Improving the hydrophilic microenvironment surrounding the catalytic site of fructosyltransferase enhances its...

The hydrophilic microenvironment surrounding an enzyme's active site can influence its catalytic activity. This study examines the effect of enhancing this environment in the Aspergillus niger fructosyltransferase, SucC. Bioinformatics analysis identified a cysteine residue (C66) near the catalytic triad (D64, D194, E271) as vital for maintaining the active site's structure and facilitating substrate transport. Simulated mutagenesis suggested that mutating cysteine to serine (C66S) could increase hydrophilicity without altering the structure significantly. This mutation was predicted to enhance substrate affinity, with binding energy changing from -3.65 to -4.14 kcal mol^-1. The C66S mutant, expressed in Pichia pastoris GS115, showed a 61.3% increase in specific activity, a 13.5% decrease in K_m (82.20/71.14 mM), and a 21.6% increase in k_cat (112.23/136.48 min^-1), resulting in a 40.1% increase in catalytic efficiency (1.37/1.92 min^-1 mM^-1). For fructooligosaccharides (FOS) production, C66S demonstrated enhanced transfructosylation, particularly in the initial stages of the reaction, achieving higher overall FOS yields. These findings highlight that modifying the active site hydrophilicity, without causing major structural changes, is a promising strategy for improving an enzyme's catalytic efficiency.