Construction Of Expansin Engineering Strain And Optimization Of Its Expresion Conditions

Cellulose was the most abundant and the most widely organics on earth.It was also a regenerative material,it can be degradated to glucose,this was the important link in nature.But because of high crystallization structure of cellulose and it was surrounded by lignin, directly influence the physico-chemical property of cellulose,so that it was difficult to hydrolyse into glucose.Expansin can soften and expand cell wall.Through many years reseach, scientists found that expansin can’t directly hydrolyse cellulose glucosidic bond,but it have synergism when cellulase hydrolyze cellulose.it can break hydrogen bond between cellulose molecules,this can destroy cellulose structure effectively, raise hydrogen efficiency of cellulase.In this study,cucumber D0462as the gene donator, extract total RNA,clone Cs-EXPA1and Cs-EXPA2gene, then construct Pichia pastoris composition expression vectors pGAPH a M-EXPA1and pGAPH a M-EXPA2, transform expression vectors into Pichia pastoris to obtain engineering strains, induct expression of expansin.Then optimization condition of expansin’s activity detection and engineering strains’expression, raise synergism hydrogen efficiency of expansin and cellulase to cellulose.The main results were as followed:1.Clone Cs-EXPA1gene and Cs-EXPA2from cucumber D0462by RT-PCR, the sequence was identical with sequence in GeneBank.2.Construct composition expression vectors pGAPHαM-EXPA1and pGAPHaM-EXPA2.3.Transform expression vectors pGAPHaM-EXPA1and pGAPHaM-EXPA2into Pichia pastoris by electric shock.Through identification of colony PCR and genome PCR indicated that exogenous gene had been integrated to the chromosome of Pichia pastoris.4.The correct transformant express protein by rocking bed cultivation,Through detection of SDS-PAGE gel electrophoresis found the interest protein at the29kD.5.Through comparison of Synergism between defferent expansin and cellulase,getting the synergistic activity of EXPA1protein and cellulase was17.7%, the synergistic activity of EXPA2protein and cellulase was59.8%, the synergistic activity of EXPA1protein EXPA2protein and cellulase was30.8%. Observation disintegration of filter paper through microscope found expansin had apparente disintegration on filter paper.6. When synergism of expansin and cellulase react36h,getting the most reducing sugar, the synergistic activity of EXPA1protein and EXPA2protein discern were41.9%and102.1%.The most compatible pH of synergism reacting of expansin and cellulase was4.0～5.0; the most compatible tempreture was50℃.7. We can get the most reducing sugar when used96h fermentation broth of engineering strains and cellulase degradat filter paper together;the most compatible pH of medium was7.0; the most compatible tenpreture of fermentation was28℃～30℃.