Characterization And Funtional Analysis Of Laccase From Monilinia Fructigena

Monilinia fructigena is one of the three major pathogens of fruit brown rot bacteria which leads to fruit rot, flower rot, and lead blight and also likely leads to fruit rot during transportation and storage, which casuing serious losses. Currently, research focuses on deveolopment of M. fructigena and incidence of fruit brown in its impact on the environment and the outside world, less of the bacteria pathogenesis.Laccase(EC1.10.3.2) as polyphenol oxidase, being found in the process of the pathogenesis of various pathogens, is an important pathogen virulence factors. Laccase not only involved in the budding of fungi, degradation of lignin, and occurrence of disease, also can protect fungal pathogen from the anti-toxin and tannic acid effect. It is infered that laccase may be related to the development and the pathogenesis of M. fructigena. Therefore, research on M. fructigena’’s laccase have great significance, but related research not yet carried out. In this work, M. fructigena’s laccase was heterologous expressed in Pichia pastoris. The enzymatic properties and function was carried out after purification. It provides the foundation for follow-up to the role of laccase in the incidence of fruit brown.The gene of Laccase of M.fructigena (M/LCC2) was synthesized chemically according to the yeast bias codon and integrated into the genome of Pichia pastoris GS115by electroporation. The expressed enzyme was recovered from the culture supernatant and purified. The result of enzyme activity assay and SDS-PAGE demonstrated that the recombinant laccase was induced and extracellularly expressed in P. pastoris. Main biochemical properties of this laccase, such as thermodependence and thermostability, optimal pH and pH stability, and the effect of metal ions and inhibitors, were characterized. With2,2’-azinobis-(3-ethylbenzothiazoline-6-sulfonate (ABTS) as the substrate, MfLcc had its optimal pH at3.5and optimal temperature at45℃. The Km values of the ABTS and guaiacol are0.012and0.016mM, respectively, and the corresponding Vmax values are 243.9and10.55Um*min-1*mg-1, respectively. The recombinant laccase degraded80%2,4,6-trichlorophenol after8h under the optimal conditions. The recombinant strain and its laccase can be considered as candidate for treating waste water polluted with trichlorophenols.