scholarly journals Mobility shift-based electrophoresis coupled with fluorescent detection enables real-time enzyme analysis of carbohydrate sulfatase activity

2020 ◽  
Author(s):  
Dominic P Byrne ◽  
James A London ◽  
Patrick A Eyers ◽  
Edwin A Yates ◽  
Alan Cartmell
Keyword(s):  
Capillary Electrophoresis ◽  
Real Time ◽  
High Performance ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Fluorescent Detection ◽  
Enzyme Analysis ◽  
Mobility Shift ◽  
Sulfatase Activity ◽  
Domains Of Life

AbstractSulfated carbohydrate metabolism is a fundamental process, which occurs in all domains of life. Carbohydrate sulfatases are enzymes that remove sulfate groups from carbohydrates and are essential to the depolymerisation of complex polysaccharides. Despite their biological importance, carbohydrate sulfatases are poorly studied and challenges remain in accurately assessing the activity, specificity and kinetic parameters. Most notably, separation of desulfated products from sulfated substrates is currently a time-consuming process. In this paper, we describe the development of rapid capillary electrophoresis coupled to substrate fluorescence detection as a high-throughput and facile means of analysing carbohydrate sulfatase activity. The approach has utility for the determination of both kinetic and inhibition parameters and is based on existing microfluidic technology coupled to a new synthetic fluorescent 6S-GlcNAc carbohydrate substrate. Furthermore, we compare this technique in terms of both time and resources, to high performance anion exchange chromatography and NMR-based methods, which are the two current ‘gold standards’ for enzymatic carbohydrate sulfation analysis. Our study clearly demonstrates the advantages of mobility shift assays for the quantification of near real-time carbohydrate desulfation by purified sulfatases, and could support the search for small molecule inhibitors of these disease-associated enzymes.One sentence summarySulfatases remove sulfate groups from biomolecules; in this study we report a rapid and robust capillary electrophoresis assay for the quantification of carbohydrate desulfation.

scholarly journals Mobility shift-based electrophoresis coupled with fluorescent detection enables real-time enzyme analysis of carbohydrate sulfatase activity

2021 ◽  
Vol 478 (4) ◽  
pp. 735-748
Author(s):  
Dominic P. Byrne ◽  
James A. London ◽  
Patrick A. Eyers ◽  
Edwin A. Yates ◽  
Alan Cartmell
Keyword(s):  
Real Time ◽  
High Performance ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Fluorescent Detection ◽  
Enzyme Analysis ◽  
Mobility Shift ◽  
Sulfatase Activity ◽  
Domains Of Life ◽  
Sulfated Carbohydrate

Sulfated carbohydrate metabolism is a fundamental process, which occurs in all domains of life. Carbohydrate sulfatases are enzymes that remove sulfate groups from carbohydrates and are essential to the depolymerisation of complex polysaccharides. Despite their biological importance, carbohydrate sulfatases are poorly studied and challenges remain in accurately assessing the enzymatic activity, specificity and kinetic parameters. Most notably, the separation of desulfated products from sulfated substrates is currently a time-consuming process. In this paper, we describe the development of rapid capillary electrophoresis coupled to substrate fluorescence detection as a high-throughput and facile means of analysing carbohydrate sulfatase activity. The approach has utility for the determination of both kinetic and inhibition parameters and is based on existing microfluidic technology coupled to a new synthetic fluorescent 6S-GlcNAc carbohydrate substrate. Furthermore, we compare this technique, in terms of both time and resources, to high-performance anion exchange chromatography and NMR-based methods, which are the two current ‘gold standards’ for enzymatic carbohydrate sulfation analysis. Our study clearly demonstrates the advantages of mobility shift assays for the quantification of near real-time carbohydrate desulfation by purified sulfatases, and will support the search for small molecule inhibitors of these disease-associated enzymes.

scholarly journals Correction: Mobility shift-based electrophoresis coupled with fluorescent detection enables real-time enzyme analysis of carbohydrate sulfatase activity

