Hidekazu Koya

Development of mediated BOD biosensor system of flow injection mode for shochu distillery wastewater.

Although microbial biochemical oxygen demand (BOD) sensors utilizing redox mediators have attracted much attention as a rapid BOD measurement method, little attempts have been made to apply the mediated BOD biosensors to the flow injection analysis system. In this work, a mediated BOD sensor system of flow injection mode, constructed by combining an immobilized microbial reactor with an electrochemical flow cell of three electrodes configuration, has been developed to estimate BOD of shochu distillery wastewater (SDW). It was demonstrated consequently that the mediated sensing was realized by employing phosphate buffer containing potassium hexacyanoferrate as the carrier. The output current was found to yield a peak with a sample injection, and to result from reoxidation of reduced mediator at the electrode. By employing the peak area as the sensor response, the effects of flow rate and pH of the carrier on the sensitivity were investigated. The sensor system using a microorganism of high SDW-assimilation capacity showed good performance and proved to be available for estimation of BOD of SDW.

Glucose Sensor Based on Ubiquinone-Modified Carbon Paste Electrode

Abstract Two types of carbon paste electrodes containing ubiquinone (CoQ) and glucose oxidase (GOD) were constructed, and their electrochemistry and amperometric response to substrate were investigated. For one type of electrode in which the surface of carbon particles was covered with a part of CoQ molecules added, it was found that the CoQ adsorbed on the carbon particles exerted an insulating effect on the electrode reaction. For the other type of electrode in which the surface was preadsorbed with GOD and most of CoQ was dissolved in the liquid paraffin phase, it was demonstrated that CoQ served as an electron transfer mediator. The latter electrode showed amperometric responses to glucose in the applied potential range 300 – 500 mV and a linear response in the range 0.2 – 4.0 mM in an electrolyte solution (pH 7.0).

Amperometric 2,4‐Dichlorophenoxyacetate Biosensor System Based on a Microbial Reactor and a Tyrosinase‐Modified Electrode

Abstract A novel 2,4‐dichlorophenoxyacetate (2,4‐D) biosensor system was constructed with a reactor for microbial degradation and a flow cell based on a tyrosinase‐modified graphite electrode for product detection. The microorganism, isolated from the agricultural soil collected at northern Kyusyu Island and identified as Ralstonia sp. was employed as the 2,4‐D degrader. Immobilization was performed with a glass column packed with silica gel particles by circulating Luria‐Bertani medium containing 2,4‐D inoculated with the bacteria. The degradation capability of the immobilized cells packed in the reactor was confirmed by circulating a mineral salt medium containing 2,4‐D and monitoring the decrease in 2,4‐D content. The tyrosinase electrode was employed to monitor phenolic and catecholic compounds, since it could be presumed that 2,4‐dichlorophenol and 3,5‐dichlorocatechol could be produced as intermediates in the degradation of 2,4‐D by Ralstonia sp. The flow cell of three electrodes configuration was assembled by using the enzyme electrode as a working electrode. Consequently, amperometric response current could be observed by injecting 2,4‐D solution with phosphate buffer as the mobile phase at the applied potential of 0.5 V vs. Ag/AgCl. The sensitivity of the system was shown to depend on the composition of the mobile phase by comparing the sensitivities obtained with phosphate buffer and mineral salt medium as the mobile phase.

Amperometric Detection of Phenol with Cytochrome C‐Modified Gold Electrode Using Dual Working Electrode System

Abstract The electrochemistry and amperometric sensor response for phenol of cytochrome c‐modified gold electrode have been investigated. The increase in cathodic current with the concentration of H2O2 observed in cyclic voltammograms at the potential more negative than 0.0 V could be considered to arise from the direct electron transfer from the Au electrode to the active site of the immobilized cyt c. The additional increase in the cathodic current with addition of phenol demonstrated the peroxidase‐like activity of cyt c. The amperometric sensor response for phenol depended strongly on the applied potential. The cathodic response current, which is usually used for phenol biosensor based on horseradish peroxidase, could not be observed due to the low peroxidatic activity of cyt c and/or the competition of the reduction of enzyme intermediates by the direct electron transfer with that by the phenol‐mediated mode. This competition could be avoided by using the dual working electrode system in which the direct electron transfer was controlled by the applied potential of the first electrode, and the reaction product was detected electrochemically with the second electrode. Consequently, a cathodic response current attributable to oxidized phenol could be observed with addition of phenol at the second electrode, indicating cytochrome c to have a weak peroxidase activity.

Immobilization of enzyme to platinum electrode and its use as enzyme electrode

A glucose electrode was fabricated by immobilizing glucose oxidase covalently onto a platinized platinum electrode. The sensor showed rapid response with response time of 2—4 s, and also the linear response to the glucose concentration, ranging from 2 x 10-3 to 5 mM. The sensitivity was found to be correlated with the surface area of a base electrode used.

