Construction of a Bifunctional Enzyme Fusion for the Combined Determination of Biogenic Amines in Foods

Abstract

Biogenic amines (BAs) are a group of low-molecular-mass organic bases derived from free amino acids. Due to the undesirable effects of BAs on human health, amine oxidase-based detection methods for BAs in foods have been developed. Here, we developed a bifunctional enzyme fusion (MAPO) using a Cu(2+)-containing monoamine oxidase (AMAO2) and a flavin adenine dinucleotide-containing putrescine oxidase (APUO) from Arthrobacter aurescens. It was necessary to activate MAPO with supplementary Cu(2+) ions, leading to a 6- to 12-fold improvement in catalytic efficiency (kcat/KM) for monoamines. The optimal temperatures of Cu(2+)-activated MAPO (cMAPO) for both tyramine and putrescine were 50 °C, and the optimal pH values for tyramine and putrescine were pH 7.0 and pH 8.0, respectively, consistent with those of AMAO2 and APUO, respectively. The cMAPO showed relative specific activities of 100, 99, 32, and 32 for 2-phenylethylamine, tyramine, histamine, and putrescine, respectively. The tyramine-equivalent BA contents of fermented soybean pastes by cMAPO were more than 90% of the total BA determined by HPLC. In conclusion, cMAPO is fully bifunctional toward biogenic monoamines and putrescine, allowing the combined determination of multiple BAs in foods. This colorimetric determination method could be useful for point-of-care testing to screen safety-guaranteed products prior to instrumental analyses.

Formation, Degradation, and Detoxification of Putrescine by Foodborne Bacteria: A Review

“…The detection limits of different biosensors for BAs varying between 0.05 μg/kg and 10 mg/kg (Kivirand and Rinken ; Erim ). Recently, putrescine alone or simultaneously with another BA was detected by biosensors, for example, during meat‐spoilage process (Bóka and others ), in alcoholoic beverages (Di Fusco and others ; Bóka and others ); fish samples (Alonso‐Lomillo and others ; Henao‐Escobar and others ), seafoods (Inaba and others ; Henao‐Escobar and others ), or fermented soybean paste (Lee and others ).…”

Analytical possibilities of Putrescine and Cadaverine enzymatic colorimetric determination in tuna based on diamine oxidase: A critical study of the use of ABTS

“…Although the same scheme is frequently used for the colorimetric determination of biochemical metabolites, replacing the charge transfer mediator by a chromogen (such as 2,2′-Azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) [17,18] or 3,3′,5,5′-Tetramethylbenzidine (TMB)), for BPA determination colorimetric methods based on this scheme [18], or on enzymatic reactions [19], are scarce. These methods are usually based on using synthetic chemical receptors.…”

Resolution of quaternary mixtures of cadaverine, histamine, putrescine and tyramine by the square wave voltammetry and partial least squares method

“…The main problem of the described biosensors come from the poor enzyme selectivity towards the different BAs and/or the high working potentials needed for operation, leading to an increase in interferences [20]. In order to improve the selectivity of biosensors more specific enzymatic systems have been used in the construction of these electrochemical biosensors [21][22][23][24][25]. However, these systems present a higher cost associated with their manufacture, and limit the analysis to only one amine.…”