title: Purification and characterization of tyrosinase from walnut leaves (Juglans regia).
abstract: Polyphenol oxidase (PPO) is a type-3 copper enzyme catalyzing the oxidation of phenolic compounds to their quinone derivates, which are further converted to melanin, a ubiquitous pigment in living organisms. In this study a plant originated tyrosinase was isolated from walnut leaves (Juglans regia) and biochemically characterized. It was possible to isolate and purify the enzyme by means of an aqueous two-phase extraction method followed by chromatographic purification and identification. Interestingly, the enzyme showed a rather high monophenolase activity considering that the main part of plant PPOs with some exceptions solely possess diphenolase activity. The average molecular mass of 39,047 Da (Asp(101)→Arg(445)) was determined very accurately by high resolution mass spectrometry. This proteolytically activated tyrosinase species was identified as a polyphenol oxidase corresponding to the known jrPPO1 sequence by peptide sequencing applying nanoUHPLC-ESI-MS/MS. The polypeptide backbone with sequence coverage of 96% was determined to start from Asp(101) and not to exceed Arg(445).
authors.affiliation: Institut für Biophysikalische Chemie, Universität Wien, Althanstraße 14, A-1090 Wien, Austria.
authors.forename: Florime
authors.initials: F
authors.lastname: Zekiri
authors.affiliation: Institut für Biophysikalische Chemie, Universität Wien, Althanstraße 14, A-1090 Wien, Austria.
authors.forename: Christian
authors.initials: C
authors.lastname: Molitor
authors.affiliation: Institut für Biophysikalische Chemie, Universität Wien, Althanstraße 14, A-1090 Wien, Austria.
authors.forename: Stephan G
authors.initials: SG
authors.lastname: Mauracher
authors.affiliation: Institut für Analytische Chemie, Universität Wien, Währinger Straße 38, A-1090 Wien, Austria.
authors.forename: Claudia
authors.initials: C
authors.lastname: Michael
authors.affiliation: Institut für Analytische Chemie, Universität Wien, Währinger Straße 38, A-1090 Wien, Austria.
authors.forename: Rupert L
authors.initials: RL
authors.lastname: Mayer
authors.affiliation: Institut für Analytische Chemie, Universität Wien, Währinger Straße 38, A-1090 Wien, Austria.
authors.forename: Christopher
authors.initials: C
authors.lastname: Gerner
authors.affiliation: Institut für Biophysikalische Chemie, Universität Wien, Althanstraße 14, A-1090 Wien, Austria. Electronic address: annette.rompel@univie.ac.at.
authors.forename: Annette
authors.initials: A
authors.lastname: Rompel
mesh_terms: D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D031324:Juglans / Q000737:chemistry* / Q000201:enzymology*; D008969:Molecular Sequence Data; D014442:Monophenol Monooxygenase / Q000737:chemistry / Q000302:isolation & purification*; D018515:Plant Leaves / Q000737:chemistry / Q000201:enzymology; D011487:Protein Conformation; D013056:Spectrophotometry, Ultraviolet; D053719:Tandem Mass Spectrometry
chemical_list: D014442:Monophenol Monooxygenase
keywords: Catechol oxidase; Juglans regia; Laccase; Polyphenol oxidase; Type-3 copper protein; Tyrosinase