|
type |
Journal Article |
authors |
Xue Z, 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. |
last changed |
2017/07/12 12:54 |
|