In our study, we produced intracellular blue laccase by growing the filamentous fungus Aspergillus ochraceus NCIM-1146 in potato dextrose broth. The enzyme was then purified 22-fold to a specific activity of 4.81 U/mg using anion-exchange and size exclusion chromatography. The molecular weight of purified laccase was estimated as 68 kDa using sodium dodecyl sulfate polyacrylamide gel electrophoresis. The enzyme showed maximum substrate specificity toward 2,2'-Azinobis, 3-ethylbenzothiazoline-6-sulfonic acid than any other substrate. The optimum pH and temperature for laccase activity were 4.0 and 60°C, respectively. The purified enzyme was stable up to 50°C, and high laccase activity was maintained at pH 5.0 ∼ 7.0. Laccase activity was strongly inhibited by sodium azide, EDTA, dithiothreitol, and L-cysteine. Purified laccase decolorized various textile dyes within 4 h in the absence of redox mediators. HPLC and FTIR analysis confirmed degradation of methyl orange. The metabolite formed after decolorization of methyl orange was characterized as p-N,N'-dimethylamine phenyldiazine using GC-MS.
- Aspergillus ochraceus NCIM-1146
- Redox mediators
- Textile dyes