Biochemical characterization and mechanism of action of a thermostable beta-glucosidase purified from Thermoascus aurantiacus.
Biochemical characterization and mechanism of action of a thermostable beta-glucosidase purified from Thermoascus aurantiacus.
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DOI：
10.1042/bj3530117
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发表时间：
2001
期刊：
影响因子：
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通讯作者：
Neil J. Parry;David E. Beever;Emyr Owen;Isabel Vandenberghe;Jozef Van Beeumen;M. K. Bhat
中科院分区：
文献类型：
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作者：
Neil J. Parry;David E. Beever;Emyr Owen;Isabel Vandenberghe;Jozef Van Beeumen;M. K. Bhat
通过DEAE-Sepharose、Ultrogel AcA 44和Mono-P柱层析，从嗜热菌（Thermobacteraurantiacus）中分离纯化了一种胞外β-葡萄糖苷酶。该酶是一种同源三聚体，单体分子量为120 kDa;只有三聚体在80 ℃和pH 4.5下具有最佳活性。在90摄氏度时，该酶显示出其最佳活性的70%。它在pH 5.2和高达70 ℃的温度下稳定48小时，但在70 ℃以上和pH值高于和低于5.0时稳定性下降。该酶水解芳基和烷基β-D-葡糖苷和纤维寡糖，并且对具有β-糖苷键的底物具有特异性。在二糖的非还原端的葡萄糖残基的位置2、4和6处的羟基似乎对于催化是必需的。该酶对对硝基苯基β-d-葡萄糖苷的K（m）最低（0.1137 mM），对纤维二糖和β，β-海藻糖的K（cat）最高（17052 min（-1））。它从纤维寡糖的非还原端每次释放一个葡萄糖单元，并且水解速率随着链长的增加而降低。葡萄糖和d-δ-葡内酯竞争性抑制β-葡萄糖苷酶，K（i）值分别为0.29 mM和8.3 nM，而甲醇、乙醇和丙-2-醇激活该酶。在甲醇、乙醇和丙-2-醇的存在下，该酶分别与葡萄糖或纤维二糖催化甲基、乙基和丙基β-d-葡糖苷的合成，尽管纤维二糖是优选的。酸性pH有利于水解和转糖基化，但高浓度的醇有利于后者的反应。纤维二糖水解的立体化学研究表明，T。aurantiacus是保留糖苷酶，而N-末端氨基酸序列比对表明其是糖苷水解酶家族3的成员。
An extracellular beta-glucosidase from Thermoascus aurantiacus was purified to homogeneity by DEAE-Sepharose, Ultrogel AcA 44 and Mono-P column chromatography. The enzyme was a homotrimer, with a monomer molecular mass of 120 kDa; only the trimer was optimally active at 80 degrees C and at pH 4.5. At 90 degrees C, the enzyme showed 70% of its optimal activity. It was stable at pH 5.2 and at temperatures up to 70 degrees C for 48 h, but stability decreased above 70 degrees C and at pH values above and below 5.0. The enzyme hydrolysed aryl and alkyl beta-d-glucosides and cello-oligosaccharides, and was specific for substrates with a beta-glycosidic linkage. The hydroxy groups at positions 2, 4 and 6 of a glucose residue at the non-reducing end of a disaccharide appeared to be essential for catalysis. The enzyme had the lowest K(m) towards p-nitrophenyl beta-d-glucoside (0.1137 mM) and the highest k(cat) towards cellobiose and beta,beta-trehalose (17052 min(-1)). It released one glucose unit at a time from the non-reducing end of cello-oligosaccharides, and the rate of hydrolysis decreased with an increase in chain length. Glucose and d-delta-gluconolactone inhibited the beta-glucosidase competitively, with K(i) values of 0.29 mM and 8.3 nM respectively, while methanol, ethanol and propan-2-ol activated the enzyme. The enzyme catalysed the synthesis of methyl, ethyl and propyl beta-d-glucosides in the presence of methanol, ethanol and propan-2-ol respectively with either glucose or cellobiose, although cellobiose was preferred. An acidic pH favoured hydrolysis and transglycosylation, but high concentrations of alcohols favoured the latter reaction. The stereochemistry of cellobiose hydrolysis revealed that beta-glucosidase from T. aurantiacus is a retaining glycosidase, while N-terminal amino acid sequence alignment indicated that it is a member of glycoside hydrolase family 3.