Mini-scaffoldin enhanced mini-cellulosome hydrolysis performance on low-accessibility cellulose (Avicel) more than on high-accessibility amorphous cellulose

A glycoside hydrolase family 5 Bacillus subtilis endoglucanase, a family 9 Clostridium thermocellum processive endoglucanase, and a family 48 Clostridium phytofermentans ISDg cellobiohydrolase were assembled together by the high-affinity interaction between three cohesins in a mini-scaffoldin (mini-CipA) and dockerins in three cellulases, forming the mini-cellulosome. This mini-cellulosome exhibited enhanced hydrolytic activity on low-accessibility cellulose (microcrystalline cellulose, Avicel) and high accessibility cellulose (regenerated amorphous cellulose, RAC) as compared to the non-complexed cellulase mixture at the same enzyme amount. The stimulation factors (SF, i.e., activity ratios of the mini-cellulosome to the non-complexed cellulase mixture) were larger on Avicel than on RAC regardless of substrate/enzyme ratios. Also, SF increased when substrate/enzyme ratio increased. The different hydrolysis patterns of the mini-cellulosome and cellulase mixture on Avicel and RAC suggested that the construction of synthetic cellulosomes would be an efficient way to significantly enhance cellulose hydrolysis rate and digestibility, especially in the case of low-accessibility recalcitrant cellulose at low enzyme usage.

► The synergistic effect of cellulosome was more significant on Avicel than on RAC.
► The synergistic effect of cellulosome increased when enzyme usage decreased.
► Construction of cellulosome would increase cellulose conversion at low enzyme usage.