Enzyme assays

Reymond, Jean‐Louis; Fluxà, Viviana S.; Maillard, Noélie

doi:10.1039/b813732c

Cited by 124 publications

(113 citation statements)

References 250 publications

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“…[e] Relative activator efficiency h = f Y max pEC 50 [mm], f = 0.37. expected to have a similarly diverse and important impact. Whereas cellular uptake of DNA-countercation complexes is extensively studied and routinely used in many variations, [9,10] their potential to function as multienzyme detectors and multianalyte sensors [12,13] remained to be clarified.…”

Section: Counterion Activatorsmentioning

confidence: 99%

“…To complete the picture and develop a multifunctionality as rich as for CPPs, we wondered whether or not countercations could activate DNA to function as cation transporters, enzyme detectors and biosensors. [3] To answer this question, we here first characterize counterion-activated DNA as ion carriers in the "U-tube" [2,11] and in vesicles, use then a collection of amphiphilic, bola-amphiphilic, aliphatic, aromatic and macrocyclic ammonium and guanidinium countercations to outline the nature of DNA activation, and explore sensing applications [12,13] with phytase and phytate as examples.…”

Section: Introductionmentioning

confidence: 99%

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Amphiphilic Counterion Activators for DNA: Stimuli‐Responsive Cation Transporters and Biosensors in Bulk and Lipid Bilayer Membranes

et al. 2009

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We report that amphiphilic counterions can enable DNA to act as cation carrier, enzyme detector and biosensor. Calf thymus DNA is used as example throughout the study. Evaluation of a series of counterion activators suggests that strong amphiphilicity, alkyl or calix[4]arene tails and guanidinium cations give best results, whereas weak amphiphilicity, bola-amphiphilicity, planar aryl tails and ammonium cations are less satisfactory for various reasons. In the U-tube, DNA-counterion complexes can carry cations such as safranin O or p-xylene-bis-pyridinium bromide (DPX) across bulk chloroform membranes, whereas anions such as carboxyfluorescein (CF) and (8-Hydroxy-1,3,6-pyrenetrisulfonate (HPTS) are not transported. Uptake of DNA-counterion complexes into intact vesicles is demonstrated by DNA trapping experiments with internal polylysine. Comparison of results from different assays suggests that DNA-counterion complexes act as cation carriers under mild conditions, whereas pore formation and lysis dominate at higher concentrations. Applicability of DNA-counterion transporters for the detection of enzyme activity is demonstrated with phytate as an inactivating substrate and phytase as a reactivating enzyme. Compatibility with biosensing is exemplified with the fluorometric monitoring of phytate levels in almond extracts. The conceptual significance of these findings is briefly discussed, as are promising perspectives such as the application of DNA chemistry to multianalyte sensing in fluorogenic vesicles.

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Section: Counterion Activatorsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Amphiphilic Counterion Activators for DNA: Stimuli‐Responsive Cation Transporters and Biosensors in Bulk and Lipid Bilayer Membranes

et al. 2009

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“…In such cases, rational protein design or directed evolution may overcome these limitations. [4] These techniques, together with the developments in analytical and assay methods, [5] promise the discovery of novel biocatalysts with improved properties.…”

Section: Introductionmentioning

confidence: 99%

Bacillus subtilis Esterase (BS2) and its Double Mutant Have Different Selectivity in the Removal of Carboxyl Protecting Groups

Barbayianni¹,

Kokotos²,

Bartsch³

et al. 2009

Adv Synth Catal

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An esterase from Bacillus subtilis (BS2) and its double mutant E188W/M193C quickly hydrolyze n-butyl, n-propyl, methoxyethyl and allyl esters. The wild-type BS2 preferentially removes such esters from the g-position of glutamate diesters, while the engineered enzyme has a reversed selectivity removing esters from the a-position of glutamate diesters.Automated docking and molecular dynamic simulations were performed to understand the molecular reason for the different regioselectivity.

