Liu Bo, Li Zhuo, Hong Ye, Ni Jinfeng, Sheng Duohong, Shen Yulong
State Key Laboratory of Microbial Technology, University of Shandong, 250100, Shandong, Jinan, P.R. China.
Biotechnol Lett. 2006 Oct;28(20):1655-60. doi: 10.1007/s10529-006-9137-0. Epub 2006 Aug 16.
An exo-beta-D-glucosaminidase gene (PH0511) was cloned from the hyperthermophilic archaeon, Pyrococcus horikoshii, and expressed in Escherichia coli. The purified protein showed a strong exo-beta-D: -glucosaminidase activity by TLC analysis. DTT (50 mM) had little effect on its homodimeric structure during SDS-PAGE. The enzyme was optimally active at 90 degrees C (over 20 min) and pH 6. It had a half-life of 9 h at 90 degrees C and is the most thermostable glucosaminidase described up to now. The activity was not inhibited by ethanol, 2-propanol, DMSO, PEG-400, denaturing agents SDS (5%, w/v), urea, guanidine hydrochloride (5 M) and Mg(2+), Mn(2+), Co(2+), Ca(2+), Sr(2+), Ni(2+) (at up to 10 mM).
从嗜热古菌火之球菌中克隆出一个外-β-D-氨基葡萄糖苷酶基因（PH0511），并在大肠杆菌中表达。通过薄层色谱分析，纯化后的蛋白显示出很强的外-β-D-氨基葡萄糖苷酶活性。在十二烷基硫酸钠聚丙烯酰胺凝胶电泳期间，50 mM二硫苏糖醇对其二聚体结构影响很小。该酶在90℃（超过20分钟）和pH 6时活性最佳。它在90℃下的半衰期为9小时，是目前所描述的最耐热的氨基葡萄糖苷酶。乙醇、2-丙醇、二甲基亚砜、聚乙二醇-400、变性剂十二烷基硫酸钠（5%，w/v）、尿素、盐酸胍（5 M）以及镁离子、锰离子、钴离子、钙离子、锶离子、镍离子（浓度高达10 mM）均不抑制该酶的活性。
