Joseph J. Jen

Preparation of a Homogeneous Tomato Fruit Peroxidase

Tomato fruit (Lycopersicon esculentum Mill cv. Walters) peroxidase was purified to apparent homogeneity by a three step procedure: hydrophobic chromatography, DEAE Sephacel chromatography and semi-preparative electrophoresis. A purification of 71 fold and a yield of 52% relative to crude extract were obtained. The pure enzyme was brown in color and showed a molecular weight of 45,000 as estimated from SDS disc gel electrophoresis and gel filtration on Ultrogel AcA 34. The pH optimum of tomato peroxidase varied with substrate dyes used and the enzyme may have some hydrophobic properties near its active site. The optimum temperature was 35 degrees C for this enzyme, and IAA oxidase activity was evident in the presence of 2,4-dichlorophenol and manganese. The apparent KM for IAA was measured to be 0.24 mM.

Characterization of the Pectic Enzymes from Monilinia fructicola1)

Summary Monilinia fructicola was grown in a chemically defined medium under conditions to give maximum pectic enzyme production. Endo-polygalacturonase (PG), exo-PG, endo-polymethylgalacturonase (PMG), endo-polymethylgalacturonate lyase (PMGL) and pectin methylesterase (PME) activities were detected in the culture filtrates. The PG exhibited a p H optimum at 5.2 and was stable at acidic p H. The PMG exhibited p H optima at 4.5 to 5.0 and 7.5. A heat stability phenomenon was associated with the PG of the two day old culture filtrate.

Purification and characterization of polyphenol oxidase in redhaven peaches

Summary Polyphenol oxidase from Redhaven peaches ( Prunus persica BATSCH) was purified by column chromatography on, Phenyl Sepharose, ECTEOLA Cellulose, Hydroxylapatite, and benzoylated DEAF Sephadex. The partially purified enzyme showed two major bands on, disc gel electrophoresis when stained for polyphenol oxidase activities, proteins, glycoproteins, and lipoproteins. Thin layer isoelectric focusing data indicated a pI between p H 4.5 and 5.5. The purified enzyme was stable for two months in buffered 20 % sucrose, glycerol, or ethylene glycol solutions at −15°C. Digestion of the enzyme with carbohydrases and proteases generated new isoenzymic forms of the enzyme.

Partial Purification and Characterization of an Endo-Polygalacturonase from Monilinia fructicola and its Implication in the Brown Rot Disease of Peaches)

Summary A polygalacturonase (PG) from cultural media of Monilinia fructicola was purified 50-fold with 30% yield by ultrafiltration followed by successive chromatography on Sephadex G-75, Ecteola cellulose, and Biogel P-150 columns. The enzyme moved as a single band on disc gel electrophoresis and exhibited a single symmetrical peak on ultracentrifugation. Optimal PG 4 ) activity was at pH 5.2 and 50 ˚C with good enzyme stability between pH 4 and 6. The active form of the enzyme appeared to be a tetramer of molecular weight near 80,000. The enzyme was stable at acid pH but dissociated at pH > 9 to form an inactive monomer with a molecular weight of 20,000. Disc gel electrophoretic patterns supported this hypothesis. Kinetic studies indicated a V max of 2,700 µmoles/min/mg protein and a km of 1 x 10- 6 M with sodium polypectate as substrate. Although viscosity and uronic acid dehydrogenase measurements indicated the endo nature of this PG, hydrolysis of pectate yielded galacturonic acid as the major end product. Anthocyanins extracted from grapes, peaches, and cranberries caused varied inhibition while various polyphenol compounds and benlate, a fungicide, did not inhibit this PG at all. The properties of this enzyme in relation to host-pathogen interaction were discussed.