Abstract

The objective of this study is to obtain new laccase and enzyme source with remarkable dye removal potential. Thirty isolates of white rot fungi were screened for extracellular laccase-production using 2,2-azinobis (3-ethylbenzothiazoline-6-sulfonate) (ABTS) assay as indicator. Among these, Polyporus pseudobetulinus strain WR77 exhibits the highestlaccase activity and its suitable enzyme production medium contains; 1% (w/v) rice chaff, 0.5 g/L di-ammonium tartrate, and 0.01 g/L peptone as the carbon; inorganic and organic nitrogen sources; respectively. The laccase was 60-fold concentrated (by ammonium sulphate precipitation, Q-sepharose anion-exchange chromatography, and Superdex G-75 gel filtration chromatography) and gave the specific activity of 617.12 U/mg. The MW of prepared enzyme is 75.2 kDa under SDS-PAGE determination. Empirical analyzing results indicate that the optimum pH and temperature of the enzyme are around 40°C and pH 4, respectively. Furthermore, this enzyme can resist to wide pH range (4.0-11.0) with more than 95% maximum activity remained. The enzyme’s K_m and V_max, with ABTS substrate, were 447.93 mM and 104.17 µmol/min/mg protein, respectively. The prepared enzyme was strongly inhibited by Hg²⁺ and Fe²⁺ but weakly (9.7%) stimulated by 10 mM Cu²⁺ ions. The strain WR77 shows good ability in decolorizing many synthetic dyes (200 mg/L initial concentration); Ambifix Blue H3R (98% in 8 days), Ambifix Yellow H3R (24% in 10 days) and Ambifix Red HE3B (50% in 18 days). The prepared laccase alone (5 U/ml)could decolorize Ambifix Blue H3R by 65% within 15 min and Malachite Green by 80% within 24 h. It can be concluded that new enzyme and source with satisfactory dye removal potential have been successfully achieved. Further studies should be attempted to evaluate their feasibility in industrial uses.

Key words: Polyporus pseudobetulinus, white-rot fungi, Laccase, dye decolorization, synthetic dyes.