Quantitative Determination of Cellulose Accessibility to Cellulase Based on Adsorption of a Nonhydrolytic Fusion Protein Containing CBM and GFP with Its Applications

Abstract

Heterogeneous cellulose accessibility is an important substrate characteristic, but all methods for determining cellulose accessibility to the large-size cellulase molecule have some limitations. Characterization of cellulose accessibility to cellulase (CAC) is vital for better understanding of the enzymatic cellulose hydrolysis mechanism (Zhang and Lynd, Biotechnol. Bioeng. 2004, 88, 797-824; 2006, 94, 888-898). Quantitative determination of cellulose accessibility to cellulase (m2/g of cellulose) was established based on the Langmuir adsorption of the fusion protein containing a cellulose-binding module (CBM) and a green fluorescent protein (GFP). One molecule of the recombinant fusion protein occupied 21.2 cellobiose lattices on the 110 face of bacterial cellulose nanofibers. The CAC values of several cellulosic materials -- regenerated amorphous cellulose (RAC), bacterial microcrystalline cellulose (BMCC), Whatman No. 1 filter paper, fibrous cellulose powder (CF1), and microcrystalline cellulose (Avicel) -- were 41.9, 33.5, 9.76, 4.53, and 2.38 m2/g, respectively. The CAC value of amorphous cellulose made from Avicel was 17.6-fold larger than that of crystalline cellulose - Avicel. Avicel enzymatic hydrolysis proceeded with a transition from substrate excess to substrate limited. The declining hydrolysis rates over conversion are mainly attributed to a combination of substrate consumption and a decrease in substrate reactivity. Declining heterogeneous cellulose reactivity is significantly attributed to a loss of CAC where the easily hydrolyzed cellulose fraction is digested first.

Comparative study of corn stover pretreated by dilute acid and cellulose solvent‐based lignocellulose fractionation: Enzymatic hydrolysis, supramolecular structure, and substrate accessibility

“…This model not only correlates disparate phenomena reported in the literature but also clearly suggests that low cellulose accessibility is the most important substrate characteristic limiting enzymatic hydrolysis rates (Zhang and Lynd, 2006). More recently, a quantitative assay for determining cellulose accessibility to cellulase (CAC) has been established based on adsorption of a non-hydrolytic fusion protein (TGC) containing a cellulose-binding module and a green fluorescence protein (Hong et al, 2007). This new approach more accurately assesses substrate characteristic related to enzymatic cellulose hydrolysis than traditional methods such as nitrogen adsorption-based Brunauer-EmmettTeller (BET), size exclusion, and small angle X-ray scattering (Hong et al, 2007;Zhang and Lynd, 2004).…”

“…The tub-ground materials were approximately 9 months old (harvested in fall 2005). The recombinant thioredoxin-green fluorescent protein-cellulose binding module (TGC) fusion protein was produced in Escherichia coli BL21 (pNT02) (Hong et al, 2007) and purified by affinity adsorption on RAC followed by modest desorption using ethyl glycol (EG) (Hong et al, 2008b). The EG was removed by using membrane dialysis in a 50 mM sodium citrate buffer (pH 6.0).…”

“…More recently, a quantitative assay for determining cellulose accessibility to cellulase (CAC) has been established based on adsorption of a non-hydrolytic fusion protein (TGC) containing a cellulose-binding module and a green fluorescence protein (Hong et al, 2007). This new approach more accurately assesses substrate characteristic related to enzymatic cellulose hydrolysis than traditional methods such as nitrogen adsorption-based Brunauer-EmmettTeller (BET), size exclusion, and small angle X-ray scattering (Hong et al, 2007;Zhang and Lynd, 2004). Regenerated amorphous cellulose (RAC) that is prepared from microcrystalline cellulose (Avicel) has $20-fold higher CAC (Hong et al, 2007(Hong et al, , 2008b and exhibits much faster enzymatic hydrolysis rates than microcrystalline cellulose (Zhang et al, 2006a), which is in agreement with the model prediction that increasing CAC is more important for increasing hydrolysis rates than decreasing DP (Zhang and Lynd, 2006).…”

“…This new approach more accurately assesses substrate characteristic related to enzymatic cellulose hydrolysis than traditional methods such as nitrogen adsorption-based Brunauer-EmmettTeller (BET), size exclusion, and small angle X-ray scattering (Hong et al, 2007;Zhang and Lynd, 2004). Regenerated amorphous cellulose (RAC) that is prepared from microcrystalline cellulose (Avicel) has $20-fold higher CAC (Hong et al, 2007(Hong et al, , 2008b and exhibits much faster enzymatic hydrolysis rates than microcrystalline cellulose (Zhang et al, 2006a), which is in agreement with the model prediction that increasing CAC is more important for increasing hydrolysis rates than decreasing DP (Zhang and Lynd, 2006). The CAC value of Avicel (m 2 per gram of Avicel) based on the TGC adsorption was only one-tenth of that based on the BET method (Marshall and Sixsmith, 1974), implying that about 90% gross surface area measure based on nitrogen adsorption cannot be accessible to largesize cellulase protein molecules, at least initially.…”

“…The CAC value of Avicel (m 2 per gram of Avicel) based on the TGC adsorption was only one-tenth of that based on the BET method (Marshall and Sixsmith, 1974), implying that about 90% gross surface area measure based on nitrogen adsorption cannot be accessible to largesize cellulase protein molecules, at least initially. Traditional size exclusion techniques are labor intensive and cannot distinguish the real cellulase binding area (110 face) or ignore the external surface area (Hong et al, 2007;Zhang and Lynd, 2004).…”