Theses and dissertations (Applied Sciences)
Permanent URI for this collectionhttp://ir-dev.dut.ac.za/handle/10321/6
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Item Cloning and characterisation of three novel lipases from Thermomyces lanuginosus(2022) Mbamali, Siphiwengesihle Kuhle Silindile; Permaul, Kugen; Mchunu, Nokuthula PeaceAlthough 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