Induction To Lignin Degradation Enzymes Activity Of Lenzites Gibbosa And Study Of Laccase Properties

In order to enhance lignin degrading enzymes activity of Lenzites gibbosa significantly,induction effects of inducers to laccase and MnP activity and their optimal levels were studied.At this condition,Laccase was isolated and its enzyme properties were studied.Considering practical application of laccase,laccase immobilization and dye decoloration of it were studied.Than we cloned and analyzed an transcription factor ace1 relating to gene expression regulation of lignin degrading enzymes in L.gibbosa.The result show that production of laccase and MnP were greatly improved when adding appropriate inducer to the liquid medium of L.gibbosa.The inducing effects of 1.5%wheat bran(biological inducer)is a very significant to laccase and MnP;chemical inducer guaiacol showed best effect to laccase production;while for MnP,the best chemical inducer was veratryl alcohol.Research about inducers combination showed that when biological inducer and chemical inducer added simultaneously,the enzymes activity were higher than that when one inducer alone added.The result:When 1.5%wheat bran and 1mmol L-1 guaiacol added simultaneously,the peak value of laccase activity would reached 3594.46 U·L-1,which was 24.9 times than that of the initial medium;When 1.5%wheat bran and 3mmol L-1 veratryl alcohol added simultaneously,the peak value of MnP activity would reached 935.48 U L-1,which was 12.2 times than that of the initial medium.Ion exchange column chromatography and gel filtration column chromatography were used for purification of Laccase,compared with the crude enzyme extract,purified laccase specific activity reached 527.28 U·mg-1,about 29 times increased,recovery rate was 15.93%.A single laccase protein band was detected in the SDS-PAGE electrophoresis which indicates that the laccase protein has been purified basically,the apparent molecular weight of laccase is 66.7 kDa.Research of laccase properties show that the optimal reaction temperature is about 55?,the optimum reaction pH 2.5,laccase can remain relatively stable in the environment pH=7,4?20?.Immobilization of laccase show that L.gibbosa laccase were all well immobilized in two methods(sodium alginate embedding method and gelatinadsorption),the sodium alginate immobilized laccase has better effect results,laccase activity reached 784.16U·g-1,the rate was 80.2%.The optimal reaction temperature of all the two kinds of immobilized laccase were 60 C,their thermal stability,pH and storage stability were also better than those of free laccase,degradation effect of immobilized laccases on dyes were also good generally.We cloned atotal length gene of 2956 bp by using degenerate PCR and gene walking technologies,which identified as L.gibbosa transcription factors acel gene after comparative analysis.