Browsing by Author "Gibbon, Jerard"
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- ItemRational Engineering of a Loop Region in the Saccharomyces cerevisiae β-fructofuranosidase(Stellenbosch : Stellenbosch University, 2019-04) Gibbon, Jerard; Volschenk, Heinrich; Trollope, Kim; Stellenbosch University. Faculty of Science. Dept. of Microbiology.ENGLISH ABSTRACT: The increased need for chemical processes to become more environmentally friendly and the pressure for these processes to be more efficient have led to the chemical industry looking to biocatalysis. Biocatalysts are organic catalysts that increase the rate of the reaction by lowering the activation energy of the chemical reaction. Although biocatalysts are highly specific and can produce enantiomerically pure products compared to inorganic catalysts, they cannot produce the yields seen with inorganic catalysts and are unable to function under the harsher conditions under which inorganic catalysts operate. With the advent of molecular techniques, these biocatalysts can be tailored to function under harsher conditions associated with the chemical industry. The engineering of enzymes has been a hit-and-miss activity as the interaction between amino acids as well as the folding of a protein is still not well understood. This has led to the search for techniques to understand the interactions of amino acids better and identify the structural features that confer activity, specificity and stability. In the GH 32 enzyme family, several studies have aimed at understanding the transfructosylating activity or hydrolytic activity present in these enzymes. Fructooligosaccharides (FOS) consist of fructose bound to sucrose as acceptor molecule by either a β-(2-6)-glycosidic bond or a β-(2-1)-glycosidic bond. The fructose can either fructosylate the glucose or the fructose of the sucrose forming neokestose for the former and 1-kestose or 6-kestose for the latter. The FOS can be polymerised with the addition of one fructosyl unit which increases the degree of polymerisation (DP). In this study higher DP FOS is classified as FOS with a DP of 4 and higher. It was previously identified that a TI loop region within the GH 32 enzymes had the potential to limit the formation of higher DP FOS. This study aimed to evaluate the effect the TI loop region had on the DP of FOS within the Saccharomyces cerevisiae β-fructofuranosidase (Suc2) making use of rational enzyme engineering. Employing four rational engineering substitutions, three of the variants were able to produce higher DP FOS. Interestingly, these three variants produced 1F-fructofuranosyl nystose, an ability which has not been documented in the literature for the S. cerevisiae enzyme. The variants were able to produce multiple isomers of higher DP FOS based on their HPLC retention time. Unfortunately, due to a lack of available standards, these could not be identified. The TI loop was able to change the activity of Suc2 as the variants were predominantly transfructosylating enzymes, moving away from the predominantly hydrolytic activity of Suc2. This was confirmed as the variants increased their total FOS production by more than fourfold. Additionally, the changes to the TI loop changed the regiospecificity of the variant Suc2 enzyme producing ten times more neokestose than Suc2. The latter indicates that the TI loop contributes to regulating the orientation of the acceptor molecule, as previously described in the literature. Lastly, the changes to the TI loop led to a change in the topology of the catalytic pockets of the variants compared to Suc2, with the variants’ catalytic pockets resembling enzymes with high transfructosylating activity. This functional knowledge of the TI loop in S. cerevisiae’s β-fructofuranosidase gained in this study through mutational analysis contributed to a new understanding of how this loop governs the hydrolytic or transfructosylating activity of β-fructofuranosidase enzymes.