Abstract: To investigate the effect of the structure of designer cellulosome on the enzymatic kinetics,the designer cellulosome with different structures were constructed through extracellular self-assembly by anchoring cellulases to the primary scaffoldin Scaf I-CBM3a or the adaptor scaffoldin Scaf II-Cip A and Scaf II-Sca B.The thermostability difference between designer cellulosome and free enzymes was analyzed comparatively,the enzymatic kinetic analysis of steam explosion corn stover was carried out,the Michaelis constant（K_m）,maximum reaction rate(V_max)and substrate feedback inhibition constant（K）were obtained finally.As the results,the binding of cellulase and scaffold protein can reduce its thermal sensitivity and improve its thermal stability.The steam explosion corn stover of designer cellulosome with secondary scaffold protein has the value of K_m=0.02 mg/m L,V_max=19 mg/（m L·min）,K=0.04 mg/L.It indicates that the affinity between enzymes and substrates enhanced by secondary scaffold proteins is higher than that of the simple structure,K_mincreases by 19.8%.The complex spatial structure of designer cellulosome can change the enzymatic kinetics characteristics,even improve the thermostability of the cellulase.