Production Of Laccase On Cassava Residue Solid State Fermentation And The Decoloirzation Of Aniline Blue With The Laccase

Screening PDA plate containing guaiacol was used to screening the fungus producinglaccase.82samples were collected and9strains formed the red colour circle on the screeningplate was achieved. Using of cassava residue as a support-substrate for the production oflaccase on solid state fermentation by the9strains were investigated and only5strains couldproduce laccase in this condition. Strain N3had the maximum laccase activity in the5strains.Through the identification of the strain N3, it has99%homology with Pleurotus sp., therebyit was named Pleurotus sp. N3. The effects of the initial pH, moisture content, fungus age, theconcentration of inorganic salt on laccase production by Pleurotus sp. N3were evaluated. Theoptimal initial pH, moisture content, fungus age, concentration of CuSO4·5H2O, MnSO4·H2O,KH2PO4for laccase production by Pleurotus sp. N3were4.0,70%,11d,0.635g·L-1,1.28g·L-1and18.3g·L-1respectively. Laccase showed a maximum activity of5.5U·(g ds)-1andthree extracellular isoenzymes on the fermentation at eleven days. An interesting differencewas noticed in the production of extracellular isoenzymes during the fermentation. Moreover,there was a strict correlation of the laccase activity and the production of isoenzymes.In addition, the laccase derived from Pleurotus sp. N3showed a good activity in syntheticdyes color removal, decolorizing92.73%aniline blue (15mg·L-1) and98.30%indigo carmine(30mg·L-1) with the addition of1mmol·L-11-hydroxybenzotriazole (HOBT). Vsible lightwavelength scanning and native polyacrylamide gel electrophoresis (Native-PAGE) analysiswas firstly carried out to determine the feasibility of aniline blue and indigo carminedecolorization with the laccase. Results of visible light wavelength scanning andNative-PAGE showed that the laccase derived from Pleurotus sp. N3could decolorize anilineblue and indigo carmine. And the mediator (HOBT) markedly enhanced the decolorization.The results showed the high potential of laccase from Pleurotus sp. N3on the solid cassavaresidues substrate fermentation for synthetic dyes decolorization, especially for aniline blueand indigo carmine. The mechanism of the indigo carmine decolorization was already studied.In order to study the mechanism of the aniline blue decolorization, the laccase isoenzymeswere separated by DEAE and SP chromatographic column. One laccase isoenzyme wasobtained from the separation.In order to study the mechanism of the aniline blue decolorization, the purified laccasewas used to decolorize the aniline blue. Technical condition including mediator (HOBT)concentration, laccase activity and reaction temperature were optimized byone-factor-at-a-time experiments. Moreover, mechanism of the decolorization was proposedon the basis of analyses of decolorizing products. The optimized condition was laccaseactivity of200U·L-1, HOBT concentration of4mmol·L-1, and reaction temperature of40℃.Under this condition, the decolorization ration of aniline blue (24mg·L-1) achieved by90.42%in the incubation of6h. These results suggest that laccase/HOBT system effectivelydecolorizes aniline blue. Moreover, aniline blue decolorizing products during thedecolorization process were further analyzed by ultra high performance liquidchromatography-mass spectrometry (UPLC-MS). Three compounds were identified, with their molecular weight was566,545, and522, respectively. The result suggests that thedecolorization of color is the function of the carbon-nitrogen double bond (C=N) rupture inthe molecule of aniline blue.