热稳定酸眭β-葡萄糖苷酶的分离纯化及其酶学性质

摘要

耐酸性黑曲霉菌株Aspergillus niger L.的菌丝体破碎液依次经过乙醇沉淀、离子交换层析和凝胶过滤等步骤处理,获得电泳纯的β-葡萄糖苷酶,SDS-PAGE显示其分子质量为125.7kD。β-葡萄糖苷酶水解对硝基苯-β-D-吡喃葡萄糖苷的最适pH值为3.0～4.0,最适温度为70℃,表观米氏常数（Km）值为2.35mmol/L,表观kcat/Km值为2.99×10^4L/（mol·s）;水解京尼平苷、水杨苷的表观kcat/Km值分别是1.26×10^4、1.37×10^4L/（mol·s）;水解活性受Mn2＋的显著激活和Fe^2＋、Zn^2＋、Cu^2＋等离子的微弱抑制。该酶活性在pH2.0～8.3保持稳定;酶在65℃时保温60min,残余酶活达到了85%,是一种热稳定酸性β-葡萄糖苷酶。%In this study,an acidic β-glucosidase（BGL） was purified from acid-tolerant Aspergillus niger L. mycelia by ethanol precipitation,DEAE-Sepharose column chromatography and Sephadex G-100 column chromatography.SDS-PAGE showed that the molecular weight of the enzyme was 125.7 kD.Further characterization revealed that it had maximal hydrolytic activity on p-nitrophenyl-β-D-glucopyranoside（pNPG） at pH 3.0 - 4.0 and 70 ℃ with a Km of 2.35 mmol/L and a kcat/Km of 2.99 × 10^4 mol/L·s.The kcat/Km values for hydrolyzing geniposide and salicin were 1.26 × 10^4 L/（mol·s） and 1.37 × 10^4 L/（mol·s）,respectively.The hydrolytic activity was activated obviously by Mn2＋ but inhibited faintly by Fe^2＋,Zn^2＋ and Cu^2＋.The BGL was highly stable at pH 2.0 - 8.5,and 85% of its original activity could be maintained after 60 min of heat treatment at 65 ℃.Thus,the enzyme was highly stable to heat.