CELLULASE IN TOBACCO CALLUS - REGULATION AND PURIFICATION

Cellulase, 1,4-beta-D-glucan 4-glucanohydrolase (EC 3.2.1.4), was purified 1500-fold from callus of Nicotiana tabacum L. cv. Petit Havana SR1 by differential extraction and by hydrophobic affinity, and gel permeation chromatography. The enzyme has a broad pH optimum between 5.5 and 6.5 and a pI of ca. 8.2. SDS-PAGE showed two polypeptides of 50 and 52 kDa, respectively. The enzyme was obviously isolated as a macromolecular aggregate. It degraded hydroxyethylcellulose, carboxymethylcellulose, Avicel, 1,3; 1,4-beta-D-glucan and tobacco xyloglucan, but Tropaeolum xyloglucan, xylan and polygalacturonic acid were not degraded by the purified enzyme. The cellulase activity in tobacco tissue was rapidly regulated in response to a morphogenetic hormone treatment. Following transfer of callus tissue to a shoot-inducing medium the enzyme activity decreased during the first 4 days to ca. 25 % of the value in tissue on callus-maintaining medium. The involvement of the downward regulation of cellulase in the early events of morphogenesis is discussed.