Thesis (PhD)--Stellenbosch University, 2015. / ENGLISH ABSTRACT: β-fructofuranosidases are hydrolytic enzymes that act on sucrose to yield the products glucose and fructose. Under high substrate conditions these enzymes display fructosyltransferase activity which results in the synthesis of fructooligosaccharides (FOS). Some enzymes display higher propensities for FOS synthesis than others, with the determinants of this activity remaining unclear. The consumption of FOS produces a prebiotic effect that positively alters the composition of the colonic microflora, and as a result is linked to improved human and animal health. The increased demand for FOS has necessitated the industrial production of these nutraceuticals. In enzymatic sucrose biotransformation processes operating at high substrate loading and temperatures between 50 and 60°C, β-fructofuranosidase activity is negatively influenced by glucose product inhibition and thermal instability. The aim of this study was therefore to engineer the Aspergillus japonicus β-fructofuranosidase, FopA, to improve a FOS synthesis bioprocess. A dual approach was employed to engineer FopA so as to increase the probability of obtaining an improved enzyme variant(s). A random mutagenesis approach was applied to harness the potential of the randomness of introduced mutations as precise structural knowledge of the enzyme regions involved in the phenotypic presentation of product inhibition, specific activity and thermal stability was unavailable. A semi-rational approach afforded the additional opportunity to reduce the number of variants to be screened, yet theoretically increased the functional content of the library. This study details the development of a method to rapidly quantify FOS using Fourier transform mid infrared attenuated total reflectance spectroscopy and multivariate data analysis. The method offers improvements over conventionally used high performance liquid chromatography in terms of reduced sample analysis times and the absence of toxic waste products. This is the first report on the direct screening of an enzyme variant library for FOS synthesis to identify improved variants and will significantly support future engineering of β-fructofuranosidases using random mutagenesis approaches. The random mutagenesis approach yielded a variant displaying limited relief from glucose inhibition. At the peak difference in performance, the variant produced 28% more FOS from the same amount of sucrose, when compared to the parent. The semi-rational approach, using a combined crystal structure and evolutionary-guided approach, yielded a four amino acid combination variant displaying improved specific activity and thermostability that was able to reduce the time to completion of an industrial-like FOS synthesis reaction by 26%. The positive outcome of the semi-rational approach showed that engineering loops regions in an enzyme is a feasible strategy to improve both specific activity and thermostability, most probably due to the modification of enzyme structural flexibility. A bioinformatic tool that enables the identification of β-fructofuranosidases displaying high-level FOS synthesis from protein sequence alone was also developed during the study. These investigations revealed conserved sequence motifs characteristic of enzymes displaying low- and high-level FOS synthesis and a structural loop, unique to the latter group, that were readily applicable identifiers of FOS synthesis capacity. The tool presented may also be useful to improve the understanding of the structure-function relationships of β-fructofuranosidases by facilitating the identification of variations in groups of enzymes that have been functionally sub-classified. / AFRIKAANSE OPSOMMING: β-fruktofuranosidases is hidrolitiese ensieme wat op sukrose inwerk en glukose en fruktose as produkte vorm. Onder toestande met hoë substraatkondisies vertoon hierdie ensieme fruktosieltransferase-aktiwiteit wat tot die sintese van frukto-oligosakkariede (FOS) lei. Sommige ensieme neig na ʼn hoër FOS-sintese as ander, maar die bepalende faktore vir hierdie aktiwiteit is nog onbekend. Die verbruik van FOS veroorsaak ʼn prebiotiese effek wat die samestelling van kolon mikroflora positief beïnvloed en met verhoogde mens- en dieregesondheid verbind word. Die verhoogde aanvraag vir FOS het die industriële produksie van hierdie nutraseutiese middel genoodsaak. Tydens ensiemgedrewe sukrose-biotransformasieprosesse by hoë substraatladings en temperature tussen 50 en 60 °C, word β-fruktofuranosidase-aktiwiteit