3. Electronic Theses and Dissertations (ETDs) - All submissions
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Item In-field and in silico bioprospecting for hydroxynitrile lyases and terpenoid synthases from flora in South Africa(2020) Tomescu, Mihai-SilviuEnzymes 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 potatoItem HIV-1 subtype C proteases: overexpression, structural, kinetic and thermodynamic characterisation(2016-05-10) Tomescu, Mihai-SilviuAccording to UNAIDS, there are ~36.9 million people infected with HIV-1 in the world. Of those, 25.8 million live in sub-Saharan Africa and 6.8 million in South Africa. HIV-1 subtype C accounts for over 95% of HIV infections in South Africa. HIV-1 retrovirus acquires mutations rapidly because of the viral reverse transcriptase. Naturally occurring polymorphisms distinguishing wild type C-SA PR from other proteases make it less susceptible to inhibitors. E35D↑G↑S is a C-SA PR variant with a double insertion in the flap region of the protease. The insertions and background mutations may decrease susceptibility to inhibitors as well as alter kinetic parameters due to increased flap flexibility. This study intended to characterise the effect of the mutations and insertions in E35D↑G↑S on structural, kinetic activity and drug susceptibility. Chemically-synthesised E35D↑G↑S autocatalyses rapidly, impeding further characterisation. There was no detectable overexpression of the E35D↑G↑S protease in Escherichia coli BL21 (DE3)pLysS and Rosetta 2® cells. If the protease is catalytically enhanced, attributed cytotoxicity may prevent overexpression of the protein. Increased autocatalytic activity could also prevent crystallisation. Inactive E35D↑G↑S D25A did not overexpress either, indicating that codon harmonisation with the expression host ought to be performed. C-SA PR was shown to be a predominantly beta-sheeted protein using circular dichroism spectroscopy. The KM of the fluorogenic substrate resembling the capsid/ p2 cleavage site for C-SA PR was 22.02 ±4.09 μM. The specific activity, catalytic turnover and catalytic efficiency of the wild-type C-SA PR protease were found to be 35.68 ±1.06 μmole.min-1.mg-1, 12.79 ±0.38 s-1 and 1.17 ±0.055 s-1.μM-1, respectively. The thermodynamics of binding of atazanavir, ritonavir and darunavir to C-SA PR were determined using isothermal titration calorimetry. The binding of atazanavir and ritonavir to C-SA PR is entropically driven and enthalpically opposed. However, the binding of darunavir to C-SA PR was found to be both entropically and enthalpically favourable. The dissociation constants of the inhibitors in the absence of substrate (Kd) are in the pico-molar range and increased by approximately one order of magnitude when saturating concentrations of substrate were introduced. Atazanavir, ritonavir and darunavir have dissociation constants (Kd) of 160.56 ±54.59 pM, 113.34 ±46.47 pM and 10.24 ±6.02 pM, respectively. Darunavir binds significantly tighter. Keywords: C-SA PR, E35D↑G↑S, insertion mutations, protease, autocatalysis, ITC.