ACTIVITY OF MUSHROOM POLYPHENOL OXIDASE IN ORGANIC MEDIUM

A kinetic study of the activity of mushroom polyphenol oxidase in an o rganic system was carried out to obtain detailed enzyme kinetic data i n relation to optimization of reaction conditions and substrate specif icity. A simple method for consistent measurement of reaction rates in the heterogeneous enzyme/organic solvent system (consisting of immobi lized polyphenol oxidase and a hydrated solution of the substrate in c hloroform) was designed. The aqueous content of the system was optimiz ed using p-cresol as the substrate. With this system, a crude extract of Agaricus bisporus was used to hydroxylate and oxidize a range of se lected p-substituted phenolic substrates, yielding o-quinone products. Michaelis-Menten kinetics were used to obtain apparent K(M) and V(max ) values with respect to each of these substrates. Results from this a nalysis indicated a correlation between the enzymic kinetic parameters obtained and the steric requirements of the substrates, which could b e rationalized in terms of the restricted flexibility of the enzyme wh en it is in chloroform and also in terms of substrate and solvent hydr ophobicity. In the course of the investigation UV molar absorption coe fficients of several o-quinones were measured by a novel method: H-1 n uclear magnetic resonance (NMR) spectroscopy was employed to determine component concentrations in reaction mixtures resulting from the tran sformation of phenols by polyphenol oxidase in chloroform. Thus the UV molar absorption coefficients could be obtained directly, avoiding th e necessity to isolate the water-sensitive, unstable o-quinones. (C) 1 993 John Wiley & Sons, Inc.