Cloning Of Cellulase CBH1Gene From Trichodcrma Koningii And Its Expression In Pichia Pastoris

Cellulose is a kind of recycled resource that accounts1/3~1/2of the plant dry weight and is themost abundant organic carbon source on earth. If these natural resources can be effectively used, itwill have practical significance for humankind to solve the energy crisis, food shortages, andenvironmental pollution problems.Using Microorganisms and cellulase to break down cellulose is an effective and non-pollutingmethod. Cellulase is a biological catalyst of high activity which can degrade cellulose. It is widelyused in feed, environmental protection, food, wine, textile, papermaking and other fields. However,the natural microbial cellulase exists many problems such as low yield, low activity and productioninhibition by glucose, which largely restrict the industrial production and application of cellulase. Inorder to obtain cellulase resources with high activity and high production rate, genetic engineeringtechnology has been paid more attention to.Cellobiohydrlases (CBH) are believed to be the most efficient enzyme which can releasecellobiose as a main product from highly crystalline cellulose. In this study, the CBHⅠgene wascloned from a high cellulase-producing Trichoderma koningii (T. koningii) and expressed in Pichapastoris (P. pastoris). The main contents were as follows.(1) CBHⅠ gene was amplified from genomic DNA of T. koningii by PCR and cloned inPMD19-T vector. Sequencing and sequence alignment results showed that the DNA sequence lengthwas1626bp and the homology reached99.63%, compared with Trichoderma viride (FJ871063.1) ingenbank.(2) CBHⅠ gene was inserted into secreting expression vector pGAPZαA to construct therecombinant pGAPZαA-CBH Ⅰ. It was linearized and transformed intoPichia pastoris byelectroporation. The recombinant Pichia pastoris was selected by zeocin-resistant, and then incubatedwith YPD medium for cellulase activity and SDS-PAGE analyses. The result showed that CMCaseand avicelase activity was1.1798and0.1276U/mL, respectively; and the molecular weight of theexpressed protein was53kDa determined by SDS-PAGE, indicating that cellulase CBHⅠ gene wassuccessfully expressed in Pichia pastoris. The optimal temperature and pH of the expressed cellulasewere45~50℃and4.5~5.0, respectively.