Construction Of Cellulase Mutant Pool And Screening High Activity Mutants

Since the problem of energy shortage is becoming more and more serious, thismajor theme is looking for a renewable energy. Cellulose is the largest resources ofcarbohydrate polymers on Earth. It is the mainly renewable sources of energy and rawmaterials. Therefore, cellulase which is able to hydrolysis cellulose gets more andmore attentions. All the researches aim to find cellulases with highly activity andstable. However, most works choose soluble substrate, whose structure is differentfrom the nature substrate.Therefore, cellulase which is screened in this way don’thave a highly activity towards the nature substrate. Unfortunately, insoluble substratecan not go through the cell membrane, and can not be hydrolyzed by the cellulaseinside. So a cell surface display-releasing system which is perfect for the directedevolution of cellulases was constructed.CpCel5A from Clostridium phytofermentans was cloned and expressed by proteinengineering technology, and constructed into cell surface display-releasing system（pET20b-INP-5A,pET20bp-INP-I-Cel5Aand pET20b INP-In-Cel5A）, respectively.Cell surface display and release function is determined by M9and transparent circle.Meanwhile， the mutant pools of endoglucanase CpCel5A and progressiveendoglucanase CpCel9from Clostridium phytofermentans was constructed by cellsurface display-controlled releasing system and screened. The database buildingmethod that we used is POE-PCR.PCR and sequencing proved that pET20bp-INP-Cel5A、pET20bp-INP-I-Cel5and pET20b INP-IN-Cel5A have been constructed. Polyacrylamide gelelectrophoresis showed the INP-Cel5A, INP-I-Cel5A and INP-IN-Cel5A protein wassuccessfully expressed in E.coli. M9and transparent circle experiments showedCpCel5A was transported to the cell surface by ice nucleoprotein protein, and then spliced by intein, so Cel5A can digested RAC around the E.coli. Compared to surfacedisplay-releasing system, cell surface display-controlled releasing system can carryhigh molecular weight protein and the effect of releasing is better. The research hasconstructed mutant libraries of CpCel5A and CpCel9using poe-pcr. It is showed thatthe pET20b-INP-In-5A and pET20b-INP-In-9was successfully constructed, andcompleted screening of mutant library.