Cloning And Expression Of Auricularia Auricula Laccase Genes In Pichia Pastoris

Laccase (EC1.10.3.2) is a kind of Cu-contained polyphenol oxidase produced by many white-rot fungi. Laccases are involved in lignification of xylem tissues,laccases can react to various substrate such as polyphenol and aromatic diamines. Interest in laccases has been fueled by their potential uses in detoxification of environmental pollutants, prevention of wine decoloration, paper processing, enzymatic conversion of chemical intermediates, and production of useful chemicals from lignin. Auricularia auricular is a saprophytic fungus belonging to the Basidiomycetes of the family Auriculariaceae, it can secret laccase constitutively, The thesis study initially cloning of laccase gene from Auricularia auricular, expression of laccase gene.In this study, loned lac1gene with gene donor Auricularia auricular, constructing and applying the laccase gene screening system based on specific point recombinant, lac1 cDNA was cloned from hyper-producing laccase Auricularia auricular strain, yeast induced expression vector and constitutional expression vector were constructed respectively, transformed into Pichia pastoris by electroporation and got engineering strain that express laccase gene effectively. results were summarized as follows:1.Screening hyper-producing laccase strainAuricularia auricular 29 and Auricularia auricular 981 were selected by oxidative band screening method, the result showed that two strains appeared obvious oxidative band, Auricularia auricular 29 product higher laccase enzyme.Auricularia auricular 29 and Auricularia auricular 981 were cultured and measured enzyme activity by liquid fermentative culture medium, the result showed that two strains had laccase activity and the highest enzyme activity period focus on 18-22 day, the higher laccase activity strain is Auricularia auricular 981.2.Screening and cloning of laccase geneSaccharomyces cerevisiae secreting expression vector pPADα-Rec based on specific point recombinant were constructed. Applying the laccase gene screening system based on specific point recombinant, lac1 cDNA was screened from Auricularia auricular. lac1 DNA and lac gene that no containing signal itself was cloned by PCR.3.Construction of expression vectorsConstruction of Pichia pastoris induced expression vector of pPIC-lac1 and constitutional expression vector of pGAPH-lac1.Construction of Pichia pastoris induced expression vector of pPIC9-lac and constitutional expression vector of pGAPHα-lac.4. The expression of laccase gene in Pichia pastorisThe linear expression vectors were transformed into Pichia pastoris by electroporation, Transformants were identified by PCR: GS115-pPIC-lac1; GS115-pPIC9-lac; GS115-pGAPH-lac1; GS115-pGAPHα-lac.Transformants are all expressed.SDS-PAGE showed that others aren't expressed except GS115-pPIC9-lac of laccase gene.5.The effect of recombinant laccase productivity by fermentative pHGS115-pPIC9-lac strain was induced Expression in differert pH fermentative liquid, then laccase activity was determined, the further research determined the optimal fermentative pH of GS115-pPIC9-lac was 4.0.6.Analysis of enzymatic characteristics of GS115-pPIC9-lacUnder optimum fermentative condition, the activity of the laccase secreting expression was 0.149U/mL. The properties of enzyme were determined. The optimum temperature and pH value were 40℃and pH 5.0, respectively. Thermostability and pH stability were very well under the optimal conditions.