Kenzo Toyama

Determination of phosphate ions with an enzyme sensor system

An enzyme sensor system for the determination of phosphate ions was constructed using immobilized enzymes and an oxygen electrode. The principle of this method is based upon the nucleoside phosphorylase catalyzed reaction for which the presence of inorganic phosphorus is indispensable. One assay could be completed within 3 min. This enzyme sensor was able to withstand at least 70 assays. This system was applicable to simple, rapid and continuous determination of phosphate ions in food.

Enzyme sensor for hypoxanthine and inosine determination in edible fish

SummaryAn enzyme sensor for hypoxanthine (Hx) and inosine (HxR), consisting of an enzyme membrane and an oxygen electrode, was constructed, Xanthine oxidase (XO) and nucleoside phosphorylase (NP) were both immobilized on a membrane prepared from cellulose triacetate, 1,8-diamino-4-aminomethyloctane and glutaraldehyde. The enzyme sensor responded to Hx and HxR in the presence of phosphate, while it responded only to Hx in the absence of phosphate. A linear correlation was observed between current decrease and the concentrations of Hx and HxR in the range 0.5–2.0 mM respectively. Correlation coefficients between the present enzyme sensor and a conventional enzymatic method were 0.98 and 0.94 for Hx and HxR respectively. The standard deviation was +-1.5 μM and 0.75 μM for Hx and HxR respectively in 100 experiments. A simple and rapid determination of Hx and HxR in fish meat was possible within 3 min with the enzyme sensor.

Simultaneous determination of hypoxanthine and inosine with an enzyme sensor

Abstract A sensor for the simultaneous determination of hypoxanthine and inosine was prepared by a combination of the enzyme system (shown below) and an oxygen electrode. Xanthine oxidase and nucleoside phosphorylase, respectively, were covalently immobilized on triacetyl cellulose membranes containing 1,8-diamino-4-aminomethyloctane. Xanthine oxidase membrane, three sheets of the triacetyl cellulose membrane described above, and nucleoside phosphorylase membrane were placed in that order on the tip of the oxygen electrode. The optimum conditions for simultaneous determination of hypoxanthine and inosine were pH 7.8, 303 K, and a flow rate of 1.4ml min −1 . Calibration curves for hypoxanthine and inosine were linear up to 0.4 mM and 4 mM, respectively. The relative errors were 6% and 1.5% for hypoxanthine and inosine, respectively, in 24 assays.

Simultaneous determination of hypoxanthine, inosine, inosine-5′-phosphate and adenosine-5′-phosphate with a multielectrode enzyme sensor

Abstract A multielectrode enzyme sensor for the simultaneous determination of adenosine-5′-phosphate (AMP), inosine-5′-phosphate (IMP), inosine (HXR) and hypoxanthine (HX)in fish meat was developed by assembling four enzyme sensors for AMP, IMP, HXR and HX in a flow cell. These compounds were determined from oxygen consumption according to the following reactions: AMP AD IMP NT HXR NP, PO 3− 4 HX XO, O 2 Uric acid where AD is AMP deaminase, NT is 5′-nucleotidase, NP is nucleoside phosphorylase and XO is xanthine oxidase. Enzymes were covalently bound to a membrane prepared from cellulose triacetate, 1,8-diamino-4-aminomethyloctane and glutaraldehyde. Sensors for HX, HXR, IMP and AMP were prepared by attaching membranes of XO, XONP, XO NPNT, and of XONPNT and AD, respectively, to four oxygen electrodes. Samples extracted from sea bass, bream, flounder, abalone and arkshell were analyzed within 5 min, from the simultaneous response curves of the four electrodes. Results obtained by the multisensor system were in good agreement with those determined by each single electrode.

