Heterologous Expression Of GH5 And GH12 Family Cellulase Of Penicillium Oxalicum And Study On The Function Of Physiological

Nature is rich in biomass resources.With the depletion of fossil fuels and the deterioration of the ecological environment,scientists from all over the world are paying attention to the renewable resources,especially bioethanol and biodiesel.Cellulose is the most abundant renewable resource.At present,acid hydrolysis and enzymatic hydrolysis were used to degrade cellulose.Compared with acid hydrolysis,the enzymatic hydrolysis is favored by mild reaction conditions and no toxic substances.Improving the activity of single enzyme components is an effective way to improve the cellulose degradability by genetic manipulation,but it is difficult to significantly improve the cellulose degradation ability of cellulase at now.It is important to study the composition of cellulase system and identify the enzymatic properties and functions of the components for reconstructing the enzyme complexes suitable for different substrates and improving the function of the cellulase system.Most cellulase genes are induced by cellulose.They also have basic expression under non-induced conditions.The oligosaccharides were produced by cellulase from basic expression,which further induces the expression of the cellulase gene.Inaddition to,there are present of constitutive expression genes,which may play the same role as cellulase from basic expression at the early stage of cellulose induction.Identification of these cellulase's functions has great significance for improving the cellulase's gene induction and regulation networks.The main research progress of this paper is as follows:1.Endoglucanase expression in Pichia pastorisThe primers for encoding gene cel5B,cel5D,cel5E,cel5F,cel12A,cel12B and cel12C were designed.According to the multiple cloning sites of pPIC9K vector,appropriate cleavage sites were added at end of the primers,and 6-8 His tags were also added at the C-terminus.Seven target fragments were amplified by using cDNA of 114-2 as template,and the target fragments and vector were treated with restriction endonuclease to construct recombinant vectors.Linearized recombinant vectors were transferred to GS115 strain by electroporation.The recombinant proteins' expression was induced by methanol and analyzed by SDS-PAGE.Recombinant endoglucanases rCel12A,rCel12B,rCel12C,rCel5B and rCel5E were successfully obtained.2.Recombinant proteins and their enzymatic properties Recombinant proteins rCel12A,rCel12B,rCel12C,rCel5B and rCel5E were obtained by affinity purification.Sodium carboxymethylcellulose,locust bean gum,xylan,pNPG,pNPX,salicylline,sucrose,pectin,starch and polygalacturonic acid were used to find their optimum substrate.The results show that they all belong to endoglucanase.The specific activities of CMCase by rCel12A,rCel12B,rCel12C,rCel5B and rCel5E were 2.46 IU/mg,0.97 IU/mg,0.63 IU/mg,2.32 IU/mg and 5.07 IU/mg,respectively.The ability of rCel5E acting on CMC-Na was twice of rCel5B and rCel12A,5.2 times of rCel12B and 8.0 times of rCel12C.The optimum temperature of rCel12A was 50? and that of the other four recombinant proteins were 60?.The optimum pH of rCel5B,rCel5E,rCel12B,rCel12C and rCel1 2A was 4,4,4,5,6,respecitively.The experiment found that rCell2A had a poor thermal stability.When it was incubated at 50? for 30 minutes,the vitality is 30%of the initial activity.However,rCel12B has 50%activity after incubation at 50 ? for 120 minutes.After incubation at 60 ? for 120 minutes,rCel5B and rCel5E can still retain 60%-70%activity.In 70 ?,all recombinant proteins lose the activity quickly.The results showed that the thermostability of rCel5B and rCel5E in the GH5 family was better than that of rCell2A and rCel12B from the GH12 family.The results of regenerated amorphous cellulose(RAC)degradated by recombinant endoglucanase showed that the main products were disaccharide and trisaccharide.3.Preliminary study on the function of constitutive cellulase Five single gene knockout strains ?cel12C,?cel5E,?cel5F,?Acel61B,?Acel61C and six double knockout strains Acell2C?cel5E,?cel5FAcel5E,?cel61BAcel5E,?cel61BAcel5F,?cel61C?cel5E and ?cel61C?cel5F were obtained by homologous recombination.The phenotype and spore germination have no difference between original strain and gene knockout strains under the culture conditions of glucose,PDA and cellulose.The biological functions of cel12C,cel5E,cel5F,cel61B and ce161C above need to be explored...