JPS6314301B2 - - Google Patents
Info
- Publication number
- JPS6314301B2 JPS6314301B2 JP54165047A JP16504779A JPS6314301B2 JP S6314301 B2 JPS6314301 B2 JP S6314301B2 JP 54165047 A JP54165047 A JP 54165047A JP 16504779 A JP16504779 A JP 16504779A JP S6314301 B2 JPS6314301 B2 JP S6314301B2
- Authority
- JP
- Japan
- Prior art keywords
- fructose
- concentration
- enzyme
- immobilized
- electrode
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired
Links
- 229930091371 Fructose Natural products 0.000 claims description 31
- 239000005715 Fructose Substances 0.000 claims description 31
- RFSUNEUAIZKAJO-ARQDHWQXSA-N Fructose Chemical compound OC[C@H]1O[C@](O)(CO)[C@@H](O)[C@@H]1O RFSUNEUAIZKAJO-ARQDHWQXSA-N 0.000 claims description 31
- 238000000034 method Methods 0.000 claims description 23
- 239000005515 coenzyme Substances 0.000 claims description 21
- FBPFZTCFMRRESA-KVTDHHQDSA-N D-Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 claims description 20
- 108090000790 Enzymes Proteins 0.000 claims description 13
- 102000004190 Enzymes Human genes 0.000 claims description 13
- 235000010355 mannitol Nutrition 0.000 claims description 10
- 238000006243 chemical reaction Methods 0.000 claims description 8
- 101710088194 Dehydrogenase Proteins 0.000 claims description 6
- 229930027945 nicotinamide-adenine dinucleotide Natural products 0.000 description 20
- BOPGDPNILDQYTO-NNYOXOHSSA-N nicotinamide-adenine dinucleotide Chemical compound C1=CCC(C(=O)N)=CN1[C@H]1[C@H](O)[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OC[C@@H]2[C@H]([C@@H](O)[C@@H](O2)N2C3=NC=NC(N)=C3N=C2)O)O1 BOPGDPNILDQYTO-NNYOXOHSSA-N 0.000 description 11
- BAWFJGJZGIEFAR-NNYOXOHSSA-N NAD zwitterion Chemical compound NC(=O)C1=CC=C[N+]([C@H]2[C@@H]([C@H](O)[C@@H](COP([O-])(=O)OP(O)(=O)OC[C@@H]3[C@H]([C@@H](O)[C@@H](O3)N3C4=NC=NC(N)=C4N=C3)O)O2)O)=C1 BAWFJGJZGIEFAR-NNYOXOHSSA-N 0.000 description 10
- 229950006238 nadide Drugs 0.000 description 9
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 7
- 229940088598 enzyme Drugs 0.000 description 7
- 239000008103 glucose Substances 0.000 description 7
- 239000007853 buffer solution Substances 0.000 description 6
- 229930006000 Sucrose Natural products 0.000 description 5
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 5
- 238000005259 measurement Methods 0.000 description 5
- 239000005720 sucrose Substances 0.000 description 5
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 description 4
- 238000002848 electrochemical method Methods 0.000 description 4
- BASFCYQUMIYNBI-UHFFFAOYSA-N platinum Chemical compound [Pt] BASFCYQUMIYNBI-UHFFFAOYSA-N 0.000 description 4
- 235000000346 sugar Nutrition 0.000 description 4
- 150000008163 sugars Chemical class 0.000 description 4
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 3
- 230000008859 change Effects 0.000 description 3
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 description 3
- 235000015203 fruit juice Nutrition 0.000 description 3
- 238000004128 high performance liquid chromatography Methods 0.000 description 3
- 238000006722 reduction reaction Methods 0.000 description 3
- 102000018832 Cytochromes Human genes 0.000 description 2
- 108010052832 Cytochromes Proteins 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 2
- 239000003431 cross linking reagent Substances 0.000 description 2
- 230000007423 decrease Effects 0.000 description 2
