Isolation, purification and partial characterization of apple polyphenol oxidase

Abstract

Polifenol oksidaza je izolirana iz jabuka sorte Idared uz stupanj pročišćenja od 9,7 i iskorištenje 61,61 %. Postupak pročišćavanja uključivao je taloženje amonijevim sulfatom, taloženje acetonom i sušenje acetonskog taloga u struji dušika pri 24 °C. Enzim je pokazivao pH optimum pri pH 7, i optimalnu temperaturu djelovanja pri 24 °C uz L-3,4-dihidroksifenilalanin kao supstrat. Od ispitanih supstrata, polifenol oksidaza je pokazala najveću aktivnost uz katehin, a potom uz epikatehin, 4-metilkatehol , klorogensku kiselinu, L-3,4- dihidroksifenilalanin, kafeinsku kiselinu te epigalokatehingalat. Najboljim inhibitorima polifenol oksidaze pokazali su se glutation, askorbinska kiselina i cistein.Polyphenol oxidase was purified from the apple variety Idared with 9.7-fold purification and 61.61% recovery by ammonium sulphate precipitation, acetone precipitation and drying of acetone precipitate under gentle stream of nitrogen at 25 °C. Optimum polyphenol oxidase activity was at pH 7.0, and optimum temperature for activity was at 24 °C, both determined with L-3,4-dihydroxyphenylalanine as substrate. Of the substrates tested, activity was greatest with catechin followed by 4-methylcatechol, chlorogenic acid, L-3,4-dihydroxyphenylalanine, caffeic acid and epigallocatechin gallate. The most effective inhibitors tested were glutathione, ascorbic acid and cysteine