Enzmye Production From Fungi With High Yield Of Laccase And Enzymatic Kinetics For Degradation Of Dyes

Laccase is a multicopper enzyme that can oxidize phenols or aromatic amines by reducingmolecular oxygen to water. This enzyme is remarkably non-specific regarding substrates, andits zone of application can be widened even further in the presence of the aromatic electrontransfer or radical-forming mediators. Laccase has the potential to be implemented in variousindustrial fields, such as pulp biobleaching, discoloration of textile dye, wastewater treatment,polymer syntheses, etc. However, a major limitation for successful industrial application oflaccase in the bioremediation processes mentioned above is the high costs of laccaseproduction. The development of approaches for reducing the costs of laccase production withhigh efficiency and environmental friendliness by optimising the fermentation medium andcondition is a top priority.In the present paper, the production of laccase by white-rot fungus Panus conchatus insubmerged cultures was investigated. Extracellular laccase formation by P. conchatus can beconsiderably stimulated by the addition of wheat bran and copper sulfate. The maximallaccase activity, approximately196.1U/mL, was obtained in the shake flasks. Submergedculture on larger scale was carried out in a7.5L stirred-tank fermentor, in which the highestactivity of laccase was about200U/mL. The good regression coefficients indicated that theLogistic model was justifiable for the expression and prediction of laccase production by P.conchatus in submerged cultures. Crude laccase of P. conchatus was also used forbiobleaching in LQP1P1sequence with reed pulp. It showed that kappa number, brightness,physical properties and yellowing number of pulp were improved in a certain extent after thebleaching by laccase. The pulp bleachability was increased comparatively after treated bylaccase/mediator system.Laccase production by P. conchatus in submerged culture using low-cost medium andcooking liquor of bagasse was investigated. Soybean meal and copper sulfate were shown tobe the excellent supporters and played positive roles in enzyme production with the maximallaccase activity of476.7U/mL in the shake flasks. Xylose was found to be the majority of themonosaccharide in liquor from the cooking with diluted H2SO4. The biomass of white-rotfungi Panus conchatus, Flammulina velutipes and Psathyrella candolleana increased during fermentation in the liquid medium based on the cooking liquor without detoxification, butonly F. velutipes showed excellent enzyme production with the maximal laccase activity ofabout200U/L in the medium.Laccase produced by P. conchatus in submerged culture was purified to electrophoretichomogeneity by precipitation, ultrafiltration, ion-exchange chromatography andsize-exclusion chromatography with a final purification fold of6.77and a yield of74.1%. Theenzyme was found to be a typical fungal laccase with molecular mass of65kDa and Kmvalueof5.7μ2for ABTS. The optimum pH and temperature for laccase activity were2.5and60oC, respectively. The purified laccase retained97.9%of its initial activity after26dinculbation at pH8.0and4oC. The extracellular crude laccase was effective in decolorizationof synthetic dyes, decolorizing95.2%of RBBR in0.5h and84.1%of Methyl Orange in2hwithout redox mediators, approximately100%of Fuchsin Acid in1h with the addition of0.1%HOBT as the meditor.It was found that anthraquinone dye RBBR could be directely degraded by the crudelaccase of P. conchatus. The degradation of more than90%for100mg/L of RBBR wasobtained in15min with1U/mL of laccase. The relationships between the degradation of dyeand time, pH, temperature, as well as the concentration of laccase were established. Aempirical model of dye degradation was developed based on the experimental results. Thegood regression coefficient indicated that the models were justifiable for the expression andprediction of RBBR degradation.A detailed investigation concerning to effects of pH, temperature, concentrations of dye,laccase and mediator on the reaction rate of dye degradation by laccase/mediator system(LMS) was conducted. Triphenylmethane dye Fuchsin Acid could be effectively degraded byLMS. The degradation of more than90%for100mg/L Fuchsin Acid was obtained in40minwith2U/mL of laccase and0.5%of HOBT. A kinetic model of reaction rate of dyedegradation by laccase/HOBT system was developed based on the experimental results. Therewas a good agreement between the calculated and measured data, indicating that the modelwas reasonable expression and predication for the reaction rate during dye degradation.An airlift bioreactor was developed and used to degradation of dye by laccase afterrepeated addition of RBBR or Fuchsin Acid. A Uv-vis spectrophotometric method was established for rapid and temporally resolved monitoring the degradation of dye in thebioreactor. The result showed that the activity of P. conchatus laccase was stable in thebioreactor and had good degradation ability for both of the two dyes. The degradations ofmore than90%and60%of RBBR and Fuchsin Acid were obtained, respectively, in every30min with8times of dye additions. A kinetic model of degradation reaction rate with everytime of dyes addition was developed based on the experimental results. The good regressioncoefficient of the calculated and measured data indicated that the model were justifiable forthe expression and predication for the reaction rate during dye degradation in the bioreactor.