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Characterization and preliminary mutation analysis of a thermostable alanine racemase from Thermoanaerobacter tengcongensis MB4
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Characterization and preliminary mutation analysis of a thermostable alanine racemase from Thermoanaerobacter tengcongensis MB4
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Characterization and preliminary mutation analysis of a thermostable alanine racemase from Thermoanaerobacter tengcongensis MB4
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Journal Article
Characterization and preliminary mutation analysis of a thermostable alanine racemase from Thermoanaerobacter tengcongensis MB4
2013
Overview
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 His
6
-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.
Publisher
Springer Science and Business Media LLC,Springer Japan,Springer Nature B.V
