Properties Of Spore Laccase From Bacillus Subtilis WN02 And Its Use In Dye Decolorization

In this work, medium supplemented with copper ions was used to enrich laccase-producing bacterial strains from activated sludge, which was collected from the secondary settling tank of a wastewater treatment plant. A strain exhibiting laccase activity was isolated and named as WN02. Laccase activity was detected by adding solutions of laccase substrates on the center or edge of the colony. The result showed that the strain WN02 could oxidize syringaldazine (SGZ),2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonate) (ABTS) and 2,6-dimethoxyphenol (DMP). The isolated strain was identified as Bacillus subtilis WN02, based on the results of morphology observation, physiological and biochemical tests and 16S rDNA sequence analysis. The strain demonstrated optimum growth temperature at 30℃, and the most favorable pH of the culture medium was observed as pH 6.0.The laccase of strain WN02 possessed catalytic ability during a broad pH range. The oxidation of 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonate) (ABTS), syringaldazine and 2,6-dimethoxyphenol showed optimum pH of 3.0,7.0 and 8.6, respectively. The spore laccase exhibited high stability under different pH ranges, especially in alkaline conditions. The optimum temperature for oxidizing SGZ by the spore laccase was 60℃. High laccase activity was observed at 40～80℃. Moreover, the spore laccase could retain more than 20% of highest activity even at 100℃, and the activity could remain for 24 h at the optimum temperature. Cysteine and dithiothreitol strongly inhibited the spore laccase activity under a concentration of 0.1 mmol/L, while NaN3 and SDS with the same concentration could promote the enzyme activity. EDTA showed only a slight inhibition effect under a higher concentration. The activity of spore laccase decreased with the increasing of concentration of the organic solvents.1% Tween 80 and Triton X-100 could strongly inhibit laccase activity. The metal ions Hg2+,Ag+,Mn2+ could strongly inhibit laccase activity, while other metal ions such as Ba2+,Co2+,Na+ activated laccase activity. The presence of Al3+ and Fe3+ demonstrated a slight inhibition effect.The spore laccase of strain WN02 could decolorize four synthetic dyes with different chemical structures in the absence of mediators. The decolorization percentages (6h) for crystal violet, indigo carmine and Remazol Brilliant Blue R (RBBR) were 81.47%,72.85% and 26.69%, respectively. The decolorization of reactive black proceeded rather slowly, with a decolorization percentage of less than 10%. Seven mediators were tested for their effects on dye decolorization by the spore laccase. The result showed that:the decolorization of crystal violet was not significantly influenced by the addition of mediators, however, some mediators including ABTS, acetosyringone, promazine and syringaldehyde could efficiently enhance the decolorization of indigo carmine, more than 90% of which was decolorized after 2 h. RBBR and reactive black could also be efficiently decolorized in the presence of ABTS, acetosyringone, or syringaldehyde. When acetosyringone was selected as mediator, the spore laccase decolorized crystal violet, indigo carmine, RBBR and reactive black in pH 9.0, with a percentage of 93.99%,95.37%,92.99% and 91.21%, respectively. The results suggested the potential applications of spore laccase in the treatment of dye effluents in alkaline conditions.