- 235000013399 edible fruits Nutrition 0.000 description 2
- 238000006911 enzymatic reaction Methods 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 235000013305 food Nutrition 0.000 description 2
- 230000003100 immobilizing effect Effects 0.000 description 2
- 229910052760 oxygen Inorganic materials 0.000 description 2
- 239000001301 oxygen Substances 0.000 description 2
- 229910052697 platinum Inorganic materials 0.000 description 2
- 244000144730 Amygdalus persica Species 0.000 description 1
- LUAZZOXZPVVGSO-UHFFFAOYSA-N Benzyl viologen Chemical compound C=1C=C(C=2C=C[N+](CC=3C=CC=CC=3)=CC=2)C=C[N+]=1CC1=CC=CC=C1 LUAZZOXZPVVGSO-UHFFFAOYSA-N 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- 244000241235 Citrullus lanatus Species 0.000 description 1
- 235000012828 Citrullus lanatus var citroides Nutrition 0.000 description 1
- 108010015776 Glucose oxidase Proteins 0.000 description 1
- 239000004366 Glucose oxidase Substances 0.000 description 1
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 description 1
- 108010093096 Immobilized Enzymes Proteins 0.000 description 1
- 241000192130 Leuconostoc mesenteroides Species 0.000 description 1
- 244000141359 Malus pumila Species 0.000 description 1
- 235000006040 Prunus persica var persica Nutrition 0.000 description 1
- 241000220324 Pyrus Species 0.000 description 1
- 241000219094 Vitaceae Species 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 235000021016 apples Nutrition 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 108010051210 beta-Fructofuranosidase Proteins 0.000 description 1
- 230000001588 bifunctional effect Effects 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 230000008878 coupling Effects 0.000 description 1
- 238000010168 coupling process Methods 0.000 description 1
- 238000005859 coupling reaction Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 238000000840 electrochemical analysis Methods 0.000 description 1
- 238000004186 food analysis Methods 0.000 description 1
- 235000011389 fruit/vegetable juice Nutrition 0.000 description 1
- 150000002303 glucose derivatives Chemical class 0.000 description 1
- 229940116332 glucose oxidase Drugs 0.000 description 1
- 235000019420 glucose oxidase Nutrition 0.000 description 1
- 235000021021 grapes Nutrition 0.000 description 1
- 150000002463 imidates Chemical class 0.000 description 1
- 239000012212 insulator Substances 0.000 description 1
- 235000011073 invertase Nutrition 0.000 description 1
- 238000004811 liquid chromatography Methods 0.000 description 1
- 229910052751 metal Inorganic materials 0.000 description 1
- 239000002184 metal Substances 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 210000003097 mucus Anatomy 0.000 description 1
- 229940101270 nicotinamide adenine dinucleotide (nad) Drugs 0.000 description 1
- 150000007524 organic acids Chemical class 0.000 description 1
- 235000005985 organic acids Nutrition 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 230000001590 oxidative effect Effects 0.000 description 1
- FIKAKWIAUPDISJ-UHFFFAOYSA-L paraquat dichloride Chemical compound [Cl-].[Cl-].C1=C[N+](C)=CC=C1C1=CC=[N+](C)C=C1 FIKAKWIAUPDISJ-UHFFFAOYSA-L 0.000 description 1
- 235000021017 pears Nutrition 0.000 description 1
