Department of Biotechnology, College of Food Science and Nutritional Engineering, China Agricultural University, Beijing 100083, China.
Carbohydr Polym. 2013 Jan 30;92(1):784-91. doi: 10.1016/j.carbpol.2012.09.086. Epub 2012 Oct 8.
A novel β-glucosidase gene (PtBglu1) from the thermophilic fungus, Paecilomyces thermophila, was cloned and expressed in Pichia pastoris. PtBglu1 contained an open reading frame of 1440-bp nucleotides and encoded a protein of 479 amino acids which showed significant similarity to other fungal β-glucosidases from glycoside hydrolase (GH) family 1. The recombinant β-glucosidase (PtBglu1) was secreted at high level of 190.2 U mL(-1) in high cell density fermentor (5L). PtBglu1 was purified to homogeneity, and was found to be a glycoprotein with molecular mass of 56.7 kDa. The purified PtBglu1 showed optimum catalytic activity at pH 6.0 and 55 °C. The enzyme exhibited broad substrate specificity with highest activity toward pNP-β-D-glucopyranoside, followed by pNP-β-D-galactopyranoside and cellobiose. The K(m) values for pNP-β-D-glucopyranoside, cellobiose, gentiobiose and salicin were 0.55 mM, 1.0 mM, 1.74 mM and 6.85 mM, respectively. These properties make PtBglu1 a potential candidate for various industrial applications.
一株嗜热真菌——Thermomyces thermophila 的新型β-葡萄糖苷酶基因（PtBglu1）被克隆并在毕赤酵母中表达。PtBglu1 含有一个 1440-bp 的开放阅读框，编码一个 479 个氨基酸的蛋白质，与其他糖苷水解酶（GH）家族 1 的真菌β-葡萄糖苷酶有显著的相似性。重组β-葡萄糖苷酶（PtBglu1）在 5L 高密度发酵罐中以 190.2 U mL(-1)的高水平分泌。PtBglu1 被纯化为均一性，被发现是一种糖蛋白，分子量为 56.7 kDa。纯化的 PtBglu1 在 pH 6.0 和 55°C 时表现出最佳的催化活性。该酶表现出广泛的底物特异性，对 pNP-β-D-葡萄糖苷的活性最高，其次是 pNP-β-D-半乳糖苷和纤维二糖。pNP-β-D-葡萄糖苷、纤维二糖、龙胆二糖和水杨苷的 K(m) 值分别为 0.55 mM、1.0 mM、1.74 mM 和 6.85 mM。这些特性使 PtBglu1 成为各种工业应用的潜在候选者。
