The productive cellulase binding capacity of cellulosic substrates

2019

Preprint

Self Cite

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 of 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 (non-productive 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 non-productive binding sites promotes hydrolysis, while maintaining an elevated productive binding capacity throughout conversion is key to preventing hydrolysis slowdown.

Section: Resultssupporting

confidence: 61%

Section: Quantification Of Solubilized Sugarsmentioning

confidence: 99%

Section: Product]mentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

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

Nill

2019

Preprint

Self Cite

“…Comparison of the measured cellobiose release after 30 seconds for reactions of 0.5 g/L of cellulose with 130 µmole/g Cel7A by biosensor and HPAEC-PAD are shown in Figure S4, and are not significantly different. From our previous work, we expected to measure values of productive binding in the range of 0.1-10 µmoles/g of cellulose 3 . Using kcat=5.4 s -1 we then expect that our experiments required that the sensor accurately measure a rate of cellobiose release, d[CB]/dt, accurately in the range of 0.02-3 µM/s.…”

Section: Validation Of Biosensor Measurementsmentioning

confidence: 99%

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

Nill

ACS Sustainable Chem. Eng.

2020

Self Cite

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.

Mechanistic kinetic models of enzymatic cellulose hydrolysis—A review

Biotech & Bioengineering

Cardona

Karuna

et al. 2017

Self Cite

119

Bioconversion of lignocellulose forms the basis for renewable, advanced biofuels, and bioproducts. Mechanisms of hydrolysis of cellulose by cellulases have been actively studied for nearly 70 years with significant gains in understanding of the cellulolytic enzymes. Yet, a full mechanistic understanding of the hydrolysis reaction has been elusive. We present a review to highlight new insights gained since the most recent comprehensive review of cellulose hydrolysis kinetic models by Bansal et al. (2009) Biotechnol Adv 27:833-848. Recent models have taken a two-pronged approach to tackle the challenge of modeling the complex heterogeneous reaction-an enzyme-centric modeling approach centered on the molecularity of the cellulase-cellulose interactions to examine rate limiting elementary steps and a substrate-centric modeling approach aimed at capturing the limiting property of the insoluble cellulose substrate. Collectively, modeling results suggest that at the molecular-scale, how rapidly cellulases can bind productively (complexation) and release from cellulose (decomplexation) is limiting, while the overall hydrolysis rate is largely insensitive to the catalytic rate constant. The surface area of the insoluble substrate and the degrees of polymerization of the cellulose molecules in the reaction both limit initial hydrolysis rates only. Neither enzyme-centric models nor substrate-centric models can consistently capture hydrolysis time course at extended reaction times. Thus, questions of the true reaction limiting factors at extended reaction times and the role of complexation and decomplexation in rate limitation remain unresolved. Biotechnol. Bioeng. 2017;114: 1369-1385. © 2017 Wiley Periodicals, Inc.