重组葡萄糖脱氢酶酶学性质研究

Enzymatic Characterization of A Recombinant Glucose 1-Dehydrogenase

目的 对重组葡萄糖脱氢酶(rGDH)酶学性质进行研究。方法 对rGDH进行纯化,取纯化后的rGDH,测定其最适反应温度、最适pH、金属离子耐受性、有机溶剂耐受性、底物偏好性、辅酶依赖性、米氏常数(K_(m))等酶学特征。结果 经测定rGDH的最适反应温度为40℃,最适反应pH为10.5,对D-葡萄糖的K_(m)为0.3 mol/L,该酶只耐受特定有机溶剂,大部分金属离子对其酶活有促进作用,且具有较强的底物特异性,是一种NAD^(+)、NADP^(+)双辅酶依赖型酶,但对NAD^(+)具有更强的亲和力。结论 本研究筛选并表达得到耐碱的rGDH,解决了大规模酶法制备熊去氧胆酸反应中辅酶再生问题,降低了生产成本。

Objective To study the enzymatic properties of recombinant glucose 1-dehydrogenase(rGDH).Methods The rGDH was purified and the optimal temperature,optimal pH,metal ion tolerance,stability in organic solvents,long-term stability,substrate preference,coenzyme dependence,Michaelis constant(K_(m))and other enzymatic properties of rGDH were determined.Results The optimal reaction temperature of rGDH was 40℃,and the optimal reaction pH was 10.5.The K_(m) for D-glucose is 0.3 mol/L.The enzyme had a strong substrate specificity and was sensitive to most organic solvents.The rGDH was an NAD^(+),NADP^(+)dual coenzyme-dependent enzyme,but had a stronger affinity for NAD^(+).Most of the metal ions could promote its activity.Conclusion In this study,an alkali-tolerant recombinant glucose dehydrogenase is screened and expressed,which can solve the problem of coenzyme regeneration in the large-scale enzymatic preparation of ursodeoxycholic acid and reduce the production cost.