In order to overproduce mushroom laccase in P. pastoris harboring pPICHOLI-Lacc6, the effects of methanol feeding frequency, in terms of cell growth and extracellular laccase activity, were investigated in batch culture. When 1% methanol was fed every 6 hr, both cell growth and production of the laccase protein decreased due to methanol toxicity (Fig.3A). Cell growth was maintained at high levels at a feeding interval of 12~24 hr, whereas extracellular laccase production decreased significantly when methanol was fed every 24 hr.Extracellular laccase activity and cell growth reached maximal levels when methanol was fed at 12-hr intervals.Next, the effects of methanol concentration were examined by feeding methanol at different concentrations every 12 hr. Cell growth and laccase activity approached maximum levels after 120 hr of incubation (Fig. 3B and 3C). Methanol was found to inhibit both cell growth and laccase production at concentration of ≥ 2%. The optimal concentration ranged from 0.5% to 1% (Fig. 3B and 3C).