Thesis (MSc (Genetics. Institute of Plant Biotechnology))--Stellenbosch University, 2008. / 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.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/2380 |
| Date | 12 1900 |
| Creators | Ohlhoff, Colin Walter |
| Contributors | Lloyd, J. R., Bauer, R., Kossmann, J. M., Stellenbosch University. Faculty of AgriSciences. Dept. of Genetics. Institute for Plant Biotechnology. |
| Publisher | Stellenbosch : Stellenbosch University |
| Source Sets | South African National ETD Portal |
| Language | English |
| Detected Language | English |
| Type | Thesis |
| Rights | Stellenbosch University |
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