The Role of Evolving Interfacial Substrate Properties on Heterogeneous Cellulose Hydrolysis Kinetics

Abstract

Interfacial enzyme reactions require formation of an enzyme–substrate complex at the surface of a heterogeneous substrate, but often multiple modes of enzyme binding and types of binding sites complicate analysis of their kinetics. Excess heterogeneous substrate is often used as a justification to model the substrate as unchanging, but using the study of the enzymatic hydrolysis of insoluble cellulose as an example, we argue that reaction rates are dependent on evolving substrate interfacial properties. We hypothesize that the relative abundance of binding sites on cellulose where hydrolysis can occur (productive binding sites) and binding sites where hydrolysis cannot be initiated or is inhibited (nonproductive binding sites) contribute to rate limitations. We show that the initial total number of productive binding sites (the productive binding capacity) determines the magnitude of the initial burst phase of cellulose hydrolysis, while productive binding site depletion explains overall hydrolysis kinetics. Furthermore, we show that irreversibly bound surface enzymes contribute to the depletion of productive binding sites. Our model shows that increasing the ratio of productive to nonproductive binding sites promotes hydrolysis, while maintaining an elevated productive binding capacity throughout conversion is key to preventing hydrolysis slowdown.

Reduced type‐A carbohydrate‐binding module interactions to cellulose I leads to improved endocellulase activity

“…However, the importance of appended CBMs to cellulase activity has been recently brought into question by their apparent insignificance under high solids biomass loadings in biorefinery settings (Pakarinen et al, 2014;Varnai et al, 2013). In addition, CBMs have been predicted to lead to nonproductive binding of cellulase enzymes to cellulose although predictions from these kinetic models were not proven experimentally (Gao et al, 2013a;Nill and Jeoh, 2020). Moreover, the impact of CBM fusion on activity of commercially relevant cellulases toward pretreated substrates is not well understood either (Kim et al, 2010).…”

Analysis of the phosphorylome of trichoderma reesei cultivated on sugarcane bagasse suggests post-translational regulation of the secreted glycosyl hydrolase Cel7A

“…We also speculate that phosphorylation is a way to control biomass degradation, since the increased hydrogen bonds might somewhat induce the immobilization of the protein. Since cellulases need to pass through the cellulose surface to break it, an immobilized enzyme might indicate a lack of productivity [ 72 , 73 ]. However, the opposite may also be inferred, since the dissociation time of Cel7A is much longer than the formation of cellobiose product itself, giving the enzyme a higher chance of feeding its active tunnel with the substrate [74] .…”

Modeling coordinated enzymatic control of saccharification and fermentation by Clostridium thermocellum during consolidated bioprocessing of cellulose

“…Our reasoning is, in part, supported by the findings of Kothari et al (2019), where cellulose utilization by C. thermocellum is unaffected by cellulose accessibility and often attains complete cellulose solubilization with little to no hindrances. Besides, Nill and Jeoh (2020) also demonstrated that productive binding sites of microcrystalline cellulose (Avicel) often decrease rapidly at low conversions (<20% conversion) during enzymatic hydrolysis and stabilize thereafter. Conversely, C. thermocellum in this case only ceases to grow in the late stages of CBP (>80% conversion; Figure 4), which begs the question of whether the decline in the accessible area for cellulase action truly played any role in this phenomenon.…”

“…This is possible through a combination of experimental procedures to estimate the evolution of the bound fraction of cellulase concentrations using sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE; Y.‐H. P. Zhang & Lynd, 2003, 2005) and estimation of productive binding sites through hydrolysis rates obtained from real‐time, sub‐second measurements of cellobiose concentrations using specialized biosensor (Nill & Jeoh, 2020). Similarly, although overflow metabolism is believed to cause the premature halt in biomass growth under high solid loadings (Holwerda et al, 2014), the possibility of flux re‐distributions to growth‐decoupled cellulosome‐secreting pathways, the induction of famine environments and mass transfer limitations due to high solid loadings should not be overlooked.…”