Theses and dissertations (Applied Sciences)
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Item Directed evolution of B-xylanase from Thermomyces lanugtnosus(2000) Stephens, Dawn Elizabeth; Permaul, Kugen; Prior, A.; Singh, SurenMost natural enzymes may be unsuitable for biotechnological processes since they have evolved over millions of years to acquire their specific biological functions. Such enzymes are often genetically altered to suit the rigours of industrial processes. Directed evolution is one such strategy and makes use of iterative rounds of random mutagenesis, screening and recombination to enhance the existing properties of enzymes. Thermomyces lanuginosus is a thermophilic fungus that produces high levels of a thermostable xylanase. The xylanase gene from T lanuginosus DSM 5826 (xynA) was functionally expressed in E. coli as a LacZ-fusion protein (Schlacher et al., 1996) and later crystallized (Gruber et al., 1998). In this study, it was undertaken to improve the thermo stability and catalytic activity of xynA using error-prone PCR with different concentrations of MnCh. The first step prior to mutagenesis was to determine the levels of xylanase that could be attained by the wild type XynA, both in the presence and absence of an inducer. IPTG, a lactose analogue, was used since xynA was expressed with a lac promoter. High amounts of IPTG were found to adversely affect xylanase production, whilst a low amount (0.1 mM) enhanced xylanase production. This amount was used to later induce xylanase production by the variants obtained after mutagenesis. IPTG was found to increase the rate and production of xylanase. After random mutagenesis of xynA, transformed colonies were first selected for xylanase production on 0.4% Remazol Brilliant Blue xylan and then screened at different temperatures for improved stability and activity. After the first round of screening, four variants, viz., IB5, IB7, IBLl and ID2, showed slight improvement in both stability and activity and were subjected to further mutagenesis, using low concentrations of MnCh. Three variants, viz., 2B7-1O, 2B7-6 and 2BIl-16, with markedly enhanced stability, were obtained. Variant 2B7-10 exhibited a five fold higher activity (3430 nkat/ug total protein) than the wild type XynA (657 nkatl ug total protein). It retained 71% of its activity after treatment at 80°C for 60 min and had a t1/2of 215 min at 70°C, which is higher than that attained by XynA. Long-term thermo stability screening at 70, 80, 90 and 100°C revealed that variants 2B7-6 and 2B11-16 were, however, the most stable enzymes generated in this study, although their activities were lower or almost comparable with their parents. Sequence analysis of variant ID2 revealed 4 amino acid substitutions within the a-helix of the protein. This region was strongly conserved with the more stable variant xylanases generated in this study. The most profound mutation seen with variant 2B7-10 was the disruption of the disulphide bridge. Most of the mutants obtained in this study displayed a trade-off between stability and activity, the exception being mutant 2B7-10. Currently, DNA shuffling techniques are being used to recombine these traits in a single xylanase.Item Evaluation of the bleach-enhancing effects of xylanases on bagasse-soda pupil(2002) Bissoon, Sadhvir; Singh, SurenThe extent of diffusion and surface modification of a purified 23.6 kDa xylanase isolated from Thermomyces lanuginosus on bagasse pulp was evaluated. Polyclonal anti-xylanase antibodies were raised in two rabbits and in conjunction with immunogold labeling and microscopic studies enzyme diffusion and degradation studies were performed. The purity of the xylanase was confirmed by SDS-PAGE and western blots confirmed the antigen-antibody hybrid on the nitrocellulose membrane.Item Application of xylanases in bleaching of industrial pulps(2000) Madlala, Andreas Muzikababa; Singh, SurenThe ever-increasing demand for a wide variety of paper products has led to the pulp and paper industry becoming one of the largest industries in the world. In 1988 the United States alone produced almost 71 million metric tonnes of paper and pulp board (Jeffries, 1992). South Africa has also become one of the major international producers of pulp and paper products. Since 1970, the production of paper and board by the South African industry achieved an average growth rate of 5.2% per annum, and in 1997 South Africa was the twelfth largest producer of pulp and 24th biggest supplier of paper and board in the world (Molony, 1999).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.Item Protein engineering of fungal xylanase(2007) Stephens, Dawn ElizabethProtein engineering technologies, such as directed evolution and DNA recombination, are often used to modify enzymes on a genetic level for the creation of useful industrial catalysts. Pre-treatment of paper pulps with xylanases have been shown to decrease the amounts of toxic chlorine dioxide used to bleach pulp. This study was undertaken to improve the thermal and alkaline stabilities of the xylanase from the fungus Thermomyces lanuginosus using ep-PCR and DNA shuffling.