Induction of Chitinases in Rice Callus Treated with Chitin Derivatives

Abstract

(2ml), containing 20mg of the TV-acetylchitosan, l ml of 0. 1 Mcitric acid/0.2 M Na2HPO4 buffer, pH 3.0, and enzyme solution, with shaking at 37°C for 1hr, and stopped by addition of 1 ml of6.7% Na2WO4in 1/3N H2SO4. After centrifugation (1500 x g, 10min), reducing sugars in the supernatant were measured by a modification of Schales' method.11} One unit (U) is denned as the amount of chitinase which produces 1 /imol of reducing sugars as vV-acetyl-D-glucosamine per min under these conditions. For separation of chitinases, crude extract was brought to 80% saturation by addition of solid (NH4)2SO4 at 4°C, and the resultant precipitate dissolved in 20ml of 25mM imidazole/HCl buffer, pH 6.8, containing 1 mMEDTA. The solution was dialyzed against the same buffer and put onto a DEAE-cellulofine column (1.5 x 14cm), equilibrated with the same buffer. Chitinases were eluted with 50ml of the buffer and then with 400ml of a linear concentration gradient (0 to 0.3 m) of NaCl in the buffer. Gel nitration was done on a Sephadex G-75 column (1.5 x 76cm), equilibrated with 0.2m sodium phosphate buffer, pH 6.0. Bovine serum albumin (66 kDa), ovalbumin (45kDa), a-chymotrypsinogen A (bovine pancreas, 26