A High-Throughput Assay to Measure Cellulase Binding and Synergism in Ternary Mixtures

Santhanam, Navaneetha; Walker, Larry P.

doi:10.13031/2013.24190

Cited by 5 publications

(5 citation statements)

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“…The results obtained for this study clearly demonstrate the successful integration of both cellulase and cellulose labeling methods to achieve high spatio-temporal resolution of cellulose-cellulase interactions. Previous studies have focused on the use of either labeled cellulases (Jeoh et al, 2002;Pinto et al, 2007;Santhanam and Walker, 2008) or labeled cellulose (Helbert et al, 2003;Jervis et al, 1997), but have not explored the use of a complete fluorescent assay. Through amine-targeted labeling of cellulases with AF647 dye, followed by purification through n-PAGE and electroelution, fluorescently labeled, highly purified, active enzymes were obtained.…”

Section: Discussionmentioning

confidence: 99%

“…A stock suspension of bacterial micro-crystalline cellulose (BMCC, Monsanto Cellulon, Monsanto, San Diego, CA) was prepared as previously described (Jeoh et al, 2002;Santhanam and Walker, 2008). Fluorescent labeling of cellulose with 5-(4,6-dicholorotriazinyl)-aminofluorescein (DTAF) was carried out as described previously (Helbert et al, 2003).…”

Section: Cellulose Labelingmentioning

confidence: 99%

“…The activities of labeled and unlabeled cellulases were measured by incubating them with 1 mg/mL BMCC in 50 mM sodium acetate buffer (pH 5.5) over 18 h at 508C, and measuring the amount of reducing sugars produced (Santhanam and Walker, 2008). Reactions were stopped by centrifugation and reducing sugars were measured using the parahydroxybenzoic acid hydrazide (PAHBAH) method (Lever, 1972).…”

Section: Cellulase Activity Assaymentioning

confidence: 99%

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Immobilization of cellulose fibrils on solid substrates for cellulase‐binding studies through quantitative fluorescence microscopy

Moran‐Mirabal

Santhanam

Corgié

et al. 2008

Biotech & Bioengineering

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Cellulases, enzymes capable of depolymerizing cellulose polymers into fermentable sugars, are essential components in the production of bioethanol from lignocellulosic materials. Given the importance of these enzymes to the evolving biofuel industry considerable research effort is focused on understanding the interaction between cellulases and cellulose fibrils. This manuscript presents a method that addresses challenges that must be overcome in order to study such interactions through high-resolution fluorescence microscopy. First, it is shown that cellulose can be immobilized on solid substrates through a polymer lift-off technique. The immobilized cellulose aggregates present characteristic morphologies influenced by the patterned feature size used to immobilize it. Thus, through a variety of pattern sizes, cellulose can be immobilized in the form of cellulose particles, cellulose mats or individual cellulose fibrils. Second, it is shown that both cellulose and Thermobifida fusca cellulases Cel5A, Cel6B, and Cel9A can be fluorescently tagged and that the labeling does not inhibit the capability of these cellulases to depolymerize cellulose. The combination of the immobilization technique together with fluorescence labeling yields a system that can be used to study cellulose-cellulase interactions with spatial and temporal resolution not available through more conventional techniques which measure ensemble averages. It is shown that with such a system, the kinetics of cellulase binding onto cellulose fibrils and mats can be followed through sequences of fluorescence images. The intensity from the images can then be used to reconstruct binding curves for the cellulases studied. It was found that the complexity of cellulose morphology has a large impact on the binding curve characteristics, with binding curves for individual cellulose fibrils closely following a binding saturation model and binding curves for cellulose mats and particles deviating from it. The behavior observed is interpreted as the effect pore and interstice penetration play in cellulase binding to the accessible surface of cellulose aggregates. These results validate our method for immobilizing nanoscale cellulose fibrils and fibril aggregates on solid supports and lay the foundation for future studies on cellulase-cellulose interactions.

