Abstract
BACKGROUND: Tyrosinase is a bifunctional enzyme that catalyzes both the hydroxylation of monophenols to o-diphenols (monophenolase activity) and the subsequent oxidation of the diphenols to o-quinones (diphenolase activity). Due to the potential applications of tyrosinase in biotechnology, in particular in biocatalysis and for biosensors, it is desirable to develop a suitable low-cost process for efficient production of this enzyme. So far, the best production yield reported for tyrosinase was about 1 g L(-1), which was achieved by cultivating the filamentous fungus Trichoderma reesei for 6 days. RESULTS: In this work, tyrosinase from Verrucomicrobium spinosum was expressed in Escherichia coli and its production was studied in both batch and fed-batch cultivations. Effects of various key cultivation parameters on tyrosinase production were first examined in batch cultures to identify optimal conditions. It was found that a culture temperature of 32 °C and induction at the late growth stage were favorable, leading to a highest tyrosinase activity of 0.76 U mL(-1). The fed-batch process was performed by using an exponential feeding strategy to achieve high cell density. With the fed-batch process, a final biomass concentration of 37 g L(-1) (based on optical density) and a tyrosinase activity of 13 U mL(-1) were obtained in 28 hours, leading to a yield of active tyrosinase of about 3 g L(-1). The highest overall volumetric productivity of 103 mg of active tyrosinase per liter and hour (corresponding to 464 mU L(-1) h(-1)) was determined, which is approximately 15 times higher than that obtained in batch cultures. CONCLUSIONS: We have successfully expressed and produced gram quantities per liter of active tyrosinase in recombinant E. coli by optimizing the expression conditions and fed-batch cultivation strategy. Exponential feed of substrate helped to prolong the exponential phase of growth, to reduce the fermentation time and thus the cost. A specific tyrosinase production rate of 103 mg L(-1) h(-1) and a maximum volumetric activity of 464 mU L(-1) h(-1) were achieved in this study. These levels have not been reported previously.