Do enzymatic hydrolyzability and Simons' stain reflect the changes in the accessibility of lignocellulosic substrates to cellulase enzymes?

In an attempt to elucidate the impact of substrate accessibility to cellulases on the susceptibility of lignocellulosic substrates to enzymatic hydrolysis, a hydrogen peroxide treated, Douglas fir kraft pulp was dried using several methods with varying levels of intensity. Oven-drying at 50 and 100 degrees C, air-drying, and freeze-drying methods were employed to remove the interfibrillar water from the pulp samples. Subsequently, the never-dried and variably dried pulps were hydrolyzed using a commercial cellulase preparation supplemented with additional beta-glucosidase. Drying reduced the susceptibility of the substrates to enzymatic hydrolysis, which can be attributed to the hornifying effect that drying has on fibers. This effect was more pronounced for the fibers that were oven-dried at 100 degrees C (23% reduction) and 50 degrees C (15% reduction), and there was a good correlation between the Simons's stain results and the enzymatic digestibility of the dried pulps. These observations indicated that drying significantly reduced the population of larger pores and that the partial closure of larger pores created a large number of smaller pores that were not accessible to the displacement dye molecules (orange dye). The inaccessibility of the cellulose to the enzymes, due to the collapse or closure of the large pores, appears to be the primary reason for the lower susceptibility of the dried pulps to enzymatic hydrolysis.

The effect of shaking regime on the rate and extent of enzymatic hydrolysis of cellulose.

In an attempt to elucidate the effect of mixing on the rate and extent of enzymatic hydrolysis of cellulosic substrates, alpha-cellulose was hydrolysed using a commercial cellulase preparation at varying levels of substrate concentration (2.5,5 and 7.5% (w/v)) and by using three shaking regimes: continuous at low-speed (25 rpm), continuous at high-speed (150 rpm) and an intermittent regime comprised of high and low-speed shaking intervals. The continuous, high-speed shaking produced the highest conversion yields, whereas the intermittent and low-speed shaking regimes resulted in lower conversions. After 72 h, at all shaking regimes (150 rpm,25 rpm and intermittent), using a low substrate concentration (2.5%) produced conversion yields (82,79 and 80%) higher than those obtained at high (7.5%) substrate concentration (68,63 and 68%). As the substrate concentration increased, the conversion yields at intermittent shaking gradually approached those resulting from high-speed shaking. Thus, it appears that intermittent shaking could be a beneficial process option as it can reduce the mixing energy requirements while producing reasonably high conversion yields.

Do cellulose binding domains increase substrate accessibility?

This article provides an overview of various theories proposed during the past five decades to describe the enzymatic hydrolysis of cellulose highlighting the major shifts that these theories have undergone. It also describes the effect of the cellulose-binding domain (CBD) of an exoglucanase/xylanase from bacterium Cellulomonas fimi on the enzymatic hydrolysis of Avicel. Pretreatment of Avicel with CBDCex at 4 and 37 degrees C as well as simultaneous addition of CBDCex to the hydrolytic enzyme (Celluclast, Novo, Nordisk) reduced the initial rate of hydrolysis owing to irreversible binding of CBD proteins to the substrate's binding sites. Nonetheless, near complete hydrolysis was achieved even in the presence of CBDCex. Protease treatment of both pure and CBDCex-treated Avicel reduced the substrates' hydrolyzability, perhaps owing to proteolysis of the hydrolyzing enzyme (Celluclast) by the residual Proteinase K remaining in the substrate. Better protocols for complete removal of CBD proteins from the substrate need to be developed to investigate the effect of CBD adsorption on cellulose digestibility.

Steam pretreatment of Douglas-fir wood chips. Can conditions for optimum hemicellulose recovery still provide adequate access for efficient enzymatic hydrolysis?

Douglas-fir sapwood and heartwood were impregnated with SO2 and steam exploded at three severity levels, and the cellulose-rich, water-insoluble component was enzymatically hydrolyzed. The high-severity conditions resulted in near complete solubilization and some degradation of hemicelluloses and a significant improvement in the efficiency of enzymatic digestibility of the cellulose component. At lower severity, some of the hemicellulose remained unhydrolyzed, and the cellulose present in the pretreated solids was not readily hydrolyzed. The medium-severity pretreatment conditions proved to be a good compromise because they improved the enzymatic hydrolyzability of the solids and resulted in the recovery of the majority of hemicellulose in a monomeric form within the water-soluble stream. Sapwood-derived wood chips exhibited a higher susceptibility to both pretreatment and hydrolysis and, on steam explosion, formed smaller particles as compared to heartwood-derived wood chips.