Abstract
To elucidate the mechanism of ethylene-enhanced degreening in satsuma mandarin (Citrus unshiu Marc. c.v. Nichinan No. 1) fruits, a water-soluble chlorophyllase (chlorophyll-chlorophyllide-hydrolase, E.C. 3.1.1.14) was prepared from the peel of ethylene-treated fruits. The maximum catabolic rate of chlorophyll a (Chl a) to chlorophyllide a occurred in the presence of 30∿40% concentration of acetone at 30°C. Optimum concentrations of substrate and protein for the reaction were 12.4 μM and 2.6 mg/4.5ml, respectively. The optimum pH value was 7.6 with a phosphate buffer, 7.86 with a borate buffer, and 7.5 with an acetate buffer. At pH 7.6 (phosphate buffer) and 25°C in the presence of 33% acetone, the tentative Km value for Chl a hydrolysis activity was approximately 6.5 μM. Moreover, when the reaction mechanism of Chl a degradation was investigated using the 3-dimensional fluorescence spectral measurements, fluorescent Chl catabolites (FCC, Ex/Em : 440/670 nm) were detected but the fluorescent Chl-ring cleavage catabolites (Ex/Em : around 320∿360/440∿460 nm) were not. The FCC formation was inhibited by p-chloromercuribenzoate (PCMB), an inhibitor of chlorophyllase, but the inhibition was reversible by the addition of reduced glutathione. These results suggest that Chl a degradation occurred through chlorophyllide a formation catalyzed by a water-soluble chlorophyllase from ethylene-treated Citrus unshiu fruits. A possible participation of the enzyme in ethylene-enhanced degreening of the fruit peel is briefly discussed.
