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
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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 2 h at 37 ◦C. The apparent Km of the
enzyme displayed on spores was 443 ± 124 M for ABTS with a Vmax of 150 ± 16 U/mg spores. Notably,
1 mg of spores displaying B. subtilis laccase (3.4 × 102 U for ABTS as a substrate) decolorized 44.6 g indigo
carmine in 2 h. The spore reactor (0.5 g of spores corresponding to 1.7 × 105 U in 50 mL) in a consecutive
batch recycling mode decolorized 223 mg indigo carmine/L to completion within 42 h 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.
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