Construction Of T.Reesei Cbh1Expression Carrier And Its Expression In A.Nigher

The key to improve the efficienty of cellulose is biodegradable of cellulose.Biodegradation has mild conditions,low cost and no pollution to the environment.Trichoderma reesei secretes series cellulolytic enzymes known to participate synergistically in the degradation of biomass,60%of total extracellular protein is CBH I.CBH I can hydrolyze the cellulose from the reducing end to release cellobiose and is considered to be the key enzyme component of cellulases in the degradation of cellulose.But secred cellobiohydrolase hydrolase gene expression level from Aspergillus niger is low,lead to the cellulose activity is not high.In this study,we tried to express cbhl in Aspergillus niger,thus attempts to establish the effective filamentous Aspergillus niger expression system for cellulases.The main results were illustrated as follows:1.Aspergillus niger had the highest enzyme activity at the fifth day,highest exoglucanase activity was19.88U/mL,highest endonuclease activity was23.40U/mL,the highest beta-glycosidase activity was87.20U/mL,the highest filter enzyme activity was27.01U/mL.The beta glycosidase enzymes were relatively high, but exoglucanase activity was low.2.The optimal preparing condition for the Aspergillus niger protoplasts was as follows:The16hour mycelium was obtained with the high-speed shaking condition at300r/min and digest with1%cellulose+l%snailase+0.1%lywallzyme in the osmotic stabilizer(lmol/L sorbitol) for2hour under30℃.3.The PUC118-cbh1as a template, cbhl target fragment amplification by PCR. The gene was conjugated with the express carrier pPTRⅡ,and was shifted into JM109, recombinant plasmid of pPTRⅡ-cbh1is obtained. The plasmid is confirmed to be constructed as expectation by enzyme digestion and sequence reaction.4.The cbh1gene expression carrier (pPTRⅡ-cbh1) was transformed into Aspergillus niger Y14protoplast with the PEG-CaCl2-mediated transformation method. The transformants that had integrated the gene were selected by PT, and selected cbhl by PCR.5.Measured that transformants exoglucanases total enzyme activity and filter paper activity reached to a maximum in the fourth days,of31.91U/mL and40.98U/mL,respectively,which were increased by1.61times and1.52times.