# Improving the hydrophilic microenvironment surrounding the catalytic site of fructosyltransferase enhances its catalytic ability

## TL;DR

Changing a specific amino acid near the active site of an enzyme improves its ability to produce fructooligosaccharides by enhancing its catalytic efficiency.

## Contribution

A novel mutation strategy is proposed to improve enzyme catalytic efficiency by modifying the hydrophilic microenvironment around the active site.

## Key findings

- The C66S mutation increased the enzyme's specific activity by 61.3% and catalytic efficiency by 40.1%.
- The mutation improved substrate affinity and transfructosylation, leading to higher fructooligosaccharide yields.
- Simulations predicted a significant increase in binding energy from -3.65 to -4.14 kcal mol−1.

## Abstract

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 Km (82.20/71.14 mM), and a 21.6% increase in kcat (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.

The online version contains supplementary material available at 10.1007/s10529-025-03566-8.