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B6db references: 23703245

type Journal Article
authors Xue Z1, Hu Y, Xu S, Ohnishi K, Ma Y, Ju J, Zhao B.
title Characterization and preliminary mutation analysis of a thermostable alanine racemase from Thermoanaerobacter tengcongensis MB4
journal Extremophiles
Activity 5.1.1.1
Family 5.1.1.1.a
sel selected
ui 23703245
year (2013)
volume 17
number 4
pages 611-21
 
keywords Alanine Racemase/genetics/*metabolism
abstract A thermostable alanine racemase from Thermoanaerobacter tengcongensis MB4 was successfully expressed in Escherichia coli and characterized. The full-length gene MBalr2 (1164 bp) encodes 388 amino acid residues including 6 out of 8 highly conserved amino acid residues at the entryway to the active site of alanine racemase. Recombinant MBAlr2 and three mutants (S171A, H359Y and double mutation S171A/H359Y) of MBAlr2 were purified by His6-tag affinity column and gel filtration chromatography. The purified protein MBAlr2 was a dimeric PLP-dependent enzyme with broad substrate specificity. The optimal racemization temperature and pH were 70-75 C and 11.0, respectively. The kinetic parameters K m and V max of MBAlr2 at 70 C, determined by HPLC, were 20.16 mM and 1414 μmol min(-1) for L-alanine, and 9.95 mM and 702.6 μmol min(-1) for D-alanine, respectively. Enzymatic assays showed that the activity of both mutants (S171A and H359Y) was lost, but the activity of mutant S171A/H359Y was recovered to 69.8 % of wild type, which suggested that residues Ser171 and His359 might be the important residues for catalytic mechanisms of MBAlr2.

PMID:

last changed 2014/03/06 10:11

B6db references