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Protocol for analyzing enzymatic hydrolysis of cellulose using surface pitting observation technology.

ABSTRACT: Assaying enzymatic degradation of water-insoluble substrates like cellulose is challenging because only the substrate surface is accessible to the enzymes resulting in low reaction rates. Here, we describe a protocol for surface pitting observation technology (SPOT), an ultra-sensitive quantitative assay for analyzing enzymatic hydrolysis of cellulose. We describe the use of a porous substrate to accelerate the hydrolysis rate of cellulose. We also detail the steps for combining inkjet patterning and optical profilometry to analyze volume loss upon hydrolysis. For complete details on the use and execution of this protocol, please refer to Tsudome et al. (2022).¹.

SUBMITTER: Tachioka M

PROVIDER: S-EPMC9898803 | biostudies-literature | 2023 Jan

REPOSITORIES: biostudies-literature

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Protocol for analyzing enzymatic hydrolysis of cellulose using surface pitting observation technology.

Tachioka Mikako M Tsudome Mikiko M Deguchi Shigeru S

STAR protocols 20230124 1

Assaying enzymatic degradation of water-insoluble substrates like cellulose is challenging because only the substrate surface is accessible to the enzymes resulting in low reaction rates. Here, we describe a protocol for surface pitting observation technology (SPOT), an ultra-sensitive quantitative assay for analyzing enzymatic hydrolysis of cellulose. We describe the use of a porous substrate to accelerate the hydrolysis rate of cellulose. We also detail the steps for combining inkjet patternin ...[more]

PMID: 36825811

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Project description:BackgroundDuring cellulosic ethanol production, cellulose hydrolysis is achieved by synergistic action of cellulase enzyme complex consisting of multiple enzymes with different mode of actions. Enzymatic hydrolysis of cellulose is one of the bottlenecks in the commercialization of the process due to low hydrolysis rates and high cost of enzymes. A robust hydrolysis model that can predict hydrolysis profile under various scenarios can act as an important forecasting tool to improve the hydrolysis process. However, multiple factors affecting hydrolysis: cellulose structure and complex enzyme-substrate interactions during hydrolysis make it diffucult to develop mathematical kinetic models that can simulate hydrolysis in presence of multiple enzymes with high fidelity. In this study, a comprehensive hydrolysis model based on stochastic molecular modeling approch in which each hydrolysis event is translated into a discrete event is presented. The model captures the structural features of cellulose, enzyme properties (mode of actions, synergism, inhibition), and most importantly dynamic morphological changes in the substrate that directly affect the enzyme-substrate interactions during hydrolysis.ResultsCellulose was modeled as a group of microfibrils consisting of elementary fibrils bundles, where each elementary fibril was represented as a three dimensional matrix of glucose molecules. Hydrolysis of cellulose was simulated based on Monte Carlo simulation technique. Cellulose hydrolysis results predicted by model simulations agree well with the experimental data from literature. Coefficients of determination for model predictions and experimental values were in the range of 0.75 to 0.96 for Avicel hydrolysis by CBH I action. Model was able to simulate the synergistic action of multiple enzymes during hydrolysis. The model simulations captured the important experimental observations: effect of structural properties, enzyme inhibition and enzyme loadings on the hydrolysis and degree of synergism among enzymes.ConclusionsThe model was effective in capturing the dynamic behavior of cellulose hydrolysis during action of individual as well as multiple cellulases. Simulations were in qualitative and quantitative agreement with experimental data. Several experimentally observed phenomena were simulated without the need for any additional assumptions or parameter changes and confirmed the validity of using the stochastic molecular modeling approach to quantitatively and qualitatively describe the cellulose hydrolysis.

Dataset Information

Protocol for analyzing enzymatic hydrolysis of cellulose using surface pitting observation technology.

Publications

Protocol for analyzing enzymatic hydrolysis of cellulose using surface pitting observation technology.

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