Blue multicopper oxidases, laccases displayed on the surface of Bacillus spores were used to decolorize a widely used textile dyestuff, indigo carmine. The laccase-encoding gene of Bacillus subtilis, cotA, was cloned and expressed in B. subtilis DB104, and the expressed enzyme was spontaneously localized on Bacillus spores. B. subtilis spores expressing laccase exhibited maximal activity for the oxidation of 2,2′-azino-bis (3-ethylthiazoline-6-sulfonate) (ABTS) at pH 4.0 and 80°C, and for the decolorization of indigo carmine at pH 8.0 and 60°C. The displayed enzyme retained 80% of its original activity after pre-treatment with organic solvents such as 50% acetonitrile and n-hexane for 2h at 37°C. The apparent K m of the enzyme displayed on spores was 443±124μM for ABTS with a V max of 150±16 U/mg spores. Notably, 1mg of spores displaying B. subtilis laccase (3.4×10 2U for ABTS as a substrate) decolorized 44.6μg indigo carmine in 2h. The spore reactor (0.5g of spores corresponding to 1.7×10 5U in 50mL) in a consecutive batch recycling mode decolorized 223mg indigo carmine/L to completion within 42h at pH 8.0 and 60°C. These results suggest that laccase displayed on B. subtilis spores can serve as a powerful environmental tool for the treatment of textile dye effluent.
All Science Journal Classification (ASJC) codes
- Applied Microbiology and Biotechnology