Studies On Cloning And Expression Of Endoglucanases Gene And Its Catalytic Domain Key Amino Acids From Trichodermalongibrah Iatum

Endoglucanase(egl)is one of the most important components of cellulose enzyme,which has been widely applied in several areas such as the biomass energy,food,feed,fabric washing,paper pulp.In this research,with genetic engineering techniques,the author constructed recombinant expression vectors of egI gene and studied the function of the catalytic domain structure.The main result were as follows,The mature peptide of egI(1386bp)was amplified with a template of the recombinant plasmid T-egI.It was inserted into vectors of pET-28a(+),pET-28-egI,pAMAL-C2X and pACYC-Dute were transformed to Escherichia coli Rosset(DE3).SDS-PAGE electrophoresis analyzes its expression products in the vicinity of a significant expression of 43kDa bands.Product of expression in E.coli was about 10%of bacterial whole proteins,and there were not any activity of endoglucanase.Althoughthe results have poor contributy to the industrial production,expression of cellulase genes in prokaryotic still provides a theoretical basis for the next studies.The gene of egl was then inserted into vectors of pPIC9K,which were transformed to Pichia pastoris GS115 by electroporation.In the culture supernatant,the activity of the degradation of carboxymethyl cellulose was certified and analysed,SDS-PAGE detection showd that the molecular weight of functional protein was 48 KD.Detection of the enzyme activity showed that the optimal temperature was 50-55 ?,and the optimal pH was 5.0.In the optimal conditions for electroporation,the author obtained 6 geneticin highly resistant recombinant expression strains in 100 randomly selected transformants using secondary shock.With methanol induction,the product of the high resistant recombinant expression vector had an activity of endoglucanase,and the expression level exceeded 21.5U/ml in P.pastoris under the optimal pH and temperature conditions,the author obtained 6 geneticin highly resistant recombinant expression strains in 100 randomly selected transformants using secondary shock.With methanol induction,the product of the high resistant recombinant expression vector had an activity of endoglucanase,and the expression level exceeded 21.5 U/ml in P.pastoris under the optimal pH and temperature conditions.In oder to figure out the key amino acid in enzyme catalytic domain structure(CD),the segments of the catalytic domain were knocked out and point mutations of the egl encoded gene were also performed.In the comparison with the nzyme activity of mutant vector transformed in Pichia pastoris,the result suggested that three amino acids including 194D,196Q,218E in the catalytic domain are important for enzyme activity.To sum up,this results can be provided with a good reference in the rational design of cellulose enzyme and the construction of cellulase high-yield strain.