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dc.contributor.advisorPermaul, Kugen
dc.contributor.advisorSingh, Suren
dc.contributor.authorWakelin, Kyle
dc.date.accessioned2009-10-13T11:32:22Z
dc.date.available2011-03-31T22:20:06Z
dc.date.issued2009
dc.identifier.other324239
dc.identifier.urihttp://hdl.handle.net/10321/480
dc.descriptionSubmitted in complete fulfillment for the Degree of Master of Technology (Biotechnology)in the Department of Biotechnology and Food Technology, Faculty of Applied Sciences, Durban University of Technology, Durban, South Africa, 2009.en_US
dc.description.abstractProtein engineering has been a valuable tool in creating enzyme variants that are capable of withstanding the extreme environments of industrial processes. Xylanases are a family of hemicellulolytic enzymes that are used in the biobleaching of pulp. Using directed evolution, a thermostable and alkaline stabl xylanase variant (S340) was created from the thermophilic fungus, Thermomyces lanuginosus. However, a host that was capable of rapid growth and high-level expression of the enzyme in large amounts was required. The insert containing the xylanase gene was cloned into a series a pET vectors in Escherichia coli BL21 (DE3) pLysS and trimmed from 786 bp to 692 bp to remove excess fungal DNA upstream and downstream of the open reading frame (ORF). The gene was then re-inserted back into the pET vectors. Using optimized growth conditions and lactose induction, a 14.9% increase in xylanase activity from 784.3 nkat/ml to 921.8 nkat/ml was recorded in one of the clones. The increase in expression was most probably due to the removal of fungal DNA between the vector promoter and the start codon. The distribution of the xylanase in the extracellular, periplasmic and cytoplasmic fractions was 17.3%, 51.3% and 31.4%, respectively. The modified enzyme was then purified to electrophoretic homogeneity using affinity chromatography. The xylanase had optimal activity at pH 5.5 and 70°C. After 120 min at 90°C and pH 10, S340 still displayed 39% residual activity. This enzyme is therefore well suited for its application in the pulp and paper industry.en_US
dc.description.sponsorshipNational Research Foundationen_US
dc.format.extent153 pen_US
dc.language.isoenen_US
dc.subjectBiotechnologyen_US
dc.subjectXylanases--Geneticsen_US
dc.subjectEscherichia coli--Geneticsen_US
dc.subjectProtein engineeringen_US
dc.subjectEnzymes--Industrial applicationsen_US
dc.subjectYeast fungi--Genetic engineeringen_US
dc.titleOverexpression and partial characterization of a modified fungal xylanase in Escherichia colien_US
dc.typeThesisen_US
dc.dut-rims.pubnumDUT-000371


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