Purification and characterization of laccase from white rot fungus WR77 and its applcation in decoloriztion of syntherization of synthetic dyes

Abstract

It is well known that laccase produced by white rot fungi were able to decolorize synthetic dyes. Therefore in this research white rot fungi were screened for high laccase activity with new characteristic. Selective white rot fungi produced high laccase activity by growing on medium supplemented with 2,2-azinobis(3-ethylbenzothiazoline-6-sulfonate) (ABTS). White rot fungus strain WR77 showed highest laccase activity among other isolates. WR77 was tested for the effect of carbon source and nitrogen source on highest laccase production. One percent (w/v) rice chaff, 0.5 g/L di-ammonium tartrate and 0.01 g/L peptone were supplemented in the medium as carbon and nitrogen sources showed highest laccase activity. Purification WR77 laccase by ammonium sulphate precipitation, Q-sapharose anion-exchange chromatography, and Sephadex G-75 gel filtration chromatography showed enzyme activity as 617.12 U/mg protein. The enzyme has molecular mass of approximately 75.2 kDa as determined by SDS-PAGE. Purified WR77 laccase remained active at pH range 4.0 to 11.0 at 40°C for up to 120 min of incubation time. The Km value of enzyme for ABTS as substrate was 447.93 mM and its corresponding Vmax value of 104.17 µmol/min/mg protein. 5.0 mM Hg²⁺ and 1.0 mM Fe²⁺ inhibited WR77 laccase activity whereas 10 mM Cu²⁺ ions stimulated WR77 laccase activity by 9.7%. WR77 could decolorized 98% Ambifix Blue H3R was in 8 days, 24% Ambifix Yellow H3R in 10 days and 50% Ambifix Red HE3B in 18 days. Crude laccase with concentration 5 U/ml could also decolorize 65% Ambifix Blue H3R within 15min and 80% Malachite Green within 24hr.