Akademik Veri Yönetim Sistemi
BIOCHEMICAL ENGINEERING JOURNAL, cilt.229, 2026 (SCI-Expanded, Scopus)
alpha-Amylase (EC 3.2.1.1) and alpha-glucosidase (EC 3.2.1.20) are key enzymes in starch hydrolysis, widely applied in biotechnological and food industries. In this study, recombinant alpha-amylase (AflAmy) and alpha-glucosidase (AflGlu) from Anoxybacillus flavithermus 2641 (T) were purified using a cobalt affinity column, yielding proteins of approximately 50 kDa as confirmed by SDS-PAGE. Both enzymes were immobilised through the cross-linked enzyme aggregate (CLEA) method. Optimal CLEA preparation involved 96 % ammonium sulfate saturation at 4 degrees C for 30 min, followed by cross-linking with 5 mM glutaraldehyde for 2-3 h at room temperature. Free AflAmy exhibited optimal activity at pH 8.0 and 70 degrees C, while immobilization shifted its optimum to pH 9.0. Free AflGlu was most active at pH 8.0 and 60 degrees C, changing to 55 degrees C upon immobilization. CLEA forms displayed lower Km value for AflAmy-IM, indicating increased substrate affinity. Reusability tests showed immobilised AflAmy retained activity over nine cycles, AflGlu over three, and their combined form (Combi-CLEA) up to nine cycles. Thermal stability of immobilised AflAmy improved, maintaining activity for 150 min at 70 degrees C. Both free and immobilized forms of the enzymes achieved similar to 50 % starch hydrolysis within 60 min, demonstrating comparable catalytic efficiency and enhanced operational stability after immobilization.