The Structure And Function Of Bacillus Subtilis Laccase

Laccases (Laccase, EC1.10.3.2), a type of multicopper-containing polyphenoloxidases，are members of multicupper oxidase family. With the specific redox potential,laccases will be capable of degrading phenolic substrate completely to carbon dioxide,bringing the by-product water only. Thus the utilizing of laccase to treatment of industrialdye wastewater is both economic and effective and environmental friendly method. As ahigh efficiency and green catalyst, laccase has always been the key research focus of thefield that environmental protection, biological detection, the paper industry(biologicalpulping and bleaching),food industry (food flavor improvement) and other.We found a laccase CotA from Bacillus subtilis, but the activity of CAR is very low.To get high activity bacterial laccase, the bacterial laccase genes were cloned and theirsequences were heterologously expressed in host cell in this study. It obtained the mutantsCAH, CAHEG and CAHEGF by Site-directed mutagenesis (CAH mutates R155H,CAHEG mutates R155H, A158E and S210G mutations, CAHEGF mutates R155H, A158E,S210G and I224F). By using ABTS as substrate, it had investigated the characterizationand catalytic effect. Based on these, the determination of oxidation potential has beenperformed. Through the determination of various dye absorption changes in the maximumabsorption wavelength, it had carried out applied research on the decoloration of syntheticdyestuff. Compared with CAR, redox potential of CAH increased from0.32V to0.42V (vs.normal hydrogen electrode), its activity increased from17.1U/mg to220.4U/mg. Theoptimum pH of mutants and wild-type laccase is4,4,4,3, adapting to alkalinity. Thethermal stability and the stability of pH of mutants were better than that of wild type. Inaddition, the decolorization of CAH were higher than that of CAR most obviously, after3h, the decolorization rate of indigo carmine and crystal violet can reach74%and63%respectively.In conclusion, this paper obtained mutants with the better thermostability, the higheractivity, the stronger tolerance to alkaline environment and the better decolorization abilityof synthetic dyes. In terms of decoloration capability for the synthetic dyestuffs, therelationship between structure and function of laccase has been significantly investigated,which thus accelerates the process of its industrial application.