Gu Na-Yeon, Kim Jung-Lim, Kim Hyun-Jung, You Dong-Ju, Kim Han-Woo, Jeon Sung-Jong
Department of Biomaterial Control (Brain Korea 21 program), Don-Eui University, 995 Eomgwangno, Busanjin-gu, Busan 614-714, Republic of Korea.
J Biosci Bioeng. 2009 Jan;107(1):21-6. doi: 10.1016/j.jbiosc.2008.10.002.
A microorganism (strain HJ6) producing extracellular beta-glycosidase was isolated from a hot springs located in Arima-cho, Hyogo, Japan. The cells were long-rods (2-4 microm) about 0.4 microm in diameter, and formed yellow-colored colonies, like most other strains of the genus Thermus. The pH and temperature for optimal growth were 6.5 and 80 degrees C. Thus, the HJ6 strain displayed a higher optimal temperature than other described Thermus sp. The gene encoding beta-glycosidase (TtbetaGly) was cloned, sequenced, and comprised of 1296 nucleotides encoding a protein (431 amino acids) with a predicted molecular mass of 48.7 kDa. TtbetaGly was expressed in Escherichia coli cells, and the recombinant protein was purified to homogeneity. The optimal temperature and pH for beta-glycosidase activity were found to be 90 degrees C and 8.5, respectively. The half-life of heat inactivation was about 30 min at 95 degrees C indicating that TtbetaGly had higher thermostability than beta-glycosidases from other Thermus sp. The results of the kinetics experiment indicated that beta-D-fucoside and beta-D-glucoside were better substrates of TtbetaGly than beta-D-galactoside. The catalytic efficiency (k(cat)/K(m)) of TtbetaGly at 80 degrees C increased 70-fold to that at 40 degrees C, indicating that this enzyme was activated at high temperatures. Thin layer chromatography showed that the enzyme had transglycosylation activity at high temperature and that various transfer products were formed in the reaction with lactose or cellobiose.
从日本兵库县有马町的一处温泉中分离出一株能产生胞外β-糖苷酶的微生物（菌株HJ6）。该细胞为长杆状（2 - 4微米），直径约0.4微米，像嗜热栖热菌属的大多数其他菌株一样形成黄色菌落。最佳生长的pH值和温度分别为6.5和80℃。因此，HJ6菌株显示出比其他已描述的嗜热栖热菌属菌株更高的最适温度。编码β-糖苷酶的基因（TtbetaGly）被克隆、测序，由1296个核苷酸组成，编码一种蛋白质（431个氨基酸），预测分子量为48.7 kDa。TtbetaGly在大肠杆菌细胞中表达，重组蛋白被纯化至同质。发现β-糖苷酶活性的最佳温度和pH值分别为90℃和8.5。在95℃下热失活的半衰期约为30分钟，表明TtbetaGly比其他嗜热栖热菌属的β-糖苷酶具有更高的热稳定性。动力学实验结果表明，β-D-岩藻糖苷和β-D-葡萄糖苷是TtbetaGly比β-D-半乳糖苷更好的底物。TtbetaGly在80℃时的催化效率（k(cat)/K(m)）比在40℃时提高了70倍，表明该酶在高温下被激活。薄层色谱显示该酶在高温下具有转糖基化活性，并且在与乳糖或纤维二糖的反应中形成了各种转移产物。
