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Overexpression and partial characterization of a modified fungal xylanase in Escherichia coli

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dc.contributor.advisor Permaul, Kugen
dc.contributor.advisor Singh, Suren
dc.contributor.author Wakelin, Kyle
dc.date.accessioned 2009-10-13T11:32:22Z
dc.date.available 2011-03-31T22:20:06Z
dc.date.issued 2009
dc.identifier.other 324239
dc.identifier.uri http://hdl.handle.net/10321/480
dc.description Submitted 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.abstract Protein 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.sponsorship National Research Foundation en_US
dc.format.extent 153 p en_US
dc.language.iso en en_US
dc.subject Biotechnology en_US
dc.subject Xylanases--Genetics en_US
dc.subject Escherichia coli--Genetics en_US
dc.subject Protein engineering en_US
dc.subject Enzymes--Industrial applications en_US
dc.subject Yeast fungi--Genetic engineering en_US
dc.title Overexpression and partial characterization of a modified fungal xylanase in Escherichia coli en_US
dc.type Thesis en_US
dc.dut-rims.pubnum DUT-000371


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