(409g) Enzymatic Hydrolysis of Cellulosic Biomass in Nonaqueous Solvents

Cellulases and xylanases are two classes of glycoside hydrolases that act upon biomass in order to produce simple sugars for later fermentation or catalytic upgrading into biofuels. Ionic liquids are organic salts that by definition have melting points below 100°C. Ionic liquids are of interest to those concerned with cellulosic biomass because they help to unravel the complex mixture of cellulose, hemicellulose, and lignin that constitute the substantial portion of most biomass. After pretreatment of the substrate with ionic liquids, cellulase and xylanase gain additional interfacial over which they can more easily and more quickly hydrolyze long polysaccharide chains. Yet, this type of pretreatment requires an additional separation step in which the ionic liquid is separated from the mixture before enzymatic hydrolysis can occur. If one could design compatible ionic liquid-enzyme systems, this separation step may be incorporated into existing downstream processes. Certain glycoside hydrolases have been known to still yield appreciable amounts of sugar as product in low concentrations of ionic liquids in water. One study discovered two hyperthermophilic cellulases that retain nearly complete activity in low concentrations of [Emim][OAc] in water even at highly elevated temperatures [1]. We have discovered that the family 11 xylanase from Trichoderma longibrachiatum also retains enzymatic activity with reduced yield at concentrations of up to 20 wt% [Emim][EtSO₄] or [Emim][OAc] in water.  We have employed molecular dynamics (MD) simulation in order to uncover the mechanisms by which these enzymes are affected by the addition of non-aqueous cosolvents.

Our simulations and the relevant experimental work of other groups do not show that enzyme denaturation is the cause of the lost activity. On the contrary, our simulations indicate that the highly ionic systems display much more fidelity to crystallographic structure when compared to water, as measured by root mean square displacement (RMSD) on time scales of up to 500ns. Our simulations suggest that ionic liquids, even in low concentrations, dampen the enzymes’ dynamic motions that may have implications in proper substrate binding and enzyme activity. In systems that remain more active in ionic liquids, these motions tend to be less affected by the presence of the solvent. In more aqueous solvents, key features of the enzymes fluctuate more rapidly and with a higher magnitude, as measured by root mean square fluctuations (RMSF). For the systems in which ionic liquids reduce the yield of the enzymes, the mixed solvents appear to disrupt a network of coordinated motions and change the pattern of intrinsic fluctuations. Along with disruption of dynamic motions, we found that cations tend to become kinetically trapped near the catalytic residues of the enzymes. Therefore, inhibition might also play a role in lowering catalytic yield. For these systems of glycoside hydrolases, our simulations suggest that dampened dynamics and inhibition are the two most likely causes of hindered enzymatic function in these ionic liquid systems, while denaturation may not play a role at all.

[1] S. Datta et al., “Ionic liquid tolerant hyperthermophilic cellulases for biomass pretreatment and hydrolysis”, Green Chemistry, 2010, 12, 338-345.