2021 ◽  
Vol 478 (13) ◽  
pp. 2537-2538
Author(s):  
Dominic P. Byrne ◽  
James A. London ◽  
Patrick A. Eyers ◽  
Edwin A. Yates ◽  
Alan Cartmell
Keyword(s):  
Real Time ◽  
Fluorescent Detection ◽  
Enzyme Analysis ◽  
Mobility Shift ◽  
Sulfatase Activity

scholarly journals Cellodextrin Utilization byBifidobacterium breveUCC2003

2011 ◽  
Vol 77 (5) ◽  
pp. 1681-1690 ◽  
Author(s):  
Karina Pokusaeva ◽  
Mary O'Connell-Motherway ◽  
Aldert Zomer ◽  
John MacSharry ◽  
Gerald F. Fitzgerald ◽  
...  
Keyword(s):  
Gene Cluster ◽  
High Performance ◽  
Amperometric Detection ◽  
Hydrolytic Activity ◽  
Anion Exchange Chromatography ◽  
Negative Control ◽  
Exchange Chromatography ◽  
Insertion Mutant ◽  
Phenotypic Analysis ◽  
Mobility Shift

ABSTRACTCellodextrins, the incomplete hydrolysis products from insoluble cellulose, are accessible as a carbon source to certain members of the human gut microbiota, such asBifidobacterium breveUCC2003. Transcription of thecldEFGCgene cluster ofB. breveUCC2003 was shown to be induced upon growth on cellodextrins, implicating this cluster in the metabolism of these sugars. Phenotypic analysis of aB. breveUCC2003::cldEinsertion mutant confirmed that thecldgene cluster is exclusively required for cellodextrin utilization by this commensal. Moreover, our results suggest that transcription of thecldcluster is controlled by a LacI-type regulator encoded bycldR, located immediately upstream ofcldE. Gel mobility shift assays using purified CldRHis(produced by the incorporation of a His12-encoding sequence into the 3′ end of thecldCgene) indicate that thecldEFGCpromoter is subject to negative control by CldRHis, which binds to two inverted repeats. Analysis by high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD) of medium samples obtained during growth ofB. breveUCC2003 on a mixture of cellodextrins revealed its ability to utilize cellobiose, cellotriose, cellotetraose, and cellopentaose, with cellotriose apparently representing the preferred substrate. ThecldCgene of thecldoperon ofB. breveUCC2003 is, to the best of our knowledge, the first described bifidobacterial β-glucosidase exhibiting hydrolytic activity toward various cellodextrins.

scholarly journals Enzymatic Synthesis and Characterization of Different Families of Chitooligosaccharides and Their Bioactive Properties

2021 ◽  
Vol 11 (7) ◽  
pp. 3212
Author(s):  
Noa Miguez ◽  
Peter Kidibule ◽  
Paloma Santos-Moriano ◽  
Antonio O. Ballesteros ◽  
Maria Fernandez-Lobato ◽  
...  
Keyword(s):  
High Performance ◽  
Chemical Characterization ◽  
Amperometric Detection ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Enzymatic Production ◽  
Bioactive Properties ◽  
Substrate Properties ◽  
Chitin Hydrolysis

Chitooligosaccharides (COS) are homo- or hetero-oligomers of D-glucosamine (GlcN) and N-acetyl-D-glucosamine (GlcNAc) that can be obtained by chitosan or chitin hydrolysis. Their enzymatic production is preferred over other methodologies (physical, chemical, etc.) due to the mild conditions required, the fewer amounts of waste and its efficiency to control product composition. By properly selecting the enzyme (chitinase, chitosanase or nonspecific enzymes) and the substrate properties (degree of deacetylation, molecular weight, etc.), it is possible to direct the synthesis towards any of the three COS types: fully acetylated (faCOS), partially acetylated (paCOS) and fully deacetylated (fdCOS). In this article, we review the main strategies to steer the COS production towards a specific group. The chemical characterization of COS by advanced techniques, e.g., high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD) and MALDI-TOF mass spectrometry, is critical for structure–function studies. The scaling of processes to synthesize specific COS mixtures is difficult due to the low solubility of chitin/chitosan, the heterogeneity of the reaction mixtures, and high amounts of salts. Enzyme immobilization can help to minimize such hurdles. The main bioactive properties of COS are herein reviewed. Finally, the anti-inflammatory activity of three COS mixtures was assayed in murine macrophages after stimulation with lipopolysaccharides.

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