Studies on cutin-esterase II. Characteristics of cutin-esterase from Botrytis cinerea and its activity on tomato-cutin

Abstract

The activity of cutin-esterase, cutinase, was detected in the mycelial homogenate of Botrytis cinerea cultured in a peptone-sucrose medium at 25°C for 7 days. The crude enzyme solution was prepared from the homogenate by centrifugation at 106,600×g, treatment with (NH4)2SO4 at 70% saturation, and dialysis against 0.01 M phosphate buffer. The optimum pH, temperature and assay duration for enzyme activity were 5.0, 25°C and 18 hr, respectively. Specific activity was 255 mµmoles/mg protein/18 hr as palmitate under optimum conditions. 83% of the activity was lost by heating the enzyme solution (pH 7.6) for 4 min at 95°C. Palmitic, stearic, oleic, 9, 10-dihydroxystearic or linoleic, dihydroxyeicosanoic and octadecanedioic acids were recognized in the enzymic hydrolysate of tomato-cutin using gas-liquid chromatography. Among these fatty acids, palmitic, oleic and octadecanedioic acids were readily liberated by the enzyme, but dihydroxyeicosanoic acid, the major component of tomato-cutin, was isolated only in small amounts. The enzyme is, therefore, an exo-type cutinase which hydrolyses minor side chains of fatty acids bound to the major structure of cutin. Cutinesterase may facilitate cuticular invasion by fungi as a result of reduction in mechanical strength of the cuticle by the enzyme