Capillary electrophoresis‐based enzyme assays for β‐lactamase enzymes

Abstract

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.

Challenging Bioanalyses with Capillary Electrophoresis

“…In one approach, 14 inhibitors for thrombin were identified using transverse diffusion of laminar flow profile-capillary electrophoresis (TDLFP-CE) in less than 2 min per run . CE has been used to compare inhibitors of native and recombinant enzymes . In the period under review, inhibitors of enzymes in free solution have been characterized with UV detection for ecto-5-nucleotidase, thrombin, histone deacetylase, glucosamine-6 phosphate synthase, and human acetyl-CoA carboxylase 2 and with laser-induced fluorescence for d -amino acid oxidase and BCR-ABL .…”

“…For example, online and off-line determinations of K m values for penicillinase were 65 ± 7 and 73 ± 6 μM, respectively. 61 The online evaluation of K m with CE is also useful to compare the activities of enzymes, such as penicillinase and recombinant metallo-β-lactamase, two enzymes responsible for the deactivation of antibiotics, which had comparable K m values. 61 The identification of enzyme inhibitors is important in developing biotherapeutics.…”

“…61 The online evaluation of K m with CE is also useful to compare the activities of enzymes, such as penicillinase and recombinant metallo-β-lactamase, two enzymes responsible for the deactivation of antibiotics, which had comparable K m values. 61 The identification of enzyme inhibitors is important in developing biotherapeutics. When inhibitors are present, the enzyme activity is decreased.…”

“…62 CE has been used to compare inhibitors of native and recombinant enzymes. 61 In the period under review, inhibitors of enzymes in free solution have been characterized with UV detection for ecto-5-nucleotidase, 54 thrombin, 52 histone deacetylase, 56 glucosamine-6 phosphate synthase, 63 and human acetyl-CoA carboxylase 2 64 and with laser-induced fluorescence for D-amino acid oxidase 58 and BCR-ABL. 59 Further, inhibition studies have been done for immobilized enzymes including acetylcholinesterase, 46,53 alanine aminotransferase, 49 β-glucosidase, 60 and sulfotransferase 1A1 48 using UV detection.…”

RhII‐Catalyzed De‐symmetrization of Ethane‐1,2‐dithiol and Propane‐1,3‐dithiol Yields Metallo‐β‐lactamase Inhibitors

“…VIM‐2 expression and purification was published elsewhere [19] . Before the measurement the VIM‐2 protein was dialyzed against 500 times the volume of buffer (50 mM Tris, 500 mM NaCl, 5 % (v/v) glycerol with a pH=8) using a dialysis membrane with a 3.5 kDa MWCO.…”