Cloning Of Cellulase Gene From Trichoderma Viride And Expression In Escherichia Coli And Saccharomyces Cerevisiae

Cellulose is the most abundant biological compound on earth.cellulase canhydrolysis cellulose to liberate the individual glucose and provide the material toferment, production and so on. How to use the gene engineering to develope thecellulase activity is the most challenge toward us.Trichoderma.viride could produce cellulase, and its cDNA was used astemplate to amplified the gene encoding endonucleases egⅢandcellobiohydrolases cbhⅡ. The PCR fragment was double digested withrestriction , and the target fragment was recovered. Then it wasinserted into pSE-380 vector and pYES2 vector, respectively. The recombinantexpression plasmids pSE-cbhⅡand pYE-cbhⅡand pYES-egⅢwere obtained,and then they were transformed into E.coli and S. cerevisiae. After the threerecombinants were induced to express, SDS-PAGE showed that the BL21(DE3)contained pSE-cbhⅡhave expressed the gene of cbhⅡand protein band of47KD were present. But there are no protein band have been detected in Saccharomyces cerevisiae。After determing The levels of CBHⅡand EGⅢactivity expressed by E. coli BL21/pSE-cbhⅡ,S. cerevisiaeINVScⅠ/pYES-cbhⅡand S. cerevisiae INVScⅠ/pYES-egⅢ, the EGⅢactivityin S. cerevisiae INVScⅠ/pYES-egⅢwere up to 0.03U/ml, the optimum inducetime was 60 hour.The biochemical properties of EGⅢshowed that it had anptimum temperature of 60℃and PH of 4.8.