Improved enzyme-substrate interactions enhance cellulose hydrolysis when using high biomass concentrations and low enzyme loadings

Although considerable progress has been made in reducing the cost of “cellulases” mixture, relatively high protein loadings are still required to achieve effective cellulose hydrolysis, restricting the economic viability of the “biomass-to-sugar” process. It is widely acknowledged that the rate and extent of the cellulolytic hydrolysis is influenced, not only by the effectiveness of enzyme cocktail, but also by the physical and chemical properties of pretreated biomass. In the work that will be described the interactions of key lignocellulosic enzymes and various biomass substrates pretreated under industrially relevant conditions were assessed.

The morphological and physicochemical changes of the pretreated biomass (e.g. gross fiber characters, lignin/hemicellulose content/location, and cellulose accessibility/crystallinity/DP) at various stages of cellulose deconstruction process were monitored. The roles and functions of major cellulases and accessory enzymes at each stage of hydrolysis were investigated. Various methods, such as Simon’s Stain and substructure-specific Cellulose Binding Modules (CBM’s) techniques were used to quantify changes in the accessibility of lignocellulosic substrate at the macroscopic (fiber), microscopic (fibril) and nanoscopic (microfibril) level. The enzyme adsorption/desorption profiles during hydrolysis were studied using various traditional enzyme/protein based assays and a lab developed enzyme-linked immunosorbent assay (ELISA). The influence of potentially inhibitory biomass-derived soluble compounds on the slowdown of enzymatic hydrolysis was also assessed.

The overall protein/enzyme loading required to achieve effective cellulose hydrolysis can be significantly reduced by optimizing the enzyme composition for a particular substrate, particularly when high biomass concentrations are used to increase the final sugar concentrations.