DD279586A3 - PROCESS FOR PRODUCING NEW SPECIFIC OXIDASES - Google Patents

Abstract

The invention relates to a process for the preparation of new specific oxidases. The aim is to react the substrates of the oxidases quantitatively and stoichiometrically with the least possible enzyme use and avoiding the denaturation of the enzymes by the intermediaer resulting hydrogen peroxide. The object of the invention is to link an oxidase and an enzyme that consumes H2O2 with one another such that the H2O2 formed at the active site of the oxidase is conducted directly to the active center of the H2O2-consuming enzyme and absorbed there. The object is achieved by a new specific oxidase of the general formula wherein A is a quinoid compound having 1-3 nuclei, E1 is an O2-consuming enzyme and E2 is an H2O2-consuming enzyme, and their method of preparation. Field of application is biochemical analysis, medical diagnostics and biotechnology. formula

Description

Field of application of the invention

The invention relates to a process for the preparation of new specific oxidases. Areas of application of the invention are all areas in which specific oxidases in mixture with hydrogen peroxide-consuming enzymes are used in solution or immobilized, for example biochemical analysis, medical diagnostics and the biotechnological production of substrate oxidation products.

Characteristic of the known technical solutions

Sequential mixtures of specific oxidases with hydrogen-acid-consuming enzymes, whether in dissolved or immobilized form, have long been known. In the case of the mixture of oxidases with peroxidatively active enzymes, they are used for the qualitative and quantitative detection of the oxidase substrates ("analytes") by determining the resulting hydrogen peroxide by means of the mostly chromogenic, but also fluorogenic oxidation catalyzed by peroxidatively active enzyme Peroxidase substrates according to the following scheme: Detection reaction:

Analyte + O ₂ --- '> Analytoxidationsprodukt + H ₂ Oj

Indicator reaction:

. _n _. peroxidative activity ". , ,, "

H ₂ O ₂ + chromogen -> chromophore + H ₂ O

This approach has a number of disadvantages attributable to the high intermediate hydrogen peroxide concentration required for the indicator reaction, in particular

Side reactions or decomposition of the resulting hydrogen peroxide,

time delay of the indicator reaction towards the analyte conversion,

- Nonlinearity of the relationship between analyte concentration and chromophore formation, as well

- lack of functional stability.

(See Wingard, L.B. et al., Biochim., Biophys, Acta 748, 211 (1983), Brooks, S.P.J., et al., Canad, J. Biochem., Cell Biol. 62 (10), 945 (19841).

In the case of mixtures of oxidases with catalatically active enzymes which have been used for the preparative recovery of specific active oxidase-substrate oxidation products, the reaction proceeds according to the following scheme: oxidation reaction:

Substrate + O2 --- ' Substrate oxidation product + H ₂ O ₂

Disproportionierungsieaktion:

_u _ catalatically active enzyme _u _, ..,

H ₂ U ₂ ^: * H ₂ U + / '. I 1

Here, the disadvantage of the sequential reaction is the deficient functional stability of the specific oxidase as a result of its destruction by the hydrogen peroxide occurring intermediately (Buchholz, K. et al., Biotechnol. Bioengng.20, 201 [1978]).

It is already known that there is a partial reduction in the abovementioned disadvantages when the enzyme consuming hydrogen peroxide is used in considerable excess over the hydrogen peroxide generating oxidase (see Leon, LP et al., Clin. Chem. 2217 | 1017 [1976]; JC et al., Biochim., Biophys. Acta 438, 23 [1976]).

This approach, however, always has the disadvantage that considerable costs are incurred by the enzyme surplus and interference by interfering with hydrogen peroxide-consuming enzymatic or non-enzymatic admixtures can only be avoided with considerable effort (see DD-AP 150256, DD-AP 130178).

It is also known and has already been proposed (Hartmeier, W. et al., Biotechn., Lett., 5, 743, 11983); DP-DE 2214422 B 2) to immobilize oxidases with hydrogen peroxide-consuming enzymes together on a support, although always with the Immobilization of the enzymes associated with changes in their catalytic) properties (activity reduction), the removal of the solubility and the close bond to the carrier must be accepted, without the kinetic disadvantages associated with the sequential Reaktionsf Jhrurig be repealed. Even with "molecule-neighborable" assembly of oxidase and peroxidase, the immobilized system does not achieve the effectiveness of the soluble mixture of enzymes (Miller, J.N. et al., Anal. Chim. Acta 93, 333 [1977]).

It is also known to use p-benzoquinone as a bifunctional reagent for attaching enzymes to solid supports (Urayama, O. et al., Anal. Biochem., 141, 194 [1984]).

