PJB-2018-865
INFLUENCE OF ADDITIONAL BINDING AND CATALYTIC DOMAINS ON EXPRESSION AND CHARACTERISTICS OF XYLANASE Z OF CLOSTRIDIUM THERMOCELLUM
Muddassar Zafar
Abstract
Xylanase Z (XynZ) is a major component of Clostridium thermocellum cellulosome. It is a multidomain enzyme comprising of N-terminus feruloyl esterase followed by substrate binding, dockerin and the catalytic domains. The purpose of this study was to observe the novelty in expression, activities and other characteristics of different variants of XynZ produced by attaching binding domain to the catalytic domain in different arrangements. XynZ variants of C. thermocellum with binding domain attached to the catalytic domain at its C-terminal (XynZ-CB), N-terminal (XynZ-BC), both N- and C-terminals (XynZ-BCB) and one with catalytic domain at both terminals of the binding domain (XynZ-CBC) were expressed in E. coli at levels around 30, 30, 33 and 15% of the total cell proteins, respectively. The specific activities of XynZ-CB, XynZ-BC, XynZ-BCB and XynZ-CBC were 72, 68, 67 and 200 U mg-1, while their activities on equimolar basis were 4176, 3944, 5092 and 19,200 U µM-1 against birchwood xylan, respectively. The overall activities of XynZ-CB, XynZ-BC, XynZ-BCB and XynZ-CBC produced in the culture were 3678, 3579, 3800 and 5400 U l-1 OD600-1, respectively. The results showed that overall yield of XynZ-C remained manifold higher than all other variants due to combined effect of its specific activity and expression level while the activity of variant XynZ-CBC was increased four folds on equimolar basis as compared to its native form.
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