(Invited) F-ALDH, a New Catalyst for Enzyme and Aptamer- Based Electrochemical Biosensors
Authors: Vasilescu Alina , Banciu Roberta Maria , Ftodiev Andreea , Necula Petrareanu Georgiana , Bala Camelia , Purcarea Cristina 
Published: February 2024

The selectivity, affinity and catalytic activity are key characteristics which recommend enzymes as components of biosensors, whether for use as main recognition element or for signal amplification. Additionally, the enzyme inhibition by various molecules can be exploited for analytical purposes. In the search for new enzymes with good stability and catalytic properties, a recombinant psychrophilic aldehyde dehydrogenase from the Antarctic Flavobacterium PL002 was recently characterized and shown to have potential in biosensing [1-5].

The enzyme is active in a wide temperature range up to 60º C and has high catalytic activity [1,2]. It accepts various aldehydes substrates with Michaelis Menten constants of e.g., Km = 0. 137 ± 0.016 mmol L−1 for benzaldehyde. The enzyme may use both nicotinamide adenin dinucleotide (NAD+) and nicotinamide adenin dinucleotide phosphate (NADP+) as cofactors and has higher affinity for NAD+ ( Km=0.033mmol L−1 and 0.431 mmol L−1 for NAD+ and NADP+, respectively [2]). As for other aldehyde dehydrogenases, the enzyme is inhibited by heavy metals and dithiocarbamate fungicides [2,3].

To explore its potential as recognition element in biosensors, enzymatic tests and electrochemical biosensors exploiting the catalytic activity of F-ALDH or its inhibition were first developed [4,5]. In biosensors, the enzyme was immobilized by cross linking in a matrix of bovine serum albumin [5] or by nickel-histidine affinity. Enzymatic inks with a stability of several months at 4º C were obtained by immobilizing the enzyme on magnetic particles functionalized with a complex of nickel-nitrilotriacetic acid (Ni-NTA) followed by mixing with different additives. Practical applications for the specific detection of (i) benzaldehyde in a pharmaceutical ingredient [5] and (ii) acetaldehyde in wines were demonstrated. In wines, acetaldehyde is present in a free form or bound to sulphur dioxide or phenolic compounds. Both free and bound acetaldehyde are relevant for wine quality and its evolution. The F-ALDH enzyme enables testing of both forms in cellar like conditions. Moreover, the detection of free acetaldehyde serves as example for aldehydes detection in vapor phase, opening the way for other similar applications.

A separate line of research explored the potential of F-ALDH for the quantitative determination of enzyme’s inhibitors. Spectrophotometry measurements emphasized a 15% reduction of enzyme’s activity in the presence of either 28 ppb thiram or 0.5 ppb mercury chloride. The solution advanced for overcoming the selectivity issues in the detection of thiram is to couple the electrochemical biosensor measuring the F-ALDH inhibition with SERS measurements of the dithiocarbamate fungicide [6].

Finally, the F-ALDH enzyme may serve as label in electrochemical aptasensors. The enzyme was attached to an aptamer for lysozyme via Ni-histidine affinity and preserved good catalytic activity for the conversion of acetaldehyde. Preliminary studies were focused on investigating the affinity of the enzyme-labeled aptamer for lysozyme in relation to the linker length.

Based on the demonstrated catalytic activity of the enzyme and its preservation upon immobilization on different supports or by linking to an aptamer, various sensing configurations can be envisaged that will exploit the potential of the new enzyme.

Acknowledgements: This work was financially supported by the UEFISCDI projects ERANET-M-ENZ4IFACES, contract 166/01.05.2020 (for AV, GNP and CP) and PN-III-P4-ID-PCE-2020-2297 E-MAP, contract 84/9.02.2021 (for RMB and AF).

References

Necula-Petrareanu G, Lavin P, Vasilescu A, Purcarea C (2018) Antarctic Flavobacterium aldehyde dehydrogenase – a cold adapted catalyst for biosensing. J Biotechnol 280:S18. https://doi. org/10.1016/j.jbiotec.2018.06.054

Necula-Petrareanu, G.; Lavin, P.; Paun, V.I.; Gheorghita, G.R.; Vasilescu, A.; Purcarea, C. Highly stable cold-active aldehyde dehydrogenase from the marine Antarctic Flavobacterium sp. PL002. Fermentation. 2022 8(1):7-24 https:// doi.org/10.3390/fermentation8010007

Vasilescu A, Titoiu A.M, Purcarea C, Necula Petrareanu G, Method for determining thiram fungicide based on enzymatic inhibition and electrochemical sensor, Romanian patent application, OSIM RO133890A2 /13.08.2018

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