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Isolation and Characterization of Novel Tyrosinase from Laceyella sacchari

[ Vol. 19 , Issue. 5 ]

Author(s):

Aleksandar Dolashki, Wolfgang Voelter, Adriana Gushterova, Jozef Van Beeumen, Bart Devreese and Bozhidar Tchorbanov   Pages 538 - 543 ( 6 )

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.

Keywords:

Tyrosinase, Laceyella sacchari, MS, MALDI

Affiliation:

Academy G. Bonchev Street, bl. 9, 1113 Sofia, Bulgaria.



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