Rational Engineering of a Loop Region in the Saccharomyces cerevisiae β-fructofuranosidase

Thesis (MSc)--Stellenbosch University, 2019.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.AFRIKAANS OPSOMMING: Die toenemende behoefte aan chemiese prosesse om meer omgewingsvriendelik te word asook die druk vir hierdie prosesse om doeltreffender te wees, het daartoe gelei dat die chemiese industrie belangstelling toon in bio-gekataliseerde prosesse. Biokatalisators is organiese katalisators wat die reaksie tempo verhoog deur die aktiveringsenergie van die chemiese reaksie te verlaag. Biokatalisators is dus aanloklike alternatiewe tot anorganiese katalisators weens hulle hoë ensiemspesifisiteit en vermoë om suiwer enantiomeriese produkte te vorm. Nietemin, produseer biokatalisators laer opbrengste as anorganiese katalisators en is nie in staat om onder die meerderheid van industriële toestande waaraan anorganiese katalisators blootgestel word te funksioneer nie. Met die aankoms van molekulêre tegnieke kan hierdie biokatalisators aangepas word om te funksioneer onder moeiliker toestande wat met die chemiese industrie geassosieer word. Die verandering van ensieme is 'n lukrake aktiwiteit aangesien die interaksie tussen aminosure sowel as die vou van proteïene nog steeds nie goed verstaan word nie. Dit het gelei tot die soeke na tegnieke om die interaksies van aminosure beter te verstaan en die strukturele eienskappe wat aktiwiteit, spesifisiteit en stabiliteit verleen, te identifiseer. In die GH 32 ensiem familie het verskeie studies daarop gefokus om die transfruktosilerende aktiwiteit of hidrolitiese aktiwiteit wat in hierdie ensieme voorkom, beter te verstaan. Frukto-oligosakkariede (FOS) is fruktose wat gebind word aan 'n akseptor molekule, sukrose, deur óf ʼn β-(2-6)-glikosidiese binding of ʼn β-(2-1)-glikosidiese binding. Die fruktose kan aan glukose of fruktose in die sukrose bind wat lei tot neokestose in die geval van glucose en 1/6-kestose vir fruktose. Die FOS kan gepolimeriseer word met toevoeging van een fruktosiel-eenheid wat die graad van polimerisasie (DP) verhoog. In hierdie studie word hoër DP van FOS geklassifiseer as FOS met 'n DP van 4 en hoër. Daar is voorheen 'n lus binne die GH 32-ensieme geïdentifiseer met die potensiaal om die vorming van hoë DP in FOS te bepaal. Die doel van hierdie studie was om die effek wat die TI-lus op die bepaling van DP van FOS het binne die Saccharomyces cerevisiae β-fruktofuranosidase (Suc2) te bepaal, deur gebruik te maak van ʼn rasionele mutasie benadering. Deur gebruik te maak van vier rasionele benaderings om Suc2 verander, kon drie van die variante hoër DP FOS produseer. Hierdie drie variante het 1F-fruktofuranosiel nystose geproduseer, wat nie in die literatuur voorheen vir Suc2 gedokumenteer was nie. Die variante kon verskeie isomere van hoër DP FOS op grond van die HPLC retensietyd produseer. Ongelukkig kon dit nie weens gebrek aan beskikbare standaarde geïdentifiseer word nie. Die TI-lus was in staat om die aktiwiteit van Suc2 te verander, van ʼn hoofsaaklik hidrolitiese ensiem na ʼn transfruktosilerende ensiem. Dit is bevestig omdat die variante hul totale FOS-produksie met meer as 4 keer verhoog het. Daarbenewens het die veranderinge aan die TI-lus die regiospesifisiteit van die Suc2-ensiem variante verander, wat daartoe gely het dat 10 keer meer neokestose produseer as Suc2. Dit dui aan dat die TI-lus bydra tot die regulering van die oriëntering van die akseptor molekule, soos voorheen in die literatuur beskryf. Laastens het die veranderinge aan die TI-lus gelei tot 'n verandering in die topologie van die katalitiese holtes van die variante in vergelyking met Suc2, met die variante se katalitiese holtes wat meer ooreenstem met ensieme met ʼn hoë transfruktosilerende aktiwiteit. Die funksionele kennis van die TI-lus in S. cerevisiae se β-fruktofuranosidase wat in hierdie studie deur middel van mutasie analise verkry is het bygedra het tot 'n nuwe begrip van hoe hierdie lus die hidrolitiese of transfruktosilerende aktiwiteit in β-fruktofuranosidase-ensieme beheer.Master