- 238000002203 pretreatment Methods 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 230000002441 reversible effect Effects 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 238000004611 spectroscopical analysis Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 230000002123 temporal effect Effects 0.000 description 1
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Description
本発明は、食品中に含まれる果糖の濃度を選択
的に定性あるいは定量分析する新規な方法、なら
びにその方法に用いる電極に関するものである。
すなわち、本発明は以下の反応式(1)に示すよう
に、Ｄ−マンニトール脱水素酵素（以下DMDH
で表す）によつて果糖を選択的にＤ−マンニトー
ルに還元し、この反応に共役して減少する還元型
補酵素濃度を電気化学的に検出し、果糖濃度を迅
速簡便に測定するものである。
食品、例えば果実中の糖類としては、ぶどう
糖、しよ糖、果糖が最も代表的なものであり、こ
れらの糖類の含有量を測定することは、食品分析
面で重要である。上記代表的糖類の中でぶどう糖
としょ糖に関しては、固定化酵素を用いて、電気
化学的に濃度を測定する方法がすでに知られてい
る。
ぶどう糖の濃度測定は、固定化グルコーススキ
シダーゼを用い、ぶどう糖を選択的に酸化するに
際し、消費される酸素あるいは生成する過酸化水
素の濃度を電気化学的に検出しようとするもので
ある。すなわち酸素の場合は、電極におけるその
還元電流を、また過酸化水素の場合は、その酸化
電流を測定するものである。またしよ糖に関して
は、β−フルクトシダーゼによつてしよ糖を加水
分解してぶどう糖を生成させ、このぶどう糖を上
記と同様の方法で電気化学的に測定するものであ
り、複合酵素を用いる以外は上記と同様の原理に
よつている。これらの電気化学的方法は選択性が
あることはもちろんであるが、次速かつ簡便であ
ることから一般的になりつつある。
一方、果糖については、現在までに、電気化学
的方法を利用して、選択的かつ迅速にその濃度を
測定する方法はまつたく知られていない。
本発明者らは、果糖と選択的に還元する酵素で
あるＤ−マンニトール脱水素酵素を用い、果糖の
還元反応に共役するこの脱水素酵素の補酵素を電
気化学的に検知することにより、果糖濃度を選択
的かつ迅速に分析できることを見出した。果糖濃
度の測定法としては、従来液体クロマトグラフイ
や分光学的方法を利用したものが知られている
が、本発明による果糖濃度の測定法は、特に迅速
性、簡便性の点でこれらの従来方法に比較して特
に優れている。
以下に本発明の実施例について述べる。
実施例 １
酵素として、リユウコノストツク メセンテロ
イデス（Leuconostoc mesenteroides）菌体より
抽出したＤ−マンニトール脱水素酵素（DMDH）
を用い、補酵素として還元型ニコチンアミドアデ
ニンジヌクレオチド（NADH）を用いる。電型
として導電性物質であるカーボンから形成された
円板状集電体の上に前記DMDHを固定化したも
のを用いる。DMDHの固定化は、グルタルアル
デヒドやイミドエステル等の２官能性架橋試薬に
よつて前記集電体表面上に直接DMDHの不溶性
膜を形成して行なう。
以上の電極を第１図に示す電極ホルダーに組み
込む。１は絶縁体よりなる支持体、２はその中に
通した金属リード、３はリード２と接続した白金
板で、前記の構成の電極４に接触している。５は
電極４を白金板と接触させて保持するネジ式とめ
具である。
このように構成した電極ホルダーを第２図に示
す測定系に組み入れる。ここで６は上記電極を組
み込んだホルダー、７は参照電極、８は対極、
９，９は緩衝液、１０は塩橋である。
第２図の測定系を用い、緩衝液９中にNADH
をあらかじめ溶解させておく。ポテンシヨスタツ
トを用い、前記ホルダー中に組み込んだ電極を参
照電極に対し−1.2Vに電位設定し、電極に流れ
るカソード電流を測定した。
第３図は緩衝液９中に果糖を添加した場合のカ
ソード電流値の時間変化を示している。緩衝液中
の果糖濃度が０から１×10-3モル／に変化する
とカソード電流が増加し、約１分で定常値に達
し、カソード電流の増加分は約4.5μAであつた。
このカソード電流の増加は、果糖がDMDHの
作用で還元され、この反応に共役して酸化された
NADHを元の還元型に還元する反応に基づくも
のである。各種濃度の果糖について同様の実験を
行うと、10-5〜10-3モル／の果糖濃度範囲で電
流増加量が直線関係を示した。
以上から本発明による方法、すなわち酵素反応
に共役して濃度が減少するNADHについて、そ
の減少分を電気化学的に測定することにより果糖
濃度の測定が可能なことがわかる。
実施例 ２
酵素として実施例１と同じDMDH、補酵素と
して酸化型ニコチンアミドアデニンジヌクレオチ
ド（NAD）を用い、まずDMDHとNADの両者
を実施例１と同じ集電体上に固定化した電極を作
製する。DMDHとNADの固定化は、両者の混合
液を集電体上に塗布乾燥した後、実施例１と同様
の架橋試薬を作用させDMDHとNADからなる不
溶性膜を集電体表面上に直接生成させることによ
り行うことができる。
作成したDMDHとNADの固定化電極は、第１
図に示したホルダー中に挿入し、第２図に示した
測定系に組み入れる。この場合、実施例１のごと
くNADHを緩衝液中に溶解させる必要はないが、
NADをNADHに変換するため、前記DMDH−
NAD固定化電極を1.2V（vs.SCE）であらかじめ
還元しておく。これによつてDMDHとNADHが
固定化された電極が構成される。
実施例１と同様緩衝液９中の果糖濃度を０から
１×10-3モル／に変化するとカソード電流が増
加し約１分で定常値が得られ、カソード電流の増
加分は約1μAであつた。各種濃度の果糖について
同様の実験を行うと、10-5〜10-3モル／の果糖
濃度範囲で電流増加量が直線関係を示し、
DMDHとNADHを固定化した本発明の電極を用
い、さらに本発明による電気化学的測定法により
果糖濃度が測定可能なことがわかつた。
実施例 ３
実施例２と同様の方法で各種果実中の果糖濃度
を測定し、得られた値を高速液体クロマトグラフ
イーによる値と比較した。サンプルはぶどう、な
し、りんご、すいか、ももの果肉のしぼり汁を用
いている。両方法による値の比較を以下の表に示
す。果汁中には他の糖（ぶどう糖、しよ糖）や有
機酸（クエン酸等）が含まれていることを考える
と、本方法によつて選択的に果糖濃度が測定でき
ていることがわかる。
さらに高速液体クロマトグラフイーでは値を得
るのに10分以上の時間を要することから１分程度
で値が得られる本発明の方法は迅速であると言え
る。また高速液体クロマトグラフイーでは、カラ
The present invention relates to a novel method for selectively qualitatively or quantitatively analyzing the concentration of fructose contained in foods, and an electrode used in the method. That is, the present invention utilizes D-mannitol dehydrogenase (hereinafter referred to as DMDH) as shown in the following reaction formula (1).
