Isolation of a latent polyphenol oxidase from loquat fruit (Eriobotrya japonica Lindl.): kinetic characterization and comparison with the active form.
Polyphenol oxidase (PPO) has been extracted from both soluble and particulate fractions of loquat fruit (Eriobotrya japonica Lindl. cv. Algerie). The soluble PPO (20% of total activity) was partially purified 3.3-fold after ammonium sulfate fractionation being in its active state. The particulate PPO fraction (80% of total activity) was purified to homogeneity in a latent form being activable by sodium dodecyl sulfate (SDS). The enzyme was purified 40.0-fold with a total yield of 15.3% after extraction by phase partitioning in Triton X-114 followed by three chromatographic steps. The molecular weight was estimated to be about 59.2 and 61.2 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and gel filtration chromatography, respectively, indicating that latent PPO is a monomer. Latent PPO catalyzed the oxidation of chlorogenic acid (CA) at a rate 50-fold faster than that of 4-tert-butylcatechol (TBC) but the soluble active counterpart only twice. Both PPOs exhibited similar Km values for TBC but Km for CA was 5-fold higher for the latent than for the active soluble PPO. Other kinetic characteristics, including sensitivity to inhibitors, substrate specificity, thermal stability, temperature, and pH profiles, were quite different between both PPOs. These results provide strong evidences that the soluble active and the particulate latent are different forms of PPO in loquat fruit flesh. The results suggest that the major PPO form for the oxidation of CA, leading to enzymatic browning under physiological conditions, is the latent one.
Sellés-Marchart
Susana
S
Grupo de Proteómica y Genómica Funcional de Plantas, Departamento de Agroquímica y Bioquímica, Facultad de Ciencias, Universidad de Alicante, Campus de San Vicente del Raspeig, Apdo. 99, E-03080 Alicante, Spain.
Casado-Vela
Juan
J
Bru-Martínez
Roque
R
D004156:Catechol Oxidase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification*; D002396:Catechols / Q000737:chemistry; D002726:Chlorogenic Acid / Q000737:chemistry; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000737:chemistry; D004795:Enzyme Stability; D031983:Eriobotrya / Q000201:enzymology*; D006361:Heating; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification; D007700:Kinetics; D013379:Substrate Specificity
D002396:Catechols; D004791:Enzyme Inhibitors; D007527:Isoenzymes; D002726:Chlorogenic Acid; C015047:p-tert-butyl catechol; D004156:Catechol Oxidase