Main content

Protein Engineering of Cel6A from Cellulomonas fimi for mechanistic studies

Show full item record

Title: Protein Engineering of Cel6A from Cellulomonas fimi for mechanistic studies
Author: Whitney, Rebecca
Department: Department of Molecular and Cellular Biology
Program: Molecular and Cellular Biology
Advisor: Clarke, Anthony
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.
URI: http://hdl.handle.net/10214/7872
Date: 2014-02
Rights: Attribution 2.5 Canada
Terms of Use: All items in the Atrium are protected by copyright with all rights reserved unless otherwise indicated.


Files in this item

Files Size Format View
Whitney_Rebecca_201402_Msc.pdf 1.743Mb PDF View/Open

This item appears in the following Collection(s)

Show full item record

Attribution 2.5 Canada Except where otherwise noted, this item's license is described as Attribution 2.5 Canada