A tyrosinase-expressing bacterium was isolated from soil, and extracellular enzymatic activity was induced by the presence of tyrosine and CuSO(4). Amplification of the 16S rDNA genes revealed a high similarity with Bacillus megaterium. The enzyme was over-expressed in Escherichia coli BL21 and purified using an affinity column. The tyrosinase was composed of 297 amino acids and was determined to be a monomer with a relative molecular mass of 31 kDa according to gel filtration. The K(m) values for 3,4-dihydroxy-L-phenylalanine (L-DOPA) and L-tyrosine were 0.35 and 0.075 mM, respectively, and the K(cat)/K(m) values were 28.9 x 10(3) and 32.9 x 10(3) (s(-1) x M(-1)). The maximum activity for both monophenolase and diphenolase was observed at 50 degrees C and pH 7.0. Enzymatic activity was enhanced in the presence of 10-50% water-miscible organic solvents, which included ethanol, methanol, 2-propanol and dimethyl sulfoxide (DMSO). The activity in 30% DMSO was 170% of the activity in water and the enantioselectivity towards L-DOPA decreased by 40%. The residual activity following an incubation period of 17 h in 0-70% methanol was constant. This newly isolated and characterized tyrosinase may have potential applications in organic synthesis due to its high activity and stability at typically denaturing conditions.
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