M. Tech. (Department of Biotechnology, Faculty of Applied and Computer Sciences), Vaal University of Technology / Lipases have been one of the important biocatalysts that catalyse the transformation of lipids
to yield very important products that can be of beneficial in food, agriculture, pharmaceutical
medicine and for the biodiesel production. In the search for novel biocatalysts, notably lipases,
the conventional culture-based techniques were used but it can only address sourcing the
biomolecule from 1-10% of the microbial population leaving the wealth of the biomolecules
packed in 90-99% of the microbial community unaccounted for. Metagenomic technique,
which is culture-independent, was developed as a comprehensive approach to address literally
100% of the microbial population thereby maximizing the chances of obtaining novel
biocatalysts with superior physico-chemical and catalytic traits. In principle, any biomolecule
including lipase could be sourced from any biologically-active environment, of which animal
rumen is one. However, among the rumenant animals goat has diverse feeding habit, thereby
laying ground for increased microbial diversity in its gastro-intestinal tract. It was thus,
postulated that goat rumen could be potential source of novel lipase isoforms. Therefore, the
aim of the study was to extract metagenomic DNA from goat rumen and construct a
metagenomic fosmid library and screen the library for lipase isoforms. The fosmid clones were
functionally screened using 1% tributyrin as a substrate and five positive clones were selected.
From the five clones, two fosmids were selected for further study. Following nucleotide
sequencing and in-silico analysis of the insert of the two selected clones, one lipase encoding
open reading frame (Lip-VUT3 and Lip-VUT5) from each fosmid clones of approximately 212
and 248 amino acids, respectively, was identified. The amino acid sequences of the Lip-VUT3
ORF contained a classical conserved lipase GSDL sequence motif while the amino acid
sequences of the Lip-VUT5 ORF contained a classical G-L-S-L-G conserved lipase/esterase
motif sequence. The two genes (Lip-VUT3 and Lip-VUT5) were successfully expressed in
Escherichia coli BL21 (DE3) and the purified enzymes exhibited respective temperature
optima of 60 °C and 70 °C, and respective pH optima of 6.0 and 10.0. Further biochemical
characterisation indicated that Lip-VUT3 and Lip-VUT5 lipases showed tolerance towards a
wide concentration (50%-100%) of methanol. Lip-VUT3 had a Km value of 0.287 mM while
Lip-VUT5 had a Km value of 0.556 Mm. This shows that Lip-VUT3 lipase has a higher affinity
for olive oil than Lip-VUT5. Lip-VUT3 and Lip-VUT5 were characterised to be true lipases
that have been recovered from the rumen environment through metagenomic approach.
Therefore, the study proved that metagenomic approach helps in recovering novel lipase
isoforms with potential down stream industrial and therapeautic applications from goat rumen metagenome, a rich but untapped source.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:vut/oai:digiresearch.vut.ac.za:10352/463 |
| Date | 09 1900 |
| Creators | Mukendi, Mujinga Grace |
| Contributors | Feto, N. A., 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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