Faculty of Applied Sciences
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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 studiesItem Cloning, expression, characterization and application of cyanase from a thermophilic fungus Thermomyces lanuginosus SSBP(2018) Ranjan, Bibhuti; Singh, Suren; Pillai, Santhosh Kumar Kuttan; Permaul, KugenRapid 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.Item Cloning and expression of xylanase variants in Pichia pastoris(2017) Govindarajulu, Natasha; Permaul, Kugen; Singh, SurenMicrobial xylanases have attracted considerable research interest because of their various applications in biotechnology including the biobleaching of kraft pulp, to increase the nutritional value of foods and animal feed as well as for their potential use in the production of ethanol and methane. In the paper and pulp industry, the bleaching process involves the use of toxic chemicals and in the interim produces harmful gases that have a negative impact on the environment. The application of enzymes for this process will potentially reduce the environmental pollution by this industry. In addition, using an enzyme that is thermostable and alkali tolerant means that they will remain active under the required processing conditions. The xylanase gene, xynA derived from Thermomyces lanuginosus DSM 5826, was previously evolved to produce a number of xylanase variants, which were further enhanced for increased thermostability and alkalinity. In this study, these variants were cloned in Pichia pastoris using the pBGP1 vector to achieve extracellular production of the recombinant proteins. The xylanase genes were isolated using PCR. Both vector and DNA inserts were linearized with restriction enzymes EcoRI and XbaI and ligated. Electroporation was employed to transform the yeast with the recombinant plasmids. This was followed by the expression of the enzymes in P. pastoris grown in yeast peptone glucose (YPD) medium. Enzyme activity was thereafter assessed and the yeast was found to produce 164, 78, 96 and 142 IU/ml of S325, S340, G41 and G53 xylanase respectively, higher levels than bacterial hosts. The enzymes were then characterized and it was established that the optimum temperatures and pH for maximum xylanase activity were, 60°C, pH 6 for S325; 40°C, pH 5 for S340; 60°C, pH 6 for G41 and 60°C, pH 7 for G53. i The pH and temperature stabilities of the respective enzymes were investigated, the S325 variant was exceptionally stable at a pH between 5 and 7 and temperature range of 40-80°C and retained a minimum of 40% of activity at higher pH and temperature after an incubation period of 90 min. The S340 variant was the least thermostable and alkali stable from all four variants, it however retained 40% of activity when subjected to conditions of pH 9, 80°C after 90 min. The G41 and G53 were highly stable under the pH and temperature conditions that they were subjected to. Thus being suitable for potential application in the pulp and paper industry. The enzymes were able to retain 80% of activity at pH 9, 80°C after 120 min. P. pastoris has been proven to be a more suitable protein expression vector than E. coli for a number of reasons, including; the ability to perform complex post-translational modifications and grow to high densities in minimal media resulting in the production of a high yield of heterologous proteins.Item Cloning, characterization and directed evolution of a xylosidase from Aspergillus niger(2016) Khan, Bibi Khadija; Permaul, Kugen; Singh, Surenβ-xylosidases catalyse the hydrolyses of xylooligosaccharides into the monosaccharide sugar, xylose. In this study we report the production of xylose under different conditions in Pichia pastoris and Saccharomyces. cerevisiae, and its conversion to bioethanol using Pichia stipitis. The aim of this study was to change the characteristics of the A. niger 90196 β-xylosidase through random mutagenesis and increase expression under the control of different promoter systems in yeasts P. pastoris and S. cerevisiae. The recombinant library created through random mutagenesis was screened for changes in activity and subsequently pH and temperature stability. One variant showed an increase in enzyme expression, thermostability, and a change in amino acid sequence at residue 226. The enzyme was then cloned, expressed and characterized in P. pastoris GS115 and S. cerevisiae. β-xylosidase was constitutively expressed in P. pastoris using the GAP promoter and the inducible AOX promoter. In S. cerevisiae the enzyme was expressed using the constitutive PGK promoter and inducible ADH2 promoter systems. Enzyme functionality with the different expression systems was compared in both hosts. The GAP system was identified as the highest-producing system in P. pastoris, yielding 70 U/ml after 72 hours, followed by the PGK system in S. cerevisiae, with 8 U/ml. A 12% SDS-PAGE gel revealed a major protein band with an estimated molecular mass of 120 kDA, and the zymogram analysis revealed that this band is a fluorescent band under UV illumination, indicating enzyme activity. Stability characteristics was determined by expressing the enzyme at different pH and temperatures. Under the control of the GAP promoter in P. pastoris, enzyme activity peaked at pH4 while retaining 80% activity between pH 3 – 5. Highest activity of 70 U/ml xylosidase was recorded at 60ºC. Due to the high enzyme production in P. pastoris, the co-expression of this enzyme with a fungal xylanase was evaluated. The xylanase gene from Thermomyces lanuginosus was cloned with the GAP promoter system and expressed together with the β-xylosidase recombinant in P. pastoris. Enzyme activities of the co-expressed recombinant revealed a decrease in enzyme activity levels. The co-expressed xylanase production decreased by 26% from 136 U/ml to 100 U/ml while the xylosidase expression decreased 86% from 70 U/ml to 10 U/ml. The xylose produced from the hydrolysis of birchwood xylan was quantified by HPLC. The monosaccharide sugar was used in a separate saccharification and fermentation strategy by P. stipitis to produce bioethanol, quantified by gas chromatography. Bioethanol production peaked at 72 h producing 0.7% bioethanol from 10 g/l xylose. In conclusion a β-xylosidase from Aspergillus niger was successfully expressed in P. pastoris and was found to express large quantities of xylosidase, that has not been achieved in any prior research to date. The enzyme was also successfully co-expressed with a Thermomyces xylanase and is now capable of bioethanol production through xylan hydrolysis. This highlights potential use in industrial applications in an effort to reduce the world dependence on petroleum and fossil fuels. However the technical challenges associated with commercialization of bioethanol production are still significant.