Theses and Dissertations (Biosciences)

Permanent URI for this collection

https://hdl.handle.net/10352/163

Browse

Browsing Theses and Dissertations (Biosciences) by Author "Feto, N. A., Dr."

Now showing 1 - 2 of 2
  • Thumbnail Image
    Item
    Engineering of an enzyme cocktail for biodegradation of petroleum hydrocarbons based on known enzymatic pathways and metagenomic techniques
    (Vaal University of Technology, 2020-07) Baburam, Cindy; Tsekoa, T., Dr.; Feto, N. A., Dr.
    Hydrocarbon pollution is becoming a growing environmental concern in South Africa and globally. This inadvertently supports the need to identify enzymes for their targeted degradation. The search for novel biocatalysts such as monooxygenases, alcohol dehydrogenases and aldehyde dehydrogenases, have relied on conventional culture-based techniques but this allows sourcing of the biomolecules from only 1-10 % of the microbial population leaving the majority of the biomolecules unaccounted for in 90-99 % of the microbial community. The implementation of a metagenomics approach, a culture-independent technique, ensures that more or less than 100 % of the microbial community is assessed. This increases the chance of finding novel enzymes with superior physico-chemical and catalytic traits. Hydrocarbon polluted soils present a rich environment with an adapted microbial diversity. It was thus extrapolated that it could be a potential source of novel monooxygenases, alcohol dehydrogenases (ADH) and aldehyde dehydrogenases (ALDH) involved in hydrocarbon degradation pathways. Therefore, the aim of the study was to extract metagenomic DNA from hydrocarbon contaminated soils and construct a metagenomic fosmid library and screen the library for monooxygenases, alcohol dehydrogenases (ADH) and aldehyde dehydrogenases (ALDH). Accordingly, the fosmid library was constructed from metagenome of hydrocarbon-contaminated soil. Then the library was functionally screened using hexadecane, octadecene and cyclohexane as substrates and fifteen positive clones were selected. The fosmid constructs of the positive clones were sequenced using PacBio next generation sequencing platform. The sequences were de novo assembled and analysed using CLC Genomic Workbench. The open reading frames (ORF) of the contigs were identified by blasting the contigs against uniport database. Accordingly, four novel genes namely amo-vut1, aol-vut3, dhy-sc-vut5 and dhy-g-vut7 that showed close similarity with our target enzymes were further analysed in silico and codon-optimized as per Escherichia coli codon preference. The codon adjusted sequences were synthesised and cloned into pET30a(+) expression vector. However, it is worth noting that expression of amo-vut1 was not successful since it was later identified to be a multi-pass member protein, which made it insoluble despite the use of detergent to the effect. There is a need to meticulously genetically engineer amo-vut1 to remove the signal and other membrane-bound peptides while maintaining its activity. Yet the other three constructs were successfully transformed and expressed in E. coli BL21 (DE3). The enzymes were purified and characterized and cocktail for hydrolysis of hexanol was succesfully engineered based on AOL-VUT3, DHY-SC-VUT5 and DHY-G-VUT7. Therefore, novel enzymes were mined from metagenome of fossil-oil contaminated soil and effective hydrocarbon-degrading enzyme cocktails containing their combination were successfully engineered.
  • Thumbnail Image
    Item
    Sequence and function-based screening of goat rumen metagenome for novel lipases
    (Vaal University of Technology, 2019-09) Mukendi, Mujinga Grace; Nelson, K. E., Prof.; Feto, N. A., Dr.
    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.