Characterization of a cellulase containing a family 30 carbohydrate-binding module (CBM) derived from Clostridium thermocellum CelJ: importance of the CBM to cellulose hydrolysis.
Clostridium thermocellum CelJ is a modular enzyme containing a family 30 carbohydrate-binding module (CBM) and a family 9 catalytic module at its N-terminal moiety. To investigate the functions of the CBM and the catalytic module, truncated derivatives of CelJ were constructed and characterized. Isothermal titration calorimetric studies showed that the association constants (K(a)) of the CBM polypeptide (CBM30) for the binding of cellopentaose and cellohexaose were 1.2 x 10(4) and 6.4 x 10(4) M(-1), respectively, and that the binding of CBM30 to these ligands is enthalpically driven. Qualitative analyses showed that CBM30 had strong affinity for cellulose and beta-1,3-1,4-mixed glucan such as barley beta-glucan and lichenan. Analyses of the hydrolytic action of the enzyme comprising the CBM and the catalytic module showed that the enzyme is a processive endoglucanse with strong activity towards carboxymethylcellulose, barley beta-glucan and lichenan. By contrast, the catalytic module polypeptide devoid of the CBM showed negligible activity toward these substrates. These observations suggest that the CBM is extremely important not only because it mediates the binding of the enzyme to the substrates but also because it participates in the catalytic function of the enzyme or contributes to maintaining the correct tertiary structure of the family 9 catalytic module for expressing enzyme activity.
Arai
Takamitsu
T
Faculty of Bioresources, Mie University, Tsu 514-8507, Japan.
Araki
Rie
R
Tanaka
Akiyoshi
A
Karita
Shuichi
S
Kimura
Tetsuya
T
Sakka
Kazuo
K
Ohmiya
Kunio
K
D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D002151:Calorimetry; D050260:Carbohydrate Metabolism; D002480:Cellulase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002482:Cellulose / Q000378:metabolism*; D003013:Clostridium / Q000201:enzymology*; D006868:Hydrolysis; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000378:metabolism; D011134:Polysaccharides / Q000378:metabolism
D001426:Bacterial Proteins; D009844:Oligosaccharides; D011134:Polysaccharides; D002482:Cellulose; C101903:CelJ protein, Clostridium thermocellum; D002480:Cellulase