Purification and activity enhancement of extracellular tyrosinase from a protease-silenced zygomycete Amylomyces rouxii strain.

Abstract

The intra- and extra-cellular monophenolase and diphenolase activities of the tyrosinase produced by Amylomyces rouxii were determined in submerged culture using Melin-Norkrans medium supplemented with 12.5 mg/L pentachlorophenol (PCP) and 0.1 g/L tyrosine. Maximal intracellular monophenolase activity was 180 U/mL while maximal extracellular monophenolase activity was 80 U/mL, both using p-cresol as substrate. For diphenolase, the highest intracellular activity was 2233 U/mL using 4-tert-butylcatechol (TBC) as substrate and extracellular diphenolase activity was 975 U/mL with catechol as substrate. The peak tyrosinase activity (mono- and diphenolase) was observed at 48 h of culture. The transformant A412-3 exhibited the highest extracellular activities, with a 2.14-fold increase in monophenolase and a 3.02-fold increase in diphenolase activity compared to the parental strain of A. rouxii. Additionally, it was confirmed that the enzyme secreted was in its active form. Extracellular tyrosinase from the transformant A412-3 was partially purified, achieving a purification factor of 10.6. SDS-PAGE analysis of partially purified tyrosinase revealed three bands of 40, 53, and 130 kDa. These bands were sequenced by LC-MS/MS, revealing eight peptides that showed similarity to tyrosinases from different fungi. It was determined that purified tyrosinase exhibited higher diphenolase activity than monophenolase activity, in line with previous studies on fungal tyrosinases.