Extraction and Purification of Tyrosinase from Environmental and Pathogenic Pseudomonas aeruginosa isolates

The current study aims to investigate the production of tyrosinase enzyme due to its importance in biotechnology. Tyrosinase was isolated for the first time in this study from pathogenic pseudomonas aeruginosa and compared its output with  that of the environmental isolate of the same enzyme in terms of enzyme activity, productivity, etc.  Pseudomonas aeruginosa produced tyrosinase when cultured in media supplemented with L-tyrosine as a substrate, CuSO4 as a cofactor,  and appropriate carbon and nitrogen sources, along with a trace salt solution. A dark or black-brown coloration was observed in 50% of the isolates, indicating positive tyrosinase production. Optimal conditions for enzyme production were pH 7, incubation at 37°C for 48 hours, and agitation in a shaker incubator at 175 rpm. Tyrosinase was extracted using ammonium sulfate precipitation at 80% saturation. This method yielded the maximum total activity of   27.5 U for the environmental isolate and  60 U for the  pathogenic isolate, with yield of 45.8% and 57% respectively .Further purification using DEAE-cellulose chromatography resulted in enzyme activity of1.5 UmL-1 with a protein concentration of 0.7 mg.mL-1 for the environmental isolate,  and 2.5 U mL-1 at 1 mg.mL -1 for the pathogenic isolate. G-75 column chromatography showed tyrosinase activity 1.6 U mL-1 with a protein concentration of 0.6 mg.mL -1 for the environmental isolate, while the pathogenic isolate 2.8 U.mL-1 at 0.8 mg.mL -1 . The highest tyrosinase activity was obtained from the pathogenic Pseudomonas aeruginosa isolate through purification via ion-exchange and gel filtration chromatography, nevertheless this difference was not statistically significant.  Purified tyrosinase from Pseudomonas aeruginosa isolates was analyzed using SDS-PAGE to determine the molecular weight of the enzyme, which was 55 kDa for both isolates.