Measuring productive and non-productive binding of Trichoderma reesei Cel7A on cellulose

The inaccessibility of cellulose to cellulases limits saccharification rates of lignocellulosic biomass. To overcome this limitation, the bioconversion of lignocellulosic biomass typically employs thermochemical pretreatment to improve biomass digestibility. We hypothesize that quantification of the change in the accessibility of cellulose to cellulase due to pretreatment can be used as an indicator to aid in predicting the impact of the pretreatment on downstream enzymatic saccharification rates of the biomass. Increasing the adsorption of cellulases to biomass alone, however, will not necessarily lead to higher saccharification rates if the enzymes are not productively bound. Cellulases bound productively to insoluble cellulose hydrolyze glycosidic bonds, while those that are non-productively bound do not. The rate of cellobiose release by cellobiohydrolases is a function of the concentration of productively bound cellobiohydrolases. Moreover, recent advances in the understanding of the cellobiohydrolase Cel7A from Trichoderma reesei (TrCel7A) has shown that once productively bound, TrCel7A hydrolyzes crystalline cellulose with a first order catalytic rate constant, k_cat ~ 5 s^-1. Applying this finding, we have developed a direct method to measure productive and non-productive binding of TrCel7A on cellulose as a means to quantify the ‘accessibility of cellulose to cellulases’. We demonstrate this method by quantifying the accessibility of different types of cellulose (filter paper, phosphoric acid swollen filter paper, microcrystalline cellulose, bacterial and algal cellulose) to TrCel7A. We further discuss the application of this method to assess productive and non-productive cellulase binding in pretreated biomass impacting minimum cellulase loadings for effective saccharification.