Purification and biochemical characterization of an extracellular β-d-fructofuranosidase from sp.

This study focused on the purification and characterization of an extracellular β-d-fructofuranosidase or invertase from JU12. The protein was purified by size exclusion chromatography with 5.41 fold and 10.87% recovery. The apparent molecular mass of the enzyme was estimated to be ~ 35 kDa using SDS-PAGE and confirmed by deconvoluted mass spectrometry. The fungal β-d-fructofuranosidase was suggested to be a monomer by native PAGE and zymography, and was found to be a glycoprotein possessing 68.92% carbohydrate content. The products of enzyme hydrolysis were detected by thin layer chromatography and revealed the monosaccharide units, d-glucose and d-fructose. β-d-fructofuranosidase showed enhanced activity at broad pH 4.0-9.0 and activity at a temperature range from 30 to 70 °C, while the enzyme was stable at pH 8.0 and 40 °C, respectively. The β-d-fructofuranosidase activity was lowered by metal ion inhibitors Ag and Hg whereas elevated by SDS and β-ME. The fungal β-d-fructofuranosidase was capable of hydrolyzing d-sucrose and the kinetics were determined by Lineweaver-Burk plot with of 10.17 mM and of 0.7801 µmol min. Additionally, the extracellular β-d-fructofuranosidase demonstrated tolerance to high ethanol concentrations indicating its applicability in the production of alcoholic fermentation processes.