Tomescu, Mihai-Silviu2020-11-162020-11-162020https://hdl.handle.net/10539/30184Thesis submitted in fulfilment of the requirements for the degree Philosophiae Doctor in Molecular and Cell Biology in the Faculty of Science, University of the Witwatersrand, Johannesburg, 2020Enzymes are useful biocatalysts capable of stereoselective biotransformation of precursors to industrially relevant products alleviating the requirement for costly hazardous chemical catalysts. Identification of new or altered enzymes is continually necessary for the improvement of current processes, some of which fall short of a usable biocatalyst. As such, there is a need for rapid bioprospecting procedures that can identify novel sources of biocatalysts. Hydroxynitrile lyases and terpene synthases are both carbon-carbon lyases with application in the fine chemical, agrochemical, pharmaceutical, flavour and fragrance industries. In this study, a methodology for bioprospecting for novel sources of hydroxynitrile lyases was established. Using this method, over 600 plants were screened and 32 were found able to degrade racemic mandelonitrile. Five of which (Achyranthes aspera, Davallia trichomonoides, Morus mesozygia, Polypodium aureum “Mandaianum”, and Thelypteris confluens) were naturally cyanogenic. In contrast, Acalypha glabrata was found to be naturally cyanogenic, however, proteinaceous extracts were unable to degrade mandelonitrile, suggesting possible affinity for a different substrate. Transcriptomic sequencing together with activity assays and LC-MS/MS were then performed on two species, Phlebodium aureum and Thelypteris confluens, resulting in prospect hydroxynitrile lyase sequences being identified. Regarding terpene synthases, the corm, leaf and flower of the phytomedicinal plant Hypoxis hemerocallidea (African potato) known to produce terpenoids were sequenced transcriptomically and proteomically. This led to the identification by functional annotation of numerous terpene synthases produced by the organism such as nerolidol synthase, germacrene D synthase and cycloartenol synthase. Transcripts were also annotated to encode for the terpene phytoalexin momilactone A synthase. Differential expression analysis revealed that the leaf upregulates linalool synthase compared to the other two tissues. Overall, this study produced a methodology for the high-throughput bioprospecting of hydroxynitrile lyases applicable to the field. Three transcriptomes were sequenced and assembled de novo from Phlebodium aureum, Thelypteris confluens and Hypoxis hemerocallidea which expands the list of biocatalysts. Prospect hydroxynitrile lyases and terpene synthase sequences were identified. This research offers a foundation for future research involving hydroxynitrile lyases from novel sources as well as and terpene synthases from the African potatoenThesis