Cloning, expression and characterization of laccase from Bacillus licheniformis NS2324 in E. coli application in dye decolorization

Laccase gene from Bacillus licheniformis NS2324 was cloned and expressed in E. coli by using pUC 18 as cloning vector and pet 15b as expression vector. The purified recombinant laccase (rLacNS2324) showed a molecular mass of 66 KDa. The optimum pH and temperature for rLacNS2324 was found to be pH 8 and 40 degrees C respectively. The half life of rLacNS2324 at pH 7, 8 and 9 is 24 h. The half life of laccase at 45 degrees C is 8 h. Laccase activity was increased in the presence of Cu2+ (135.3%), Mn2+ (283.76%), and Co2+ (199.96%) at 5 mM of concentration, but inhibited to 17.01% in the presence of 5 mM Zn2+ ions. rLacNS2324 was found tolerant to NaCl and NaI. Among the inhibitors, it was found to be tolerant to EDTA, however, its activity was inhibited in the presence of sodium azide, dithiothreitol and beta-mercapethanol. rLacNS2324 was able to decolorize a bromophenol blue by 85% and phenol red by 75% in 1 h without any mediator. Methylene blue was almost completely degraded (99.28% decolorization) by 10 IUml(-1) of laccase at 40 degrees C, pH 8.0 and in time 4 h. Overall rLacNS2324 showed ability to be used industrially to decolorize dyes in an eco-friendly and cost effective way.