Abstract
The current work focused on the purification and biochemical characterization of an extracellular β-d-fructofuranosidase from a novel fungus identified genetically using 18 S rRNA molecular sequencing, as Aspergillus sp. DHE1. The fungus was cultured under solid state fermentation using the Cotton (Gossypium herbaceum) seed waste as substrate. β-d-fructofuranosidase was purified through fractionation by ammonium sulphate salt, ion-exchange chromatography on DEAE-cellulose, and Sephadex G-100 gel filtration, yielding a 13.3-purification fold, 22.6% recovery and 192.9 U/mg protein specific activity. The apparent molecular mass of Aspergillus sp. DHE1 β-d-fructofuranosidase was 78 kDa as estimated on sodium dodecyl sulphate polyacrylamide gel electrophoresis. Optimal pH and temperature were observed at 6.0 and 50 °C, respectively, and the enzyme was quite thermostable at neutral/alkalophilic conditions, retaining 72% of its original activity after 1 h of incubation at 60 °C. The activity of the purified Aspergillus sp. DHE1 β-d-fructofuranosidase was stimulated by 1 mM of KCl (138.5%), NaCl (125.9%), CoCl2 (121.2%), and CaCl2 (112.3%), whereas the same concentration of AgCl, FeCl3, HgCl2, and CuCl2 inhibited the activity by 48.7%, 30.5%, 29.9%, and 26.2%, respectively. The kinetic studies using d-sucrose as the substrate were investigated using a Lineweaver-Burk plot, with Km and Vmax of 0.85 mM and 47.62 U/mL, respectively, indicating the high substrate affinity of β-d-fructofuranosidase. The ability of Aspergillus sp. DHE1 β-d-fructofuranosidase to tolerate ethanol suggests that it may have numerous uses in the food industry, including the production of alcoholic beverages.
Published Version
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