNJL143-2
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Publisher
Database: Elsevier - ScienceDirect
Journal: Journal of Bioscience and Bioengineering - Volume 114, Issue 4, October 2012, Pages 391–397
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
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