Rearch On Induction, Production And Application Of Bacterial Laccase

In this study,we have isolated a forest soil bacterium which was grown in Luria-Bertani (LB) medium supplementing with 0.4mM Cu²⁺.The spores of the strain showed laccase-like activity,oxidizing syringaldazine,2,6-dimethoxyphenol and ABTS.Sequence analysis of 16S rDNA plus bacterial morphology observation and physiological biochemistry classified it as Bacillus subtilis.Hence this strain was named as Bacillus subtilis WD23.On solid agar media, the red pigmentation of spores was showed by incubation at 37℃for 72 h.The laccases were bound to B.subtilis WD23 spores,laccase activities clearly correlated well with spore count and the production of laccase in liquid media was variable and in most cases less than that on solid agar media.B.subtilis WD23 was removed from the solid-sporulation media,washed and finally suspended in distilled water(pH 7.0).Using the oxidation of syringaldazine determined the effective activities of laccase and laccase activity of the spore suspension,the optimum temperature and pH were 60℃and 6.8, respectively.It showed higher activity within a temperature range from 40 to 70℃.Especially, laccase activity could be determined at 0℃and 100℃.The spore-bound laccase exhibits a higher thermal stability and pH-stabilities:The temperature half-life of the laccase was 2.5 h at 80℃and the laccase activity was disappeared after 18h.It showed higher stabilities over a broad pH range and the pH half-life was more than 6 months at pH 6.8.The spore laccase was used for the decolorization of alizarin red,90%of the dye was removed in 24 h.In addition,B. subtilis WD23 also against UV lights and hydrogen peroxide.Bacteria could produce a high amount of laccase in a short time due to their fast growth speed;compared with fungal laccases,there is no glycosylation modification in spore-bound laccases,and they have higher thermal stability and enzyme activity in alkaline pH environments.Especially,the spores could live in wastewater without any nutrition.Due to the these unusual properties,the spore-bound laccase from B.subtilis WD23 is of important significance in industrial applications such as environmental protection,food industry,paper biobleaching,and so on.In this study,time-extended immobilization method was used to analyze a variety of enzymatic properties,The optimum pH and temperature of immobilized laccase were 6.8,70℃.Thermal stability property of immobilized laccase was more stable than the free laccase. The immobilized enzyme has good recycled use stability and the residual activity.Fungi and bacteria was co-cultured to reduce COD of Black liquor,and total metabolism can better improve the removal rate of COD.