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 approximately 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 approximately 5-fold greater than that of the intact enzyme. The total activity increased from 1925Ul(-1)OD(600)(-1) for XynC-BC to 3050Ul(-1)OD(600)(-1) for XynC-C. However, the overall increase in activity was approximately 9-fold higher for XynZ-C (32,900Ul(-1)OD(600)(-1)) versus XynZ-BDC (3665Ul(-1)OD(600)(-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 2h as compared to 42% for XynZ-BDC. However, XynC-BC retained 70% activity on incubation at 70 degrees C for 2h 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.
University of the Punjab, Lahore, Pakistan.
Muhammad
M
Sajjad
M Imran M
MI
Khan
Nadeem S
NS
Akbar
Sajjad
S
Ahmad
Imran
I
Ali
M Waheed
MW
Akhtar
D000595:Amino Acid Sequence; D001483:Base Sequence; D020134:Catalytic Domain / Q000235:genetics*; D003001:Cloning, Molecular / Q000379:methods*; D048013:Acetivibrio thermocellus / Q000201:enzymology* / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D043364:Endo-1,4-beta Xylanases / Q000235:genetics* / Q000378:metabolism; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D016014:Linear Models; D008969:Molecular Sequence Data; D013696:Temperature
D043364:Endo-1,4-beta Xylanases