PURIFICATION AND CHARACTERIZATION OF AN EXTREMELY THERMOSTABLE BETA-GLUCOSIDASE FROM THE HYPERTHERMOPHILIC ARCHAEON PYROCOCCUS-FURIOSUS
PURIFICATION AND CHARACTERIZATION OF AN EXTREMELY THERMOSTABLE BETA-GLUCOSIDASE FROM THE HYPERTHERMOPHILIC ARCHAEON PYROCOCCUS-FURIOSUS
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DOI：
10.1111/j.1432-1033.1993.tb17763.x
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发表时间：
1993-04-01
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影响因子：
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通讯作者：
ZEHNDER, AJB
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文献类型：
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作者：
KENGEN, SWM;LUESINK, EJ;ZEHNDER, AJB
超嗜热性热球菌（Pyrococcus furiosus）的纤维二糖培养细胞的无细胞提取物含有非常高的β -葡萄糖苷酶活性（19.8 U/mg）。细胞质酶被纯化了22倍，达到明显的均匀性，表明该酶占细胞总蛋白的近5%。天然β -葡萄糖苷酶分子量为230 +/- 20 kDa，由58个+/- 2-kDa亚基组成。酶的pI为4.40。巯基不是活性所必需的，酶也不依赖于二价阳离子或高离子强度。该酶在pH 5.0和102-105℃条件下具有最佳活性。从Lineweaver-Burk图中，纤维素二糖（K(m) = 20 mM）和对硝基苯- β - d -葡萄糖苷（K(m) = 0.15 mM）的V（max）值分别为470 U/mg和700 U/mg。纯化后的酶也表现出高的-半乳糖苷酶活性和-木糖苷酶活性，但对-连接的双糖或-连接的聚合物（如纤维素）没有活性。纯化的β -葡萄糖苷酶表现出良好的热稳定性，在100℃下半衰期为85 h，在110℃下半衰期为13 h。
Cell-free extracts of cellobiose-grown cells of the hyperthermophile Pyrococcus furiosus contain very high activities (19.8 U/mg) of a beta-glucosidase. The cytoplasmic enzyme was purified 22-fold to apparent homogeneity, indicating that the enzyme comprises nearly 5% of the total cell protein. The native beta-glucosidase has a molecular mass of 230 +/- 20 kDa, composed of 58 +/- 2-kDa subunits. The enzyme has a pI of 4.40. Thiol groups are not essential for activity, nor is the enzyme dependent on divalent cations or a high ionic strength. The enzyme shows optimum activity at pH 5.0 and 102-105-degrees-C. From Lineweaver-Burk plots, V(max) values of 470 U/mg and 700 U/mg were found for cellobiose (K(m) = 20 mM) and p-nitrophenyl-beta-D-glucopyranoside (K(m) = 0.15 mM), respectively. The purified enzyme also exhibits high beta-galactosidase activity and beta-xylosidase activity, but shows no activity towards alpha-linked disaccharides or beta-linked polymers, like cellulose. The purified beta-glucosidase shows a remarkable thermostability with a half life of 85 h at 100-degrees-C and 13 h at 110-degrees-C.