从纤维单胞菌属β-葡萄糖苷酶活性部位的动力学分析。

Kinetic analysis of the active site of an intracellular beta-glucosidase fromCellulomonas biazotea.

机构信息

National Institute for Biotechnology and Genetic Engineering, P.O.Box 577, Faisalabad, Pakistan.

出版信息

Folia Microbiol (Praha). 1997 Feb;42(1):53-8. doi: 10.1007/BF02898646.

Abstract

Purified beta-glucosidase fromCellulomonas biazotea had an apparentK (m) andV for 2-nitrophenyl beta-D: -glucopyranoside (oNPG) of 0.416 mmol/L and 0.22 U/mg protein, respectively. The activation energy for the hydrolysis of pNPG of beta-glucosidase was 65 kJ/mol. The inhibition by Mn(2+) vs. oNPG of parental beta-glucosidase was of mixed type with apparent inhibition constants of 0.19 and 0.60 micromol/L for the enzyme and enzyme-substrate complex, respectively. Ethanol at lower concentrations activated while at higher concentrations it inhibited the enzyme. The determination of apparent pK (a)'s at different temperatures and in the presence of 30 % dioxane indicated two carboxyl groups which control theV value. The thermal stability of beta-glucosidase decreased in the presence of 10 % ethanol. The half-life of beta-glucosidase in 1.75 mol/L urea at 35 degrees C was 145 min, as determined by 0-9 mol/L transverse urea gradient-PAGE.

摘要

从纤维单胞菌属中纯化得到的β-葡萄糖苷酶对 2-硝基苯-β-D：-吡喃葡萄糖苷（oNPG）的表观 K（m）和 V 值分别为 0.416mmol/L 和 0.22U/mg 蛋白。β-葡萄糖苷酶水解对硝基苯-β-D-葡萄糖苷的活化能为 65kJ/mol。Mn（2+）对亲本β-葡萄糖苷酶的抑制作用为混合型，对酶和酶-底物复合物的表观抑制常数分别为 0.19 和 0.60μmol/L。乙醇在较低浓度下激活该酶，而在较高浓度下则抑制该酶。在不同温度下和在 30%二氧六环存在下测定的表观 pK（a）值表明，有两个羧基基团控制 V 值。β-葡萄糖苷酶在 10%乙醇存在下的热稳定性降低。在 35°C 时，1.75mol/L 尿素中β-葡萄糖苷酶的半衰期通过 0-9mol/L 横向尿素梯度-PAGE 测定为 145min。