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    Production and characterization of DNA ligases isolated from Kogelberg Biosphere metagenomics library.
    (2021) Zuma, Lindiwe Khumbuzile.; Pooe, Ofentse Jacob.
    Microbial enzymes have been described as an underutilized source of novel enzymes with potential economic advantages. Recently discovered enzymes such as DNA ligase from metagenomic studies, have been shown to achieve great potential in transforming the reagent market specifically in the African continent. Reagent proteins are frequently utilized in the research field widely and are prone to protein degradation and shelf-life reduction. Hence, this study sought to improve biological activity, shelf life and stability of the two DNA ligases identified from Kogelberg Biosphere metagenomics library. Two recombinant DNA ligases expression studies were done using E.coli BL21 and purification studies were done subsequently using affinity chromatography. Both recombinant DNA ligases (Ligsv081 & LigpET30) were successfully expressed and purified as homogenous proteins. In this study two approaches were used to enhance the biological DNA ligases, the first approach used was PEGylation. The purified proteins were conjugated to PEG using site-specific PEGylation and non-specific PEGylation. FTIR and UV-VIS spectroscopy were used to analyze the secondary structure of the PEG conjugated DNA ligases. Thermal stability assays were then employed to assess protein stability in the conjugation with PEG. Site-specific PEGylation enhanced ligase activity and reduced the formation of protein aggregates. The second approach involved DNA ligase co-expression in the presence of PfHsp70 or chimeric transcription factor, respectively. Protein co-expression and co-purification assays were conducted. The co-expression and co-purification assays of both proteins with chimeric transcription factor (cTF) were successful, followed by co-expression and co-purification of LigpET30-PfHsp70. Ligation assays were conducted to assess bioactivity of proteins. All DNA ligase complexes were functional and their melting point was increased. Taken together, site-specific PEGylation and protein co-expression with PfHsp70 potentially extended the shelf-life and stability of the proteins. PEGylation strategies and co-expression strategies can potentially be used to enhance reagents in diagnostic and therapeutic tools in molecular biology field.
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    The isolation and characterisation of proteases from Euphorbia tirucalli, E. triangularis, and Carica papaya latex.
    (2023) Boodhoo, Akira.; Coetzer, Theresa Helen Taillefer.
    Plant proteases play an important role in the food and industrial sectors from meat tenderisers to milk clotting agents and even anti-parasitic agents. Proteases have been identified in plant latex, but many proteases have not been isolated and characterised. This research aimed to isolate and characterise proteases from the latex of Euphorbia tirucalli, E. triangularis, and Carica papaya. Three-phase partitioning (TPP) of E. tirucalli plant latex revealed the presence of two active proteases on gelatin-containing zymograms, that were subsequently separated by size exclusion chromatography. These proteases were classified as a 75 kDa serine protease (E. tiru SP), inhibited by PSMF, and SBTI and a 37 kDa cysteine protease (E. tiruCP), inhibited by E-64. Analysis of E. triangularis latex by TPP and p-aminobenzamidine affinity chromatography showed the presence of three serine proteases inhibited by PMSF and SBTI, E. laris SP1 (>97.4 kDa), E. laris SP2 (68 kDa) and E. laris SP3 (38 kDa). These proteases showed stability in constant ionic strength buffers from pH 4 to 9, both with and without the presence of the reducing agent cysteine. Papain was isolated from the latex of Carica papaya for use as a control protease in zymograms on account of the anomalous behaviour of commercial papain preparations on these gels. Papain was isolated by two methods: ammonium sulfate precipitation followed by TPP and CM-cellulose cation exchange chromatography. The isolated papain was detected by rabbit anti-papain antibodies in a dot blot and western blot and showed inhibition by E-64. The latex from all three plant species showed milk clotting activity with higher activity in the presence of calcium chloride. These findings suggest that isolated plant latex proteases from the Euphorbia species can be used in the food industry as milk clotting agents. Further characterisation of these isolated proteases should identify further uses in biotechnology.