negatief deur glukose produkonderdrukking en termiese onstabiliteit beïnvloed. Die doel van hierdie studie was dus om die Aspergillus japonicus β-fruktofuranosidase, FopA, vir ʼn verbeterde FOS-sintese bioproses te manipuleer. ʼn Tweeledige benadering is vir FopA manipulasie gevolg om die waarskynlikheid van verbeterde variant(e) te verhoog. ʼn Lukrake mutagenese benadering, wat die potensiaal van ingevoegde mutasie ewekansigheid inspan, is in die lig van onvoldoende akkurate kennis van die strukturele gedeeltes betrokke by produkinhibisie-, spesifieke aktiwiteit- en termiese stabiliteit fenotipes gevolg. Die toepassing van ʼn semi-rasionele benadering het ook geleentheid vir die sifting van ʼn kleiner variantbibioloteek geskep, terwyl die funksionele inhoud teoreties verhoog word. Die studie beskryf die ontwikkeling van ʼn metode vir die vinnige kwantifisering van FOS, gebaseer op Fourier transform middel infrarooi geattenueerde totale refleksie spektroskopie en meerveranderlike data-analise. Dit is die eerste melding van ʼn direkte sifting van ʼn ensiemvariantversameling vir FOS-sintese om verbeterde variante te identifiseer, en kan die toekomstige manipulasie van β-fruktofuranosidases deur middel van lukrake mutagenese-benaderings beduidend ondersteun. Die lukrake mutagenese-benadering het ʼn variant met beperkte opheffing van glukose-onderdrukking gelewer. By die punt waar die prestasie die meeste verskil, het die variant 28% meer FOS vanaf dieselfde hoeveelheid sukrose geproduseer in vergelyking met die ouer-ensiem. Die semi-rasionele benadering, gegrond op ʼn kombinasie van kristalstruktuur en evolusionêre-geleide benaderings, het ʼn vier-aminosuurkombinasie variant met hoër spesifieke aktiwiteit en termostabiliteit gelewer wat die voltooiingstyd van ʼn tipiese industriële FOS sintesereaksie met 26% kon verkort. Die positiewe uitkoms van die semi-rasionele benadering het aangedui dat manipulasie van die lusgedeeltes in ʼn ensiem ʼn lewensvatbare strategie is om beide spesifieke aktiwiteit en termostabiliteit te verbeter, moontlik as gevolg van wysigings in die buigsaamheid van die ensiemstruktuur. ʼn Bioïnformatika-hulpmiddel vir die identifikasie van β-fruktofuranosidases met hoë vlakke van FOS-sintese op grond van proteïenvolgordes is ook tydens die studie ontwikkel. Motiewe met gekonserveerde volgordes kenmerkend van lae- en hoë-vlak FOS-produserende ensieme en ʼn strukturele lus, uniek tot die laasgenoemde groep, is tydens die ondersoek onthul wat as maklike identifiseerders van FOS-sintesekapasiteit kan dien. Die voorgestelde hulpmiddel kan ook nuttig wees om die struktuur-funksie-verwantskap van β-fruktofuranosidases beter te verstaan deur die identifikasie van variasie in ensiemgroepe wat funksioneel gesubklassifiseer is.

Uma dieta rica em gordura leva sabidamente a efeitos deletérios, associados a síndrome metabólica, por outro lado carboidratos como os frutooligossacarídeos (FOS) estão associados à melhora da saúde gastrointestinal e à prevenção do excesso de gordura corporal e das alterações metabólicas associadas. O presente estudo avaliou os efeitos em longo prazo de quantidades elevadas de FOS em parâmetros metabólicos, na esteatose hepática não alcoólica (EHNA) e nos ácidos graxos de cadeia curta (AGCC) de camundongos C57BL recebendo dieta normolipídica e hiperlipídica. Foram utilizados 60 animais divididos em seis grupos que receberam por quatro meses as seguintes dietas experimentais: controle (C), normolipídica rica em fibra (F), normolipídica suplementada com FOS (FOS), hiperlipídica (HL), hiperlipídica rica em fibras (HLF) e hiperlipídica com FOS (HLFOS). A dieta controle continha 5% de celulose microcristalina, enquanto a rica em fibras continha 15%; já as suplementadas com FOS, ambas continham 15% desse prebiótico. Foram analisados: peso dos animais; composição corporal; ingestão alimentar; glicemia de jejum; perfil lipídico sérico e hepático; histologias hepática e intestinal; malondialdeído (MDA), retinol e ?- tocoferol hepáticos; AGCC nas fezes. Ao final do período experimental não foi verificada alteração no consumo calórico entre os grupos, mas a suplementação com FOS na dieta hiperlipídica promoveu menor ganho de peso corporal e redução dos pesos do fígado e do tecido adiposo retroperitoneal em comparação ao HL e HF. O FOS preveniu a EHNA e diminuiu os níveis de da alanina aminotransferase e de colesterol sérico nos modelos experimentais animais de obesidade e síndrome metabólica (SM), mas sem alterações significativas no perfil lipídico hepático, na glicemia de jejum, na capacidade antioxidante total ou nas dosagens hepáticas de MDA, de retinol e de ?