Modeling and evaluation of the sucrose-degrading activity of recombinantly produced oligo-1,6-glucosidase from A....

Fructose is the most preferred sugar to provide benefits for sweetening and health. As many industrial enzymes are used to produce High Fructose Syrup (HFS), it is vital to explore alternative enzymes for fructose production. Oligo-α-1,6-glucosidase (O-1-6-glucosidase) hydrolyzes non-reducing ends of isomaltooligosaccharides, panose, palatinose, and an a-limit dextrin by breaking α-1,6-glucoside bonds, although it generally has no activity on α-1,4-glucoside bonds of maltooligosaccharides. In this study, sucrose-hydrolyzing activity of O-1-6-glucosidase of thermophilic A. gonensis was evaluated. For this purpose, O-1-6-glucosidase gene region of A. gonensis was cloned in the pET28(a)+ expression vector, the expression product was purified, modeled, and biochemically characterized. The optimal activity of the enzyme was to be at pH 7.0 and 60 °C. The enzyme activity was halved at the end of the 276^th h at 60 °C. The enzyme maintained its activity even after 300 h at pH 6.0-10.0. The values of K, V, k_cat, and k_cat/K were determined as 44.69 ± 1.27 mM, 6.28 ± 0.05 µmoL/min/mg protein, 6.70 1/s and 0.15 1/mMs^-1, respectively. While Zn²⁺, Cu²⁺, Pb²⁺, Ag²⁺, Fe³⁺, Hg²⁺, and Al²⁺ metal ions inhibited O-1-6-glucosidase, Mn²⁺, Fe²⁺, and Mg²⁺ ions activated the enzyme. Consequently, A. gonensis O-1-6-glucosidase (rAgoSuc2) has interesting properties, especially for HFS production.