ISOLATION AND PROPERTIES OF POLYPHENOL OXIDASE FROM BASIDIOCARPS OF Lactarius pergamenus Fr. (Fr.) FUNGI

• Published in: Ukrainian biochemical journal 2015-03, Vol.87 (2), p.56-65
• Main Authors: Tsivinska, M V, Antonyuk, V O, Stoika, R S
• Format: Article
• Language: English
• Subjects: Agaricales - chemistry; Agaricales - enzymology; Aminophenols - chemistry; Ascorbic Acid - chemistry; Catechol Oxidase - antagonists & inhibitors; Catechol Oxidase - chemistry; Catechol Oxidase - isolation & purification; Catechol Oxidase - metabolism; Catechols - chemistry; Chromatography, Gel; Chromatography, Ion Exchange; Enzyme Assays; Enzyme Inhibitors - chemistry; Fruiting Bodies, Fungal - chemistry; Fruiting Bodies, Fungal - enzymology; Fungal Proteins - antagonists & inhibitors; Fungal Proteins - chemistry; Fungal Proteins - isolation & purification; Fungal Proteins - metabolism; Hydrogen-Ion Concentration; Isoenzymes - antagonists & inhibitors; Isoenzymes - chemistry; Isoenzymes - isolation & purification; Isoenzymes - metabolism; Kinetics; Molecular Weight; Phloroglucinol - chemistry; Resorcinols - chemistry; Substrate Specificity; Sulfates - chemistry; Thiourea - chemistry; Tyrosine - chemistry
• ISSN: 2409-4943; 2413-5003
• DOI: 10.15407/ubj87.02.056

Fresh juice of basidiocarps of Lactarius pergamenus Fr. (Fr.) fungi was subjected to ion exchange chromatography with used DEAE-toyopearl and CM-cellulose columns, as well as preparative electrophoresis in 7.5% polyacrylamide gels (pH 8.6). Three isoforms of polyphenol oxidase (PPO) were discovered and two isoforms (1-l and 1-2) were purified with a release of protein 0.42 mg/kg and 0.15 mg/kg of basidiocarps, respectively. These isoforms differ in the mobility at disc-electrophoresis in 7.5% PAGE in alkaline buffer system (pH 8.6). Specfic activity of isoform 1-2 is 4.8 times higher than that of the isoforms 1-1. The molecular weight determination by gel chromatography on the Toyopearl HW-55 demonstrated that both isoforms 1-1 and 1-2 have the same 64 ± 2 kDa molecular mass. Electrophoresis in 15% PAGE in the presence of sodium dodecylsulphate and β-mercaptoethanol revealed one band with molecular mass of 64 ± 1 kDa which suggests the presence of one polypeptide chain in the molecule ofthe enzyme. The enzyme has demonstrated the highest activity at pH 6.0 and temperature +10 °C, and at +70 °C the enzyme was inactivated. The PPO activity was the highest in young mushrooms and it decreased with their age and positively correlated with the content ofthe milky juice. Ortho-aminophenol was most effective among all the tested substrates to determine the activity of PPO (o-, m- and p-aminophenol, catechol, tyrosine, resorcinol, phloroglucinol) and its relative activity was 129% of the activity of catechol. Ascorbic acid was the most effective inhibitor of the polyphenol oxidase activity which was completely blocked at 1 mM concentration, whereas the same concentration of thiourea and sodium sulphite decreased the enzymatic activity by 40-45%. The PPO in L. pergamenus fungi basidiocarps was mainly localized in the mushroom milky juice where its high activity may be associated with protection of basidiocarps against various pathogens.