Response surface methodology study of laccase production in Panus tigrinus liquid cultures

The growth medium for Panus tigrinus laccase production was studied in shaken culture through response surface methodology. The impact of five crucial variables on enzyme production, including glucose and nitrogen concentrations and three distinct putative inducers, namely, copper sulphate, 2,5-xylidine and olive-mill wastewater was thoroughly investigated. Among them, 2,5-xylidine remarkably stimulated enzyme production while Cu2+ ions within the tested concentration range (0–0.25 mM) had an opposite effect. The maximal laccase activity (about 2 IU/mL on day 11) was observed in the medium containing glucose and nitrogen at concentrations of 12.5 g/L and 20 mM, respectively, and 0.25 mM 2,5-xylidine as the sole inducer. A 35-fold increase in laccase activity was obtained with respect to the same medium where 2,5-xylidine had been omitted. With the medium thus optimised, further experiments were performed in a 3-L stirred-tank reactor (STR) leading to a laccase activity of 2200 IU/L on day 9 with a productivity of 10.2 IU/(L h) and a specific activity of 26.7 IU/mg protein. RT-PCR analyses of total RNA from both non-induced and putatively induced cultures conducted in STR showed that the transcript levels of only one out of two constitutively expressed laccase genes were markedly increased by the presence of 0.25 mM 2,5-xylidine in the optimised medium.