Production of laccase by Botrytis cinerea and fermentation studies with strain F226

After induction, seven strains of Botrytis cinerea released into the culture broth considerable amounts of laccase in a brief production time. The set-up of a suitable production process was studied with a selected strain in a 10-L fermenter. The optimum fermentation conditions were a 3% inoculum with a high degree of sporulation, a simple medium containing 20 g L(-1) of glucose and 2 g L(-1) of yeast extract at pH 3.5, 2 g L(-1) gallic acid as inducer, added after 2 days of growth, an agitation speed of 300 rpm, an aeration rate of 1.2 vvm and a temperature of 24 degrees C. By optimizing the culture conditions, the enzyme activity reached 28 U ml(-1) in 5 days with a specific activity of 560 U mg(-1) protein. The best procedure to obtain a suitable crude enzyme preparation was concentration of the supernatant medium to 10% of the initial volume by ultrafiltration, followed by a fractional precipitation with ethanol. The optimum pH and temperature for laccase activity were 5.5 and 40 degrees C, respectively, with syringaldazine as the substrate.