Abstract
A novel β-glucosidase gene (designated PtBglu3) from Paecilomyces thermophila was cloned and sequenced. PtBglu3 has an open reading frame of 2,557 bp, encoding 858 amino acids with a calculated molecular mass of 90.9 kDa. The amino acid sequence of the mature polypeptide shared the highest identity (70%) to a glycoside hydrolase (GH) family 3 characterized β-glucosidase from Penicillium purpurogenum. PtBglu3 without the signal peptides was cloned into pPIC9K vector and successfully expressed in Pichia pastoris as an active extracellular β-glucosidase (PtBglu3). High activity of 274.4 U/ml was obtained by high cell-density fermentation, which is by far the highest reported yield for β-glucosidase. The recombinant enzyme was purified to homogeneity with 3.3-fold purification and a recovery of 68.5%. The molecular mass of the enzyme was estimated to be 116 kDa by SDS-PAGE, and 198.2 kDa by gel filtration, indicating that it was a dimer. Optimal activity of the purified enzyme was observed at pH 6.0 and 65 °C, and it was stable up to 60 °C. The enzyme exhibited high specific activity toward pNP-β-D-glucopyranoside, cellooligosaccharides, gentiobiose, amygdalin and salicin, and relatively lower activity against lichenan and laminari...Continue Reading
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