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Protein Engineering of Cel6A from Cellulomonas fimi for mechanistic studies

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dc.contributor.advisor Clarke, Anthony Whitney, Rebecca 2014-03-13T12:59:04Z 2014-03-13T12:59:04Z 2014-02 2014-02-14 2014-03-13
dc.description.abstract Cellulolytic enzymes commonly use one of two different mechanisms to achieve cellulose hydrolysis, differentiated by the stereochemical orientation of the anomeric carbon post reaction. The purpose of this study was to alter the reaction mechanism of Cel6A from Cellulomonas fimi using a catalytic cys variant in combination with chemical alkylation. To achieve this goal, the catalytic cys variant was purification under reducing conditions to preclude the 56% autooxidation that occurred to the introduced sulfhydryl under environmental oxygen levels. The inclusion of reducing agents in the purification buffers had a negative impact on the activity of the WT protein due to disruption of the native disulfide bonds. Additionally it was discovered that Tris-HCl has an inhibitory effect on the activity of Cel6A. Preliminary alkylation attempts indicated that the propionylation of the introduced cysteine may be able to rescue catalytic activity, but more definitive experimentation is required. en_US
dc.language.iso en en_US
dc.rights Attribution 2.5 Canada *
dc.rights.uri *
dc.subject cellulase en_US
dc.subject reaction mechanism en_US
dc.subject cellulomonas fimi en_US
dc.title Protein Engineering of Cel6A from Cellulomonas fimi for mechanistic studies en_US
dc.type Thesis en_US Molecular and Cellular Biology en_US Master of Science en_US Department of Molecular and Cellular Biology en_US
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Attribution 2.5 Canada Except where otherwise noted, this item's license is described as Attribution 2.5 Canada