| Cellobiohydrolases (CBH) are one of the most important components of Trichoderma reesei cellulase in the efficient enzymatic hydrolysis of lignocelluloses and key to the enzymatic hydrolysis of microcrystalline cellulose catalyzed by cellulase. T. reesei cbh2 gene was cloned and heterologously or homologously expressed in Escherichia coli, Pichia pastoris and T. reesei, respectively.T. reesei cbh2 gene was obtained by reverse transcription PCR and used for recombinant vector construction which was subsequently transformed into E. coli. The result of enzyme production induced by IPTG showed that T. reesei cbh2 gene was successfully expressed inside E. coli cells.cbh2 gene and cbh2s whose codons were optimized were used to construct recombinant vectors for heterologous expression in P. pastoris, respectively. Two excellent P. pastoris transformants PC-1 and PC-2, encompassing cbh2 and cbh2s respectively, were obtained after screening. The SDS-PAGE result of fermentation broth showed single band with molecular weight 58 kDa that is recombinant CBH II with about 5 kDa glycosylation by P. pastoris.Enzyme production of PC-1 and PC-2 induced for 96 h was compared and PC-2 was found to have 2.02 times higher cellobiohydrolase activity and 1.66 times higher soluble proteins than PC-1. The result indicated that the strategy of codon optimization was able to enhance the heterologous expression of cbh2 gene in P. pastoris.The optimal pH and temperature of the CBH Ⅱ produced by recombinant P. pastoris were found to be 5.0 and 50℃, respectively. The 3D models of CBD and CD regions were made in this work.In order to improve the synergistic degradation ability of T. reesei cellulase, the gene cbh2 was placed onto the downstream position of the T. reesei strong promoter Pcbhl and its signal peptide gene, further using pCAMBIA1300 as vector skeleton, forming the recombinant vector pCAMBIA1300-hph-PsCT with hygromycin B as resistance marker. I was transformed into T. reesei by Agrobactrium tumefaciens-mediated transformation (AMT). The factors affecting the AMT of T. reesei were optimized and the transformation efficiency was significantly enhanced. As to the specific requirement of the selection of T. reesei transformants with high cellobiohydrolase activity, the two steps of screening, which is hygromycin B resistance secreening in combination with growth rate secreening of colonies on microcrystalline cellulose agar plate, was designed and employed, by which 10 T. reesei transformants with high cellobiohydrolase activity were obtained from 326 T. reesei transformants.Time courses and enzymatic activities of the T. reesei transformant C10 were investigated using the medium and the conditions optimized. After 120 h fermentation in shaking flasks, the endoglucanase and cellobiase activities of C10 were at the same level as ZU-02, but the cellobiohydrolase activity of C10 was 122.44 U/mL,5.4-fold higher than that of ZU-02. Because the cellobiohydrolase activity in the T. reesei cellulase was significantly enhanced, the total enzymatic activity (filter paper activity) was elevated to 28.92 IU/mL,4.3 times higher than that of ZU-02.The cellulase from recombinant T. reesei was applied to the enzymatic hydrolysis of lignocelluloses using the cellulase from original strain ZU-02 as control. Under same conditions, the cellulase from recombinant T. reesei C10 had a yield of 93.8% in the enzymatic hydrolysis of corn stover, while the cellulase from ZU-02 was only 81.1%. This work has an important role in promoting the directed evolution of cellulase and the conversion and utilization of lignocelluloses. |
PDF Full Text Request