| Laccase (benzenediol:oxygen oxidoreductases; EC 1.10.3.2) is a type of copper-containing polyphenol oxidase that is first discovered in the exudates of the Japanese lacquer tree. Laccase is one of a small group of enzymes called the blue copper oxidases, the other members of which are the plant ascorbate oxidases and the mammalian plasma protein ceruloplasmin. Laccases are widespread in higher plants and fungi, which are divided into two major groups Rhus laccase and fungal laccase. The substrate range of laccase is fairly broad and it can oxidate phenolic compounds and other kinds of compounds contain hydroxy, acidyl and amino group. Because of its strong ability of oxidation, laccase is widely used in paper pulp industry, food industry, biological detection and biosynthesis.In this thesis, the fermentation medium of Pycnoporus sanguineus (Fr.) Murr. for laccase production was optimized. The laccases produced by Pycnoporus sanguineus (Fr.) Murr. were purified and characterized. The applications in decolorization of different dyestuffs were studied. The results were as follows:The important factors influencing laccase production, which identified by Plackett-Burman design were wheat bran, glucose and peptone. On this basis, the path of steepest ascent was undertaked to approach the optimal region of the three significant factors. Box-Behnken design and response surface analysis were adopted for further investigating. The optimal parameters of fermentation medium obtained from the regression equation were wheat bran 2.5%, glucose 2.0%, peptone 1.2%, natural pH. Under this fermentation condition, the maximum laccase activity was 330.66U/mL which increased 38.9% than that (238 U/mL) in the basal fermentation medium.The laccases produced by Pycnoporus sanguineus (Fr.) Murr. were in the sediment, the dissolubility of that is very poor. The laccase activity in the supernatant is 72.1% of that in the solution which is the dissolved crude laccase sediment using 0.1 mol/L NaOH aq.. The supernatant of dissolved laccase sediment was perified by dialysis, concentration and Sephadex G-100 filtration. After the purification, a pure laccase was obtained and the purity and yield were 1.94 folds and 19.53% of the fermentation broth respectively.The general stability is very good, and its remaining activity is 24.5% at 4℃in 60 days. The optimal pH was 5.2 and 6.4 in phosphate and acetate buffers respectively, and the remaining activity is more than 80% at pH 5.8～8 incubated 12 hours. The optimal temperature was 40℃and the remaining activity is more than 80% at 60℃incubated 50 minutes. It showed that the laccase adapted to a wide range of pH and had good thermal stability. The laccase could be activated by Zn2+ and inhibited weakly by Co2+,Ca2+ and strongly by Al3+,Ba2+,Na+,Mg2+. The Km of ortho-tolidine was 0.772mmol/L.The crude laccases produced by Pycnoporus sanguineus (Fr.) Murr. could decolorize the anthraquinone dye (alizarin red), azo dye (methyl red) and triarylmethane dye (bromocresol green) effectively without mediators.The best decoloration rate of dyes could be obtained in 8 hours, after then the growth rate slowed down. The optimal temperature was 30℃, and the decoloration rate reduced with increase in temperature. The effect of pH on the three dyes decolorization was complicated. The red shift or blue shift of maximum absorption wavelength occurred when the pH was changed. The decoloration rate increased with increase in initial laccase activity, reached the top when the activity was 75.68U/mL, and then the growth rate slowed down.
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