热稳定6-磷酸-β-葡萄糖苷酶TteBglB异源表达、分离纯化及酶学性质分析

Heterologous Expression,Purification and Characterization of Thermo-stable 6-phosphate-β-glucosidase TteBglB

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Ttebgl B,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p H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Mn^2＋、Ni^2＋、Co^2＋或Fe^2＋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。

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 Ttebgl B was isolated from this strain and then heterologously expressed in E.coli BL21（DE3）.The optimum p H and temperature of TteBglB were p H 6.0 and 70℃,respectively,and the enzyme kept stable at a p H 4 ~ 10 at 70℃.Additionally,it was also confirmed that TteBglB belonged to glycosyl hydrolase family 1（GH1） and could act without Mn,2 ＋,Ni,2 ＋,Co,2 ＋,Fe,2 ＋and other divalent metal ions.Using p NPβG6P as substrate,the values of Km,Kcat,and Vmaxfor 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.