Please use this identifier to cite or link to this item: https://hdl.handle.net/10321/5523
Title: Increasing catalytic activity of a fructosyltransferase using site-directed mutagenesis
Authors: Wang Fanzhi 
Keywords: Fructooligosaccharides;Prebiotics;Transfructosylation;Enzyme engineering;Fructosyltransferase
Issue Date: May-2024
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.
Description: 
Submitted in complete fulfilment for the Degree of Master of Applied Science in Biotechnology, University of Technology, Durban, South Africa, 2024.
URI: https://hdl.handle.net/10321/5523
DOI: https://doi.org/10.51415/10321/5523
Appears in Collections:Theses and dissertations (Applied Sciences)

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