Improving the hydrophilic microenvironment surrounding the catalytic site of fructosyltransferase enhances its catalytic ability
Year: 2025
Type: article
Abstract: The hydrophilic microenvironment surrounding the active site of an enzyme may affect its catalytic activity. In the present study, the impact of improving the hydrophilic microenvironment on the catalytic ability of Aspergillus niger fructosyltransferase, SucC, was investigated. By comprehensive and comparative bioinformatic analysis, it was predicted that a cysteine residue (C66) close to the catalytic triad (D64, D194 and E271) in SucC could play an important role in maintaining the spatial structure of the active site and facilitate the transport of substrates through the substrate tunnel. By simulating saturated mutagenesis, tertiary structure alignment, and molecular docking, it was predicted that a mutation from cysteine to serine (C66S) could increase the hydrophilic environment surrounding the active site without major changes in its structure. Two more amino acid residues (E296 and H310), in addition to D64, D122, R193, D194, and E271 in mutant C66S were predicted to be interacting with sucrose with the binding energy changing from -3.65 to -4.14 kcal/mol. Subsequently, mutant C66S was constructed by site-directed mutagenesis and expressed in Pichia pastoris GS115. The purified C66S mutant showed improved enzymatic activity with a 61.3% increase in its specific activity. Its Km value was decreased by 13.5% while the kcat value increased by 21.6%. When the mutant enzyme was used for FOS production, transfructosylation activity increased in the initial stages. The increase of hydrophilicity surrounding the active site enhanced the transfructosylating activity of the enzyme. Therefore, modification of the hydrophilic microenvironment surrounding the active site could be an alternative way to artificially evolve an enzyme’s catalytic efficiency.
Source: Biotechnology Letters
Authors: Fanzhi Wang, Suren Singh, Kugen Permaul
Institution: Durban University of Technology
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: hybrid
Estimated APC paid: $3,890
Award: G787789904
OpenAlex ID: W4407961720