Trimethylamine biosensor with flavin-containing monooxygenase type 3 (FMO3) for fish-freshness analysis

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Abstract

Trimethylamine (TMA) biosensor was constructed by immobilizing flavin-containing monooxygenase type-3 (FMO3), as one of drug metabolizing enzymes in human liver, onto a sensitive area of a dissolved oxygen electrode.

The FMO3 immobilized sensor with flow injection analysis (FIA) was calibrated against trimethylamine solutions (putrefactive substance of fish) from 1.0 to 50.0 mmol/l, with good reproducibility in the repeatedly measurements (coefficient of variation: 4.39%, n = 5).

By applying the TMA sensor for a fish-freshness analysis with extractions of horse-mackerel, the sensor output increased with the holding time of fish samples at 25 °C because of their decomposition. The TMA sensor with FMO3 would be convenient device for evaluating fish freshness.

Introduction

An assessment of food freshness is important in the field of food industries. Fish freshness has been evaluated chemically and expressed as K-value which is useful index of raw-fish grade [1]. However, the K-value approach require the sample preparation and the complicated sensor system with several kinds of biochemical substances because the K-value was calculated from the concentrations of inosine 5′-monophosphate (IMP), inosine (HxR) and hypoxanthine (Hx) in the fish-extract solution, with several kinds of biochemical process and reagents [2], [3]. Then a newly approach is required at fish markets, restaurant kitchens and tables, i.e. non-destructive methods with simple biochemical reaction, such as smell evaluation of putrid fish-odor with higher sensitivity of human smell sense.

Trimethylamine (TMA) is typical and common fish-odor substance in seafood, and is produced by the decomposition of trimethylamine N-oxide (TMAO) in sea creatures [4], [5], [6]. The fact is that fresh marine products contain little TMA (i.e. Bay scallop: 0.41 mmol/l, Red rice prawn: 0.15 mmol/l, others: small amount or not detected), but plenty of odorless trimethylamine N-oxide (i.e. Starspotted smooth-hound: 18.77 mmol/l, Red halibut: 5.31 mmol/l, Olive flounder: 4.17 mmol/l, Red rice prawn: 4.14 mmol/l, Bay scallop: 3.58 mmol/l, etc.) as a precursor substance [7], [8], [9]. TMA is produced by decomposition of TMAO by microorganism and its concentration is rapidly increased in marine products after their death [4], [5], [10]. The TMA measurement in seafood has been reported to be one of indicators for the evaluation of fish freshness (fresh: 0–1 mg/100g, initial corruption: 1–5 mg/100g, rotting fish [uneatable in the raw]: more than 6 mg/100g) [6], [11], [12], [13], [14]. Then, TMA is expected to be freshness index in fish [12], [13], [14], [15].

In humans, TMA is metabolized exclusively to TMAO with up to 60 mg/day excreted in the urine of healthy people and less than 5% excreted as the parent compound [16], [17]. The reaction is widely considered to be catalyzed by flavin-containing monooxygenase (FMO, EC1.14.13.8) as one of xenobiotic metabolizing enzymes [18], [19], [20] which can decompose and detoxicate most chemicals in vivo, even the inhaled VOC. Trimethylaminuria or “fish-odor syndrome” is a human disorder characterized by an impaired ability to convert the malodorous TMA to its odorless N-oxide [21]. A mutation in the FMO gene, which decreases TMA metabolism, has been described recently [22]. FMO3 (type 3 of the polymorphic FMO family enzymes) has been recognized to be the major hepatic form in adults and catalyze the majority of TMAO formation from TMA [22], [23], [24] in vivo with the following reaction:TMA+NADPH+H++O2FMO3TMAO+H2O+NADP+

FMO3 is possible to be expressed from human FMO3 cDNA using a baculovirus expression system [22], [25] and commercialized. Oxygen consumption accompanied the enzyme reaction has been used for analyzing the enzyme activity or the substrate concentration [22], [26].

In this research, a fish-freshness sensor was developed using flavin-containing monooxygenase type-3 as a major enzyme for metabolizing TMA in human liver [24]. The sensor was also applied for evaluating the fish freshness with extract samples from horse-mackerel as sample fish.

Section snippets

Construction of TMA biosensor

Fig. 1 shows the structure of the FMO3 immobilized biosensor and the reaction cell for the flow injection analysis (FIA) of TMA solutions. The TMA sensor was constructed by attaching an FMO3 immobilized membrane onto a sensitive area of a dissolved oxygen electrode (Model BO-P, ABLE Corp., Tokyo, Japan) using a nylon net and a silicon O-ring [27]. Flavin-containing monooxygenase type-3 (FMO3, E.C.1. 14.13.8, P233, from Adult human liver; 30,200 pmol/mg min, Gentest Corporation, MA, USA) catalyzes

Evaluation of the TMA biosensor

Fig. 3 indicates the typical response curves of the FMO biosensor by applying the injection (100 μl) of the standard TMA solutions or phosphate buffer solution in the flow injection analysis with 0.15 ml/min of buffer flow rate. In the figure, the sensor output was presented by the absolute value of the current difference from the initial current value for phosphate buffer solution.

As the figure indicates, the sensor outputs increased rapidly following the injections of the TMA solution (0, 0.01,

Conclusions

The TMA biosensor was constructed by immobilizing flavin-containing monooxygenase type-3, as one of drug metabolizing enzymes in human liver, to a dissolved oxygen electrode.

The FMO3 immobilized biosensor was calibrated against TMA solutions from 1.0 to 50.0 mmol/l with good reproducibility. As the results of the experiments with extractions of horse-mackerel, the sensor output increased with the holding time of fish samples at 25 °C because of their decomposition. The FMO3 biosensor was expected

Acknowledgements

This study was supported in part by The Hoso-Bunka Foundation Inc. (HBF) Assistance Grants, by Tokyo Ohka Foundation Grants-in-Aid for promotion of science and technology and SECOM Science and Technology Foundation for Research Grants.

Kohji Mitsubayashi is a professor of Tokyo Medical and Dental University (Department of Biomedical Devices and Instrumentation). His research interests include wearable chemical sensors for human monitoring, a newly olfactometric system using biological materials, biomolecular and medical devices, microsystem technology, etc.

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  • Cited by (0)

    Kohji Mitsubayashi is a professor of Tokyo Medical and Dental University (Department of Biomedical Devices and Instrumentation). His research interests include wearable chemical sensors for human monitoring, a newly olfactometric system using biological materials, biomolecular and medical devices, microsystem technology, etc.

    Yohei Kubotera had been an undergraduate student at Tokai University (Mitsubayashi Laboratory) from 2000 to 2001. He had investigated a fish-freshness sensor.

    Kazuhisa Yano had been an undergraduate student at Tokai University (Mitsubayashi Laboratory) from 2001 to 2002. He had investigated a fish-freshness sensor and a smell reproducer.

    Yuki Hashimoto had been a graduate student at Tokai University (Mitsubayashi Laboratory) from 2000 to 2002. He had investigated electrical and optical bio-sniffers and a smell communication system.

    Takuo Kon is a graduate student at Tokai University (Mitsubayashi Laboratory). He has investigated an optical bio-sniffer and cell informatics.

    Shinya Nakakura is a graduate student at Tokai University (Mitsubayashi Laboratory). He has investigated cell informatics and optical and micro system for flow injection analysis.

    Yoshitake Nishi is a professor of Tokai University (Department of Material Science). He has investigated intelligent materials and applications.

    Hideaki Endo is an associate professor of Tokyo University of Marine Science and Technology (Department of Ocean Science). His research interests include newly biosensors and flow cytometer for fish analysis, etc.

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