A novel bacterium strain exhibiting high laccase activity was separated and purified from the forest soil of the Liangshui National Nature Reserve in Heilongjiang Province with Cu2+ as the enrichment culture reagent. The strain was identified as Bacillus subtilis WD23 by morphological observation, physiological and biochemical tests and 16S rDNA sequence analysis. The cotA gene was cloned by primers designed according to B. subtilis cotA gene sequence on the GenBank. The cotA gene was inducibly expressed by the system of pET-22b (+)/E. coli BL21 (DE3). The laccase activity of the culture extraction was 1700 U/mL. The result suggests that the laccase activity is derived from the spore coat protein. The immobilized spore laccase could decolorize black pulping liquid with a decoloration rate of 24.9% and a COD reduction by 31.2% after seven days of treatment.
