title: Production of recombinant Agaricus bisporus tyrosinase in Saccharomyces cerevisiae cells.
abstract: It has been demonstrated that Agaricus bisporus tyrosinase is able to oxidize various phenolic compounds, thus being an enzyme of great importance for a number of biotechnological applications. The tyrosinase-coding PPO2 gene was isolated by reverse-transcription polymerase chain reaction (RT-PCR) using total RNA extracted from the mushroom fruit bodies as template. The gene was sequenced and cloned into pYES2 plasmid, and the resulting pY-PPO2 recombinant vector was then used to transform Saccharomyces cerevisiae cells. Native polyacrylamide gel electrophoresis followed by enzymatic activity staining with L-3,4-dihydroxyphenylalanine (L-DOPA) indicated that the recombinant tyrosinase is biologically active. The recombinant enzyme was overexpressed and biochemically characterized, showing that the catalytic constants of the recombinant tyrosinase were higher than those obtained when a commercial tyrosinase was used, for all the tested substrates. The present study describes the  recombinant production of A. bisporus tyrosinase in active form. The produced enzyme has similar properties to the one produced in the native A. bisporus host, and its expression in S. cerevisiae provides good potential for protein engineering and functional studies of this important enzyme.
authors.affiliation: Dipartimento di Scienze e Tecnologie Biologiche ed Ambientali, Università del Salento, Via Prov. Lecce-Monteroni, 73100 Lecce, Italy.
authors.forename: Chiara
authors.initials: C
authors.lastname: Lezzi
authors.forename: Gianluca
authors.initials: G
authors.lastname: Bleve
authors.forename: Stefano
authors.initials: S
authors.lastname: Spagnolo
authors.forename: Carla
authors.initials: C
authors.lastname: Perrotta
authors.forename: Francesco
authors.initials: F
authors.lastname: Grieco
mesh_terms: D000364:Agaricus / Q000201:enzymology* / Q000235:genetics; D001483:Base Sequence; D055162:Biocatalysis; D015153:Blotting, Western; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D014442:Monophenol Monooxygenase / Q000096:biosynthesis* / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D010084:Oxidation-Reduction; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000235:genetics* / Q000378:metabolism*; D013379:Substrate Specificity
chemical_list: D011994:Recombinant Proteins; D014442:Monophenol Monooxygenase