Biochemical characterisation of the coexisting tyrosinase and laccase in the soil bacterium Pseudomonas putida F6

Two phenol oxidases, a tyrosinase and a laccase were isolated from cell extracts of the soil bacterium Pseudomonas putida F6. Both oxidases were purified to homogeneity separately using a combination of anion exchange chromatography, gel filtration and gel slicing. The purified tyrosinase is a monomer with a relative molecular mass (M,) of approximately 39,000 while the purified laccase has a relative molecular mass (M,) of approximately 59,000. Maximum activity, for tyrosinase with L-tyrosine (monophenolase) and L-dopa (diphenolase) and for laccase with syringaldazine, was observed at 30 degrees C and pH 7.0. Both enzymes were stable over a broad range of temperatures and were most stable at 30 degrees C. Both the monophenolase and diphenolase activities of tyrosinase were relatively stable across a broad range of pH values with maximum stability at pH 5.0 and 4.0, respectively. Laccase was most stable at pH 7.0 with a narrow bell shaped curve over a range of pH values. P. putida F6 tyrosinase has a 1.5-fold higher affinity for L-tyrosine compared to L-dopa. Furthermore P. putida F6 tyrosinase exhibits a 2.7-fold higher affinity for alpha-methyl-DL-tyrosine compared to alpha-methyl-L-tyrosine. (c) 2006 Elsevier Inc. All rights reserved.