JPH088865B2 - Method for producing chemically modified enzyme - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION (Industrial field of application) The present invention relates to an analytical reagent for clinical diagnosis, a biosensor,
The present invention relates to an enzyme used in a bioreactor or the like, and particularly to a chemically modified oxidase using flavin adenine dinucleotide (hereinafter abbreviated as FAD) as a coenzyme, which has a property of catalyzing redox reaction even in a system having a small amount of dissolved oxygen.

(Prior Art) Among oxidoreductases existing in nature, those having a coenzyme that exchanges electrons at the active center as a functional group can be seen. For example, in the case of glucose oxidase (hereinafter abbreviated as GOD), one molecule of glucose oxidase contains two molecules of FAD. When this GOD is in the reaction system,
It catalyzes the reaction of oxidizing glucose to glucono-δ-lactone (reaction formula (1)). On the other hand, the GOD that has become the reductant in reaction formula (1) reacts with oxygen to become the original oxidant (reaction formula (2)).

Glucose + GOD _ox → Glucono-δ-lactone + GOD _red
(1) GOD _red + O ₂ → H ₂ O ₂ + GOD _ox (2) (where GOD _ox and GOD _red represent oxidant and reductant, and G
It represents the oxidation or reduction state of FAD in OD. ) The reaction rate formula derived from the above reaction formula is Becomes

(Here, υ represents the reaction rate, that is, the amount of glucono-δ-lactone or H ₂ O ₂ produced per unit time or the amount of decrease of O _2. Vmax is the maximum rate, Cg and Co are glucose and O ₂
The concentrations of Kg and Ko are GO and G for glucose and O _2, respectively.
The Michaelis constant of D is shown. ) This reaction formula is generally established not only in GOD but also in the reaction system catalyzed by oxidase. In this case, the reaction rate υ is not only influenced by the concentration Cg of the substrate glucose, but is also controlled by the oxygen concentration Co in the reaction system. Therefore, it is not so remarkable when the glucose concentration Cg is low, but when the glucose concentration is high and the dissolved oxygen concentration of the system is low, the oxygen concentration Co
The reaction speed υ is limited by this, and there is a drawback that the reaction speed cannot be further increased.

This problem becomes a problem when the oxidase is used for sample analysis or bioreactor. Especially in a biosensor using an immobilized enzyme, accurate analysis cannot be performed when the amount of dissolved oxygen is small when analyzing a specific compound, and it is an important issue to overcome this problem. There is.

To address these issues, FA, which has recently been at the center of enzyme activity,
In order to exchange electrons with D, a method of replacing oxygen and allowing a mediator to coexist in the reaction system or inside the electrode has been proposed.

Incidentally, since FAD exists in the high molecular weight polypeptide chain of oxidase, it is considered that it does not come into direct contact with electrodes such as platinum. Therefore, in order to cause electron transfer between the FAD and the electrode when the amount of dissolved oxygen is small, it is necessary to bridge the transfer of electrons by the mediator. Therefore, when an appropriate mediator is allowed to coexist in the reaction system or the electrode, the enzyme in solution or the immobilized enzyme can easily carry out the redox reaction even when the amount of dissolved oxygen is small. This method is believed to be useful for analysis, in particular for improving the characteristics of biosensors, and for increasing the efficiency of reactors. In addition, there are expectations for implantable biosensors that operate in living organisms with low dissolved oxygen content.

However, there are still some problems in stable supply of these mediators. That is, a large amount of mediator is required to dissolve the mediator in the reaction system at a uniform concentration, which is not economical. When a mediator is present in the electrode, for example, when a relatively low-solubility mediator is dip-coated on the solid electrode, some or all of the mediator dissolves into the reaction solution with the reaction, and the mediator concentration decreases. As a result, the reaction speed is reduced. So for biosensors and reactors,
There is also a method of providing an overwhelmingly large amount of mediator storage (Japanese Patent Laid-Open No. 61-61049), but a decrease in the reaction rate due to the outflow of the mediator into the reaction solution cannot be avoided.

Further, a method in which a conductive thin film such as polypyrrole is made to play the role of a mediator (JP-A-62-115284, 62-115).
No. 285) has been proposed, but it is considered difficult to directly contact the active center of the enzyme with the conductive thin film, and a sufficient current density has not been obtained by this method, and it has not reached a practical level.

Furthermore, a method has been proposed in which electrons are directly transferred to and from an electrode using a GOD in which ferrocenecarboxylic acid, which is recognized as a mediator, is directly chemically bound (Y. Degani, A. Heller, J. phys. Chem, 91 (6), 1285-12
89 (1987); C & E News, March 16, 24-26 (1987)), this method treats GOD with urea and reacts it with ferrocenecarboxylic acid in a denatured state, so that only about 50% of the activity is recovered. In addition, the current density is insufficient and this has not reached a practical level.

