Increasing catalytic activity of a fructosyltransferase using site-directed mutagenesis

Abstract

Fructooligosaccharides (FOS) are naturally occurring metabolites that have a wide application in the food industry. They are one of the most well-studied prebiotics and have been used as an alternative sweetener to sucrose, as the modern diet demands healthier and calorie-reduced foods. FOS is commercially produced either by hydrolysis of inulin into inulin-type FOS or by sucrose transfructosylation into levantype FOS. The levan-type FOS are short-chain FOS and are produced under the catalysis of fructosyltransferase (FTase) or fructofuranosidase (FFase). In this study, FOS production was studied using a fructosyltransferase, SucC, which was originally isolated from Aspergillus niger and was functionally expressed in Pichia pastoris. The tertiary structure of SucC was determined by bioinformatics analysis and catalytic sites were verified and validated by wet and dry experiments where the amino acid residues D64, D194 and E271 were proved to form the catalytic triad. Three mutants, C66S, G273V, L313H were constructed aiming to improve the enzyme performance. Only the C66S mutant showed improved enzymatic activity which was 61% increase in specific activity. The other mutants, G273V and L313H, led to a complete loss of enzyme activity. By simulating saturated mutagenesis, tertiary structure alignment, and molecular docking, it was predicted that the C66S mutation could increase the hydrophilic environment surrounding the active site without visible changes in its structure. Two more amino acid residues (E296, H310) in addition to D64, D122, R193, D194, E271 in mutant C66S were predicted to be interacting with sucrose, and the binding energy changed 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 mutant C66S 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%. Its transfructosylation efficiency significantly improved during the initial reaction stages of FOS production. These results clearly revealed that the increase of hydrophilicity surrounding the active site enhanced the transfructosylating activities. Therefore, modification of the hydrophilic micro-environment surrounding the active site could be an alternative way to artificially evolve an enzyme’s catalytic efficiency.