PHYSICOCHEMICAL AND FUNCTIONAL-CHARACTERIZATION OF A HIGH-MOLECULAR-WEIGHT CARBOXYMETHYLCELLULASE FROM CUSCUTA-REFLEXA

Carboxymethylcellulase (CMCase) was extracted and purified from an ang iosperm parasite Cuscuta reflexa free from beta-glucosidase and other enzyme activities. The molecular mass and Stokes' radius of the purifi ed enzyme are 144 kDa and 44 Angstrom, respectively. The diffusion coe fficient and frictional ratio of the enzyme were 5.15 x 10(-7) cm(2)/s ec and 1.27. The SDS-PAGE revealed homotetrameric nature of the enzyme with a subunit molecular mass of 35+/-1 kDa. Titration against DTNB a nd NBS revealed 19 sulfhydryl groups and 8 tryptophan groups, respecti vely, per mole of the enzyme. A sharp pH optimum at 5.0 was obtained. Cuscuta CMCase activity is unique amongst plant endoglucanases in bein g stimulated by Mg2+ and Mn2+ ions and by various thiols. Reaction pro duct analysis, mode of enzyme action and substrate specificity test su ggest the endo-nature of the purified CMCase. The enzyme showed K-m va lue of 26+/-1 mg/ml for carboxymethylcellulose (sodium salt).