Cloning, purification and biochemical characterization of oligo-1,4-1,6-alpha-glucosidase (sucrase-maltase-isomaltase) from Anoxybacillus gonensis

Fructose is one the most preferred sugar, as fructose are provides the sweetening and health benefits for human. Therefore, it is an important requirement to explore alternative production methods of fructose recognized as safe. Today, many industrial enzymes are used in the production of HFS. Oligo-𝛼-1,6-glucosidase hydrolysis 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, it was aimed to evaluate sucrose hydrolyzing activity of oligo-𝛼-1,6-glucosidase belonged to A. gonensis G2T, which is a thermophilic bacterium. For this purpose, oligo-𝛼-1,6-glucosidase gene region of A. gonensis G2T was cloned in pET28(a)+ expression vector, the expression product was purified by heat-shock treatment, ion exchange chromatography, hydrophobic interaction colon chromatography techniques. Purified enzyme was characterized biochemically. The purified enzyme showed optimal activity at 60 °C and pH 7.00. It was determined that the enzyme activity was halved at the end of the 276th hour at 60 °C. The enzyme maintained its activity even after 300 hours in the pH range of 6.00-10.00 at 60 °C. The values of Km, Vmax, kcat and kcat/Km were determined as 44,69±1,27 mM, 6,28±0,05 µmoL/min/mg protein, 6,70 1/sec and 0,15 1/mMsec-1, respectively. While Zn2+, Cu2+, Pb2+, Ag2+, Fe3+, Hg2+ and Al2+ metal ions inhibited the enzyme activity even at low concentrations, Mn2+, Fe2+ and Mg2+ ions behaved as activators on the enzyme. In conclusion, A. gonensis G2T oligo-1,6-glucosidase is an enzyme with interesting properties especially for High Fructose Syrup (HFS) production. Keywords: Anoxybacillus gonensis G2T, oligo-1,4-1,6-𝛼-glucosidase, HFS, sucrase.