Characterization of a thermostable family 1 Glycosyl Hydrolase enzyme from Putranjiva roxburghii seeds.

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.