Properties of cellulosomal family 9 cellulases from Clostridium cellulovorans.
The cellulosomal family 9 cellulase genes engH, engK, engL, engM, and engY of Clostridium cellulovorans have been cloned and sequenced. We compared the enzyme activity of family 9 cellulosomal cellulases from C. cellulovorans and their derivatives. EngH has the highest activity toward soluble cellulose derivatives such as carboxymethylcellulose (CMC) as well as insoluble cellulose such as acid-swollen cellulose (ASC). EngK has high activity toward insoluble cellulose such as ASC and Avicel. The results of thin-layer chromatography showed that the cleavage products of family 9 cellulases were varied. These results indicated that family 9 endoglucanases possess different modes of attacking substrates and produce varied products. To investigate the functions of the carbohydrate-binding module (CBM) and the catalytic module, truncated derivatives of EngK, EngH, and EngY were constructed and characterized. EngHDeltaCBM and EngYDeltaCBM devoid of the CBM lost activity toward all substrates including CMC. EngKDeltaCBM and EngMDeltaCBM did not lose activity toward CMC but lost activity toward Avicel. 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.
Section of Molecular and Cellular Biology, University of California, Davis, CA 95616, USA.
Takamitsu
T
Arai
Akihiko
A
Kosugi
Helen
H
Chan
Roger
R
Koukiekolo
Hideaki
H
Yukawa
Masayuki
M
Inui
Roy H
RH
Doi
D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D020134:Catalytic Domain / Q000235:genetics; D002480:Cellulase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D002482:Cellulose / Q000378:metabolism*; D046974:Clostridium cellulovorans / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D009097:Multienzyme Complexes / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D013379:Substrate Specificity
D001426:Bacterial Proteins; D009097:Multienzyme Complexes; C473132:cellulosome, Clostridium; D002482:Cellulose; D002480:Cellulase