A gallotannin degrading esterase from leaves of pedunculate oak

Abstract

An enzyme from leaves of pedunculate oak ( Quercus robur, syn. Q. pedunculata) that catalysed the efficient hydrolysis of galloylglucose esters and related compounds was purified more than 1900-fold in 67% yield to apparent homogeneity. For the purified native enzyme, M r values of 150 000 and 300 000, respectively, were estimated by gel filtration, while a single polypeptide band of M r 75 000 was detected by SDS-PAGE, suggesting that the native enzyme existed both as a dimer and a tetramer of apparently identical subunits. The enzyme had a temperature optimum of 35–40°; it was inactivated above ca 55°, but exhibited a relative activity of 42% even at 0°. Maximal reaction rates occurred between pH 4.3 and 5.0, while no activity was observed above pH 7.8 and below pH 3.8; the enzyme was most stable at pH 5.0. The enzyme was absolutely inactive with variously substituted methyl cinnamates, 1- O-sinapoylglucose, substrates with nitro-substitution of the aromatic acid moiety, and with phenolic glucosides. Hydrolysis occurred with simple galloyl esters (methyl, ethyl, propyl gallate), naphthyl acetate (but not with naphthyl propionate or butyrate), mono- to hexa- substituted galloyl-β- d-glucoses, variously ring-substituted 1- O-benzoyl-β- d-glucoses, and with depsides like meta-digallic acid or chlorogenic acid. The enzyme exhibited both pronounced esterase and depsidase activities, thus closely resembling the properties of fungal tannases. By analogy to these enzymes, the esterase from oak leaves was classified as a plant ‘tannase’ (tannin acyl hydrolase, EC 3.1.1.20). © 1997 Elsevier Science Ltd. All rights reserved