A mechanistic model for rational design of optimal cellulase mixtures

Abstract

A model-based framework is described that permits the optimal composition of cellulase enzyme mixtures to be found for lignocellulose hydrolysis. The rates of hydrolysis are shown to be dependent on the nature of the substrate. For bacterial microcrystalline cellulose (BMCC) hydrolyzed by a ternary cellulase mixture of EG2, CBHI, and CBHII, the optimal predicted mixture was 1:0:1 EG2:CBHI:CBHII at 24 h and 1:1:0 at 72 h, at loadings of 10 mg enzyme per g substrate. The model was validated with measurements of soluble cello-oligosaccharide production from BMCC during both single enzyme and mixed enzyme hydrolysis. Three-dimensional diagrams illustrating cellulose conversion were developed for mixtures of EG2, CBHI, CBHII acting on BMCC and predicted for other substrates with a range of substrate properties. Model predictions agreed well with experimental values of conversion after 24 h for a variety of enzyme mixtures. The predicted mixture performances for substrates with varying properties demonstrated the effects of initial degree of polymerization (DP) and surface area on the performance of cellulase mixtures. For substrates with a higher initial DP, endoglucanase enzymes accounted for a larger fraction of the optimal mixture. Substrates with low surface areas showed significantly reduced hydrolysis rates regardless of mixture composition. These insights, along with the quantitative predictions, demonstrate the utility of this model-based framework for optimizing cellulase mixtures.

Induction of lignocellulose-degrading enzymes in Neurospora crassa by cellodextrins

“…The Δ3βG and Δ3βGΔcre strains, which lack three β-glucosidases, produced mostly cellobiose. These data are consistent with the hypothesis that the three β-glucosidase enzymes provide the bulk of the glucose-generating activity in WT cultures (37).…”

Systems-level Modeling with Molecular Resolution Elucidates the Rate-limiting Mechanisms of Cellulose Decomposition by Cellobiohydrolases

“…Via this calibration procedure, the desorption rate constant of TrCel7A was estimated to be in the range 1 ϫ 10 Ϫ3 to 1 ϫ 10 Ϫ2 s Ϫ1 , consistent with the values inferred from the bulk measurements of reaction kinetics (21)(22)(23)(24). The reference value of the complexation rate constant employed in our SLATE simulations was set to 1 ϫ 10 Ϫ3 s…”

Designing novel cellulase systems through agent-based modeling and global sensitivity analysis

“…Modeling results suggest enzyme engineering efforts focused on improving the catalytic action of exoglucanase enzymes would be valuable while metabolic engineering strategies to optimize the exoglucanase to endoglucanase ratio is another area of importance. Few efforts to optimize these ratios are currently under way 41,42 though more efforts are anticipated in the future. Improving enzyme half-life is another area of great significance.…”

“…It has been shown recently that the endoglucanase is more important to the cellulase mixture when hydrolyzing cellulose with higher degree of polymerization. 41 An upper bound on the concentration of endoglucanase in the enzyme cocktail has been suggested due to the balancing act of exoglucanases as more chain ends become available. The model presented in this research does predict this upper bound on endoglucanase concentration and the balancing act of the endo-exo-synergy is evident in Figure 4.…”