Heterologous Expression Of Bacillus Amyloliquefaciens Laccase Gene And Characterization Of Recombinant Enzyme

A novel bacterium strain exhibiting high laccase activity was separated and purified from the forest of the Liangshui National Nature Reserve in Heilongjiang Province,by constructing vectors and achieving laccase gene heterologous expression in Pichia pastoris,thus purified laccase protein,study enzymatic properties and dye decolorization.First,using extraction kit to extrat LSOl's DNA,amplified 16S rDNA fragments.Subsequent gel recovery and purification,the purified target gene attach to T vector,transformed into E.coli JM109 competent cells,execute blue-white screening and plasmid extraction and verification.After verification,the double digestion the plasmids and pPICZaA of the carrier,the laccase gene fragment connecte to expression vector digested under T4DNA ligase,introduced into E.Top10 competent cells,PCR validation.At the same time,the preparation of Pichia pastoris competent cells,recombinant plasmid was linearized,electroporated into Pichia pastoris successfully constructed recombinant yeast strain.Develop recombinant yeast,to produced its growth curve,found that biomass and protein content has been increased,pH remains unchanged,the enzyme activity reached its peak in the first 11 days and then decreased.The pure enzyme of the size of about 65KD was obtained after SDS-PAGE electrophoresis,recombinant yeast producing crude enzyme solution by ultrafiltration,dialysis,DEAE-sepharose Fast Flow and Sephadex G75,its activity for 990.45U/L.Experimental results show that the purified recombinant laccase with ABTS and SGZ substrate when the optimum pH of 4.6 and 6.6,respectively,with good pH stability in neutral and alkaline conditions.The recombinant laccase optimum reaction temperature of 80 ?,but the experiment showed that the higher the temperature stability of the worse.Metal ion Na+?Mg2+ and Fe3+ on the activity promoted,other metal ions have a more or less inhibition.Common inhibitor,organic solvent and NaCl to inhibit the activity of the recombinant laccase is larger with the increasing concentration,most intense L-Cys and DTT.The participation of the mediator acetosyringone,recombinant laccase under acidic and alkaline conditions,three kinds of industrial dye RB Brilliant Blue,isatin and activity black decolorization experiments,results show that under basic conditions,exhibited good bleaching effect,6h after decolorization rate reached 70.45%,78.98%and 85.46%,respectively,its dye degradation in the future there will be good prospects.