Effects of culture conditions on production of extracellular laccase by Rhizoctonia praticola.

It was found that the soil-dwelling fungus Rhizoctonia praticola 93A was capable to produce laccase in submerged cultures. Effects of culture conditions on the enzyme biosynthesis in shaken flask and aerated bioreactor cultures were evaluated to improve the yields of the process. Production of extracellular laccase was considerably intensified by the addition of Cu2+ to a carbon-limited and nitrogen-sufficient culture medium (C/N = 0.98). When an optimized medium containing glucose (2 g/l) and L-asparagine (1.5 g/l) was used and enzyme synthesis was stimulated by addition of 5 microM Cu2+ before inoculation, maximal laccase activities obtained in a batch cultivation were, approximately, 1000 nkat/l. Under these conditions, addition to the medium of the aromatic inducer 2,5-xylidine (1 mM) led to a 10-fold increase in laccase activity. Laccase productivity in shaken flask cultures was also enhanced (to more than 4000 nkat/l on day 3) by using a medium with the initial pH of 7.5. Such a high value of the optimal medium pH for laccase production by R. praticola is exceptional among the ligninolytic fungi. In fermenter fungal cultures supplemented with cupric ions, the highest laccase activity (about 4000 nkat/l after 3 days' cultivation) was reached after 24-h incubation using a bioreactor with the aeration rate of 21/min, the agitation speed of 200 rpm, and a constant medium pH of 8.0.