Bioethanol production from immobilized amylase produced by marine Aspergillus flavus AUMC10636

Abstract

Aspergillus flavus, an amylase-producing fungus, was isolated from the Mediterranean Sea, Alexandria, Egypt. It was selected according to the formation of a clear zone using the Dox-medium containing 1% starch for enzyme production. The maximum amount obtained of amylase production was 22.68 U/mL after 7 days of incubation under static condition. Partially purified amylase with 70% ethanol provoked a single protein band with 54 kDa molecular weight using SDS-PAGE giving a specific activity of 307.35 U/mg. The purified α-amylase exhibited optimal activity of 65.17 and 71.5 U/mL at pH = 6 and 50 °C, respectively. The calculated enzyme kinetics were K_m = 5 mg/mL and V_max = 100 U/mL. Residual α-amylase retained optimum stability at 50 °C by 67.74% and at pH = 6 by 100% after 60 min of incubation. The highest enzyme tolerance occurred at 1.5 mM NaCl concentration. The highest enzyme yield (165.24 U/mL) was provided by the immobilized amylase with 1% of covalent bonding agents solution, providing double the sugars’ byproduct than from the free amylase form, and double the bioethanol fermentation yield with 0.12 g/g sugar/l. Highly specific activity of such enzyme in the immobilized form could offer a highly effective approach for bioethanol production on the industrial scale than the free and other reported forms.