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Theses and dissertations (Applied Sciences)

Permanent URI for this collectionhttp://ir-dev.dut.ac.za/handle/10321/6

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Has files: YesSubject: search.filters.subject.Enzymes--Biotechnology

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    Cloning and characterisation of three novel lipases from Thermomyces lanuginosus
    (2022) Mbamali, Siphiwengesihle Kuhle Silindile; Permaul, Kugen; Mchunu, Nokuthula Peace
    Although other Thermomyces lanuginosus lipases have already been reported in the literature, genome sequencing resulted in three different lipases being identified. Thus this study aimed to characterise these novel T. lanuginosus lipases. The three lipase gene sequences were analysed to investigate their novelty, similarities and to compare them to existing lipases. It was found that they were different from each other and had low identity to existing lipases. Conserved domain analysis showed that all three genes belong to the abhydrolase superfamily, the family in which lipases and esterases belong. Furthermore, lipase C was also part of another family, PLNO2877 superfamily, another conserved protein domain family specifically for triacylglycerol lipases. Protein sequence alignment analysis also revealed that lipases A and B are more similar to each other compared to lipase C. SWISS protein models were also created using the best template matches for each protein sequence, the protein models further indicated the distinctness of lipase C and the similarity between lipases A and B were further demonstrated by superimposing their ribbon. The cDNA of Thermomyces lanuginosus SSBP was used to amplify the lipase A and lipase B genes using primers designed for pPICZαA and pPIC9K cloning and expression vectors. Lipase B gene was also cloned into pPBG1. When the PCR products were analysed for amplification with gel electrophoresis. Lipase B amplification produced a single distinct band of approximately 1100 bp which was the expected PCR product for all three Pichia cloning vectors. Amplification for lipase A proved to be unsuccessful as three bands were produced instead of a single distinct band. Plasmid pET100/DTOPO containing the artificially synthesised three putative lipases were synthesised for expression in E.coli BL21 (DE3). This method yielded higher expression levels for all three lipases when compared to Pichia. After purification, the recombinant lipases from E. coli produced lipase yields of 176.2 ± 1.2 for lipase A; 184.1 ± 0.46 for lipase B; and 181 ± 0.13 for lipase C. This was much higher than the activity obtained from P. pastoris expression. Enzyme characterisation was performed using E. coli only. The temperature optimum of all three lipases was identical at 60˚C. All three lipases had preference for alkaline conditions, with an optimum of pH 8, and activity was stable between pH 7.0-10.0. All three lipases preferred longer chain substrates, with p-nitrophenyl palmitate (C16) being the most favourable, with an exception of lipase C which preferred p-nitrophenyl stearate (C18) with activity 7% higher than that on p-nitrophenyl palmitate. These lipases therefore have temperature and pH properties that will be useful for thnumerous industrial applications of lipases which will be investigated in future studies
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    Cloning, expression, characterization and application of cyanase from a thermophilic fungus Thermomyces lanuginosus SSBP
    (2018) Ranjan, Bibhuti; Singh, Suren; Pillai, Santhosh Kumar Kuttan; Permaul, Kugen
    Rapid industrialization and proliferative development of chemical and mining industries have resulted in increased global pollution and environment deterioration, due to the release of numerous toxic substances. This has extreme relevance in the South African context due to the high amount of cyanide used by local mines in comparison to that utilized globally. This has created the need for the development of novel approaches viz., using microbial enzymes for its remediation because of lower process times, lower energy requirements, and their cost-effective, nontoxic and eco-friendly characteristics. From previous work in our lab, the whole genome sequencing and secretome analysis of the industrially-important fungus Thermomyces lanuginosus SSBP revealed the presence of a cyanate hydratase gene and enzyme, respectively. Cyanate hydratase detoxifies cyanate in a bicarbonate-dependent reaction to produce ammonia and carbon dioxide. The cyanate hydratase gene (Tl-Cyn) from this fungus was therefore cloned, overexpressed, purified, characterized and its potential in cyanate detoxification has also been evaluated. The recombinant cyanate hydratase (rTl-Cyn) showed high catalytic efficiency, suggesting that it could be used for bioremediation applications. Though, cyanate hydratase catalyzes the decomposition of cyanate, the requirement of bicarbonate is a major drawback for its effective utilization in large-scale applications. Hence, a novel strategy was developed to limit the bicarbonate requirement in cyanate remediation, by the combinatorial use of two recombinant enzymes viz., cyanate hydratase (rTl-Cyn) and carbonic anhydrase (rTl-CA) from T. lanuginosus. This integrative approach resulted in the complete degradation of cyanate using 80% less bicarbonate, compared to the cyanate hydratase alone. In addition, co-immobilization of these recombinant enzymes onto magnetic nanoparticles and evaluation of their potential in bio-remediation of cyanurated wastes together with their reusability resulted in more than 80% of cyanate detoxification in wastewater samples after 10 cycles. Another novel strategy was also developed for the simultaneous removal of heavy metals and cyanate from synthetic wastewater samples, by immobilizing the rTl-Cyn on magnetic multi- walled carbon nanotubes (m-MWCNT-rTl-Cyn). The m-MWCNT-rTl-Cyn simultaneously reduced the concentration of chromium (Cr), iron (Fe), lead (Pb) and copper (Cu) by 39.31, 35.53, 34.48 and 29.63%, respectively, as well as the concentration of cyanate by ≥85%. The crystal structure of Tl-Cyn in complex with inhibitors malonate or formate at 2.2 Å resolution was solved for the first time to elucidate the molecular mechanism of cyanate hydratase action. This structure enabled the creation of a mutant enzyme with ~1.3-fold enhanced catalytic activity as compared to the wild-type Tl-Cyn. In addition, the active site region of Tl-Cyn was found to be highly conserved among fungal cyanases. Information from the 3D structure could enabled the creation of novel fungal cyanases, which may have potential for biotechnological applications, biotransformation and bioremediation.