A colorimetric assay for the screening and kinetic analysis of nucleotide sugar 4,6-dehydratases

Vogel, Ulrike; Beerens, Koen; Desmet, Tom

doi:10.1016/j.ab.2022.114870

Cited by 2 publications

(2 citation statements)

References 34 publications

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“…The detection and characterization of biotransformations is an important aspect in the development of new biocatalytic methods to access glycan building blocks. In this regard, a simple phenol-based detection method toward nucleotide sugar 4,6-dehydratases (NSDs) was recently developed, which enabled high-throughput analysis using crude enzymes for the first time . Using resorcinol, a phenolic compound which reacts with the ketone within the oxidized product of NSD action, absorbance could be monitored at 510 nm.…”

Section: Using Biocatalysis To Provide the Building Blocks For Bioche...mentioning

confidence: 99%

“…In this regard, a simple phenol-based detection method toward nucleotide sugar 4,6-dehydratases (NSDs) was recently developed, which enabled high-throughput analysis using crude enzymes for the first time. 113 Using resorcinol, a phenolic compound which reacts with the ketone within the oxidized product of NSD action, absorbance could be monitored at 510 nm. Compared with previous methods, which monitored product formation at 320 nm, less background interference was observed.…”

Section: Using Biocatalysis To Provide the Building Blocks For Bioche...mentioning

confidence: 99%

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Biocatalytic Approaches to Building Blocks for Enzymatic and Chemical Glycan Synthesis

Dolan

Cosgrove

Miller

2022

JACS Au

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While the field of biocatalysis has bloomed over the past 20−30 years, advances in the understanding and improvement of carbohydrate-active enzymes, in particular, the sugar nucleotides involved in glycan building block biosynthesis, have progressed relatively more slowly. This perspective highlights the need for further insight into substrate promiscuity and the use of biocatalysis fundamentals (rational design, directed evolution, immobilization) to expand substrate scopes toward such carbohydrate building block syntheses and/or to improve enzyme stability, kinetics, or turnover. Further, it explores the growing premise of using biocatalysis to provide simple, cost-effective access to stereochemically defined carbohydrate materials, which can undergo late-stage chemical functionalization or automated glycan synthesis/polymerization.

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Section: Using Biocatalysis To Provide the Building Blocks For Bioche...mentioning

confidence: 99%

Section: Using Biocatalysis To Provide the Building Blocks For Bioche...mentioning

confidence: 99%

Biocatalytic Approaches to Building Blocks for Enzymatic and Chemical Glycan Synthesis

Dolan

Cosgrove

Miller

2022

JACS Au

View full text Add to dashboard Cite

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Ultrasensitive and colorimetric small extracellular vesicles (sEVs) analysis via dual-cycle signal tool

et al. 2023

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For the clinical diagnosis of diseases and for basic biological research, it is crucial to develop a trustworthy and efficient method for detecting small extracellular vesicles (sEVs) in multiple experimental conditions. Here, we create a colorimetric assay that enables sensitive and precise sEVs identification without the need for pricey equipment. In this assay, the exonuclease III (Exo III)-assisted signal recycle is activated by the released single-strand DNA (ssDNA) from SMBs (streptavidin magnetic beads)-aptamer-ssDNA complex after identification of sEVs. By integrating with the strand displacement amplification (SDA) process, a significant amount of double-strand DNA products with G-rich tails is produced. The G-rich tails fold to G-quadruplex under the assistance of hemin to catalyze the oxidation of TMB, yielding a color change. The approach offers a broad detection range of 5 orders of magnitudes based on the signal recycles and SDA. In addition, single-stranded DNA binding protein (SSB) is exploited in this method to minimize the background signal from non-specific digestion of Exo-III, making the method a robust tool for sEVs detection and disease diagnosis.

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A colorimetric assay for the screening and kinetic analysis of nucleotide sugar 4,6-dehydratases

Cited by 2 publications

References 34 publications

Biocatalytic Approaches to Building Blocks for Enzymatic and Chemical Glycan Synthesis

Biocatalytic Approaches to Building Blocks for Enzymatic and Chemical Glycan Synthesis

Ultrasensitive and colorimetric small extracellular vesicles (sEVs) analysis via dual-cycle signal tool

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