Isolation, cloning and characterization of a tyrosinase with improved activity in organic solvents from Bacillus megaterium.

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. Copyright 2009 S. Karger AG, Basel.