Presentation Information
[P01-115]Development of the bio-fluorometric sensor using nicotinoprotein carveol dehydrogenase
○Kentaro Yazaki1, Houcheng Xue1, Kurea Ikegai1, Mao Fukushi1, Ryutaro Asano1, Kazunori Ikebukuro1, Kenta Ichikawa2, Kenta Iitani2, Kohji Mitsubayashi2, Wakako Tsugawa1 (1. Department of Biotechnology and Life Science, Graduate School of Engineering, Tokyo University of Agriculture and Technology, Tokyo, Japan (Japan), 2. Department of Biomedical Devices and Instrumentation, Laboratory for Biomaterials and Bioengineering, Institute of Integrated Research, Institute of Science Tokyo (Japan))
Keywords:
NAD-dependent,enzyme sensor,fluorescence,nicotinoprotein
The demand for natural flavor and active ingredient, including carveol and carvone in food, beverages and oral care products is increasing year by year. To ensure the quality of these naturally derived products, there is a growing need for rapid and simple biosensing systems.
Unlike typical dehydrogenases that require the external addition of NAD(H), nicotinoproteins, a unique NAD+-dependent dehydrogenase, contain a tightly but non-covalently bound, non-exchangeable NAD(H) molecule within their active site.1,2,3 This structural feature allows the enzyme to function as a self-contained catalytic unit, enabling the design of "cofactor-less" biosensors that operate efficiently without the continuous supply of expensive external cofactors. Carveol dehydrogenase (CADh) is a nicotinoprotein catalyzing oxidation of carveol to carvone. We will report the bio-fluorometric sensor for carvone detection, using nicotinoprotein CADh from Rhodococcus erythropolis DCL14.2
The recombinant CADh was expressed in Escherichia coli and purified by Ni-affinity chromatography. The purified CADh showed activity using DCIP as an external electron acceptor, as Vmax : 4.0 U/mg, Km : 50 µM for (–)–carveol. The enzyme showed fluorescence peak at 440 nm (Ex 340 nm) indicating that NADH was tightly bound to CADh.3 Building upon these biochemical characteristics, a bio-fluorometric sensor system was constructed.
The integrated system consisted of a 340 nm bandpass filter as an excitation source, a UV-LED equipped with a 490 nm bandpass filter for fluorescence measurement, and a detection unit featuring a CADh-immobilized membrane on the optical fiber. These components were connected via a bifurcated optical fiber probe, allowing for real-time monitoring of the NADH fluorescence within the enzyme matrix.4
The sensor's performance was evaluated by immersing the enzyme-immobilized fiber-optic probe into a buffer solution and monitoring fluorescence changes upon the addition of various concentrations of carveol or carvone.
The addition of carveol resulted in a slight decrease in fluorescence at 1 mM carveol, whereas the addition of carvone caused a clear decrease, likely due to the oxidation of NADH. The integrated bio-fluorometric sensor successfully achieved the detection of carvone within a linear range of 10 to 300 μM.
These findings provide critical insights into the behavior of nicotinoproteins in biosensing applications. Since the calibration curve for authentic carvone allows for the measurement of carvone even at low concentrations, this biosensor can be used for the rapid and simple detection of raw materials in products containing naturally derived carvone.
References
[1] V. Bystrykh et al, J Bacteriol, 1993, 175, 6, 1814-1822
[2] M. van der Werf et al., J Biol. Chem, 1999, 274, 37, 26296-26304
[3] H. Xue, et al., Mol. Sci., 2026, 27, 2367
[4] X. Zhang, et al., Sens Actuators, B., 2025, 427, 137220
Unlike typical dehydrogenases that require the external addition of NAD(H), nicotinoproteins, a unique NAD+-dependent dehydrogenase, contain a tightly but non-covalently bound, non-exchangeable NAD(H) molecule within their active site.1,2,3 This structural feature allows the enzyme to function as a self-contained catalytic unit, enabling the design of "cofactor-less" biosensors that operate efficiently without the continuous supply of expensive external cofactors. Carveol dehydrogenase (CADh) is a nicotinoprotein catalyzing oxidation of carveol to carvone. We will report the bio-fluorometric sensor for carvone detection, using nicotinoprotein CADh from Rhodococcus erythropolis DCL14.2
The recombinant CADh was expressed in Escherichia coli and purified by Ni-affinity chromatography. The purified CADh showed activity using DCIP as an external electron acceptor, as Vmax : 4.0 U/mg, Km : 50 µM for (–)–carveol. The enzyme showed fluorescence peak at 440 nm (Ex 340 nm) indicating that NADH was tightly bound to CADh.3 Building upon these biochemical characteristics, a bio-fluorometric sensor system was constructed.
The integrated system consisted of a 340 nm bandpass filter as an excitation source, a UV-LED equipped with a 490 nm bandpass filter for fluorescence measurement, and a detection unit featuring a CADh-immobilized membrane on the optical fiber. These components were connected via a bifurcated optical fiber probe, allowing for real-time monitoring of the NADH fluorescence within the enzyme matrix.4
The sensor's performance was evaluated by immersing the enzyme-immobilized fiber-optic probe into a buffer solution and monitoring fluorescence changes upon the addition of various concentrations of carveol or carvone.
The addition of carveol resulted in a slight decrease in fluorescence at 1 mM carveol, whereas the addition of carvone caused a clear decrease, likely due to the oxidation of NADH. The integrated bio-fluorometric sensor successfully achieved the detection of carvone within a linear range of 10 to 300 μM.
These findings provide critical insights into the behavior of nicotinoproteins in biosensing applications. Since the calibration curve for authentic carvone allows for the measurement of carvone even at low concentrations, this biosensor can be used for the rapid and simple detection of raw materials in products containing naturally derived carvone.
References
[1] V. Bystrykh et al, J Bacteriol, 1993, 175, 6, 1814-1822
[2] M. van der Werf et al., J Biol. Chem, 1999, 274, 37, 26296-26304
[3] H. Xue, et al., Mol. Sci., 2026, 27, 2367
[4] X. Zhang, et al., Sens Actuators, B., 2025, 427, 137220
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