Purification and characterization of a new laccase from Shiraia sp.SUPER-H168

A new laccase from Shiraia sp.SUPER-H168 was purified by ion exchange column chromatography and gel permeation chromatography and the apparent molecular mass of this enzyme was 70.78 kDa, as determined by MALDI/TOF-MS. The optimum pH value of the purified laccase was 4, 6, 5.5 and 3 with 2,6-dimethoxyphenol (DMP), syringaldazine, guaiacol and 2,2′-azinobis-(3-ethylbenzthiazoline-6-sulphonate) (ABTS) as substrates, respectively. The optimum temperature of the purified laccase was 50 °C using DMP, syringaldazine and guaiacol as substrates, but 60 °C for ABTS. Inhibitors and metal ions of SDS, NaN₃, Ag⁺ and Fe³⁺ showed inhibition on enzyme activity of 10.22%, 7.86%, 8.13% and 67.50%, respectively. Fe²⁺ completely inhibited the purified laccase. The K_cat/K_m values of the purified laccase toward DMP, ABTS guaiacol and syringaldazine were 3.99 × 10⁶, 3.74 × 10⁷, 8.01 × 10⁴ and 2.35 × 10⁷ mol^?1 L S^?1, respectively. The N-terminal amino acid sequence of the purified laccase showed 36.4% similarity to Pleurotus ostrestus. Approximately 66% of the Acid Blue 129 (100 mg L^?1) was decolorized by 2.5 U of the purified laccase after a 120 min incubation at 50 °C. Acid Red 1 (20 mg L^?1) and Reactive Black 5 (50 mg L^?1) were decolorized by the purified laccase after the addition of Acid Blue 129 (100 mg L^?1).