Chirico W J, Brown R D
Eur J Biochem. 1987 Jun 1;165(2):333-41. doi: 10.1111/j.1432-1033.1987.tb11446.x.
A beta-glucosidase has been purified from culture filtrates of the fungus Trichoderma reesei QM9414 grown on microcrystalline cellulose. The beta-glucosidase was purified using two successive DEAE-Sephadex anion-exchange chromatography steps, followed by SP-Sephadex cation-exchange chromatography and concanavalin-A--agarose chromatography. Evidence for homogeneity is provided by polyacrylamide disc gel electrophoretic patterns, which show a single protein band. Sedimentation equilibrium analysis yielded a molecular mass of 74.6 +/- 2.4 kDa. Sodium dodecyl sulfate/polyacrylamide gel electrophoresis yielded a single protein band with a molecular mass of 81.6 kDa. Thus, the enzyme appears to be a single, monomeric polypeptide. The beta-glucosidase is isoelectric at pH 8.5. The enzyme is rich in basic amino acids and contains few half-cystine and methionine residues. The purified beta-glucosidase contains less than 1% by weight of neutral carbohydrate. The beta-glucosidase catalyzes the hydrolysis of cellobiose, p-nitrophenyl beta-D-glucopyranoside and 4-methylumbelliferyl beta-D-glucopyranoside; the values of V/Km for each substrate were determined to be 2.3 X 10(4), 6.9 X 10(5) and 2.9 X 10(6) M-1 S-1 respectively. The enzyme is optimally active from pH 4.5 to 5.0 and is labile at higher hydrogen ion concentrations. The beta-glucosidase has an unusually high affinity for D-glucose (Ki = 700 microM). Comparison of inhibition constants for cello-oligosaccharides suggests that the substrate-binding region of the beta-glucosidase comprises multiple subsites.
已从在微晶纤维素上生长的里氏木霉QM9414真菌的培养滤液中纯化出一种β-葡萄糖苷酶。该β-葡萄糖苷酶通过连续两步DEAE-葡聚糖阴离子交换色谱法进行纯化，随后进行SP-葡聚糖阳离子交换色谱法和伴刀豆球蛋白A-琼脂糖色谱法。聚丙烯酰胺圆盘凝胶电泳图谱显示为单一蛋白条带，证明其均一性。沉降平衡分析得出分子量为74.6±2.4 kDa。十二烷基硫酸钠/聚丙烯酰胺凝胶电泳产生一条分子量为81.6 kDa的单一蛋白条带。因此，该酶似乎是一种单一的单体多肽。β-葡萄糖苷酶在pH 8.5时呈等电点。该酶富含碱性氨基酸，半胱氨酸和甲硫氨酸残基较少。纯化的β-葡萄糖苷酶中中性碳水化合物含量按重量计不到1%。β-葡萄糖苷酶催化纤维二糖、对硝基苯基β-D-吡喃葡萄糖苷和4-甲基伞形酮基β-D-吡喃葡萄糖苷的水解；每种底物的V/Km值分别测定为2.3×10⁴、6.×10⁵和2.9×10⁶ M⁻¹ S⁻¹。该酶在pH 4.5至5.0时活性最佳，在较高氢离子浓度下不稳定。β-葡萄糖苷酶对D-葡萄糖具有异常高的亲和力（Ki = 700 μM）。对纤维寡糖抑制常数的比较表明，β-葡萄糖苷酶的底物结合区域包含多个亚位点。
