Engineering dextranase for the directed preparation of low-molecular-weight dextran

Dextranase acts as a biocatalyst for the production of isomaltooligosaccharides (IMOs) and low-molecular-weight dextran (LMWD) in the food and pharmaceutical industries. Nevertheless, the naturally derived dextranase from Chaetomium gracile (CgDEX) suffers from unsatisfactory catalytic activity, which hinders its industrial utilization. We hypothesized that modification of non-conserved residues distal to the catalytic center could enhance the enzym's catalytic performance. In this work, candidate mutation sites were screened via multiple sequence alignment (MSA), followed by site-directed mutagenesis to engineer CgDEX and heterologous expression in Pichia pastoris. The resultant mutant exhibited a specific activity of 5185 ± 70 U/mg, representing an increase of 17.5 ± 2.3% relative to the wild-type enzyme. Molecular docking revealed strengthened hydrogen-bond interactions between catalytic residues and substrate upon mutation, consistent with the markedly elevated substrate affinity determined by enzyme kinetic assays. Hydrolysis of dextran by the mutant predominantly yielded IMOs with a degree of polymerization ranging from 2 to 4; additionally, the engineered enzyme could work synergistically with dextransucrase to synthesize LMWD. Collectively, this study verifies the feasibility of green manufacturing of functional oligosaccharides and LMWD using engineered CgDEX.