M. Tech. (Department of Biotechnology, Faculty of Applied and Computer Sciences), Vaal University of Technology. / During one of our preliminary studies in 2015, metagenomic DNA extracted from the goat
rumen was sequenced and the in silico mining of the biorefining enzyme showed the presence
of significant number of different biocatalysts, such as amylases (E.C 3.2.1.1), xylanases (E.C
3.2.1.8), pectinases (E.C 3.2.1.15) and cellulases (E.C 3.2.1.4). Hence, a subsequent study was
conducted which is aimed at extracting metagenomic DNA from the goat rumen, constructing
the metagenomic library using pCC2-FOS™ plasmid vector (Epicentre®), and eventually
screening the constructed library for potential novel amylases using soluble starch as a
substrate. Accordingly, rumen digesta was aseptically collected from four compartments of
each goat and pulled before extraction of metagenomic DNA. The conventional CTAB
protocol was modified to extract the metagenomic DNA from the rumen digesta. As a result,
high molecular weight DNA was obtained and used to construct the metagenomic fosmid
library. Since the host (Escherichia coli EPI 300-T1r) supplied with CopyControlHTP Fosmid
Library Production Kit has background amylase expression we opted for a knockout E. coli
strain with deleted starch hydrolysis (amylase expression) pathway. The library was
subsequently screened for the presence of amylase isoforms using soluble starch as a substrate.
Therefore, for the purpose of this study, four fosmids clones showing amylase activity were
selected, recombinant vector isolated and MiSeq-sequenced. Out of four recombinant proteins,
only one (pET30a(+)-amy-vut12) was successfully expressed. Subsequently, pET30a(+)-amyvut12
was further characterize physicochemically. Interestingly, the recombinant enzyme
showed maximum activity in the pH and temperature ranges of 6.0 - 8.0 and 70 - 90oC,
respectively. Hence, this implies that novel recombinant protein has sound activity from acidic
to alkaline pH range and potently thermostable. Further work should be done to optimize and
improve the solubility of three other recombinant proteins (pET30a(+)-amy-vut2, pET30a(+)-
amy-vut9 and pET30a(+)-amy-vut14) studied, which might harbour important traits. Most
importantly, immobilization as well as crystallographic studies of pET30a(+)-amy-vut12 and
downstream applications should further be investigated.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:vut/oai:digiresearch.vut.ac.za:10352/484 |
| Date | 09 1900 |
| Creators | Rabapane, Kgodiso Judith |
| Contributors | Feto, Naser Aliye, Dr, Nelson, K. E., Prof. |
| Publisher | Vaal University of Technology |
| Source Sets | South African National ETD Portal |
| Language | English |
| Detected Language | English |
| Type | Thesis |
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