Flow Injection Biosensor System for 2,4-Dichlorophenoxyacetate Based on a Microbial Reactor and Tyrosinase-modified Electrode

Synthetic chlorinated organic compounds have been used extensively as herbicides and pesticides and the contamination of ecosystems with these compounds has stimulated great interest in investigation of these frequently toxic or bioaccumulatable compounds. 2,4-Dichlorophenoxyacetate (2,4-D), one of these compounds, is a synthetic phytohormone that has been widely used as a herbicide for controlling broadleaf weeds, and huge amount of 2,4-D has been released into the environment. 2,4-D is known, on the other hand, to be susceptible to rapid biological degradation in natural environments, and has been used as a model for genetic and biochemical study on the chloroaromatic degradation. A number of its degrading bacteria have been isolated worldwide from a variety of environments and extensively examined on a molecular basis (Amy et al., 1985; Sinton et al., 1986; Perkins et al., 1990; Fulthorpe et al., 1992, 1995; Ka et al., 1994; Tonso et al., 1995; Top et al., 1995; Maltseva et al., 1996; Suwa et al., 1996; Vallaeys et al., 1996; Kamagata et al., 1997; Cavalca et al., 1999; Laemmli et al., 2000; Itoh et al., 2002). One of the most extensively studied strain is Ralstonia eutrophus JMP134, which carries a 2,4-D-degrading gene cluster on the transmissible plasmid pJP4 (Don & Pemberton, 1981; Neilson et al., 1992; Fukumori & Hausinger, 1993a; Laemmli et al., 2000). On the other hand, reliable determination of 2,4-D is indispensable to investigate its biological degradation. A biosensor is a device utilizing a biological sensing element, and a variety of biosensors for 2,4-D detection have been developed (Table 1). Most of sensors reported so far, however, can be classified into either immunoassay or immunoenzymatic assay. They are based on the specific interaction between 2,4-D (antigen) and its antibody. These biosensors have been demonstrated to show very high sensitivities (e.g. the lower detection limit of less than 1 μg/L), while these assays are generally said to be somewhat cumbersome to perform and require considerably expensive reagents. A biosensor based on the inhibition of catalytic activity of enzyme alkaline phosphatase in the presence of 2,4-D has been also proposed and a detection limit of 0.5-6 μg/L has been obtained. The enzyme inhibition effect has been also observed for another pesticide, indicating the analyte selectivity of the sensor to be not expected. On the other hand, very few attempts have been made on the biosensor employing microorganisms as the sensing component.

Hydrolysis of soluble starch by rotary disc of immobilized glucoamylase

Abstract Immobilized glucoamylase sheet was prepared using soluble collagen prepared from cow hide powder as the support material. The immobilized glucoamylase sheet was attached to the rotary disc and the rates of hydrolysis of maltose and soluble starch in the tank were measured. Qualitative discussions are made of the effect of stirring speed of immobilized enzyme disc on the overall reaction rate.

Immobilization of Two Enzyme System of Glucoamylase and Glucoseoxidase onto Platinum Electrode and its Characteristics as Maltose Sensor.

白金電極をアミノシラン化処理してアミノ基を導入し, このアミノ基を介してグルコアミラーゼ (GA) とグルコースオキシダーゼ (GOD) の2酵素を, 固定化用担体を用いることなく, 同時に白金電極に固定化して2酵素固定化白金電極 (GA-GOD-Pt) を作製した.得られたGA-GOD-Ptと対極としてAg/AgClを組み合わせてマルトースセンサを構成し, その特性を検討した.マルトース濃度検量線はpHが5.5から7.5の範囲においてより高いpHでよりよい直線性を示した.この傾向を理論的考察を援用して説明し, 直線的な検量線を得るためにはGA活性を抑制して使用すべきであることを示した.ステップ応答法による動的特性の測定からGA-GOD-Ptはむだ時間おくれのない一次おくれ系とみなせることを示した.

Kinetic studies on the decomposition rate of uric acid by uricase.

酵母から得たウリカーゼによる尿酸の分解に関して, pH7.0のトリス・ホウ酸緩衝液を用いてアラントインおよび過酸化水素の影響, ウリカーゼの活性経時変化および尿酸濃度の経時変化の測定と結果の解析を行った.尿酸分解実験は温度15, 25および35℃, 尿酸初濃度1.0×10-3, 3.0×10-3および5.0×10-3mg/cm-3, ウリカーゼ初濃度3.7×10-4～7.4×10-3mg/cm3で行い, 尿酸の最終分解率は約34～92%の範囲であった.結果はつぎのとおりである.1) アラントインおよび過酸化水素は尿酸分解に阻害作用を及ぼさない.2) ウリカーゼの活性経時変化を分数式あるいは一次式で表すことができた.3) ウリカーゼの活性経時変化を考慮した速度式と尿酸分解の実測値を用いて式中のパラメータの値を推定した.その速度式と得られたパラメータ値を用いた尿酸分解過程のシミュレーションは実測値と比較的よく一致した.

Rotary Multidisc Reactor of Collagen Supported Immobilized Glucoamylase

A rotary multidisc reactor of immobilized glucoamylase was constructed; and the hydrolysis rates of maltose (Katayama Kagaku Co., Japan) and soluble starch (Katayama Kagaku Co., Japan) were measured. The parameters included in the kinetic equation were estimated for these reaction systems.