A high-throughput screening assay for hydroxynitrile lyase activity

Abstract

A high-throughput screening assay for hydroxynitrile lyase activity accepting a wide range of HNL-substrates is presented, which is useful either for enzyme fingerprinting or screening of huge variant libraries generated in metagenome or directed evolution approaches.

Uneven twins: Comparison of two enantiocomplementary hydroxynitrile lyases with α/β-hydrolase fold

“…The substrate range for the cleavage reaction was investigated using a microtiter plate assay based on the detection of HCN (Andexer et al, 2006). Commercial available cyanohydrins (acetone cyanohydrin, lactonitrile, cyclohexanone cyanohydrin, m-phenoxy-benzaldehyde cyanohydrin, and propionaldehyde cyanohydrin) were employed as substrates, which were in some cases (e.g.…”

“…Since the substrate range of the cleavage reaction of MeHNL has only partially been investigated (Hughes et al, 1994;Bühler et al, 2003), a qualitative comparison of the enzymes' substrate ranges concerning the cleavage reaction of various commercially available cyanohydrins was carried out employing an HCN-based microtiter plate assay (Andexer et al, 2006). The substrate ranges of MeHNL and AtHNL are almost equal concerning cyclic and aromatic cyanohydrins (mandelonitrile and cyclohexanone cyanohydrin), which are transformed with moderate to high activities in both cases.…”

Contemporary molecular tools in microbial ecology and their application to advancing biotechnology

“…Generally, an assay is designed to detect the enzymatic activity either in agar colonies or crude cell lysates by alteration of chromophores or fluorophores (Bornscheuer 2002, Goddard and Reymond 2004, Andexer, Guterl et al 2006. Most agar-plate-based methods suffer from low sensitivity due to the diffusion of the soluble products.…”

Directed Evolution of Industrial Biocatalysts

“…Finally in the K ö nig reaction, with a primary amine and barbituric acid as a coupling reagent, a colored compound that can be detected at 580 nm, is formed. Recently, this method was further developed for activity measurements in microtiter plates, and is thus suitable for high -throughput systems [111] . The assay is theoretically useful to detect the activity and enantioselectivity of HNLs towards any cyanohydrin substrate.…”