Capillary electrophoresis‐based enzyme assays for β‐lactamase enzymes

Generic in‐capillary as well as offline CE‐based enzyme assays were developed for serine‐β‐lactamases and metallo‐β‐lactamases. The hydrolysis of benzylpenicillin to benzylpenicilloic acid was analyzed using 100 mM sodium phosphate solution, pH 6.0, as a background electrolyte. In‐capillary assays e...

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Bibliographic Details
Published in:Electrophoresis 2019-09, Vol.40 (18-19), p.2375-2381
Main Authors: Chen, Chunyang, Kramer, Jan S., Brunst, Steffen, Proschak, Ewgenij, Scriba, Gerhard K.E.
Format: Article
Language:English
Subjects:
Assaying
Bacillus cereus - enzymology
Bacterial Proteins - analysis
Bacterial Proteins - antagonists & inhibitors
Bacterial Proteins - metabolism
beta-Lactamase Inhibitors - pharmacology
beta-Lactamases - analysis
beta-Lactamases - metabolism
Capillary electrophoresis
Electrophoresis
Electrophoresis, Capillary - methods
Enzyme assay
Enzyme Assays - methods
Enzymes
Limit of Detection
Linear Models
Linearity
Metallography
Metallo‐β‐lactamase
Penicillinase
Polyethylenes
Reproducibility of Results
Sodium phosphate
Substrates
Tazobactam - pharmacology
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Summary:Generic in‐capillary as well as offline CE‐based enzyme assays were developed for serine‐β‐lactamases and metallo‐β‐lactamases. The hydrolysis of benzylpenicillin to benzylpenicilloic acid was analyzed using 100 mM sodium phosphate solution, pH 6.0, as a background electrolyte. In‐capillary assays employed an uncoated as well as a polyethylene oxide‐coated capillary, while the offline assays employing long end and short end injection were performed in an uncoated capillary. Using procaine hydrochloride or 4‐hydroxybenzoic acid as internal standard, the respective assays were validated with regard to linearity, LOD and LOQ, repeatability, precision, and accuracy. The assays were applied to the determination of the Michaelis‐Menten parameters Km and Vmax of Bacillus cereus penicillinase as well as New Delhi metallo‐β‐lactamase 1 and Verona integrin‐encoded metallo‐β‐lactamase 2. Furthermore, the inhibition of the enzymes by irreversible and competitive inhibitors was evaluated. Comparable data were obtained with all assays. The use of a simple substrate ensured broad applicability to the various types of β‐lactamases.
ISSN:0173-0835
1522-2683
DOI:10.1002/elps.201900104