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Two Variants of a High-Throughput Fluorescent Microplate Assay of Polysaccharide Endotransglycosylases.
- Source :
-
Applied biochemistry and biotechnology [Appl Biochem Biotechnol] 2016 Apr; Vol. 178 (8), pp. 1652-65. Date of Electronic Publication: 2016 Jan 11. - Publication Year :
- 2016
-
Abstract
- Polysaccharide endotransglycosylases (PETs) are the cell wall-modifying enzymes of fungi and plants. They catalyze random endo-splitting of the polysaccharide donor molecule and transfer of the newly formed reducing sugar residue to the nonreducing end of an acceptor molecule which can be a polysaccharide or an oligosaccharide. Owing to their important role in the cell wall formation, the inhibition of PETs represents an attractive strategy in the fight against fungal infections. We have elaborated two variants of a versatile high-throughput microplate fluorimetric assay that could be used for effective identification of PETs and screening of their inhibitors. Both assays use the respective polysaccharides as the donors and sulforhodamine-labeled oligosaccharides as the acceptors but differ from each other by mode of how the labeled polysaccharide products of transglycosylation are separated from the unreacted oligosaccharide acceptors. In the first variant, the reactions take place in a layer of agar gel laid on the bottoms of the wells of a microtitration plate. After the reaction, the high-Mr transglycosylation products are precipitated with 66 % ethanol and retained within the gel while the low-Mr products and the unreacted acceptors are washed out. In the second variant, the donor polysaccharides are adsorbed to the surface of a microplate well and remain adsorbed there also after becoming labeled in the course of the transglycosylation reaction whereas the unused low-Mr acceptors are washed out. As a proof of versatility, assays of heterologously expressed transglycosylases ScGas1, ScCrh1, and ScCrh2 from the yeast Saccharomyces cerevisiae, CaPhr1 and CaPhr2 from Candida albicans, and of a plant xyloglucan endotransglycosylase (XET) are demonstrated.
- Subjects :
- Candida albicans enzymology
Cell Wall enzymology
Fluorescence
Fungal Proteins biosynthesis
Fungal Proteins chemistry
Gene Expression Regulation, Fungal
Glycoside Hydrolases biosynthesis
Glycoside Hydrolases chemistry
Glycosyltransferases biosynthesis
Glycosyltransferases chemistry
High-Throughput Screening Assays methods
Membrane Glycoproteins biosynthesis
Membrane Glycoproteins chemistry
Mycoses drug therapy
Polysaccharides chemistry
Polysaccharides metabolism
Saccharomyces cerevisiae enzymology
Saccharomyces cerevisiae Proteins biosynthesis
Saccharomyces cerevisiae Proteins chemistry
Fungal Proteins isolation & purification
Glycoside Hydrolases isolation & purification
Glycosyltransferases isolation & purification
Membrane Glycoproteins isolation & purification
Saccharomyces cerevisiae Proteins isolation & purification
Subjects
Details
- Language :
- English
- ISSN :
- 1559-0291
- Volume :
- 178
- Issue :
- 8
- Database :
- MEDLINE
- Journal :
- Applied biochemistry and biotechnology
- Publication Type :
- Academic Journal
- Accession number :
- 26754421
- Full Text :
- https://doi.org/10.1007/s12010-015-1973-8