Isolation,Identification Of Several Wild Polypores And Purification,Cloning Of A Novellaccase

Polypores are a group of macro-basidiomycetes with poroid hymenophores and leathery to woody basidiocarps.They are widely distributed all over the world and occurs on trees.Many polypores possess great potential applications including functional enzymes,medicinal activities,biodegradation,etc.In the present study,we focused on screening,isolation,identification of polypores species and their functional activities.It is good to exploration and applications of wild polypores resources.In the present study,fruiting bodies of wild polypores were collected.Pure cultures were obtained.Culture conditions,antioxidant activity,extracellular laccase,and biodegradation activities were further investigated,with details as follows.1.Two pure culture strain SS and BJ were obtained from fruiting bodies of two wild polypore species.Further studies were investigated including classification,optimum culture conditions,and bioactives of fermentation broth.The result shows that: Strain SS was classified as Phellinus tuberculosis based on morphology and ITS identification.Culture conditions of strain SS showed that the best carbon sources was glucose;the best nitrogen source was soybean powder;the best C/N ratio was 20/1;the best growth factor is Vitamin C;the optimal temperature was 28 °C;and the optimal p H was 7.0.Total antioxidant of fermentation broth of strain SS activity was 0.102 mM(FeSO4);Strain BJ was classified as Fomitiporia punicata.Culture conditions showed that the best carbon sources was glucose and maltose;the best nitrogen source was yeast extract;the best C/N ratio was 10/1;the optimal temperature was 28 °C;and the optimal pH was 7.0.Total antioxidant activity of fermentation broth was 0.517 mM towards Trolox.Total superoxide dismutase was 770.37 U/g.2.Laccase high production strain(numbered as F1635)was obtained.It was classified as Trametes trogii based on morphological and ITS molecular identification.A novel laccase of electrophoresis grade(TsL)was gained following a purification protocol including DEAE-Sepharose,SP-Sepharose,and gel filtration chromatography.Enzymetic properties,degradation function,and gene cloning of TsL were subsequently preformed.The result showed that TsL was a monomeric protein with molecular weight of 64.8 kDa.Tsl manifested optimal pH of 2.6,optimal temperature of 50 °C,and enzyme kinetic constants Km of 18.58 ?M,Vmax of 1.125 ?mol/min respectively,using ABTS as substrate.TsL was effective in the decolorization of bromothymol blue,methyl orange,chrome black and evans blue.After 12-hours incubation,degradation ratios of all samples reached more than 78%.However,TsL as avoid of obvious degradation effects on acephate and beta cypermethrin.Two partial laccases cDNA sequences,1246 bp and 1264 bp respectively,were obtained using conserved regions of fungal laccases.