title: Secretory expression, characterization and docking study of glucose-tolerant β-glucosidase from B. subtilis.
abstract: The thermostable, glucose tolerant β-glucosidase gene (bgl) of Glycoside hydrolase family 1, isolated from Bacillus subtilis, was cloned and overexpressed in Escherichia coli. The bgl has open reading frame of 1,407 bp, encoding 469 amino acids with predicted molecular weight of 53 kDa. The recombinant protein (BGL) was purified 10.76 fold to homogeneity with specific activity of 54.04U/mg and recovery of 38.67%. The purified BGL was optimally active at pH 6.0 and temperature 60°C. The enzyme retained more than 85% of maximum activity after 1h preincubation at 60°C. The kinetic analysis indicated that BGL has highest catalytic efficiency (Kcat/Km) against p-nitrophenyl-β-d-xylopyranoside (654.58 mM(-1)s(-1)) followed by p-nitrophenyl-β-d-glucopyranoside (292.53 mM(-1)s(-1)) and p-nitrophenyl-β-d-galactopyranoside (61.17 mM(-1)s(-1)). The Ki value for glucose and δ-gluconolactone was determined to be 1.9 mM and 0.018 mM, respectively. The BGL exhibited high tolerance against detergents and organic solvents. The homology modeling revealed that protein has 19 α-helices and 4 β-sheets and adopted (α/β)8 TIM barrel structure. Substrate docking and LigPlot analysis depicted the amino acids of active site involved in hydrogen bonding and hydrophobic interactions with substrates. The efficient BGL secretion with exploration of structural and functional relationship offer vistas for large scale production and various industrial applications.
authors.affiliation: Department of Biochemistry, C.B.S.H., Govind Ballabh Pant University of Agriculture and Technology, Pantnagar, Uttarakhand 263145, India.
authors.forename: Shivangi
authors.initials: S
authors.lastname: Chamoli
authors.affiliation: Chemical Biology Laboratory, Department of Biotechnology, Indian Institute of Technology Roorkee, Roorkee, Uttarakhand 247667, India.
authors.forename: Piyush
authors.initials: P
authors.lastname: Kumar
authors.affiliation: Chemical Biology Laboratory, Department of Biotechnology, Indian Institute of Technology Roorkee, Roorkee, Uttarakhand 247667, India.
authors.forename: Naveen Kumar
authors.initials: NK
authors.lastname: Navani
authors.affiliation: Department of Biochemistry, C.B.S.H., Govind Ballabh Pant University of Agriculture and Technology, Pantnagar, Uttarakhand 263145, India. Electronic address: akv72@rediffmail.com.
authors.forename: Ashok Kumar
authors.initials: AK
authors.lastname: Verma
mesh_terms: D000595:Amino Acid Sequence; D001412:Bacillus subtilis / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D003001:Cloning, Molecular; D004789:Enzyme Activation; D004795:Enzyme Stability; D005947:Glucose / Q000737:chemistry / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D062105:Molecular Docking Simulation; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D013379:Substrate Specificity; D013696:Temperature; D001617:beta-Glucosidase / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: D011994:Recombinant Proteins; D001617:beta-Glucosidase; D005947:Glucose
keywords: Extracellular expression; Molecular docking; Thermostable-glucose tolerant β-glucosidase