Isolation and characterisation of novel enzymes producing fructo- and galacto-oligosaccharides through a metagenomic approach
Year: 2018
Type: dissertation
Abstract: 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).
Author: Marthinus Luther Smith
Language: English
Subfield: Nutrition and Dietetics
Field: Nursing
Domain: Health Sciences
SDG: Zero hunger
Open Access status: green
OpenAlex ID: W2800374214