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Section: Discussionmentioning

confidence: 99%

Section: Cellulose Labelingmentioning

confidence: 99%

Section: Cellulase Activity Assaymentioning

confidence: 99%

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Immobilization of cellulose fibrils on solid substrates for cellulase‐binding studies through quantitative fluorescence microscopy

Moran‐Mirabal

Santhanam

Corgié

et al. 2008

Biotech & Bioengineering

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“…Despite the current uncertainty and challenges to the development of second generation biofuels and bioproducts, there remains considerable global interest in the biochemical conversion of biomass into fermentable sugars (Harris et al, 2013). Progress has been made in lowering the cost of biomass saccharification through process engineering and the innovative application of genomics, protein engineering, and other molecular biology approaches (Gusakov et al, 2005;Irwin et al, 1993;Karlsson et al, 2001;Santhanam and Walker, 2008;Snow and O'Dea, 1981;Wilson, 2012). However, the saccharification of lignocellulosic biomass remains one of the most expensive steps in the production of advanced biofuels (Luterbacher et al, 2010;Lynd et al, 2008;Wang et al, 2012).…”

Section: Introductionmentioning

confidence: 99%

Investigation of the porous structure of cellulosic substrates through confocal laser scanning microscopy

Yang

Moran‐Mirabal

Parlange

et al. 2013

Biotech & Bioengineering

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At the most fundamental level, saccharification occurs when cell wall degrading enzymes (CWDEs) diffuse, bind to and react on readily accessible cellulose fibrils. Thus, the study of the diffusive behavior of solutes into and out of cellulosic substrates is important for understanding how biomass pore size distribution affects enzyme transport, binding, and catalysis. In this study, fluorescently labeled dextrans with molecular weights of 20, 70, and 150 kDa were used as probes to assess their diffusion into the porous structure of filter paper. Fluorescence microscopy with high numerical aperture objectives was used to generate high temporal and spatial resolution datasets of probe concentrations versus time. In addition, two diffusion models, including a simple transient diffusion and a pore grouping diffusion models, were developed. These models and the experimental datasets were used to investigate solute diffusion in macro- and micro-pores. Nonlinear least squares fitting of the datasets to the simple transient model yielded diffusion coefficient estimates that were inadequate for describing the initial fast diffusion and the later slow diffusion rates observed; on the other hand, nonlinear least squares fitting of the datasets to the pore grouping diffusion model yielded estimations of the micro-pore diffusion coefficient that described the inherently porous structure of plant-derived cellulose. In addition, modeling results show that on average 75% of the accessible pore volume is available for fast diffusion without any significant pore hindrance. The method developed can be applied to study the porous structure of plant-derived biomass and help assess the diffusion process for enzymes with known sizes.

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“…They are classified as either random or processive depending on whether they catalyze single or multiple bond cleavages in succession. Cellulases are key in the production of soluble sugars from lignocellulosic material that can be used for biofuel and bioproducts production and have become the focus of many fluorescence studies that explore their catalytic activity, processivity, and interaction with cellulosic materials. − Yet, these studies have lacked the resolution to elucidate processivity and catalysis during cellulose depolymerization at the nanoscale, which could be provided by SMFS.…”

mentioning

confidence: 99%

Labeling and Purification of Cellulose-Binding Proteins for High Resolution Fluorescence Applications

et al. 2009

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The study of enzymatic reactions through fluorescence spectroscopy requires the use of bright, functional fluorescent molecules. In the case of proteins, labeling with fluorescent dyes has been carried out through covalent reactions with specific amino acids. However, these reactions are probabilistic and can yield mixtures of unlabeled and labeled enzymes with catalytic activities that can be modified by the addition of fluorophores. To have meaningful interpretations of results from the study of labeled enzymes, it is then necessary to reduce the variability in physical, chemical, and biological characteristics of the labeled products. In this paper, a solid phase labeling protocol is described as an advantageous alternative to free solution labeling of cellulose-binding proteins and is applied to tag cellulases with three different fluorophores. The products from the labeling reactions were purified to remove the unreacted dye and separate labeled and unlabeled enzymes. Characterization of the catalytic and spectroscopic properties of the isolated labeled species confirmed that highly homogeneous populations of labeled cellulases can be achieved. The protocol for the separation of labeled products is applicable to any mixture of labeled proteins, making this an attractive methodology for the production of labeled proteins suitable for single molecule fluorescence spectroscopy.

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A High-Throughput Assay to Measure Cellulase Binding and Synergism in Ternary Mixtures

Cited by 5 publications

References 0 publications

Immobilization of cellulose fibrils on solid substrates for cellulase‐binding studies through quantitative fluorescence microscopy

Immobilization of cellulose fibrils on solid substrates for cellulase‐binding studies through quantitative fluorescence microscopy

Investigation of the porous structure of cellulosic substrates through confocal laser scanning microscopy

Labeling and Purification of Cellulose-Binding Proteins for High Resolution Fluorescence Applications

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