Abstract:

6-phosphate-β-glucosidase (EC 3.2.1.86), an enzyme catalyzes 6-phosphate-glucoside compounds (such as 6-phosphate-cellobiose, 6-phosphate-cellulose oligosaccharides) to produce 6-phosphate-glucose, plays an important role in carbon utilization for microbes. Thermoanaerobacter tengcongensis MB4, a strain of thermophilic anaerobic microorganism, is an excellent resource for thermo-stable protein. In this study, gene TtebglB was isolated from this strain and then heterologously expressed in E.coli BL21 (DE3). The optimum pH and temperature of TteBglB were pH 6.0 and 70℃, respectively, and the enzyme kept stable at a pH 4~10 at 70℃. Additionally, it was also confirmed that TteBglB belonged to glycosyl hydrolase family 1 (GH1) and could act without Mn2+, Ni2+, Co2+, Fe2+ and other divalent metal ions. Using pNPβG6P as substrate, the values of Km, Kcat, and Vmax for this enzyme was 0.054 mmol/L, 81.47/min and 0.003 992 mmol/min, respectively, and specific activity of the enzyme reached to 18093 U/mg.

Key words: TteBglB, 6-phosphate-β-glucosidase, glycosyl hydrolase

摘要：

6-磷酸-β-葡萄糖苷酶（EC 3.2.1.86）催化6-磷酸-葡萄糖苷类化合物（如6-磷酸-纤维二糖、6-磷酸-纤维素寡糖）产生6-磷酸-葡萄糖而使纤维素完全分解，在微生物碳源利用过程中起重要作用。腾冲嗜热厌氧杆菌是一株嗜热厌氧微生物，提供了丰富的热稳定性蛋白基因资源。从该菌株中克隆编码6-磷酸-β-葡萄糖苷酶的基因TtebglB，并在E.coli BL21（DE3）进行了异源表达。结果表明，TteBglB催化反应的最适pH为 6.0、最适温度为70℃，在pH 4~10或 70℃时有着良好的稳定性；同时证实，TteBglB属于糖基水解酶家族1（GH1）成员，可不依赖Mn2+、Ni2+、Co2+ 或Fe2+ 等二价金属离子而发挥催化作用。以pNPβG6P为底物时，催化反应的Km为0.054 mmol/L，Kcat为81.47/min，Vmax为0.003992 mmol/min，酶比活为18.093 U/mg。

关键词: TteBglB；6-磷酸-β-葡萄糖苷酶；糖苷水解酶