title: ISOLATION AND PROPERTIES OF POLYPHENOL OXIDASE FROM BASIDIOCARPS OF Lactarius pergamenus Fr. (Fr.) FUNGI.
abstract: 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.
authors.forename: M V
authors.initials: MV
authors.lastname: Tsivinska
authors.forename: V O
authors.initials: VO
authors.lastname: Antonyuk
authors.forename: R S
authors.initials: RS
authors.lastname: Stoika
mesh_terms: D000363:Agaricales / Q000737:chemistry* / Q000201:enzymology; D000627:Aminophenols / Q000737:chemistry*; D001205:Ascorbic Acid / Q000737:chemistry; D004156:Catechol Oxidase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D002396:Catechols / Q000737:chemistry; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D057075:Enzyme Assays; D004791:Enzyme Inhibitors / Q000737:chemistry; D048690:Fruiting Bodies, Fungal / Q000737:chemistry* / Q000201:enzymology; D005656:Fungal Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D007700:Kinetics; D008970:Molecular Weight; D010696:Phloroglucinol / Q000737:chemistry; D012118:Resorcinols / Q000737:chemistry; D013379:Substrate Specificity; D013431:Sulfates / Q000737:chemistry; D013890:Thiourea / Q000737:chemistry; D014443:Tyrosine / Q000737:chemistry
chemical_list: D000627:Aminophenols; D002396:Catechols; D004791:Enzyme Inhibitors; D005656:Fungal Proteins; D007527:Isoenzymes; D012118:Resorcinols; D013431:Sulfates; C012036:sodium sulfate; D014443:Tyrosine; D010696:Phloroglucinol; D004156:Catechol Oxidase; D013890:Thiourea; C034221:catechol; D001205:Ascorbic Acid; C031389:resorcinol