title: High level expression of extracellular secretion of a β-glucosidase gene (PtBglu3) from Paecilomyces thermophila in Pichia pastoris.
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 laminarin. The present results should contribute to improving industrial production of β-glucosidase.
authors.affiliation: Bioresource Utilization Laboratory, College of Engineering, China Agricultural University, Beijing 100083, China.
authors.forename: Qiaojuan
authors.initials: Q
authors.lastname: Yan
authors.forename: Chengwei
authors.initials: C
authors.lastname: Hua
authors.forename: Shaoqing
authors.initials: S
authors.lastname: Yang
authors.forename: Yinan
authors.initials: Y
authors.lastname: Li
authors.forename: Zhengqiang
authors.initials: Z
authors.lastname: Jiang
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D018744:DNA, Plant; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D005285:Fermentation; D005656:Fungal Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D010143:Paecilomyces / Q000201:enzymology* / Q000235:genetics; D010843:Pichia / Q000201:enzymology / Q000235:genetics*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D001617:beta-Glucosidase / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D018744:DNA, Plant; D005656:Fungal Proteins; D011994:Recombinant Proteins; D001617:beta-Glucosidase