Browsing by Author "Fouche, Nicolette"
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- ItemExpression of mannanases in fermentative yeasts.(Stellenbosch : University of Stellenbosch, 2009-03) Fouche, Nicolette; Van Zyl, Willem Heber; Den Haan, Riaan; University of Stellenbosch. Faculty of Science. Dept. of Microbiology.ENGLISH ABSTRACT: The search for a cost-effective, environmentally friendly replacement for fossil fuels resulted in bio-ethanol production receiving a lot of attention. Lignocellulose, is considered to be the most abundant renewable source on earth, and consists of cellulose, hemicellulose and lignin. Exploitation thereof as a substrate for ethanol production, can serve as solution in producing bio-ethanol as an adequate replacement for fossil fuels. Hemicelluloses, contributing up to a third of the lignocellulosic substrate, consists mainly of xylan and mannan and can be degraded by hemicellulolytic enzymes that are produced by plant cell wall degrading organisms. Galactoglucomannan is the most complex form of mannan and requires a consortium of enzymes for complete hydrolysis. These enzymes include β-mannanase, β-mannosidase, α-galactosidase, β-glucosidase and galactomannan acetylesterases. Saccharomyces cerevisiae is a well-known fermentative organism that has been used in various industrial processes and is able to produce ethanol from hexose sugars. Although this organism is unable to utilize complex lignocellulosic structures, DNA manipulation techniques and recombinant technology can be implemented to overcome this obstacle. Strains of S. cerevisiae pose other shortcomings like hyperglycosylation and therefore other non-conventional yeasts (such as Kluyveromyces lactis) are now also being considered for heterologous protein production. The mannanase gene (manI) of Aspergillus aculeatus was expressed in K. lactis GG799 and S. cerevisiae Y294. K. lactis transformants were stable for two weeks in consecutive subcultures and secreted a Man1 of 55 kDa. The recombinant Man1 displayed an optimum temperature of 70°C and a pH optimum of 5 when produced by K. lactis. Activity levels of about 160 – 180 nkat/ml was obtained after 86 hours of cultivation, which was similar to the activity observed with S. cerevisiae under the same conditions. Disruption of the ku80 gene did not contribute to the stability of the cultures and a heterogeneous culture developed for 10 days of consecutive subculturing. The mannosidase gene (man1) from A. niger and mannanase gene (manI) from A. aculeatus were constitutively expressed in S. cerevisiae Y294 and S. cerevisiae NI-C-D4. The MndA and Man1 proteins appeared as a 140 kDa and 58 kDa species on the SDS-PAGE analysis when expressed in S. cerevisiae Y294, respectively. MndA had an optimum temperature of 50°C and optimum pH 5. Man1 produced by S. cerevisiae Y294 indicated a pH optimum of 6 and temperature optimum of 70°C. The MndA displayed low levels of endomannanase activity and no β-mannosidase activity could be detected. Co-expression of man1 and mndA in either S. cerevisiae Y294 and S. cerevisiae NI-C-D4, resulted in less hydrolysis of galactoglucomannan. An increase in the size of the plasmid generally results in a decrease in the copy number, leading to a decrease in the amount of ManI protein being produced. The co-expression of ManI and MndA could also have resulted in a higher metabolic burden on the cell, hence the amount of ManI are produced. This study confirms that more research should be done on the evaluation of alternative hosts for expression of foreign proteins. Furthermore, producing enzymes cocktails for industrial application should be considered rather than co-expression of various enzymes in one host.