Biopolymer gene discovery and characterization using metagenomic libraries

Ohlhoff, Colin Walter (Stellenbosch : Stellenbosch University, 2008-12)

Thesis

Traditional methods used for the discovery of novel genes have previously relied upon the ability to culture the relevant microbes and then demonstrate the activity of a specific enzyme. Although these methods have proved successful in the past, they severely limit our access to the genomes of organisms which are not able to be cultured under laboratory conditions. It was therefore the aim of this project to use metagenomic strategies for the identification of novel polymer-producing genes with the prospect of commercial exploitation. In this study, soil-derived metagenomic libraries were functionally screened for potential -glucan producing clones using aniline blue staining. Positive reacting clones were selected and sequenced. Initial sequencing revealed a gene with high homology to previously described glucan synthases, the products of these genes all having significant industrial value. The clone was transformed into a suitable bacterial host, cultured and allowed to produce the polymer of interest. The polysaccharide was purified and subjected to various chemical analyses so as to confirm its monosaccharide composition. Data suggests that this polymer is composed mainly of glucose units and that it may be secreted out of the cell. Purification of the active enzyme was attempted using classical protein purification methods with faint activity being detected using Native polyacrylamide gel electrophoresis (PAGE). Further attempts to demonstrate activity were made through the construction of a GST (glutathione S-transferase) tagged fusion protein. The second part of this study focuses on the construction and screening of a metagenomic DNA library from whey, a by-product of the cheese manufacturing process. It was envisaged that this could provide a resource for the identification of high value polymers when lactose is provided as a sole carbon source. The library was screened for function using Congo Red for the detection of extra-cellular polysaccharides.

Please refer to this item in SUNScholar by using the following persistent URL: http://hdl.handle.net/10019.1/2380
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