Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or...
Patent
1982-12-02
1984-12-25
Niebling, John F.
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
204403, 204290R, 204 1T, 429 13, 435174, 435175, 435177, 435180, 435817, B01D 5940
Patent
active
044904649
DESCRIPTION:
BRIEF SUMMARY
TECHNICAL FIELD
The invention relates to electrodes, perferably of graphite, the surface of which has been so modified that an electrochemical oxidation of co-enzyme or co-enzyme analogs occurs at the electrode surface with far greater ease than at an unmodified electrode surface. The modification implies that mediator molecules which are described in more detail below, are immobilized on the graphite by spontaneous adsorption.
The invention described may be used for regenerating the co-enzymes in different biotechnical, microbiological or biochemical processes. Regeneration is effected electrochemically at the surface of the electrode described; the circuit is closed by means of a counterelectrode of known type. Improved supervision is obtained if also a reference electrode is provided and a potentiostatic circuit is utilized.
The electrodes according to the present invention may also be utilized as the anode in so-called biochemical fuel cells, in which case the cathode is an electrode of known type, such as an oxygen or air electrode. Within the cell, a substrate, for example alcohol, is oxidized by enzyme catalysis, whereby the co-enzymes are reduced. The resulting, reduced co-enzymes can be oxidized at the modified electrode surface, and the half-cell can supply an electric current.
Furthermore, the modified electrodes according to the invention may be utilized in analysis systems together with co-enzyme-dependent enzyme. The analysis method may be utilized for the analysis of different substrates, depending upon the choice of enzyme. The modified electrode can supply a current which is approximately proportional to the substrate content of the sample. Measurement may also be effected potentiometrically, in which case the electrode potential will be approximately proportional to the logarithm of the substrate content in the sample.
BACKGROUND
Enzyme-catalysed reactions of the type ##STR1## play an important part in biological cells and in biotechnical and analytical reactions. Several hundreds of different hydrogenasas are known which selectively catalyse the conversion of different substrates into products. When the substrate is oxidized, the co-enzymes NAD.sup.+ and NADP.sup.+, respectively, are at the same time reduced to NADH and NADPH, respectively. The major part of the hydrogenases make highly specific demands on the co-enzyme, for which reason NAD.sup.+ and NADP.sup.+, respectively, are necessary to bring about a reaction.
The co-enzymes NAD.sup.+ and NADP.sup.+ are very expensive chemicals which are difficult to obtain. The possibilities of regenerating them by reoxidation therefore are of great economical importance. Regeneration may also be necessary for displacing the equilibrium (1) to the right. In this manner, the substrate can be completely converted into a product, which facilitate the isolation or increase the yield of the desired synthesis products. The displacement may also be a prerequisite for the analytical use of the reaction.
Co-enzyme may be regenerated in different ways:
Oxidation of co-enzymes with retained activity can be effected only with certain specific oxidizing agents. Such compounds are frequently termed mediators. In FIG. 1, compounds I (phenazine methosulphate, PMS), II (phenazine ethosulphate, PES), III (thionine) and IV (1,2-benzoquinone) are examples of known mediators.
Hitherto known mediators have been used to a limited extent because they, too, are expensive and their stability in the solution may be low. Furthermore, difficulties may arise when the mediators on their reaction and decomposition products are to be isolated from the desired product.
Use is made of an auxiliary reaction, for instance the following: ##STR2## Besides the enzyme according to equation (1), the enzyme lactate dehydrogenase must be added which catalyses a reaction in the opposite direction. If pyruvate is added in excess, NAD.sup.+ will be regenerated.
The use of two separate enzymes makes the biotechnical process more complicated. The reagent of the auxiliary system is mixed wit
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Gorton Lo G.
Jaegfeldt Hans .ANG..
Johansson Gillis R.
Torstensson Arne B.
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