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“…However, for the identification, characterization, and optimiza-tion of these valuable biocatalysts, only a very limited number of fundamentally different methods are described in the literature with their respective strengths and weaknesses. Unfortunately, the combination of AHL structure and specificity profiles of the QQ enzymes prevents any obvious chromogenic or fluorogenic AHL derivatization; this approach would otherwise enable easy monitoring of the reaction progress (19). Hence, QQ activity measurements have to be performed indirectly, preferentially by the detection of a reaction product or, alternatively, the remaining AHL.…”

mentioning

confidence: 99%

Fast, Continuous, and High-Throughput (Bio)Chemical Activity Assay forN-Acyl-l-Homoserine Lactone Quorum-Quenching Enzymes

Krüger

Dörr

Bornscheuer

2016

Appl Environ Microbiol

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Quorum sensing, the bacterial cell-cell communication by small molecules, controls important processes such as infection and biofilm formation. Therefore, it is a promising target with several therapeutic and technical applications besides its significant ecological relevance. Enzymes inactivating N-acyl-L-homoserine lactones, the most common class of communication molecules among Gram-negative proteobacteria, mainly belong to the groups of quorum-quenching lactonases or quorum-quenching acylases. However, identification, characterization, and optimization of these valuable biocatalysts are based on a very limited number of fundamentally different methods with their respective strengths and weaknesses. Here, a (bio)chemical activity assay is described, which perfectly complements the other methods in this field. It enables continuous and high-throughput activity measurements of purified and unpurified quorum-quenching enzymes within several minutes. For this, the reaction products released by quorum-quenching lactonases and quorum-quenching acylases are converted either by a secondary enzyme or by autohydrolysis to L-homoserine. In turn, L-homoserine is detected by the previously described calcein assay, which is sensitive to ␣-amino acids with free N and C termini. Besides its establishment, the method was applied to the characterization of three previously undescribed quorum-quenching lactonases and variants thereof and to the identification of quorum-quenching acylaseexpressing Escherichia coli clones in an artificial library. Furthermore, this study indicates that porcine aminoacylase 1 is not active toward N-acyl-L-homoserine lactones as published previously but instead converts the autohydrolysis product N-acyl-Lhomoserine. IMPORTANCEIn this study, a novel method is presented for the identification, characterization, and optimization of quorum-quenching enzymes that are active toward N-acyl-L-homoserine lactones. These are the most common communication molecules among Gram-negative proteobacteria. The activity assay is a highly valuable complement to the available analytical tools in this field. It will facilitate studies on the environmental impact of quorum-quenching enzymes and contribute to the development of therapeutic and technical applications of this promising enzyme class. The bacterial cell-cell communication by small molecules known as quorum sensing (QS) enables these single-celled organisms to coordinate their gene expression among a local population, depending on environmental conditions (1-4). Important processes such as infection and biofilm formation are controlled by QS. Hence, QS is a highly relevant target for naturally occurring and human-made interference termed quorum quenching (QQ), and it is mediated in particular by small-molecule inhibitors and QQ enzymes (5). From an ecological point of view, QQ has a significant influence on the interactions between bacteria (6, 7) and also bacteria and eukaryotes (8). Prospective therapeutic applications of QQ include the prevention a...

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Enzyme assays

Cited by 124 publications

References 250 publications

Amphiphilic Counterion Activators for DNA: Stimuli‐Responsive Cation Transporters and Biosensors in Bulk and Lipid Bilayer Membranes

Amphiphilic Counterion Activators for DNA: Stimuli‐Responsive Cation Transporters and Biosensors in Bulk and Lipid Bilayer Membranes

Bacillus subtilis Esterase (BS2) and its Double Mutant Have Different Selectivity in the Removal of Carboxyl Protecting Groups

Fast, Continuous, and High-Throughput (Bio)Chemical Activity Assay forN-Acyl-l-Homoserine Lactone Quorum-Quenching Enzymes

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