A pH-stable laccase from alkali-tolerant γ-proteobacterium JB:: Purification, characterization and indigo carmine degradation

gamma-Proteobacterium JB, an alkali-tolerant soil isolate, produced laccase constitutively in unbuffered medium. The enzyme was purified to homogeneity by ammonium sulphate precipitation, DEAE-sepharose anion exchange chromatography and preparatory polyacrylamide gel electrophoresis. The purified enzyme was a monomeric polypeptide (MW 120 kDa) and absorbed at 590 nm indicating the presence of Type I Cu2+-centre. It worked optimally at 55 degrees C and showed different pH optima for different substrates. The enzyme was highly stable in the pH range 4-10 even after 60 days at 4 degrees C. K-m and V-max values for syringaldazine, catechol, pyrogallol, p-phenylenediamine, L-methyl DOPA and guaiacol substrates were determined. Inhibitors, viz. azide, diethyldithiocarbamate, thioglycollate and cysteine-hydrochloride all inhibited laccase non-competitively using guaiacol as substrate at pH 6.5. The enzyme degraded indigo carmine (pH 9, 55 degrees C to anthranilic acid via isatin as determined spectrophotometrically and by HPLC analysis. Degradation was enhanced in the presence of syringaldehyde (571%), vanillin (156%) and p-hydroxybenzoic acid (91.6%) but not HOBT. (C) 2007 Elsevier Inc. All rights reserved.