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 (Mr) of approximately 39,000 while the purified laccase has a relative molecular mass (Mr) 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 °C and pH 7.0. Both enzymes were stable over a broad range of temperatures and were most stable at 30 °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 α-methyl-dl-tyrosine compared to α-methyl-l-tyrosine.