Fructose is selectively reduced to D-mannitol by D-mannitol (represented by ), and the reduced coenzyme concentration coupled with this reaction is electrochemically detected to quickly and easily measure fructose concentration. . The most typical sugars in foods, such as fruits, are glucose, sucrose, and fructose, and measuring the content of these sugars is important in food analysis. Regarding glucose and sucrose among the above-mentioned representative sugars, a method of electrochemically measuring the concentration using immobilized enzymes is already known. Glucose concentration measurement attempts to electrochemically detect the concentration of oxygen consumed or hydrogen peroxide produced when glucose is selectively oxidized using immobilized glucose oxidase. That is, in the case of oxygen, its reduction current at the electrode is measured, and in the case of hydrogen peroxide, its oxidation current is measured. Regarding sucrose, β-fructosidase hydrolyzes sucrose to produce glucose, and this glucose is electrochemically measured using the same method as above. It is based on the same principle as above except that it is used. These electrochemical methods are not only highly selective, but also fast and simple, so they are becoming popular. On the other hand, as for fructose, there is currently no known method for selectively and quickly measuring its concentration using an electrochemical method. The present inventors used D-mannitol dehydrogenase, an enzyme that selectively reduces fructose, and electrochemically detected the coenzyme of this dehydrogenase, which is conjugated to the reduction reaction of fructose. We have found that the concentration can be analyzed selectively and quickly. Conventionally, methods for measuring fructose concentration have been known that utilize liquid chromatography and spectroscopic methods, but the method for measuring fructose concentration according to the present invention is superior to these methods in particular in terms of speed and simplicity. Particularly superior to conventional methods. Examples of the present invention will be described below. Example 1 As an enzyme, D-mannitol dehydrogenase (DMDH) extracted from Leuconostoc mesenteroides cells
and reduced nicotinamide adenine dinucleotide (NADH) as a coenzyme. As the electrode type, a disk-shaped current collector made of carbon, which is a conductive substance, on which the DMDH is immobilized is used. Immobilization of DMDH is carried out by directly forming an insoluble film of DMDH on the surface of the current collector using a bifunctional crosslinking reagent such as glutaraldehyde or imidoester. The above electrode is assembled into the electrode holder shown in FIG. 1 is a support made of an insulator, 2 is a metal lead passed through the support, 3 is a platinum plate connected to the lead 2, and is in contact with the electrode 4 having the above structure. 5 is a screw type fastener that holds the electrode 4 in contact with the platinum plate. The electrode holder constructed in this manner is incorporated into the measurement system shown in FIG. Here, 6 is a holder incorporating the above electrode, 7 is a reference electrode, 8 is a counter electrode,
9, 9 is a buffer solution, and 10 is a salt bridge. Using the measurement system shown in Figure 2, NADH was added to buffer solution 9.
