Hirokazu Okuma

Development of a system with double enzyme reactors for the determination of fish freshness

Abstract A continous system for the determination of fish freshness with double enzyme reactors was developed and applied to the determination of the freshness indicator ( K i =[(HxR + Hx)/(IMP + Hx)/IMP + Hxr + Hx)]× 100) in many types of fish, where IMP, HxR and Hx are inosine monophosphate, inosine and hypoxanthine, respectively. The system was prepared from two combinations of oxygen electrodes and reactors. One reactor for the determination of the total amount of HxR and Hx was packed with nucleoside phosphorylase (NP) and xanthine oxidase (XOD) immobilized simultaneously on chitosan porous beads. Similarly, another reactor for IMP, HxR and Hx was packed with 5-nucleotidase (NT), NP and XOD immobilized simultaneously on chitosan beads. The system was prepared from two combinations of oxygen electrodes and reactors. One assay could be completed within 5 min. The system for the determination of fish freshness was reproducible within 2.1% ( n =30). The immobilized enzymes were sufficiently stable for at least 7 months at 4°C. More than 200 samples could be analysed in about 1 month by using these enzyme reactors. The results for fish meat (13 types) correlated satisfactorily with those obtained by liquid chromatography ( r =0.989, n ==253) and ion-exchange column chromatography ( r =0.973, n =50). These results suggest that the proposed sensor system provides a simple, rapid and economical method for the determination of fish freshness ( K i ).

Mediated amperometric biosensor for hypoxanthine based on a hydroxymethylferrocene-modified carbon paste electrode

An amperometric enzyme electrode for the determination of hypoxanthine in fish meat is described. The hypoxanthine sensor was prepared from xanthine oxidase immobilized by covalent binding to cellulose triacetate and a carbon paste electrode containing hydroxymethylferrocene. The xanthine oxidase membrane was retained behind a dialysis membrane at a carbon paste electrode. The sensor showed a current response to hypoxanthine due to the bioelectrocatalytic oxidation of hypoxanthine, in which hydroxymethyiferrocene served as an electron-transfer mediator. The limit of detection is 6 × 10−7 M, the relative standard deviation is 2.8% (n=28) and the response is linear up to 7 × 10−4 M. The sensor responded rapidly to a low hypoxanthine concentration (7 × 10−4 M), the steady-state current response being achieved in less than 1 min, and was stable for more than 30 days at 5 ° C. Results for tuna samples showed good agreement with the value determined by the conventional method.

Biosensor system for continuous flow determination of enzyme activities. I. Determination of glucose oxidase and lactic dehydrogenase activities

Abstract A biosensor system for continuous flow determination of enzyme activity was developed and applied to the determination of glucose oxidase and lactic dehydrogenase activities. The glucose oxidase activity sensor was prepared from the combination of an oxygen electrode and a flow cell. Similarly, the lactic dehydrogenase activity sensor was prepared from the combination of a pyruvate oxidase membrane, an oxygen electrode, and a flow cell. Pyruvate oxidase was covalently immobilized on a membrane prepared from cellulose triacetate, 1,8-diamino-4-aminomethyloctane, and glutaraldehyde. Glucose oxidase activity was determined from the oxygen consumed upon oxidation of glucose catalyzed by glucose oxidase. Lactic dehydrogenase activity was determined from the pyruvic acid formed upon dehydrogenation of lactic acid catalyzed by lactic dehydrogenase. The amount of pyruvic acid was determined from the oxygen consumed upon oxidation of pyruvic acid by pyruvate oxidase. Calibration curves for activity of glucose oxidase and lactic dehydrogenase were linear up to 81 and 300 units, respectively. One assay could be completed within 15 min for both sensors and these were stable for more than 25 days at 5°C. The relative errors were ±4 and ±6% for glucose oxidase and lactic dehydrogenase sensors, respectively. These results suggest that the sensor system proposed is a simple, rapid, and economical method for the determination of enzyme activities.