It has also been proposed, after immobilization of glucose oxidase and peroxidase in gelatin, to subsequently crosslink the gelatin with quinone so as to avoid the dissolution (DD-WP 0152808).

However, a specific coupling of two enzymes via a quinone acting as an electron mediator does not occur in these processes in any way, so that the o.g. Disadvantages of sequential systems are preserved.

It is also known that quinones can act as electron mediators between oxidases and peroxidase, in particular when measuring the substrate conversion by means of electrochemical methods (see Ikeda, T. et al., Agric., Biol. Chem. 48 [8] 1969 [1984]).

However, in this case the quinones are freely present as soluble constituents of the mixtures of the enzymes, so that although they can increase the electrode current, they do not eliminate the disadvantages of sequential reaction control.

Ziol of the invention

The object of the invention is to react the substrates of the oxidases quantitatively and stoichiometrically with the lowest possible enzyme conversion and while avoiding the denaturation of the enzymes by the intermediately formed H ₂ O ₂ .

Explanation of the essence of the invention

The invention is based on the object, a OxidasR and a H ₂ O _2- consuming enzyme to link together so that the resulting at the active site of the oxidase H ₂ O ₂ directly to the active site of the H ₂ O _2- consuming enzyme and there is recorded

 The object is achieved by a new specific oxidase of the general formula

E ₁ 4 A -

where A is a quinoid compound having 1-3 nuclei, egg is an emollient-consuming enzyme and E _{2 is} an H ₂ O _2- consuming enzyme.

The process for the preparation of the novel compound is characterized in that an aqueous 0-30% by volume of a solution of the enzyme E ₁ (or E ₂ ) containing water-coarse solvent with at least 2 to 10000 molar excess of a quinoid compound, having at least two positions capable of 1,4-addition, reacted in the range of pH 7-9 within 0.5-20 hours, the reaction product enriched by known methods and then in an analogous manner with the enzyme E ₂ (or E ₁ ) to (I) is implemented.

+ HpO Zv;

The new oxidases are prepared from two, independently acting enzymes, which successively reduced in the implementation of their substrates and undergo oxidized states. However, since a product of the enzyme E _{1 is} hydrogen peroxide, which in turn is a substrate of the enzyme E ₂ , there is a sequential reaction in which the overall yield depends crucially on the properties of the intermediate hydrogen peroxide (Equation 1 a ... 1 b ).

a) S ₁ + O ₂ --- P ₁ + H ₂ O ₂

b) S ₂ + H ₂ O ₂

E ₂ ,

c) H ₉ Op - HpO + · ρ-0 _ο HpOp disassembly

CL. O. (Z. O. L ~ C C-

Hydrogen peroxide is a slightly diffusible, aggressive compound, which spreads quickly in the environment with free diffusion. Therefore, the desired main reaction with the enzyme E _{2 occurs} only after a certain delay time ("lag time"), its intermediate concentration increases and side reactions are favored, The side reactions include in particular the activity-destroying action of H ₂ O ₂ on parts of the hydrogen peroxide-forming Enzymes that are not identical to the active site or the reaction with impurities that adhere in particular technical products.Mixes of oxidases with H ₂ O _2- consuming, substrate-oxidizing enzymes ("peroxidic activity") are usually used for the quantitative, specific determination of the oxidase Used substrates, the peroxidatively oxidized substrates easily detectable oxidation products, such as dyes form.

In the industrially important production of gluconic acid from glucose by glucose oxidase-catalyzed air-acid oxidation, the H ₂ O ₂ -related destruction of the enzyme by catalase, which disproportionates the H ₂ O ₂ , must be delayed.

Surprisingly, it has now been found that free transfer of the intermediate hydrogen peroxide into the medium surrounding the oxidase can be prevented if the hydrogen-generating and hydrogen peroxide-consuming enzyme are linked by a quinone. Suitable for linking are in principle all oxidoreductases which either produce hydrogen peroxide (Ei) or consume (E ₂ ). Examples of egg are alcohol oxidase, cholesterol oxidase, choline oxidase, glucose oxidase, glutamate oxidase, uricase and xanthine oxidase; for E ₂ all peroxidatively or catalytically active proteins, such as hemoglobin, catalase, lactoperoxidase and horseradish peroxidase. The enzymes linked in this way form a new oxidase corresponding to the general formula (!), In which the components no longer run independently of one another but in coordinated, successively reduced and oxidized states. Hydrogen peroxide no longer appears as a freely diffusible intermediate, eliminating the associated disadvantages. The reaction of the substrates takes place with the new oxidases according to the following general reaction equation 2:

a) S ₁ + S ₂ + O ₂ -ÜLL *. P ