Issue 2, 2021

Development of an active site titration reagent for α-amylases

Abstract

α-Amylases are among the most widely used classes of enzymes in industry and considerable effort has gone into optimising their activities. Efforts to find better amylase mutants, such as through high-throughput screening, would be greatly aided by access to precise and robust active site titrating agents for quantitation of active mutants in crude cell lysates. While active site titration reagents designed for retaining β-glycosidases quantify these enzymes down to nanomolar levels, convenient titrants for α-glycosidases are not available. We designed such a reagent by incorporating a highly reactive fluorogenic leaving group onto unsaturated cyclitol ethers, which have been recently shown to act as slow substrates for retaining glycosidases that operate via a covalent ‘glycosyl’-enzyme intermediate. By appending this warhead onto the appropriate oligosaccharide, we developed efficient active site titration reagents for α-amylases that effect quantitation down to low nanomolar levels.

Graphical abstract: Development of an active site titration reagent for α-amylases

Supplementary files

Article information

Article type
Edge Article
Submitted
28 Sep 2020
Accepted
03 Nov 2020
First published
03 Nov 2020
This article is Open Access

All publication charges for this article have been paid for by the Royal Society of Chemistry
Creative Commons BY-NC license

Chem. Sci., 2021,12, 683-687

Development of an active site titration reagent for α-amylases

R. P. Sweeney, P. M. Danby, A. Geissner, R. Karimi, J. Brask and S. G. Withers, Chem. Sci., 2021, 12, 683 DOI: 10.1039/D0SC05380E

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