Cellulose Reducing Ends (DPr) Play No Role in Cellulose Recalcitrance to Biological Conversion with T. reesei Enzymes

In the literature reports, mostly applying soluble substrates, cellulose reducing ends (REs) have been shown to play a major role in the activity of cellobiohydrolase I, to some extent, of cellobiohydrolase II, and synergy between them (Teeri et al. 1995; Zhang and Lynd 2006). However, hydrolysis studies applying insoluble substrate are limited. In one such report, Xu et al. recently reported that oxidation of REs and primary hydroxyl groups (PHGs), significantly affected cellulose conversion (Xu et al. 2009). However, cellulose REs following oxidation were not reported in this study. On the contrary, a few other studies reported a limited effect of REs on the cellulose conversion (Kurašin and Väljamäe 2011). Therefore, in this study, the role of REs in insoluble cellulose recalcitrance to biological conversion with T.ressei derived enzymes was investigated. Pure cellulose compounds were subjected to sodium chlorite-acetic acid (SC/AA) and bicinchoninic acid (BCA) oxidation and sodium borohydride (NaBH₄) reduction to block the REs. SC/AA treated cellulose solids had >60% less REs than for the untreated controls, while BCA applied for various cycles oxidized cellulose to various extents and NaBH₄ treatment was able to reduce >95% of the REs. Enzymatic hydrolysis of resulting solids with complete cellulase at protein loadings as low as 5 mg/g glucan and with purified cellulase revealed that cellulose REs and corresponding degree of polymerization (DP_r) played no role in cellulose recalcitrance to biological conversion. On the other hand, oxidation of the PHGs significantly affected cellulose digestibility but only at low cellulase loadings.