Karnchanatat Aphichart, Petsom Amorn, Sangvanich Polkit, Piaphukiew Jittra, Whalley Anthony J S, Reynolds Colin D, Sihanonth Prakitsin
Biotechnology Programme, Faculty of Science, Chulalongkorn University, Bangkok, Thailand.
FEMS Microbiol Lett. 2007 May;270(1):162-70. doi: 10.1111/j.1574-6968.2007.00662.x.
An extracellular beta-glucosidase was purified from culture filtrates of the wood-decaying fungus Daldinia eschscholzii (Ehrenb.:Fr.) Rehm grown on 1.0% (w/v) carboxymethyl-cellulose using ammonium sulfate precipitation, ion-exchange, hydrophobic interaction and gel filtration chromatography. The enzyme is monomeric with a molecular weight of 64.2 kDa as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and has a pI of 8.55. The enzyme catalyzes the hydrolysis of p-nitrophenyl-beta-D-glucopyranoside (PNPG) as the substrate, with a K(m) of 1.52 mM, and V(max) of 3.21 U min mg(-1) protein. Glucose competitively inhibited beta-glucosidase with a K(i) value of 0.79 mM. Optimal activity with PNPG as the substrate was at pH 5.0 and 50 degrees C. The enzyme was stable at pH 5.0 at temperatures up to 50 degrees C. The purified beta-glucosidase was active against PNPG, cellobiose, sophorose, laminaribiose and gentiobiose, but did not hydrolyze lactose, sucrose, Avicel or o-nitrophenyl-beta-d-galactopyranoside. The activity of beta-glucosidase was stimulated by Ca(2+), Co(2+), Mg(2+), Mn(2+), glycerol, dimethyl sulfoxide (DMSO), dithiothreitol and EDTA, and strongly inhibited by Hg(2+). The internal amino acid sequences of D. eschscholziibeta-glucosidase have similarity to the sequences of the family 3 beta-glucosyl hydrolase.
从在1.0%（w/v）羧甲基纤维素上生长的腐朽木真菌埃氏炭团菌（Ehrenb.:Fr.）Rehm的培养滤液中，通过硫酸铵沉淀、离子交换、疏水相互作用和凝胶过滤色谱法纯化出一种胞外β-葡萄糖苷酶。经十二烷基硫酸钠-聚丙烯酰胺凝胶电泳估计，该酶为单体，分子量为64.2 kDa，其等电点为8.55。该酶以对硝基苯基-β-D-吡喃葡萄糖苷（PNPG）为底物催化水解反应，米氏常数（K(m)）为1.52 mM，最大反应速度（V(max)）为3.21 U min mg(-1)蛋白。葡萄糖对β-葡萄糖苷酶有竞争性抑制作用，抑制常数（K(i)）值为0.79 mM。以PNPG为底物时，最佳活性在pH 5.0和50℃。该酶在pH 5.0且温度高达50℃时稳定。纯化的β-葡萄糖苷酶对PNPG、纤维二糖、槐糖、层二糖和龙胆二糖有活性，但不水解乳糖、蔗糖、微晶纤维素或邻硝基苯基-β-D-吡喃半乳糖苷。β-葡萄糖苷酶的活性受到Ca(2+)、Co(2+)、Mg(2+)、Mn(2+)、甘油、二甲基亚砜（DMSO）、二硫苏糖醇和EDTA的刺激，并受到Hg(2+)的强烈抑制。埃氏炭团菌β-葡萄糖苷酶的内部氨基酸序列与3家族β-葡萄糖基水解酶的序列相似。
