极耐热性乳酸脱氢酶高效表达、纯化及酶学性质

Expression, purification and characterization of a thermostable lactate dehydrogenase from Thermotoga maritima

摘要:

从海栖热袍菌扩增出编码乳酸脱氢酶的基因并将其插入热激载体pHsh构建表达质粒，在大肠杆菌Escherichia coli中进行表达产生极耐热性乳酸脱氢酶Tm-LDH。基因表达产物通过热处理，可以一步获得接近电泳纯的重组酶。酶学性质研究表明，Tm-LDH的最适反应温度为95 ℃，最适pH 7.0；纯酶在90 ℃的半衰期为2 h，在pH 5.5–8.0之间最稳定；SDS-PAGE结果显示分子量为33 kDa，与理论推算值相吻合。以丙酮酸和NADH为底物时，相对于丙酮酸的Km值1.7 mmol/L，Vmax为3.8×104 U/mg；相对于NADH的Km值7.2 mmol/L，Vmax值为1.1×105 U/mg。Tm-LDH基因在T7载体中未能实现高效表达，但是在热激载体pHsh中得到了可溶性超量表达，表达水平达到340 mg/L。该酶在65 ℃反应条件下，活性达到最高活性的50％，并能保持活性不变，这使该酶能够与常温酶匹配，在辅酶NAD再生体系的建立中具有广泛的用途。

Abstract:

The gene encoding thermostable lactate dehydrogenase (Tm-LDH) was cloned into the plasmid pHsh from Thermotoga maritima, and expressed in Escherichia coli JM 109. The recombinant protein was purified to homogeneity by a simple step, heat treatment. The recombinant enzyme had a molecular mass of 33 kDa. The optimal temperature and pH of Tm-LDH were observed 95 ℃ and 7.0. The purified enzyme had a half-life of 2 h at 90 ℃, and exhibited better stability over a pH range from 5.5 to 8.0. The Km and Vmax values were 1.7 mmol/L, 3.8×104 U/mg of protein for pyruvate, and 7.2 mmol/L and 1.1×105 U/mg for NADH, respectively. The expression of Tm-LDH in T7 system could not obtain high efficiency, but it has been soluble over-expression in pHsh system and reached 340 mg/L. The superior stability and productivity of Tm-LDH will lay the foundation of its industrial-scale fermentation and application in the NAD regeneration.