Structural Analysis and Molecular Engineering of Oligo-1,6-Glucosidase to Alleviate Product Inhibition

Oligo-1,6-glucosidase efficiently hydrolyzes α-1,6 glycosidic bonds in isomaltooligosaccharides but suffers product inhibition. To tackle this challenge, the crystal structure of an oligo-1,6-glucosidase from Paenibacillus sp. STB16 (pspOGA) was determined by X-ray diffraction. The results revealed the topological characteristics of its substrate channel and the regulatory mechanism by which residue Val219 modulates substrate specificity. Based on mutant V219A, rational design was performed by promoting product release and weakening product binding. The mutant V219A/K311M/E405A showed alleviated product inhibition. At 500 mg/mL glucose solution, its activity retained 49.36% of the initial activity, substantially higher than V219A (6.25%). The product inhibition constant (K_i value) increased 1.3-fold, with improved performance in glucose mother liquor. The underlying mechanism was further elucidated by circular dichroism and molecular dynamics simulation. These findings can provide effective strategies and a theoretical basis for the industrial application of oligo-1,6-glucosidase in glucose production and the reuse of the glucose mother liquor.