Dissolve it in advance. Using a potentiostat, the potential of the electrode incorporated in the holder was set to -1.2V with respect to the reference electrode, and the cathode current flowing through the electrode was measured. FIG. 3 shows the change in cathode current value over time when fructose is added to the buffer solution 9. When the fructose concentration in the buffer solution was changed from 0 to 1 x 10 -3 mol/, the cathode current increased and reached a steady value in about 1 minute, and the increase in cathode current was about 4.5 μA. This increase in cathodic current is due to fructose being reduced by the action of DMDH and oxidized coupled to this reaction.
It is based on a reaction that reduces NADH to its original reduced form. When similar experiments were conducted with various concentrations of fructose, the amount of increase in current showed a linear relationship in the fructose concentration range of 10 -5 to 10 -3 mol/. From the above, it can be seen that the fructose concentration can be measured by the method according to the present invention, that is, by electrochemically measuring the decrease in NADH, which decreases in concentration by coupling with an enzyme reaction. Example 2 Using DMDH as the enzyme and oxidized nicotinamide adenine dinucleotide (NAD) as the coenzyme, an electrode was prepared in which both DMDH and NAD were immobilized on the same current collector as in Example 1. Create. For immobilization of DMDH and NAD, a mixture of both is applied onto the current collector and dried, and then a crosslinking reagent similar to that in Example 1 is applied to form an insoluble film consisting of DMDH and NAD directly on the surface of the current collector. This can be done by letting The created DMDH and NAD immobilized electrodes were
Insert it into the holder shown in the figure and incorporate it into the measurement system shown in Fig. 2. In this case, it is not necessary to dissolve NADH in the buffer solution as in Example 1, but
To convert NAD to NADH, the DMDH-
Reduce the NAD-immobilized electrode with 1.2V (vs.SCE) in advance. This constitutes an electrode in which DMDH and NADH are immobilized. As in Example 1, when the fructose concentration in buffer solution 9 was changed from 0 to 1 × 10 -3 mol/, the cathode current increased and a steady value was obtained in about 1 minute, and the increase in cathode current was about 1 μA. Ta. When similar experiments were conducted with various concentrations of fructose, the amount of increase in current showed a linear relationship in the fructose concentration range of 10 -5 to 10 -3 mol/.
