Purification and characterization of a cellulase from Catharanthus roseus stems

Cellulase was purified to homogeneity from stems of Catharanthus roseus (pe riwinkle), by anion exchange, gel filtration and affinity chromatography. T he gel filtration on Sephacryl S-200 indicated M-r 96 kDa, diffusion coeffi cient 5.4 x 10(-7) cm(2)/s, Stokes' radius 40 x 10(-8) cm and frictional ra tio of 1.37. The subunit M-r was 25 kDa by SDS-PAGE. The purified enzyme co ntained 7% carbohydrate. The K-m of the enzyme was 0.44 mg/ml for CM-cellul ose. The enzyme exhibited optimum activity between 30 and 35 degrees and at pH 5.2. The enzyme was strongly inhibited by Hg2+ and Teepol, but cellobio se had no effect. Reaction product analysis suggests endo-nature of the pur ified enzyme. The most striking feature of Catharanthus cellulase is its ab ility to hydrolyze suspensions of crystalline (cellulose powder and filter paper) and partially (phospho-, alkali-) swollen cellulose, although at rat es lower than CM-cellulose. (C) 1999 Elsevier Science Ltd. All rights reser ved.