3-36: The influence of sugar/ethanol concentration on enzyme adsorption, hydrolysis performance, and the feasibility of enzyme recycle as means of reducing the cost of cellulose hydrolysis

Relatively high enzyme/protein loadings are still required for effective hydrolysis of biomass to its constituent sugars. The relatively good thermostability of the enzymes present in a “cellulase mixture” makes reusing them for multiple rounds of hydrolysis an attractive strategy to further reduce enzyme costs. We and other groups have explored various enzyme recycle strategies. A central theme emerging from these studies suggests that a better understanding of the dynamics of enzyme-substrate interactions will be crucial if an effective and economical enzyme recycle strategy is to be developed. The enzyme-substrate interactions occurring during hydrolysis represents complex protein adsorption phenomena on a solid surface. Three main groups of factors influence this dynamic interaction including reaction conditions, substrate characteristics, and the nature of the hydrolytic proteins/enzymes.  Among these factors, the influence of glucose and ethanol on enzyme-substrate interaction, enzyme recycle, enzyme recycle insertion point, reactor designs, etc., has not been explored in detail. When the influence of glucose and ethanol on enzyme adsorption and recycle (using readsorption to fresh substrate) was assessed it was found that high glucose concentrations not only limited hydrolysis performance it also restricted enzyme readsorption to fresh substrates. Converting glucose to ethanol prior to enzyme recycle resulted in ~15% increase in enzyme recovery and  20% higher cellulose hydrolysis in a 2^nd round of hydrolysis using recycled enzymes. By limiting the glucose concentrations in the reaction mixture this reduced product inhibition, increased cellulose hydrolysis and improved the effectiveness of the enzyme recycle strategy.