Purification and biochemical characterization of β-d-fructofuranosidase from Bacillus subtilis LYN12

The purification and biochemical characterization of extracellular β-d-fructofuranosidase from Bacillus subtilis LYN12 was carried out. The enzyme was purified 6.94 folds over the crude extract by gel filtration chromatography with recovery of 15.58%. The molecular mass of ∼66 kDa estimated by SDS-PAGE was confirmed by LC-MS as 64512.31 Da. Bacterial β-d-fructofuranosidase was found to be a glycoprotein with 62.64% carbohydrate content, and exhibited enhanced activities at broad pH, temperature and stable at pH 7.0, 40°C, respectively. The enzyme showed high affinity for d-sucrose. Kinetic parameters Km and Vmax were 41.98 mM and 1.184 µmol/min, respectively. β-d-fructofuranosidase activity was inhibited by the divalent metal ions Cu2+ and Hg2+, whereas improved by Mg2+, Fe2+ and few sulphydryl group reagents. β-d-fructofuranosidase demonstrated ethanol tolerance up to 15% with 76.4% of activity. B. subtilis LYN12 invertase is suggested as a potential enzyme with suitable characteristics for numerous industrial applications. Practical applications. β-d-fructofuranosidases are one of the industrially important carbohydrases utilized in many applications such as beverages, baking, confectionaries, nutraceuticals and also medicinal formulations. The production of β-d-fructofuranosidase from Bacillus subtilis LYN12 by solid-state fermentation demonstrates the utilization of agro-industrial wastes, such as wheat bran and molasses. The bacterial β-d-fructofuranosidase was produced extracellularly which aids in down-streaming processes. Most of the industrial alcohol fermentations generally operate at 10%–14% (v/v) of ethanol at the end of fermentation. Interestingly, β-d-fructofuranosidase exhibited a significant tolerance towards ethanol, the tolerance level of the bacterial invertase indicates its potentiality in the alcoholic fermentation processes. On the other hand, B. subtilis LYN12 β-d-fructofuranosidase was found to be active at a broad pH and temperature range possessing high affinity for d-sucrose. The biochemical characterization of the bacterial β-d-fructofuranosidase is crucial to understand the enzymic nature and properties.