Enzyme assays for high-throughput screening

Goddard, Jean‐Philippe; Reymond, Jean‐Louis

doi:10.1016/j.copbio.2004.06.008

Cited by 242 publications

(132 citation statements)

References 61 publications

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“…These assays generally require application of UV-visible or fluorescence spectroscopy in order to follow the color or the fluorescence of a substrate or a product [1]. Unfortunately, the natural substrates or products of many enzymes such as phosphatases, proteases, or kinases, do not have a color or are not fluorogenic, and thus, synthetically labeled substrates have to be used.…”

Section: Introductionmentioning

confidence: 99%

Fast and accurate enzyme activity measurements using a chip-based microfluidic calorimeter

Schie

Ebrahimi

Hagen

et al. 2018

Analytical Biochemistry

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Section: Introductionmentioning

confidence: 99%

Fast and accurate enzyme activity measurements using a chip-based microfluidic calorimeter

Schie

Ebrahimi

Hagen

et al. 2018

Analytical Biochemistry

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“…The method can be carried out in aseptic conditions and should be adaptable to high-throughput assays. Other highthroughput assays utilize fluorescent cellulosic derivatives and chromogenic substrates that are expensive and unsuitable to study digestibility of pre-treated biomass (Boyer et al, 2002;Goddard and Reymond, 2004;Helbert et al, 2003).…”

Section: Introductionmentioning

confidence: 99%

High‐throughput microplate technique for enzymatic hydrolysis of lignocellulosic biomass

Chundawat

Balan

Dale

2008

Biotech & Bioengineering

118

113

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Several factors will influence the viability of a biochemical platform for manufacturing lignocellulosic based fuels and chemicals, for example, genetically engineering energy crops, reducing pre-treatment severity, and minimizing enzyme loading. Past research on biomass conversion has focused largely on acid based pre-treatment technologies that fractionate lignin and hemicellulose from cellulose. However, for alkaline based (e.g., AFEX) and other lower severity pre-treatments it becomes critical to co-hydrolyze cellulose and hemicellulose using an optimized enzyme cocktail. Lignocellulosics are appropriate substrates to assess hydrolytic activity of enzyme mixtures compared to conventional unrealistic substrates (e.g., filter paper, chromogenic, and fluorigenic compounds) for studying synergistic hydrolysis. However, there are few, if any, high-throughput lignocellulosic digestibility analytical platforms for optimizing biomass conversion. The 96-well Biomass Conversion Research Lab (BCRL) microplate method is a high-throughput assay to study digestibility of lignocellulosic biomass as a function of biomass composition, pre-treatment severity, and enzyme composition. The most suitable method for delivering milled biomass to the microplate was through multi-pipetting slurry suspensions. A rapid bio-enzymatic, spectrophotometric assay was used to determine fermentable sugars. The entire procedure was automated using a robotic pipetting workstation. Several parameters that affect hydrolysis in the microplate were studied and optimized (i.e., particle size reduction, slurry solids concentration, glucan loading, mass transfer issues, and time period for hydrolysis). The microplate method was optimized for crystalline cellulose (Avicel) and ammonia fiber expansion (AFEX) pre-treated corn stover.

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“…A sufficient bathochromic or hypsochromic shift can also be induced by formation or breakdown of conjugated systems, e.g., in homogeneously catalyzed Heck reactions (26), or in biocatalytic lipid peroxidation (28). Catalytic cleavage of a covalently bound (tethered) quenching group also allows selective detection of a single-product molecule against a background of excess reactant (29) (Fig. 3a).…”

Section: Probes For Smfs In (Bio)catalysismentioning

confidence: 99%

Single-molecule fluorescence spectroscopy in (bio)catalysis

Roeffaers

Cremer

Uji‐i

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

146

114

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The ever-improving time and space resolution and molecular detection sensitivity of fluorescence microscopy offer unique opportunities to deepen our insights into the function of chemical and biological catalysts. Because single-molecule microscopy allows for counting the turnover events one by one, one can map the distribution of the catalytic activities of different sites in solid heterogeneous catalysts, or one can study time-dependent activity fluctuations of individual sites in enzymes or chemical catalysts. By experimentally monitoring individuals rather than populations, the origin of complex behavior, e.g., in kinetics or in deactivation processes, can be successfully elucidated. Recent progress of temporal and spatial resolution in single-molecule fluorescence microscopy is discussed in light of its impact on catalytic assays. Key concepts are illustrated regarding the use of fluorescent reporters in catalytic reactions. Future challenges comprising the integration of other techniques, such as diffraction, scanning probe, or vibrational methods in single-molecule fluorescence spectroscopy are suggested.S ingle-molecule fluorescence spectroscopy (SMFS) has recently developed into a powerful tool for studying biophysical and biochemical phenomena. In studies of enzymatic catalysis, SMFS has revealed that there are large differences between the catalytic activity of individual enzymes within a population (''static disorder'') and that the rate constant (k cat ) of an individual enzyme may strongly fluctuate over time (''dynamic disorder''), the latter resulting from conformational changes of the enzyme. The recent application of SMFS to catalysis by solid materials has shown that heterogeneities in k cat also exist between individual catalytic crystals of one powder sample and even between the sites of an individual crystal. In this case, heterogeneity might arise from different chemical environments within the catalyst sample. The observation of heterogeneity in the k cat of those different catalytic systems suggests that the parallel introduction and evolution of SMFS techniques in bioand chemocatalysis will deepen our insights in almost any type of catalytic conversion. Indeed, the challenge to derive overall kinetics from the contributions of individuals within a population is essentially the same for biological, heterogeneous and even homogeneous systems, as discussed in From Populations to Individuals.From a technical viewpoint, SMFS requires strongly fluorescent probe molecules. The concepts to use such probes and even the probes themselves can be exchanged freely between heterogeneous, homogeneous, and biocatalysis, as discussed in Probes for SMFS in (Bio)Catalysis. If one wants to map in even more detail the contributions of the individual enzymes or catalytic sites to the overall kinetics, further improvements of the spatial and temporal resolution of SMFS will be required (see Spatial Resolution: Micro-and Nanoscopy and Time Resolution and Dynamics). Finally, to complement the information from S...

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Enzyme assays for high-throughput screening

Cited by 242 publications

References 61 publications

Fast and accurate enzyme activity measurements using a chip-based microfluidic calorimeter

Fast and accurate enzyme activity measurements using a chip-based microfluidic calorimeter

High‐throughput microplate technique for enzymatic hydrolysis of lignocellulosic biomass

Single-molecule fluorescence spectroscopy in (bio)catalysis

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