Purification and Characterization of the Laccase Involved in Dye Decolorization by the White-Rot Fungus Marasmius scorodonius
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Marasmius scorodonius secretes an extracellular laccase in potato dextrose broth, and this enzyme was purified up to 206-fold using (NH4)2SO4 precipitation and a Hi-trap Q Sepharose column. The molecular mass of the purified laccase was estimated to be ~67 kDa by SDSPAGE. The UV/vis spectrum of the enzyme was nontypical for laccases, and metal content analysis revealed that the enzyme contains 1 mole of Fe and Zn and 2 moles of Cu per mole of protein. The optimal pH for the enzymatic activity was 3.4, 4.0, and 4.6 with 2,2’-azino-bis(3- ethylbenzothazoline-6-sulfonate) (ABTS), guaiacol, and 2,6-dimethoxy phenol as the substrate, respectively. The optimal temperature of the enzyme was 75°C with ABTS as the substrate. The enzyme was stable in the presence of some metal ions such as Ca2+, Cu2+, Ni2+, Mg2+, Mn2+, Ba2+, Co2+, and Zn2+ at a low concentration (1 mM), whereas Fe2+ completely inhibited the enzymatic activity. The enzymatic reaction was strongly inhibited by metal chelators and thiol compounds except for EDTA. This enzyme directly decolorized Congo red, Malachite green, Crystal violet, and Methylene green dyes at various decolorization rates of 63–90%. In the presence of 1-hydroxybenzotriazole as a redox mediator, the decolorization of Reactive orange 16 and Remazol brilliant blue R was also achieved.
Abstract
Marasmius scorodonius secretes an extracellular laccase in potato dextrose broth, and this enzyme was purified up to 206-fold using (NH4)2SO4 precipitation and a Hi-trap Q Sepharose column. The molecular mass of the purified laccase was estimated to be ~67 kDa by SDSPAGE. The UV/vis spectrum of the enzyme was nontypical for laccases, and metal content analysis revealed that the enzyme contains 1 mole of Fe and Zn and 2 moles of Cu per mole of protein. The optimal pH for the enzymatic activity was 3.4, 4.0, and 4.6 with 2,2’-azino-bis(3- ethylbenzothazoline-6-sulfonate) (ABTS), guaiacol, and 2,6-dimethoxy phenol as the substrate, respectively. The optimal temperature of the enzyme was 75°C with ABTS as the substrate. The enzyme was stable in the presence of some metal ions such as Ca2+, Cu2+, Ni2+, Mg2+, Mn2+, Ba2+, Co2+, and Zn2+ at a low concentration (1 mM), whereas Fe2+ completely inhibited the enzymatic activity. The enzymatic reaction was strongly inhibited by metal chelators and thiol compounds except for EDTA. This enzyme directly decolorized Congo red, Malachite green, Crystal violet, and Methylene green dyes at various decolorization rates of 63–90%. In the presence of 1-hydroxybenzotriazole as a redox mediator, the decolorization of Reactive orange 16 and Remazol brilliant blue R was also achieved.
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