Enhanced Production, Purification, and Characterization of α-Glucosidase from NTG-Mutagenized Aspergillus niger for Industrial Applications

α-Glucosidase is an essential enzyme widely used in the food and biotechnology industries for the production of isomaltose oligosaccharides (IMOs). In this study, Aspergillus niger ASP004 was subjected to two rounds of nitrosoguanidine (NTG) mutagenesis, yielding a high-producing strain, NTG-II-3-64, with a 3.7-fold increase in enzyme activity under optimized fermentation conditions. The enzyme was purified through ultrafiltration, ethanol precipitation, DEAE ion-exchange, and gel filtration chromatography, resulting in an electrophoretically pure heterodimeric protein with a native molecular mass of approximately 230 kDa, composed of two subunits. The purified α-glucosidase exhibited optimal activity at 60 °C and pH 4.5, maintaining over 90% activity within a pH range of 3.0–6.0. Kinetic analysis using p-nitrophenyl-α-D-glucopyranoside as the substrate showed a Km of 0.17 mM and a Vmax of 18.7 µmol min−1 mg−1. Additionally, the enzyme displayed transglucosidase activity, converting maltose into isomaltose oligosaccharides (IMOs), including isomaltose, panose, and isomaltriose. These findings highlight the effectiveness of NTG mutagenesis for enhancing α-glucosidase production and support its industrial application.