Isolation and characterisation of novel enzymes producing fructo- and galacto-oligosaccharides through a metagenomic approach

Smith, Marthinus Luther (2018-03)

Thesis (PhD)--Stellenbosch University, 2018.

Thesis

ENGLISH ABSTRACT: Prebiotic oligosaccharides and polymers are a form of dietary carbohydrate that are selectively fermented by the microbiota of the lower gastro intestinal tract and confer a health benefit to the host other than that of nutrition. Galacto-oligosaccharides (GOS) are a prime example of a dietary prebiotic and closely imitate human milk oligosaccharides. They consist of galactose moieties, often linked to a terminal glucose. GOS promote the growth of beneficial bacteria that aid in the prevention and treatment of gastro intestinal dysbiosis which has been linked to various diseases such as auto-immune disorders, certain cancers and metabolic disorders. They are often synthesised from lactose by the glycoside hydrolase enzyme, β-galactosidase. A cheap substrate for the production of GOS is whey permeate, an industrial waste product. Another prebiotic dietary carbohydrate is Fructo-oligosaccharides (FOS), which consist of fructose moieties often linked to a terminal glucose, and much like GOS is associated with the numerous health benefits of prebiotics. FOS is synthesised from the substrate sucrose by fructosyl-transferases. There is a great demand for new enzymatic biocatalysts with improved capabilities of producing GOS and FOS. In this study the construction and screening of numerous metagenomic libraries for prebiotic oligosaccharide and polymer synthesising enzymes are presented. The metagenomic DNA was sampled from locations rich in the substrate used for the production of the before mentioned prebiotics. The isolation, amplification and in silico analysis of three novel genes encoding proteins belonging to the Glycoside Hydrolase families 1, 2, and 68 are presented here. Their respective polypeptides were heterologously expressed in E. coli, purified and subsequently characterised biochemically in vitro through various means, and so too their respective products. Glycoside hydrolases often have two innate but distinct activities namely hydrolysis and trans-glycosylation. All three the glycoside hydrolases described here, MS-βGluc1, HW-βGal1 and SAS-Ls, had high trans-glycosylation activity apart from their hydrolytic activity. They are all excellent candidates for the industrial production of prebiotics, whether it be GOS (MS-βGluc1 and HW-βGal1) or FOS (SAS-Ls).

AFRIKAANSE OPSOMMING: Prebiotiese oligo-sakkariede en polimere is ’n tipe voedings koolhidraat wat selektief gefermenteer word deur die mikro-biota van die laer gastro-intestinale sisteem, en lewer ’n positiewe gesondheids eienskap bydrae aan die gasheer anders as die oog op liggende voedings waarde. Galakto-oligosakkariede (GOS) is ’n uitstekende voorbeeld van prebiotiese oligo-sakkariede en is ’n nouliks nabootsing van menslike melk oligosakkariede. Hulle bestaan uit opeenvolgende galaktose eenhede wat baie keer aan ’n terminale glukose molekuul gekoppel is. GOS bevorder die groei van voordelige bakterieë wat help met die voorkoming en behandeling van gastro-intestinale disbiose wat verwant hou met verskeie siektetoestande soos auto-immuniteit wanorde, sekere kankersoorte asook metaboliese wanorde. GOS word vervaardig vanaf melk laktose deur die glikosied hidrolase ensiem β-galaktosidase. ’n Goedkoop substraat vir die produksie van GOS bestaan in die vorm van industriële wei invulling. Nog ’n goeie voorbeeld van ’n prebiotiese voedings koolhidraat is Frukto-oligosakkariede (FOS), wat bestaan uit opeenvolgende fruktose eenhede wat dikwels gekoppel is aan ’n terminale glukose molekuul. Net soos GOS word FOS geassosieer met die verskeie gesondheids voordele van prebiotiese aanvulling. FOS word gesintetiseer vanaf die substraat sukrose deur ensieme genaamd fruktosiel-transferases. Daar is ’n groot aanvraag vir nuwe bio-katalise met verbeterde eienskappe vir die produksie van GOS en FOS. In hierdie studie lê ons ten toon die konstruksie en toetsing van verskeie meta-genoom biblioteke waarvan die meta-genoom DNA verkry is van gebiede wat ryk is in die substrate vir die produksie van prebiotiese oligo-sakkariede en polimere, naamlik laktose en sukrose. Vêrder beskryf ons ook die isolasie, amplifikasie en in silico analise van drie eensoortige glikosied hidrolase familie 1, 2 en 68 proteïene, onderskeidelik. Die drie proteïene genaamd HW-βgal1, MS-βgluc1 en SAS-Ls is heteroloog uitgedruk in E. coli, gesuiwer en biochemies gekarakteriseer tesame met hul onderskeie substraat produkte. Glikosied-hidrolases het dikwels twee ingebore onafhanklike en onderskeibare aktiwiteite genaamd hidrolise en trans-glikolisering. Al drie die glikosied-hidrolases wat hier beskryf word, het besondere hoë trans-glikolisering aktiwiteite hetsy vir GOS (MS-βGluc1 and HW-βGal1) of vir FOS (SAS-Ls), wat hul uitstekende kandidate maak vir industriële implementering en toepassing.

Please refer to this item in SUNScholar by using the following persistent URL: http://hdl.handle.net/10019.1/103718
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