title: Properties of diphenolase from Vanilla planifolia (Andr.) shoot primordia cultured in vitro.
abstract: Properties of diphenolase (PPO, EC1.10.3.1) from vanilla (Vanilla planifolia Andr.) shoot primordia culture were investigated. Two pH optima of the enzyme extraction at pH 6 and 8 were found. Nevertheless, the enzymes shared the same optimum pH of activity-between pH 3 and 4. Sodium dodecyl sulfate slightly improved diphenolase extraction but caused a 3-fold increase in its specific activity. The extracts of pH 6 and 8.0 revealed three isozyme bands after polyacrylamide gel electrophoresis-two of them were similar in both extracts and two distinct. The enzyme showed high thermal stability-no loss was observed after 120 min at 50 degrees C. Diethyldithiocarbamic acid, ethylenediaminetetracetic acid disodium salt, ethylene glycol bis(beta-aminoethyl ether) N,N,N',N'-tetraacetic acid, L-ascorbic acid, dithiothreitol, glutathione (reduced), and beta-mercaptoethanol were found to be potent inhibitors of the diphenolase studied. The enzyme showed also monophenolase activity. Km and Vmax were calculated with monophenols [p-coumaric acid, 3-(p-hydroxyphenyl)propionic acid, 4-hydroxybenzyl alcohol, 4-hydroxybenzaldehyde, 4-hydroxybenzoic acid] and with diphenols (caffeic acid, hydrocaffeic acid, chlorogenic acid, 4-methylcatechol, protocatechuic aldehyde and acid, and 3,4-dihydroxyphenylalanine). The highest Vmax was found with 4-hydroxybenzyl alcohol and the greatest affinity to protocatechuic acid, respectively-the most abundant monophenol and one of the least abundant o-diphenols in the studied Vanilla tissue.
authors.affiliation: Institute of Experimental Plant Biology, University of Warsaw, Miecznikowa 1, 02-096 Warsaw, Poland.
authors.forename: R
authors.initials: R
authors.lastname: Debowska
authors.forename: A
authors.initials: A
authors.lastname: Podstolski
mesh_terms: D004156:Catechol Oxidase / Q000302:isolation & purification / Q000378:metabolism*; D046508:Culture Techniques; D004587:Electrophoresis, Agar Gel; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D010944:Plants / Q000201:enzymology*; D013379:Substrate Specificity
chemical_list: D004156:Catechol Oxidase