NDLI: Two Variants of a High-Throughput Fluorescent Microplate Assay of Polysaccharide Endotransglycosylases

Content Provider	Springer Nature Link
Author	Kováčová, Kristína Farkaš, Vladimír
Copyright 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-M$_{r}$ transglycosylation products are precipitated with 66 % ethanol and retained within the gel while the low-M$_{r}$ 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-M$_{r}$ 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.
Starting Page	1652
Ending Page	1665
Page Count	14
File Format	PDF
ISSN	02732289
Journal	Applied Biochemistry and Biotechnology
Volume Number	178
Issue Number	8
e-ISSN	15590291
Language	English
Publisher	Springer US
Publisher Date	2016-01-11
Publisher Place	New York
Access Restriction	One Nation One Subscription (ONOS)
Subject Keyword	Cell walls Fluorescence assay High-throughput Polysaccharide endotransglycosylases Transglycosylation Biotechnology Biochemistry
Content Type	Text
Resource Type	Article
Subject	Medicine Molecular Biology Environmental Engineering Biochemistry Bioengineering Applied Microbiology and Biotechnology Biotechnology

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