Decolorization Process Of Aspergillus Sp.TS-A And Performance Of Recombinant Decolorization Enzymes

The effluents of printing and dyeing are one of the most difficult to degrade industrial wastewater,which have the characteristic of complex structures and poor biodegradability.Currently,bacteria,algae and fungi are considered to be functional microorganisms of degradation azo dyes.Fungi have great potential to degrade dyes and the intermediates of dyes are less toxic because of fungi own developed enzymes system and strong adaptability.Biosorption and biodegradation are often occurred in the process of dye decolorization because of the large specific surface area of fungi.However,the ability of biosorption and biodegradation are different for various microorganisms.In order to study the degradation ability of fungi and degradation mechanism of Mordant Yellow 1,the decolorization mechanism of mycelium and extracellular enzymes?MnP,LiP and Lac?were researched under different conditions in this paper through the Aspergillus sp.TS-A decolorization process.The results of this reaearch were following:?1?Study of decolorization process:Extracellular enzymes were isolated from TS-A as degradation system and the adsorption systems were chosen from live mycelium,sterilized mycelium and powder mycelium.The experiment results showed that over 72.29%of dye?50 mg/L?was degraded by extracellular enzymes and 18.8%was removed by live mycelium.The powder mycelium generally got the adsorption equilibrium?the removal rate of 44.2%?at 30 min,and its decolorization rate was higher than that of the living mycelium and sterilized mycelium?the removal rate of 30.6%?.The results of UV-Vis and FTIR illustrated that the Mordant Yellow 1 had been effectively degraded by extracellular enzymes.The enzymes biodegradation determined the decolorization process under weak acidic condition.Meanwhile,the behavior of powder mycelium fitted the models of Langmuir isotherm and second order kinetic.?2?Construction the recombinant decolorization enzymes:According to the reports of three lignin oxidase genes in NCBI,primers were designed to amplify the DNA of three oxidases.These results showed that the length of MnP encoding sequence was 1134 bp,LiP CDS sequence was 1119 bp and the size of Lac code sequence was 1827 bp.Three enzymes gene were cloned into the expression vector pPIC9K and successfully constructed the recombinant plasmids pPIC9K-MnP,pPIC9K-LiP and pPIC9K-Lac.Recombinant plasmids were successfully transferred into Pichia pastoris GS115 by electroporation and induced expressed enzyme by methanol.Three stable transformants were obtained by transferred into Pichia pastoris GS115.The specific activities of MnP,LiP,and Lac were 390 U/mg,385 U/mg and 200U/mg,which illustrated three recombinant enzymes were active.Recombinant enzymes MnP,Lac and LiP were more stable than original enzymes to temperature,pH and metal ions.?3?Performance of recombinant decolorization enzymes:The decolorization rate reached 51%,41%and 40%respectively when the engineering fungi reached the equilibrium for the degradation Mordant yellow 1 at 96 h.,which GS115/Lac was more tolerant to dye than other two strains with the changes of dye concentration.The optimum concentration of Mn²⁺and H₂O₂ were 0.4 mM and 0.2 mM for GS115/MnP degradation dyes.The optimal concentration of H₂O₂ was 0.3 mM for GS115/LiP.However,Cu²⁺had a strongly inhibitory effect on GS115/Lac.The MnP played a dominant role,which was consistent with original Aspergillus sp.TS-A extracellular enzymes.The LiP and MnP were two major enzymes involved in the degradation of Disperse Blue and Crystal Violet.The results of GC-MS analysis showed that MnP could degrade the Mordant Yellow 1 into a benzene-containing intermediate and further oxidase to trimethyl butanol and other small molecules.The degradation intermediate productions by Lac were further oxidized into an acidic small molecule substance such as trimethyl butyrate.The toxicity of dyes were reduced through the Lignin oxidase systems.