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Characterization of endogenous pyridoxal 5′-phosphate-dependent alanine racemase from Bacillus pseudofirmus OF4

Paper ID Volume ID Publish Year Pages File Format Full-Text
22250 43264 2009 5 PDF Available
Title
Characterization of endogenous pyridoxal 5′-phosphate-dependent alanine racemase from Bacillus pseudofirmus OF4
Abstract

An open reading frame of 1100 bp in the partially sequenced genome sequence of alkaliphilic Bacillus pseudofirmus OF4 was identified as a putative alanine racemase gene (dadXOF4), which was cloned and expressed in Escherichia coli BL21 (DE3). The encoded protein DadXOF4 was purified to homogeneity by His6-tag affinity column, gel filtration and ion-exchange chromatography. The amino acid sequence has highest identity with the known alanine racemase from Oceanobacillus iheyensis HTE831 (48%). The protein was a dimeric, endogenous PLP-dependent enzyme, which was demonstrated by absorption spectra and enzyme activity with or without PLP. The racemization temperature optimum was 40 °C and the optimal pH was 10.5. The kinetic parameters Km and Vmax at 40 °C of alanine racemase, determined by HPLC analysis, were 41.79 mM, 10,500 units/mg for L-alanine and 14.91 mM, 3708 units/mg for D-alanine, respectively.

Keywords
Alanine racemase; Bacillus pseudofirmus; Endogenous; Dimeric; Pyridoxal 5′-phosphate
First Page Preview
Characterization of endogenous pyridoxal 5′-phosphate-dependent alanine racemase from Bacillus pseudofirmus OF4
Publisher
Database: Elsevier - ScienceDirect
Journal: Journal of Bioscience and Bioengineering - Volume 107, Issue 3, March 2009, Pages 225–229
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering