Biochemical characterization and activity enhancement of a GH13 thermostable oligo-α-1,6-glucosidase from Geobacillus...

Oligo-α-1,6-glucosidase (OGL) catalyzes the hydrolysis of α-1,6-glycosidic linkages in oligosaccharides and plays a vital role in the production of high-glucose syrup, isomaltooligosaccharides (IMOs), and bioethanol. Industrial IMO production demands enzymes with both high thermostability and catalytic efficiency. Although thermophilic bacterial OGLs meet thermal requirements, their catalytic performance remains suboptimal. While random mutagenesis can identify beneficial mutations, traditional protein engineering often faces challenges such as stability trade-offs and extensive screening. Recently, computational tools like HotSpot Wizard and molecular docking have shown promise in semi-rational enzyme design. In this study, we aimed to enhance the catalytic activity of a thermostable OGL from Geobacillus stearothermophilus (Gst) via site-directed mutagenesis. Five point mutations (D59A, N61F, V101I, T257A, and P258N) were selected based on bioinformatics analysis and literature review. Wild-type and mutant enzymes were expressed in Escherichia coli BL21(DE3) and purified. All variants retained activity across a broad pH and temperature range, with optimal activity at 60 °C and pH 6.5. Compared to the wild-type enzyme, mutants D59A, V101I, T257A, and P258N exhibited 1.7- to 2.3-fold increases in catalytic efficiency (k_cat/K_m), with V_max values increasing by 2.2- and 1.9-fold for D59A and P258N, respectively. Molecular docking with panose and isomaltose confirmed enhanced substrate binding, particularly for GstOGLD59A. These findings demonstrate the potential of rationally engineered GstOGL variants as efficient biocatalysts for industrial IMO production and underscore the value of integrating computational and experimental approaches in enzyme optimization.