title: Isolation and characterization of novel tyrosinase from Laceyella sacchari.
abstract: We here describe the isolation and characterization of a tyrosinase from a newly isolated soil bacterium. 16S rDNA sequence analysis revealed that the bacterium most probably belongs to the species Laceyella sacchari (Ls) ( > 99.9 % identity). The tyrosinase extracellular enzymatic activity was induced in the presence of L-methionine and CuSO4. The crude enzyme was first purified by centrifugation followed by ammonium sulphate precipitation and ultrafiltration. After removal of a brown pigment, probably melanin, a purified enzyme was obtained by further separation of the crude protein mixture using size exclusion chromatography. Some 10.5 mg of pure tyrosinase (LsTyr) was isolated with a molecular mass of 30 910 Da, based on MALDI mass spectrometry. Together with the observed enzymatic activity, N-terminal chemical sequence analysis confirmed that the isolated enzyme is homologous to other tyrosinases. The kinetic parameters for the diphenol substrates L-DOPA and dopamine and for the monophenol substrate L-tyrosine were determined to be KM = 4.5 mM , 1.5 mM and 0.055 mM, and kcat/KM = 261.5 mM-1 s -1 , 30.6 mM-1 s-1 and 56.3 mM-1 s-1, respectively. Maximal activities of the purified enzyme were found to occur at pH 6.8.
authors.affiliation: Academy G. Bonchev Street, bl. 9, 1113 Sofia, Bulgaria. adolashki@yahoo.com
authors.forename: Aleksandar
authors.initials: A
authors.lastname: Dolashki
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Voelter
authors.forename: Adriana
authors.initials: A
authors.lastname: Gushterova
authors.forename: Jozef
authors.initials: J
authors.lastname: Van Beeumen
authors.forename: Bart
authors.initials: B
authors.lastname: Devreese
authors.forename: Bozhidar
authors.initials: B
authors.lastname: Tchorbanov
mesh_terms: D056449:Bacillales / Q000201:enzymology*; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D001483:Base Sequence; D004269:DNA, Bacterial / Q000235:genetics; D004298:Dopamine / Q000378:metabolism; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D007980:Levodopa / Q000378:metabolism; D008969:Molecular Sequence Data; D014442:Monophenol Monooxygenase / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D016415:Sequence Alignment; D014443:Tyrosine / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D004269:DNA, Bacterial; D012336:RNA, Ribosomal, 16S; D014443:Tyrosine; D007980:Levodopa; D014442:Monophenol Monooxygenase; D004298:Dopamine