Heterologous Expression Of Laccase From Volvariella Volvacea

Laccase is an oxygen oxidoreductase with multi copper. Containing copper as thecofactor, it has the ability to oxidize phenolic compounds with molecular oxygen as theco-substrate. Laccase has a non-specific molecular stracture, extending to a variety ofsubstrates. Therefore, laccase play an important role in nature, such as plants, annimals andfungi in vivo, as well as in modern industry, including bioremediation, paper industry, foodindustry and analytical tools et al.. However, the product of laccase is a low volume in nature,which keeps obstacles of its way to application on an enlarged scale in industry.Volvariella volvacea, one of the laccase producing white rot fungi, is an importantcommercially cultivated mushroom. Under the circumstances that a draft of the V. volvaceaV23genome has been constructed and11laccase genes also have been predicted, theexploitation and utilization of laccase in V. volvacea V23will be more convenient than ever.In this study, six laccase genes were seleceted to clone and transform into heterologousexpression system.1. Extract genome RNA of V. volvacea V23and obtain the cDNA.2. Transconduct of target genes of vv-lac1, vv-lac4, vv-lac5, vv-lac6, vv-lac8andvv-lac9into vector PMD19-T and expression vector pPIC9K.constructed with each laccasegene.3. Expression vector pPIC9K with target genes was transformed into Pichia pastorisGS115by electroporation. After screening by ABTS, Pichia strains with vv-lac6(L6/G) couldexpress laccase.4. Screened by geneticin, two stains(L6/G-1, L6/G-2) were fermented in flask to obtainprotein, respectively0.22U/L and0.35U/L.