Biosensor system for continuous flow determination of enzyme activities. II. Simultaneous determination of plural enzyme activities

A biosensor system for continuous flow determination of plural enzyme activities was prepared from the combination of two pyruvate sensors, a prereactor and a flow cell. This system was applied to the simultaneous determination of lactic dehydrogenase (LDH) and glutamic-pyruvic transaminase (GPT) activities in the same sample. These enzyme activities can be determined by measuring pyruvate produced by the enzyme reactions as follows. The amount of pyruvic acid can also be determined from the amount of oxygen consumed upon oxidation of pyruvic acid by pyruvate oxidase. (Formula: see text). Therefore, both of the detectors for the determination of lactic dehydrogenase and glutamic-pyruvic transaminase activities were prepared from the combination of a pyruvate oxidase membrane and an oxygen electrode. Pyruvate oxidase was covalently immobilized on a membrane prepared from cellulose triacetate. A linear relation was obtained between the output current and LDH or GPT activities in the range of 50 to 3,600 IU l-1 or 6 to 1,000 IU l-1, respectively. Each assay of these enzyme activities was completed within 15 min. The results obtained had a precision of ca. 4%. The sensor was stable for more than 25 days at 5 degrees C.

Development of D-allose sensor on the basis of three strategic enzyme reactions.

Rare sugars are defined as monosaccharides and their derivatives that rarely exist in nature, according to the International Society of Rare Sugars. D-Allose (3-epi d-glucose) is one of the rare sugars, for which various physiological activities have recently been found, with increasing attention to its applications to bio-industry. Until now, however, there is no convenient method of measuring these sugars in a specific manner. For detecting D-allose, three consecutive enzyme reactions were devised by fabricating of a reaction batch chamber packed with L-rhamnose isomerase (LRI), D-tagatose 3-epimerase (DTE) and a screen-printed electrode, on which D-fructose dehydrogenase (DFDH) was immobilized. To obtain a substantial sensing system, extensive experimental parameters were optimized. These included the concentration of photo-crosslinkable poly (vinyl alcohol) bearing stilbazolium groups (PVA-SbQ), reaction ratios, and temperature of the batch chamber. By adopting the three consecutive enzyme reactions, an undesirable reverse reaction was minimized. As a result, the developed sensor system exhibited a good linear response on D-allose in the range from 0.1 to 50 mM (r(2)=0.998). The system has an apparent advantage over the previous chromatography approach in terms of simplicity and inexpensiveness.

Determination of L-Ascorbic Acid in Green Tea with an Enzyme Reactor Electrode System.

(1) リアクター型バイオセンサシステムによる,ビタミンC(L-アスコルビン酸)の定量システムを構築し,マイクロ波茶葉乾燥火入機と従来法とにより製造した,緑茶中のビタミンCの定量に応用した.(2) このバイオセンサシステムは固定化酵素リアクター.酸素電極,A/Dコンバータ,マイクロコンピュータ,ペリスタティックポンプより構成されている.固定化酵素リアクターは多孔質キトサンビーズにアスコルビン酸オキシダーゼを固定化し,これをミニカラム(φ 2×50mm)に充填して調製した.1検体の測定は,3分以内であった.(3) このシステムでの検出限界は,1mg/100mlで1000mg/100mlまで直線的応答が得られた.また,繰り返しの測定精度は相対標準偏差で2%以内(n=40)であった.固定化酵素は室温で3ケ月以上安定であった.(4) 茶抽出液および各種飲料の測定結果は,酵素分析法(Fキット)及びインドフェノール法と良好な相関を得た.これらの結果,本システムを用いることにより,食品中のビタミンCを簡便,迅速,経済的に定量できることが判明した.(5) 試料として静岡県産やぶきた茶を用い,煎茶製造工程の火入れ段階でマイクロ波乾燥火入機と従来方法とを行い,これらが及ぼす茶葉中のビタミンC含量への影響について検討したところ,茶葉中に残存するビタミンC含量は,煎茶製造時の火入れ方法の違いにより大きく異なり,マイクロ波乾燥火入機で製造した茶葉には,従来方法で製造したものに比較し,多量のビタミンCが残存していた.