(Problems to be Solved by the Present Invention) The present invention relates to an analytical reagent for clinical diagnosis, a biosensor,
In order to solve the above problems in the oxidase with FAD as a coenzyme widely applied to bioreactors,
It is an object of the present invention to provide a chemically modified oxidase capable of performing a redox reaction even in a system having a small amount of dissolved oxygen.

(Means to solve the problem) The mediator is easily free of FAD in the enzyme in the free state.
It has the property that it can approach and exchange electrons. However, when a free mediator is used, there is a problem of elution into the system as described above. Therefore, the present inventors have paid particular attention to benzoquinones as mediators having high chemical reactivity and capable of rapidly progressing electron exchange with oxidase having FAD as a coenzyme in a free state,
It was examined to covalently bind to oxidase (ie, flavin oxidase) that uses as a coenzyme. To date, no examples of effective covalently chemically modified enzymes using benzoquinones have been reported. Many of the flavin oxidases have low activity in a high pH range, and were conventionally used only in a weakly acidic range, but the present inventors have found that in a relatively high pH range, benzoquinones are covalently bonded to the flavin oxidase, The present invention has been completed in which it was found that benzoquinones do not elute in the reaction system when an oxidase is used, and that the obtained chemically modified enzyme exhibits high activity.

The present invention is a method for producing an oxidase covalently bound to benzoquinones, which comprises reacting an oxidase having flavin adenine dinucleotide as a coenzyme with a buffer solution containing benzoquinones at a temperature of 4 ° C to 50 ° C.

(Action) Hereinafter, a method for covalently bonding benzoquinones to an oxidase having flavin adenine dinucleotide as a coenzyme will be described. First, GOD is dissolved in a buffer solution, benzoquinones are added thereto, and they are mixed well and then reacted.

As the buffer for dissolving GOD, sodium phosphate buffer, Tris-hydrochloric acid buffer, sodium carbonate buffer and the like are used. In the present invention, the pH range higher than the normally used action range of GOD, that is, pH 7.0 to pH 9.0, preferably pH
It is characterized in that GOD is dissolved in a buffer solution of 8.0 to pH 9.0. Next, benzoquinones dissolved in a solvent such as methanol, ethanol, or acetone are added so as to have a concentration of 0.001 to 0.1 M, mixed well, and then reacted at a temperature of 4 ° C to 50 ° C. At this time, in order to prevent the inactivation of the enzyme, preferably 4 ° C to
Perform the reaction at 10 ° C.

The benzoquinones referred to in the present invention are benzoquinone, naphthoquinone and derivatives thereof, and specific examples thereof include p-benzoquinone, o-benzoquinone, toluquinone, naphthoquinone and hydroxynaphthoquinone, which have a large electron exchange rate and are chemically modified. P-benzoquinone is most preferable because it has a relatively high solubility in an aqueous solution system.

If the reaction time is short, the reaction does not proceed sufficiently, and if the reaction time is long, the activity yield of the enzyme decreases, so 1 hour to 1 week, preferably 1
The reaction is carried out for a period of time to 3 days, more preferably 1 to 24 hours. The reaction yield is 70% when the reaction time is 1 day, and when the reaction time is 3 days,
50-60%.

Then, excess benzoquinones are completely removed by a method such as dialysis, gel filtration, and membrane separation to obtain GOD to which benzoquinones are covalently bound.

In addition, flavin oxidases other than GOD, such as glutamate oxidase, alcohol oxidase, cholesterol oxidase and the like, similarly benzoquinones by dissolving the oxidase in a pH 7.0 to pH 9.0, preferably pH 8.0 to pH 9.0 buffer solution. Can be covalently bound.

The reason why benzoquinones bind to flavin oxidase in a specific pH region is not always clear, but it is speculated as follows. Benzoquinones undergo a nucleophilic addition reaction commonly found in α, β unsaturated carbonyl compounds with compounds having a free amino group. By the way, oxidase has side chain amino groups such as glutamine, asparagine, and lysine, but these have a positive charge due to the addition of a proton in the neutral to acidic region. However, in a relatively high pH region, the abundance ratio of free amino groups increases, and it is considered that the lone electron pair of this free amino group causes a nucleophilic addition reaction and covalently bonds with benzoquinones.

(Examples) Hereinafter, the present invention will be described in more detail with reference to Examples.
Of course, it is not limited to this.

