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首页> 外文期刊>Extremophiles: Life under extreme conditions >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

机译：腾康热厌氧菌MB4的热稳定性丙氨酸消旋酶的表征和初步突变分析

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摘要

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.
机译：来自腾格热厌氧菌MB4的热稳定的丙氨酸消旋酶已在大肠杆菌中成功表达并鉴定。全长基因MBalr2（1164 bp）编码388个氨基酸残基，其中包括丙氨酸消旋酶活性位点入口处8个高度保守的氨基酸残基中的6个。通过His_6-tag亲和柱和凝胶过滤色谱纯化重组的MBAlr2和MBAlr2的三个突变体（S171A，H359Y和双突变S171A / H359Y）。纯化的蛋白MBAlr2是具有二聚体PLP依赖性的酶，具有广泛的底物特异性。最佳外消旋温度和pH分别为70-75°C和11.0。 HPLC测定的MBAlr2在70°C的动力学参数K _m和V _max（max）对L-丙氨酸分别为20.16 mM和1414μmolmin〜（-1），而9.95 mM和702.6μmolmin〜（-1） ）分别用于d-丙氨酸。酶分析表明，两个突变体（S171A和H359Y）的活性均丧失，但突变体S171A / H359Y的活性已恢复至野生型的69.8％，这表明Ser171和His359残基可能是其催化作用的重要残基。 MBAlr2。

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