title: Characterization of a thermostable family 1 Glycosyl Hydrolase enzyme from Putranjiva roxburghii seeds.
abstract: A 66-kDa thermostable family 1 Glycosyl Hydrolase (GH1) enzyme with β-glucosidase and β-galactosidase activities was purified to homogeneity from the seeds of Putranjiva roxburghii belonging to Euphorbiaceae family. N-terminal and partial internal amino acid sequences showed significant resemblance to plant GH1 enzymes. Kinetic studies showed that enzyme hydrolyzed p-nitrophenyl β-D: -glucopyranoside (pNP-Glc) with higher efficiency (K (cat)/K (m) = 2.27   x 10(4) M(-1) s(-1)) as compared to p-nitrophenyl β-D: -galactopyranoside (pNP-Gal; K (cat)/K (m) = 1.15 x 10(4) M(-1) s(-1)). The optimum pH for β-galactosidase activity was 4.8 and 4.4 in citrate phosphate and acetate buffers respectively, while for β-glucosidase it was 4.6 in both buffers. The activation energy was found to be 10.6 kcal/mol in the temperature range 30-65 °C. The enzyme showed maximum activity at 65 °C with half life of ~40 min and first-order rate constant of 0.0172 min(-1). Far-UV CD spectra of enzyme exhibited α, β pattern at room temperature at pH 8.0. This thermostable enzyme with dual specificity and higher catalytic efficiency can be utilized for different commercial applications.
authors.affiliation: Department of Biotechnology, Indian Institute of Technology Roorkee, Roorkee-247 667, Uttarakhand, India.
authors.forename: Girijesh Kumar
authors.initials: GK
authors.lastname: Patel
authors.forename: Bibekananda
authors.initials: B
authors.lastname: Kar
authors.forename: Ashwani Kumar
authors.initials: AK
authors.lastname: Sharma
mesh_terms: D000595:Amino Acid Sequence; D002021:Buffers; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D019665:Euphorbiaceae / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010940:Plant Proteins / Q000302:isolation & purification / Q000378:metabolism*; D012639:Seeds / Q000201:enzymology*; D013379:Substrate Specificity; D013696:Temperature; D013816:Thermodynamics; D001616:beta-Galactosidase / Q000302:isolation & purification / Q000378:metabolism*; D001617:beta-Glucosidase / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: D002021:Buffers; D010940:Plant Proteins; D001617:beta-Glucosidase; D001616:beta-Galactosidase