Isolating and identifying of a bacterium exhibiting spore laccase activity and research on treatment of papermaking black liquid

A novel strain exhibiting high laccase activity was screened and purified from forest soil of Liangshui National Nature Reserve in Heilongjiang with Cu²⁺ as the enrichment culture reagent. The strain was identified as Bacillus subtilis WD23 by the morphological, physiological, biochemical characteristics and the comparative analysis of 16S rDNA sequence. The strain showed optimum growth at 30°C and an optimum pH of 7.0. The strain showed strength of tolerance to NaCl and resistance to UV-C and H₂O₂. The CotA gene was cloned by upstream and downstream primers designed according to Bacillus subtilis CotA gene on GenBank. CotA gene was transformed to E.coli BL21(DE3) competent cell successfully. The recombined CotA protein was expressed by IPTG induced method. The laccase activity of the extraction of the fermentation broth was 1700 U/mL·min. The result suggested that the laccase activity derived from the spore coat protein. The immobiled spore laccase could decolorize black pulping liquor with 24.9% efficiency and decrease COD by 31.2% in 7 days.