Enhanced expression and activity yields of Clostridium thermocellum xylanases without non-catalytic domains

Two major xylanase components, XynC and XynZ from the anaerobic thermophilic bacterium, Clostridium thermocellum cellulosome were cloned and expressed with and without non-catalytic domains in E. coli. Two constructs of XynC, one with its cellulose binding domain and the catalytic domain (pXynC-BC) and the other with only the catalytic domain (pXynC-C) were produced. For XynZ the constructs produced were pXynZ-BDC, which included the dockerin domain, and pXynZ-C, which did not. E. coli cells transformed with pXynC-BC or pXynZ-BDC gave xylanase expression of 30% and 25% total cell proteins, respectively. Transformation of E coli cells with the constructs carrying only the catalytic domains gave expression levels of similar to 45% in each case. The specific activities of XynC with and without the non-catalytic domains were similar, but for XynZ the specific activity of the enzyme without the non-catalytic domains was similar to 5-fold greater than that of the intact enzyme. The total activity increased from 1925 U l(-1) OD600-1 for XynC-BC to 3050 U l(-1) OD600-1 for XynC-C. However, the overall increase in activity was similar to 9-fold higher for XynZ-C (32,900 U l(-1) CD600-1) versus XynZ-BDC (3665 U l(-1) OD600-1). Both the enzymes with and without non-catalytic domains were found to be quite stable over a broad pH range (pH 4-9). XynZ-C was more thermostable than XynZ-BDC as it retained 87% of xylanase activity when incubated at 70 degrees C for 2 h as compared to 42% for XynZ-BDC. However, XynC-BC retained 70% activity on incubation at 70 degrees C for 2 h but XynC-C lost all activity under the same conditions. K-m values for XynC-BC and XynC-C determined on soluble xylan were 3.1 and 3.6 mg ml(-1), respectively, whereas these values for XynZ-BDC and XynZ-C were 33.3 and 15.4 mg ml(-1), respectively. Thus the production of xylanase activity by expressing only the catalytic domains of XynC and XynZ is significantly enhanced. (c) 2009 Elsevier B.V. All rights reserved.