Phage Display Of The Cellobiohydrolase I Produced By Trichoderma Reesei

Cellobiohydrolase I from Trichoderma reesei is a most widely studied kind of exoglucanase. Studies about CBHI mostly focus on two aspects: one is the further explanation for the mechanisms of CBHI hydrolyzing crystalline cellulose; the other is to improve the properties, specificity, stability or catalytic activity of CBHI to make it suitable for biotechnological applications. The phage display of CBHI lays the foundation for these researches.This thesis constructed two recombinant phagemids pCANTAB 5E-cbhl and pCANTAB 5E-cd. Then transformed them into E.coli TGI and expressed CBHI on the phage tip by the infection of M13K07 helper phage.The cbh1 and cd (the DNA sequence of CBHI and the catalytic domain of CBHI) DNA fragments with Sfi I and Not I at 5'and 3'termini were prepared by PCR using plasmid pUCmT-cbh1 as template and two pairs of designed primers both carried Sfi I in upstream and Not I in downstream. After digested by both Not I and Sfi I, cbhl and cd DNA fragments were linked with phagemid PCANTAB 5E. The ligation products were transformed into competent TGI. Positive clones were screened. The recombinant phagemids pCANTAB 5E-cbhl and pCANTAB 5E-cd isolated andpurified from positive clones were confirmed by digesting with both Not I and Sfi I. The results showed that cbhl and cd DNA were linked with phagemid pCANTAB 5E successfully.The helper phage M13K07 was used to rescue the phagemid with cbhl gene insert from the transformed E.coli TGI cells. Enzyme linked immunosorbent assay (ELISA) showed that the recombinant phage specifically bound to filter paper; the exoglucanase activity of the displayed CBHI estimated with pNPC as the substrate was 2.625x 10" 14IU/CBHI molecule. The recombinant phage was also used to infect E.coli HB2151 cells for large-scale production of soluble recombinant CBHI. The molecular weight of the soluble CBHI protein was measured to be 54kDa, which was the same with the wild type CBHI from Trichoderma reesei. All these results showed that Cellobiohydrolase I was displayed by the Ml3 phage successfully.