CelI, a noncellulosomal family 9 enzyme from Clostridium thermocellum, is a processive endoglucanase that degrades crystalline cellulose.
The family 9 cellulase gene celI of Clostridium thermocellum, was previously cloned, expressed, and characterized (G. P. Hazlewood, K. Davidson, J. I. Laurie, N. S. Huskisson, and H. J. Gilbert, J. Gen. Microbiol. 139:307-316, 1993). We have recloned and sequenced the entire celI gene and found that the published sequence contained a 53-bp deletion that generated a frameshift mutation, resulting in a truncated and modified C-terminal segment of the protein. The enzymatic properties of the wild-type protein were characterized and found to conform to those of other family 9 glycoside hydrolases with a so-called theme B architecture, where the catalytic module is fused to a family 3c carbohydrate-binding module (CBM3c); CelI also contains a C-terminal CBM3b. The intact recombinant CelI exhibited high levels of activity on all cellulosic substrates tested, with pH and temperature optima of 5.5 and 70 degrees C, respectively, using carboxymethylcellulose as a substrate. Native CelI was capable of solubilizing filter paper, and the distribution of reducing sugar between the soluble and insoluble fractions suggests that the enzyme acts as a processive cellulase. A truncated form of the enzyme, lacking the C terminal CBM3b, failed to bind to crystalline cellulose and displayed reduced activity toward insoluble substrates. A truncated form of the enzyme, in which both the cellulose-binding CBM3b and the fused CBM3c were removed, failed to exhibit significant levels of activity on any of the substrates examined. This study underscores the general nature of this type of enzymatic theme, whereby the fused CBM3c plays a critical accessory role for the family 9 catalytic domain and changes its character to facilitate processive cleavage of recalcitrant cellulose substrates.
Gilad
Rachel
R
Department of Food Engineering and Biotechnology and Institute of Catalysis Science and Technology, Technion-Israel Institute of Technology, Haifa 32000.
Rabinovich
Larisa
L
Yaron
Sima
S
Bayer
Edward A
EA
Lamed
Raphael
R
Gilbert
Harry J
HJ
Shoham
Yuval
Y
D000595:Amino Acid Sequence; D001483:Base Sequence; D002480:Cellulase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002482:Cellulose / Q000378:metabolism*; D003001:Cloning, Molecular; D003013:Clostridium / Q000201:enzymology* / Q000235:genetics; D003460:Crystallization; D008969:Molecular Sequence Data; D010209:Paper; D011994:Recombinant Proteins / Q000378:metabolism; D017422:Sequence Analysis, DNA
D011994:Recombinant Proteins; D002482:Cellulose; D002480:Cellulase