Purification and properties of a novel type of exo-1,4-beta-glucanase (avicelase II) from the cellulolytic thermophile Clostridium stercorarium.
Avicelase II was purified to homogeneity from culture supernatants of Clostridium stercorarium. A complete separation from the major cellulolytic enzyme activity (avicelase I) was achieved by FPLC gel filtration on Superose 12 due to selective retardation of avicelase II. The enzyme has an apparent molecular mass of 87 kDa and a pI of 3.9. Determination of the N-terminal amino acid indicates that avicelase II is not a proteolytically processed product of avicelase I. Maximal activity of avicelase II is observed between pH 5 and 6. In the presence of Ca2+, the enzyme is highly thermostable, exhibiting a temperature optimum around 75 degrees C. Hydrolysis of avicel occurs at a linear rate for three days at 70 degrees C. Avicelase II is active towards unsubstituted celluloses, cellotetraose and larger cellodextrins. It lacks activity towards carboxymethylcellulose and barley beta-glucan. Unlike other bacterial exoglucanases, avicelase II does not hydrolyze aryl-beta-D-cellobiosides. Avicel is degraded to cellobiose and cellotriose at a molar ratio of approximately 4:1. With acid-swollen avicel as substrate, cellotetraose is also formed as an intermediary product, which is further cleaved to cellobiose. The degradation patterns of reduced cellodextrins differ from that expected for a cellobiohydrolase attacking the non-reducing ends of chains; cellopentaitol is degraded to cellobiitol and cellotriose, while cellohexaitol is initially cleaved into cellobiitol and cellotetraose. These findings, taken together, indicate that avicelase II represents a novel type of exoglucanase (cellodextrinohydrolase), which, depending on the accessibility of the substrate, releases cellotetraose, cellotriose, or cellobiose from the non-reducing end of the cellulose chains.
Bronnenmeier
K
K
Institute for Microbiology, Technical University Munich, Federal Republic of Germany.
Rücknagel
K P
KP
Staudenbauer
W L
WL
D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid; D003013:Clostridium / Q000201:enzymology*; D004591:Electrophoresis, Polyacrylamide Gel; D044643:Glucan 1,4-beta-Glucosidase; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D008969:Molecular Sequence Data; D013379:Substrate Specificity; D013696:Temperature; D001617:beta-Glucosidase / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism
D001617:beta-Glucosidase; D044643:Glucan 1,4-beta-Glucosidase