Engineering a fungal β-fructofuranosidase

Trollope, Kim Mary (2015-04)

Thesis (PhD)--Stellenbosch University, 2015.

Thesis

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.

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