Construction And Expression Of Cellulase Engineering Bacteria

Renewable resource,cellulose,is a kind of carbon source in living nature.The dry wei get of plants about 220 a hundred million ton are produced by photosynthesis in a year,cellul ose is about 50%.The fiber of haulm is about 2 a hundred million ton.So it's very important to us e cellulase to decompose cellulose.Environmental pollution,food shortage,energy crisis,sustainable development of human society can be solved in a day.The filamentous fungus Trichoderma viride is a well-known cellulolytic organism and produces a family of different cellulolytic enzymes,including endoglucanases,exocellobiohydrolases,andβ-glucosida ses which were most important to the biodegradation of the cellulosic biomass.Some of the cellulase s have been shown to cooperate in the hydrolysis of cellulose,they act in synergy.The cloning of ce llulose genes and their high expressing in Schizosaccharomyces pombe is of great significance in bi odegradation of the cellulosic biomass(cellulose and hemicellulose),such as agricultural and forestry r esidues.The CBHII,EGI and BGLI from T.viride were transferred to Schizosaccharomyces pombe,a nd cellulases-producing S.pombe engineering strains with stable heredity were constructed.It's complic to express and control the inducing Erzymes of cellulase,and exoglucanase(CBHâ…¡) is important.The expression of cellulose is effected by gene deletion of CBHâ…¡.But gene deletio n of others can effect expression of oneself.In study CBHâ…¡is expressed firstly in cel lulase system,and the production can induce other genes expression.The expression of CBHâ…¡is inhi bited by glucose,so the ability of producing cellulase can be raised by gene cloning of CBHâ…¡,and i s cloneded to the downstream of eukaryon promoter.Expression vector was constructed,it can promot e coordinate expression of other cellulose genes and the cativity of producing cellulase.In this study,primer can be designed by cDNA sequence of Trichoderma viride,gene order of CBHâ…¡amplificated by PCR.The gene was conjugated with the carrier of pUT,and was shifted i nto DH5α,Recombinant plasmid of pUT-CBH is obtained.Recombinant plasmid of pUT-CBH and f ragment of strong promoter PKI A can be shift in DH5α,then recombinant plasmid of pUT-CE is obtained.By using AcLi and electrotransformation,The expression plasmid were transform into Tric hoderma viride cell.Then it was qualitative assayed by Ben A and Congo Red method,the enzyme a ctivity was assayed by fermentation..Highe expression,enzyme activity,genetic stability can be obtain ed.50℃,200rpm,culturing 72h,the activity of enzyme is the highest The enzyme activity of total enzy me,Cx,C₁,glucosidase is 45u/ml/h,1600u/ml/h,160u/ml/(24h),15u/ml/h.The cellulose engineering bacteria which had been obtained provids the important material to the study of the expression of autogenic transformation for the T.viride cellulase genes and the a nalyses of characteristics of recombinant.From the results of the SDS-PAGE,the enzyme activity a nd characteristic of enzyme for converter,the quantity of expression,enzyme activity,genetic stability are higher than host.It can provid theory for autogenic transformation of cellulose gene and deg radation of cellulose.