琥珀酸半醛脱氢酶GabD4的表达、纯化及酶学性质分析

摘要

人工合成琥珀酸半醛脱氢酶GabD4基因,经蛋白表达和纯化,对其酶学性质进行研究.结果表明,GabD4的最适反应温度为37℃;最适pH为7.5(磷酸盐缓冲液)或9.0(甘氨酸-氢氧化钠缓冲液);金属离子中,Cu2对GabD4酶活力的影响较大;GabD4对3～5个碳的醛底物具有更高的催化效率;以乙醛为底物,计算得到米氏常数(Km)、最大反应速率(Vmax)分别为6.86 mmol/L和20.6 μmol/(min·mg protein).该结果为深入了解GabD4的酶学性质提供参考.%The synthesis of succinate-semialdehyde dehydrogenase GabD4 gene,its expression in Escherichia coli and purification were described herein.The enzymatic characterization was analyzed.The study showed that the optimal reaction temperature of GabD4 was 37 ℃.The optimal reaction pH was pH 7.5 in phosphate buffer or pH 9.0 in glycine-NaOH buffer.Among several divalent metal ions,Cu2+ had stimulative effect on enzyme activity.Furthermore,GabD4 preferred to catalyze aldehyde substrates containing 3 ～5 carbon,and it had a Michaelis constant (Km) of 6.86 mmol/L and Vmax of 20.6 μmol/(min · mg protein) with acetaldehyde as substrate.These results could provide more information for research on enzymatic properties of GabD4.