It was found that fructose concentration can be measured by the electrochemical measurement method of the present invention using the electrode of the present invention in which DMDH and NADH are immobilized. Example 3 Fructose concentrations in various fruits were measured in the same manner as in Example 2, and the obtained values were compared with values determined by high performance liquid chromatography. The samples used are squeezed juices from grapes, pears, apples, watermelons, and peach pulps. A comparison of the values obtained by both methods is shown in the table below. Considering that fruit juice contains other sugars (glucose, sucrose) and organic acids (citric acid, etc.), it is clear that this method can selectively measure fructose concentration. . Furthermore, since it takes 10 minutes or more to obtain a value using high performance liquid chromatography, the method of the present invention can be said to be rapid as it can obtain a value in about 1 minute. In high performance liquid chromatography, color
【表】
ムのつまりを防止するため、果汁の濾過処理が必
要であるが、本方法では果汁サンプルの前処理は
必要なく、簡便性の点でもすぐれている。
以上、DMDHとしてはNADHを補酵素とする
ものをとり上げたが、DMDHの種類によつては
シトクロムを補酵素とするものもある。この場合
は、メチルビオロゲン、ベンジルビオロゲンのご
とき酸化還元色素の還元型をさらに共役させると
果糖の還元反応が集電体に有効に伝えられ、大き
な電流応答が得られる。この際シトクロムは還元
型のフエロシトクロムを用いる必要がある。
また実施例には、酵素を固定化し補酵素を溶解
状態で用いる方法（実施例１）、酵素、補酵素の
両者を固定化する方法（実施例２）について述べ
たが、酵素、補酵素ともに溶解状態で用いること
も可能である。しかしこの場合は実施例１、２の
方法に比較して電流が定常値に達するまでの時間
が５分程度となり、分析の迅速性の点で、望まし
くない。酵素、補酵素は少なくとも一方を固定し
た方が再使用、経済性の面でも有利である。実施
例１と２では、分析の迅速性という点では同程度
（ともに定常電流値を得るのに１分程度）である
が、感度（同一果糖濃度の増加に対する電流増加
分）の点では実施例１の方が良好である。一方、
実施例２は、酵素も補酵素も再使用できる点や、
補酵素を溶解させなくてもよい簡便性の点でまさ
つている。
電気化学的に還元型補酵素の濃度変化を測定す
る方法としては、実施例１、２には酵素反応で酸
化型に変換された補酵素を、電気化学的に還元す
るためのカソード電流を測定する方法を採つた
が、この他にも還元型補酵素を電気化学的に酸化
するためのアノード電流を測定する方法を採るこ
ともできる。
さらに実施例２に述べた酸化型補酵素（NAD）
とDMDHを共に固定した電極を、固定化された
NADを電気化学的にNADHに還元せずそのまま
用いるとＤ−マンニトールの濃度を測定すること
が可能にもなる。これは前記反応式(1)の逆反応が
進行し、この反応に共役するNADの濃度変化が
電気化学的に測定可能なためである。Ｄ−マンニ
トールの測定は、当然のことながらNADを溶解
状態で用いて行うこともできる。したがつて本発
明による少なくともDMDHあるいはその補酵素
（還元型あるいは酸化型）を固定した電極を用い
ると果糖あるいはＤ−マンニトールの測定が可能
となる。
以上のように本発明は、果糖濃度の新しい電気
化学的分析法を提供するものであり、迅速性、選
択性がすぐれたものである。[Table] Although fruit juice must be filtered to prevent mucus from clogging, this method does not require any pre-treatment of the fruit juice sample and is superior in terms of simplicity. Above, we have discussed DMDH that uses NADH as a coenzyme, but some types of DMDH also use cytochrome as a coenzyme. In this case, if a reduced form of a redox dye such as methyl viologen or benzyl viologen is further conjugated, the reduction reaction of fructose is effectively transmitted to the current collector, and a large current response can be obtained. At this time, it is necessary to use reduced cytochrome ferrocytochrome. In addition, in the Examples, a method of immobilizing an enzyme and using a coenzyme in a dissolved state (Example 1) and a method of immobilizing both an enzyme and a coenzyme (Example 2) were described. It is also possible to use it in a dissolved state. However, in this case, compared to the methods of Examples 1 and 2, it takes about 5 minutes for the current to reach a steady value, which is not desirable in terms of speed of analysis. It is advantageous in terms of reuse and economy to fix at least one of the enzymes and coenzymes. Examples 1 and 2 are comparable in terms of quickness of analysis (about 1 minute for both to obtain a steady current value), but in terms of sensitivity (increase in current for the same increase in fructose concentration), Example 1 is better. on the other hand,
Example 2 has the advantage that both enzymes and coenzymes can be reused,
It is superior in terms of convenience as it does not require dissolving the coenzyme. As a method for electrochemically measuring changes in the concentration of reduced coenzymes, Examples 1 and 2 involve measuring cathode current for electrochemically reducing coenzymes that have been converted to oxidized coenzymes through enzymatic reactions. In addition to this method, a method of measuring the anode current for electrochemically oxidizing the reduced coenzyme can also be used. Furthermore, the oxidized coenzyme (NAD) described in Example 2
and DMDH were immobilized together.
