The Synergistic Effect Of The Rumen Bacterial Expansion Protein And Cellulase

Expansin is a small molecule protein without enzyme activity.It can improvethe degradation efficiency of cellulase when it acts on the compact cellulose.The synergistic effect of expansin from different sources on cellulase was different.In this study,six expansin sequences with high to low homology to EXLX1 were screened from rumen bacteria of Ruminantia,and the function of these proteins and the relationship between their structure and function were studied,the results are as follows:(1)When the amount of expansin was 600 μg/g substrate,the synergistic effect of expansin and cellulase reached the maximum.The maximum synergistic effect of expansin TPD2 and cellulase was 127%.(2)When cellulase was added to 0.01 FPU/g substrate,the synergetic effect of each expansin and cellulase was the maximum,with the increase of the amount of cellulase,the synergistic effect of expansin on cellulase decreased gradually.(3)When p H increased from 5.5 to 7.0,expansins Y189,UV64 and S152 still had synergistic effect with cellulase,while expansins R2F0,TPD2,5IG3 and 4918 inhibited the hydrolysis of cellulase.After 8 hours at 40°C,the residual activity of expansin Y189 was the highest.(4)The synergetic effect of rumen bacterial expansin and cellulase was fully confirmed by scanning electron microscope of filter paper.Moreover,the affinity of expansin R2F0,TPD2,5IG3 and 4918 to substrate was higher than that of expansin Y189,UV64 and S152,by comparing the binding parameters of the expansin to the substrate.The maximum binding capacity of expansin UV64,5IG3,TPD2 and R2F0 to substrates is greater than that of expansin Y189,4918 and S152.(5)When the amount of protein is less than or equal to 600 μg/g substrate,the synergistic effect between the expansin TPD2 and cellulase is greater than that of TPD2ΔDM1.This result shows that the deletion of domain DM1 reduces the activity of TPD2ΔDM1,and the single domain DM2 still has the function of expansin.(6)When the addition of cellulase is increased to 0.06 FPU/g substrate,the synergistic effect of TPD2ΔDM1 and cellulase does not change much.The results show that the loss of DM1 is more beneficial to the function of TPD2ΔDM1 under the condition of high concentration cellulase.At the initial increase of enzyme concentration to 0.06 FPU/g substrate,the synergistic effect between TPD2-DM2 and cellulase did not immediately decrease like that of expansin TPD2.However,when the dosage of cellulase continued to increase to 0.8 FPU/g substrate,TPD2-DM2 became an obstacle for cellulase to function.It shows that the domain DM2 has the ability to bind to the substrate,which makes TPD2-DM2 with two domains DM2 and cellulase competitively bind to the substrate.(7)Compared with the expansin TPD2,the stability of TPD2ΔDM1 and TPD2-DM is higher,while the affinity of TPD2ΔDM1 and TPD2-DM2 to the substrate is lower,but the maximum binding amount to the substrate has no significant change.