Example 1 Chemical modification treatment of GOD As an enzyme, GOD (Sigma, Type VII-S, Aspergil
Lus niger origin, 146000units / g) 10mg is dissolved in 0.1M sodium phosphate buffer (pH8.0) 1ml, into which 0.5M p-
0.1 ml of a solution of benzoquinone in methanol was added. This mixed solution was allowed to stand at 10 ° C. for 24 hours and then dialyzed against a large amount of 0.1 M sodium phosphate buffer (pH 5.0) at room temperature for 48 hours. The content solution after dialysis was washed out with the above pH 5.0 buffer solution to a total volume of 5 ml. Using the chemically modified enzyme solution thus obtained, the rate of hydrogen peroxide production in an air-saturated glucose aqueous solution was measured by the dye-peroxidase method to examine the residual activity.
A high activity of 0% was recovered.

Preparation of Electrode Next, a platinum electrode with a diameter of 3 mm was polished using emery paper, and then in 0.1M sulfuric acid, a saturated calomel reference electrode (hereinafter SCE) was used.
The surface was cleaned by repeating cyclic sweep for 10 minutes at a speed of 1.0V / sec in the range of -0.3 to + 1.5V. This electrode was subjected to an oxidation treatment in 0.1 M sulfuric acid by holding it at SCE +1.4 V for 5 minutes. Then, it was thoroughly washed with water and dried at 40 ° C. for 15 minutes. Immerse in a toluene solution of 10% γ-aminopropyltriethoxysilane for 1 hour, wash well with toluene,
It was dried at 40 ° C. for 15 minutes and subjected to aminosilanization treatment. Next, it was immersed in a 10% glutaraldehyde aqueous solution for 1 hour, washed with water, and then washed with the above enzyme solution covalently bonded to p-benzoquinone.
After immersion for a period of time, the enzyme was chemically fixed on the platinum electrode surface.

This electrode was thoroughly washed in a 0.1 M sodium phosphate buffer (pH 5.0) for 24 hours to wash off excess physically adsorbed enzyme and residual p-benzoquinone that could not be removed.

Measurement Immobilized enzyme electrode prepared in this way was set to 37 ° C
It was immersed in a 0.1 M sodium phosphate buffer (pH 5.0), and a 1 cm square platinum was used as a counter electrode, and a voltage of SCE + 0.6 V was applied. A glucose solution was sequentially added to this system with a microsyringe, and the output current value was recorded. The results are shown in FIG.

Further, the buffer solution was replaced, high-purity nitrogen was aerated at a flow rate of 50 ml / min, and after deoxidizing for 1 hour, the output current value was similarly recorded. The results are shown in FIG. (The current value was recorded 1 minute after the addition of glucose in order to remove the influence of oxygen brought in by the glucose solution.) Comparative Example 1 A similar GOD-immobilized electrode was prepared except that the chemical modification treatment with p-benzoquinone was omitted. Then, the measurement was performed. The measurement results are shown in FIG. 1 (in the presence of dissolved oxygen) and FIG. 2 (after deoxidation).

In the presence of oxygen, chemically modified GOD (Example 1) gave a calibration curve with a slope of about 70% compared to unmodified GOD (Comparative Example 1). This result is consistent with the result of residual activity measurement of 70% performed in a solution system after chemical modification as described in Example 1.

In the presence of dissolved oxygen, the GOD electrode (Comparative Example 1) not chemically modified shows a linear relationship between the glucose concentration and the output current value when the glucose concentration is up to about 1.5 mM.
In the region of 1.5 mM or more, the increase of the output current value slowed down, whereas the chemically modified GOD electrode of Example 1 maintained linearity up to 2.5 mM, and it was found that high concentration glucose can be accurately measured. It was This is because in Comparative Example 1, the reaction rate was limited by the amount of dissolved oxygen in the system, and accurate measurement could not be performed in the high-concentration glucose region, whereas it was chemically modified.
This is because in the GOD electrode (Example 1), p-benzoquinone exchanges electrons with the electrode in place of oxygen, and is not limited by the amount of dissolved oxygen.

In the deoxygenated system (Fig. 2), Comparative Example 1 showed almost no current response, but in Example 1, the output current value showed a linear relationship with the glucose concentration, and even in the system without oxygen, it was chemically modified. It was found that the GOD catalyzed the redox reaction.

(Effect) The present invention provides an excellent chemically modified flavin oxidase that can be applied to an analytical reagent, a biosensor, and a bioreactor that perform a redox reaction even in a system having a small amount of dissolved oxygen.

[Brief description of drawings]

FIG. 1 shows the output current value for each glucose concentration in the presence of dissolved oxygen, which was chemically modified according to the present invention.
The output current value (Example 1) of the electrode on which GOD was fixed is indicated by ◯, and the output current value of the electrode on which GOD is not chemically modified (Comparative Example 1) is indicated by Δ. FIG. 2 shows each output current value when the same measurement was performed after deoxidizing the measurement system.

Claims (1)

[Claims] 1. A method for producing an oxidase to which benzoquinones are covalently bound, which comprises reacting an oxidase containing flavin adenine dinucleotide as a coenzyme with a buffer containing benzoquinones at a temperature of 4 ° C. to 50 ° C.