If NAD is used as it is without being electrochemically reduced to NADH, it is also possible to measure the concentration of D-mannitol. This is because the reverse reaction of the reaction formula (1) proceeds and the change in the concentration of NAD conjugated to this reaction can be measured electrochemically. Of course, the measurement of D-mannitol can also be carried out using NAD in a dissolved state. Therefore, fructose or D-mannitol can be measured by using the electrode according to the present invention on which at least DMDH or its coenzyme (reduced or oxidized) is immobilized. As described above, the present invention provides a new electrochemical analysis method for fructose concentration, which is excellent in speed and selectivity.
第１図は本発明の電極を組み込んだホルダーの
縦断面図、第２図は同ホルダーを組み込んだ電気
化学測定系を示す図、第３図は果糖濃度の増加に
ともなうカソード電流の時間変化を示す。
Figure 1 is a longitudinal cross-sectional view of a holder incorporating the electrode of the present invention, Figure 2 is a diagram showing an electrochemical measurement system incorporating the holder, and Figure 3 shows the temporal change in cathode current as the fructose concentration increases. show.
Claims (1)
電体に固定化したことを特徴とする酵素電極。 ２ 酵素が、その補酵素とともに集電体に固定化
されている特許請求の範囲第１項記載の酵素電
極。 ３ 少なくともＤ−マンニトール脱水素酵素を集
電体に固定化したものとその還元型補酵素を用
い、果糖を含有する溶液中で前記酵素により果糖
を還元し、この反応に共役する前記還元型補酵素
の濃度変化を電気化学的に測定することにより果
糖濃度を求めることを特徴とする果糖濃度の測定
方法。 ４ 酵素を単独でもしくはその還元型補酵素とと
もに固定化した状態で用いる特許請求の範囲第３
項記載の果糖濃度の測定方法。[Scope of Claims] 1. An enzyme electrode characterized in that at least D-mannitol dehydrogenase is immobilized on a current collector. 2. The enzyme electrode according to claim 1, wherein the enzyme is immobilized on the current collector together with its coenzyme. 3 Using at least D-mannitol dehydrogenase immobilized on a current collector and its reduced coenzyme, fructose is reduced by the enzyme in a solution containing fructose, and the reduced coenzyme is conjugated to this reaction. A method for measuring fructose concentration, characterized by determining fructose concentration by electrochemically measuring changes in enzyme concentration. 4 Claim No. 3 in which the enzyme is used alone or in an immobilized state together with its reduced coenzyme
Method for measuring fructose concentration as described in section.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP16504779A JPS5687853A (en) | 1979-12-18 | 1979-12-18 | Enzyme electrode and measurement of fruit sugar concentration |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP16504779A JPS5687853A (en) | 1979-12-18 | 1979-12-18 | Enzyme electrode and measurement of fruit sugar concentration |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPS5687853A JPS5687853A (en) | 1981-07-16 |
| JPS6314301B2 true JPS6314301B2 (en) | 1988-03-30 |
Family
ID=15804820
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP16504779A Granted JPS5687853A (en) | 1979-12-18 | 1979-12-18 | Enzyme electrode and measurement of fruit sugar concentration |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPS5687853A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2025154550A1 (en) * | 2024-01-17 | 2025-07-24 | パナソニックＩｐマネジメント株式会社 | Reduction method, method for producing mannitol, reduction device, and electrode |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US7867740B2 (en) * | 2006-08-11 | 2011-01-11 | Board Of Trustees Of Michigan State University | Thermotoga maritima mannitol dehydrogenase |
-
1979
- 1979-12-18 JP JP16504779A patent/JPS5687853A/en active Granted
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2025154550A1 (en) * | 2024-01-17 | 2025-07-24 | パナソニックＩｐマネジメント株式会社 | Reduction method, method for producing mannitol, reduction device, and electrode |
Also Published As
| Publication number | Publication date |
|---|---|
| JPS5687853A (en) | 1981-07-16 |
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