Characterization of the extracellular cellulase from a mesophilic clostridium (strain C7).
An extracellular, 700,000-Mr multiprotein complex that catalyzed the hydrolysis of crystalline cellulose (Avicel) was isolated from cultures of Clostridium sp. strain C7, a mesophile from freshwater sediment. In addition to cellulose (Avicel, ball-milled filter paper), the multiprotein complex hydrolyzed carboxymethylcellulose, cellodextrins, xylan, and xylooligosaccharides. Hydrolysis of cellulose or cellotetraose by the complex yielded cellobiose as the main product. Cellopentaose or cellohexaose was hydrolyzed by the complex to cellotriose or cellotetraose, respectively, in addition to cellobiose. Xylobiose was the main product of xylan hydrolysis, and xylobiose and xylotriose were the major products of xylooligosaccharide hydrolysis. Activity (Avicelase) resulting in hydrolysis of crystalline cellulose required Ca2+ and a reducing agent. The multiprotein complex had temperature optima for Avicelase, carboxymethylcellulase, and xylanase activities at 45, 55, and 55 degrees C, respectively, and pH optima at 5.6 to 5.8, 5.5, and 6.55, respectively. Electron microscopy of the 700,000-Mr enzyme complex revealed particles relatively uniform in size (12 to 15 nm wide) and apparently composed of subunit structures. Elution of strain C7 concentrated culture fluid from Sephacryl S-300 columns yielded an A280 peak in the 130,000-Mr region. Pooled fractions from the 130,000-Mr peak had carboxymethylcellulase activity but lacked Avicelase activity. Except for the inability to hydrolyze cellulose, the 130,000-Mr preparation had a substrate specificity identical to that of the 700,000-Mr protein complex. A comparison by immunoblotting techniques of proteins in the 130,000- and 700,000-Mr preparations, indicated that the two enzyme preparations had cross-reacting antigenic determinants.
Cavedon
K
K
Department of Microbiology, University of Massachusetts, Amherst 01003.
Leschine
S B
SB
Canale-Parola
E
E
D001426:Bacterial Proteins / Q000648:ultrastructure; D002118:Calcium / Q000494:pharmacology; D002480:Cellulase / Q000302:isolation & purification / Q000378:metabolism*; D002846:Chromatography, Affinity; D003013:Clostridium / Q000201:enzymology*; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D015151:Immunoblotting; D007700:Kinetics; D046911:Macromolecular Substances; D008854:Microscopy, Electron; D008970:Molecular Weight; D046912:Multiprotein Complexes; D013379:Substrate Specificity; D013696:Temperature
D001426:Bacterial Proteins; D046911:Macromolecular Substances; D046912:Multiprotein Complexes; D002480:Cellulase; D002118:Calcium