Protein Engineering of Cel6A from Cellulomonas fimi for mechanistic studies

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.