Determination of adenosine 5'-monophosphate in fish and shellfish using an enzyme sensor

Abstract An enzyme sensor for the determination of adenosine-5′-monophosphate (AMP) concentration in the muscle of fish and shellfish has been developed. The AMP sensor consisted of two immobilized enzyme membranes and an oxygen probe. AMP was oxidized to uric acid by AMP-deaminase, 5′-nucleotidase, nucleoside phosphorylase and xanthine oxidase, and oxygen consumed was monitored amperometrically by an oxygen electrode. The optimum conditions for the enzyme electrode were pH 7.8 and 30°C. Output current was reproducible within 4% of the relative error when a solution containing 10 m m AMP was used. One assay could be completed within 4 min and the sensor was stable for 100 assays over 30 days at 5°C. The sensor was used to determine AMP concentration in bream , Pagrosomus unicolor Quoy and Gaimard; sea bass , Lateolobrax japonicus ; flounder , Lepidopsetta bilineata ; abalone , Haliotis discus hannai ; and arkshell , Anadara broughttoni (Shrenk). AMP in a sample solution was also determined by a conventional method, giving satisfactory comparative results .

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.

Microbial sensor system for nondestructive evaluation of fish meat quality

A microbial sensor system consisting of the bacterium (Alteromonas putrefaciens) immobilized within membranes, a flow cell, an oxygen electrode, peristaltic pumps, a buffer tank, a thermostatically controlled bath and a recorder, was constructed for the nondestructive quality evaluation of bluefin tuna. The chemical compounds on fish meat surfaces which are the indicators of fish meat quality were rapidly determined by using the proposed sensor system. Fish meat quality was determined from the rate of current decrease of the sensor. Good correlations were obtained between fish meat quality and sensor response. One assay could be completed within one minute.

Multifunctional Biosensor for the Determination of Fish Meat Freshness

The estimation of fish freshness is very important in the food industry for the manufacture of high-quality products. Indicators of fish freshness, such as nucleotides,’ ammonia,’ amines,’ volatile acids: catalase activity,’ and P H , ~ have been proposed so far. However, the determination of these indicators requires complicated and time-consuming procedures. Recently, sensors consisting of immobilized enzymes and electrochemical devices have been developed for estimation of organic compound~’.~ In the present study, a multifunctional enzyme sensor system was proposed for estimation of fish freshness. The freshness indicator K, was represented by Equation I .

The effect of temperature on the electrochemical properties of copper—DNA membranes

The influence of temperature, electrode plate metals and protamine on the membrane potential of an electrochemically prepared copper-DNA (Cu-DNA) membrane (size, 2.5 X 6 cm; thickness, 80 micron; Cu/P molar ratio, 0.4) was investigated. The results obtained showed that the membrane potential increased with temperature as well as with increasing order of ionization tendency of the divalent metals used, and decreased with an increase of protamine bound to the membrane. These results indicated that electrons accumulated on the anode side, and positive holes formed on the cathode side, of a Cu-DNA membrane prepared by electrolysis.

ON THE OIL OF FISH MEAL-I

It is well known that the oxidation of oil in fish meal results in not only accumulation of toxic peroxidual oxygen deterimental to animal or fish but also the reduction protein digestibility of the meal. However, about such deteriorative changes of the meal, especially peroxide value of fish meal oil which is possible to procceed in the manufacturing process and also in the storage period, a reliable information have scarcely been presented as yet. In addition, even though several investigations suggested the protection of fish meal from these deterioration with the aid of some chemical antioxidants, none of them did not distinctly point out the merit or behavior of these additives. To throw light on these problems, the authors tentatively prepared brown fish meal samples in the laboratory under the conditions described in Table 1, and investigated the stability of oil in the presence or absence of antioxidant added there to. The results shown in Tables 2-4 indicate that the oxidation of oil in fish meal advances most readily in the drying process, the products proving just after the completion of drying to have peroxide values 90-120meq/kg even when prepared from comparatively fresh raw fish. These undesirable changes, however, could be inhibited to a satisfactory extent by adding BHA to the boiling water in 0.10% concentration (samples No. 2 and 4), although in a lower concentration, 0.01% (No. 6), the result was rather negative. Since the results of estimation of BHA (Table 5) indicate that the amount of BHA found in the meal and in pressed oil were equal to 1% and 10%, respectively, referred to the total amount of BHA applied, a large amount of BHA may certainly remained in boiling water and be discarded. So, to save the loss of BHA, it may be better that an antioxidant is applied to the fish cake rather than to boiling water.