Purification and characterization of a new endoglucanase from Clostridium thermocellum.
An endoglucanase (1,4-beta-D-glucan glucanohydrolase, EC 3.2.1.4) from the thermophilic anaerobe Clostridium thermocellum was purified to apparent homogeneity without the use of denaturants. No carbohydrate is associated with the endoglucanase. A molecular mass of 76,000 Da was determined by SDS/PAGE. The optimal pH is 7.0 and the enzyme is isoelectric at pH 5.05. The enzyme has a temperature optimum of 70 degrees C and retains approx. 50% of its activity after 48 h at 60 degrees C. Hydrolysis of CM-cellulose takes place with a rapid decrease in viscosity but a slow liberation of reducing sugars, indicating an endoglucanase type of activity. The endoglucanase shows little ability to hydrolyse highly ordered cellulose. Cellobiose inhibits whereas Mg2+ and Ca2+ stimulate the activity. The enzyme is completely inactivated by 1 mM-Hg2+ and is inhibited by a thiol-blocking reagent.
Romaniec
M P
MP
Department of Biology, Massachusetts Institute of Technology, Cambridge 02139.
Fauth
U
U
Kobayashi
T
T
Huskisson
N S
NS
Barker
P J
PJ
Demain
A L
AL
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000187:drug effects / Q000302:isolation & purification* / Q000378:metabolism; D002480:Cellulase / Q000187:drug effects / Q000302:isolation & purification* / Q000378:metabolism; D002482:Cellulose / Q000378:metabolism; D003013:Clostridium / Q000201:enzymology*; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D007526:Isoelectric Point; D008670:Metals / Q000494:pharmacology; D008969:Molecular Sequence Data; D013696:Temperature
D001426:Bacterial Proteins; D008670:Metals; D002482:Cellulose; D002480:Cellulase