In silico sequential mutagenesis of the Carbohydrate Binding Module Family 32 (CBM32) enhances ligand binding affinity

Alginate lyase is a promising target for genetic modification for its degrading biofilm, contributing to bacterial proliferation and antimicrobial resistance. Apart from the main enzyme, the carbohydrate binding module (CBM) component can also be modified to enhance alginate lyase’s activity. This study aimed to perform sequential in silico mutagenesis, molecular docking of selected amino acid residues of Vibrio splendidus CBM32 and performed molecular dynamics (MD) simulations of the mutated structure to validate its ligand-binding efficacy. Seven residues were selected for mutagenesis based on the predicted bonds that formed between the CBM32 and the glucuronic acid ligand (LGU9). Four of seven sequential residue substitutions increased the ligand binding affinity cumulatively from -5.4 Kcal/mol to -6.9 Kcal/mol. The mutated CBM32 had similar MolProbity scores to the original V. splendidus CBM32 structure. From the post-MD simulation analysis, the mutated CBM32 had higher structural stability in a solvent system, a greater number of hydrogen bonds formed with ligand but a lower solvent-accessible surface area than the original structure. The sequential mutagenesis process significantly increased the ligand binding affinity of CBM32 while incurring a minimal change in the overall CBM32 structure. The information on these substituted residues would be beneficial for designing subsequent in vitro mutagenesis and enzymatic assays.