(228x) Artificial Cellulosome Designed from Cellobiohydrolase Module Library on Streptavidin

Celluloses is naturally occurring plant cell wall components and most abundant polysaccharides on earth. An economical conversion from cellulosic biomass materials to water-soluble sugar units is expected to produce food, feed, chemicals and fuels. Microbial cellulolytic enzymes, called cellulases, can convert cellulose to water-soluble sugar units with low energetic and environmental loads. However, highly crystallized structure of cellulose in nature makes efficient hydrolysis hard, even using cellulases from cellulolytic fungus Trichoderma reesei, so that drastic improvement of enzyme activity is one of serious issues for efficient cellulose conversion. Many cellulases are consist of modular units of independent catalytic domains (CDs) and carbohydrate-binding domeins (CBDs), and on some rumen bacteria, CDs with different functions are clustered on a giant scaffold protein containing a CBD, via cohesin-dockerin interaction, called cellulosome, to efficiently degrade cellulose. To degrade cellulose effectively, two kinds of cellulase are essential, such as endoglucanases (EG), which randomly cleave internal bonds at amorphous cellulose, and cellobiohydrolases (CBH), which move processively along the cellulose chain and liberate cellobiose unit from the end of cellulose chain. Especially, CBH is a key enzyme capable of degrading crystalline cellulose. Recently, we proposed a new design of artificial cellulosome to drastically enhance cellulase activity.^[1-3] The clustering of CDs of EG with CBD on streptavidin via biotin-avidin interaction the reducing sugar produced by free CD. In this study, we clustered various CDs of CBH and CBD on a streptavidin to enhance the degradation activity. To identify the clustered enzyme with optimal CBH and CBD combination, 5 kinds of CBH and 8 kinds of CBD were clustered on streptavidin and analyzed the reducing sugar produced from phosphoric acid swollen cellulose. Consequently, 19 kinds of clustering enzymes with CBH showed the increase of produced reducing sugar, while 21 kinds of clustered enzymes decrease of the amount of sugar. The results showed that the clustered enzymes with CBH have a different behavior from those with EG, and indicates that module structure in CBH might be repuired to express the function in cellulose degradation.

[1] Kim et al., Small., 7 (5), 656-664(2011)

[2] Kim et al., Catalysis Science and Technology., 2 (3), 499-503(2012)

[3] Nakazawa et al., ACS Catalysis., 3, 1342-1348, (2013)