Point mutations enhance catalytic efficiency of<i> Geobacillus</i><i> stearothermophilus</i> α-glucosidase: A biochemical characterization study

alpha-Glucosidases are important enzymes with a broad spectrum of industrial applications. However, traditional protein engineering often struggles to improve activity without compromising stability and usually demands extensive screening. Here, we targeted the alpha-glucosidase (AG) from Geobacillus stearothermophilus (Gst), using in silico analyses and literature precedent to select point mutations N61H and N258P. We cloned the mutated gstAG gene into the pET-28a(+) vector and expressed in Escherichia coli. After expressing and purifying both wild-type and mutant enzymes, we performed detailed biochemical assays. Both mutants maintained GstAG's optimum temperature (60 degrees C) and pH (6.5). However, each displayed enhanced catalytic efficiency: N61H lowered the Michaelis constant (Km) by 1.5-fold and raised the turnover number (kcat) by 1.7-fold relative to the wild type. These results offer a blueprint for engineering alpha-glucosidases with improved performance, unlocking new commercial and biotechnological applications.