-tocoferol. Os grupos que receberam dieta rica em fibras não apresentaram melhora nos parâmetros metabólicos ou na EHNA como ocorreu com a suplementação de FOS, sendo que o grupo HLF apresentou aumento do MDA no fígado. Já na dieta normolipídica, o FOS e a celulose foram eficazes na preservação da vitamina E hepática. A histologia intestinal evidenciou que a dieta hiperlipídica aumentou o diâmetro total da luz intestinal e reduziu a espessura muscularentérica, sendo que tanto a suplementação com FOS como com celulose reverteram esses achados, sendo o FOS o mais efetivo. Já com relação aos grupos que receberam dieta normolipídica, houve diminuição da espessura muscular entérica tanto no grupo F como no FOS, sendo a celulose a que apresentou a maior redução, e o aumento do diâmetro da luz intestinal só foi verificado no grupo F. Por fim, não houve alteração nas dosagens dos três principais AGCC nas fezes. A suplementação em longo prazo com altas doses de FOS foi efetiva na redução do peso, da adiposidade, da EHNA e do colesterol sérico em camundongos C57BL com obesidade e SM induzidas por dieta hiperlipídica, além de prevenir parcialmente as alterações morfológicas do intestino presentes nesses modelos experimentais. Os benefícios encontrados com a oferta de altas doses de FOS não foram verificados nos grupos enriquecidos com celulose microcristalina / A high-fat diet is known to lead to deleterious effects associated with metabolic syndrome, on the other hand carbohydrates such as fructooligosaccharides (FOS) are associated with improved gastrointestinal health and prevention of excess body fat and associated metabolic abnormalities. The present study evaluated the long-term effects of high doses of FOS on metabolic parameters, non-alcoholic hepatic steatosis (NASH) and short chain fatty acids (SCFAs) of C57BL mice receiving normolipid and hyperlipidic diets. A total of 60 animals were divided into six groups, which received the following experimental diets for four months: control (C), fiber-rich normolipid (F), normolipid supplemented with FOS (FOS), hyperlipidic (HL), high-fiber hyperlipid (HLF) and hyperlipidemic with FOS (HLFOS). The control diet contained 5% microcrystalline cellulose, while the high fiber contained 15%; and those supplemented with FOS, both contained 15% of this prebiotic. It was analyzed: weight of the animals; body composition; food intake; fasting blood glucose; serum and hepatic lipid profile; hepatic and intestinal histologies; malondialdehyde (MDA), hepatic ?-tocopherol and retinol; AGCC in feces. At the end of the experimental period, no change in caloric intake between groups was observed, but supplementation with FOS in the hyperlipid diet promoted a lower body weight gain and reduced weights of the liver and retroperitoneal adipose tissue compared to HL and HF. FOS prevented NASH and decreased levels of alanine aminotransferase and serum cholesterol in experimental animal models of obesity and metabolic syndrome (MS), but without significant changes in hepatic lipid profile, fasting glycemia, total antioxidant capacity or dosages of MDA, retinol and ?-tocopherol. The groups that received a high fiber diet showed no improvement in metabolic parameters or NASH as occurred with FOS supplementation, and the HLF group presented increased MDA in the liver. In the normolipid diet, FOS and cellulose were effective in the preservation of hepatic vitamin E. The intestinal histology evidenced that the hyperlipidic diet increased the total diameter of the intestinal lumen and reduced enteric muscle thickness, and both FOS and cellulose supplementation reversed these findings, with FOS being the most effective. Regarding the groups that received a normolipid diet, there was a decrease in enteric musclethickness in both the F and FOS groups, with the cellulose being the one with the greatest reduction, and the increase in the diameter of the intestinal lumen was only verified in the F group. Finally, there was no change in the dosages of the three main SCFAs in the feces. Long-term supplementation with high doses of FOS was effective in reducing weight, adiposity, NASH and serum cholesterol in C57BL mice with obesity and MS induced by a hyperlipid diet, in addition to partially preventing the morphological alterations of the intestine present in these models experiments. The benefits found in the supply of high doses of FOS were not verified in groups enriched with microcrystalline celulose