Water as a hydrolysis substrate: cellulase inhibition at high dissolved solids content

Cellulases are able to degrade cellulose at very low water content, but the hydrolysis yield decreases as the dry matter content increases, a phenomenon known as the high solids effect. Studies have shown that end product inhibition and mass transfer limitations are some of the factors involved in this process. In this study, we show that decreasing yields can also be caused by a decrease in the amount of free water available to the hydrolytic enzymes. By increasing the concentration of buffer salts and monosaccharides in the liquid fraction, these soluble molecules start interfering with the protein hydration shell and decrease cellulase performance regardless of the pH stability of the hydrolysate. The decrease in water availability is linked to the solubility and partial molar volume of the soluble molecules present, as well as the T₂ relaxation time of the liquid fraction. Low-field NMR showed linear correlations between the T₂ relaxation time of the liquid fraction and the hydrolysis yield. The depressed conversions were then linked to structural and chemical properties of the solutes studied. The only case of end product inhibition was observed with the β-glucosidases, where glucose production decreased only in the presence of glucose. Unlike other monosaccharides, glucose also prevented the cellulases from binding to the substrate. Therefore, cellulases are intrinsically inhibited by glucose and non-competitively inhibited in the presence of highly concentrated soluble molecules which decrease the water availability for hydrolysis to occur.