Colorimetric determination of the plant toxin β-cyano-l-alanine using a multistep enzyme reaction

The non-proteinogenic amino acid β-cyano-l-alanine (AlaCN) is a plant toxin formed during HCN detoxification. The presence of AlaCN and its dipeptide, γ-glutamyl-AlaCN (Glu-AlaCN), in the agriculturally important vetch (Vicia sativa) is a matter of concern, as these compounds can be harmful to animals fed with vetch. In addition, adulteration of lentils with vetch has occurred, posing a risk for human consumers. AlaCN and Glu-AlaCN have been determined by HPLC which, however, does not meet the general need for low-cost, simple analytical methods suitable for developing assay kits or sensors. Therefore, the aim of this study was to propose a simple, rapid and inexpensive colorimetric assay for AlaCN. The assay is based on an artificial cascade reaction catalyzed by nitrilase NIT4 (EC 3.5.5.1), NAD-dependent aspartate dehydrogenase (EC 1.4.1.21) and, optionally, asparaginase (EC 3.5.1.1). The NADH formed in the final step is determined at 460 nm using 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium monosodium salt (WST-8) and 1-methoxy-5-methylphenazinium methyl sulfate. In summary, the first colorimetric AlaCN test based on a multistep enzymatic reaction was proposed, with parts of it applicable as a colorimetric L-Asp test. Both tests have a limit of detection of approximately 1.4 μM. The AlaCN test enabled us to clearly distinguish between the spiked and non-spiked samples, as well as between lentils and vetch. Future work may focus on optimizing sample preparation and assay conditions to maximize responses and eliminate matrix effects, while saving material and time. This may be extended to the development of a new test for L-Asn, a precursor of acrylamide.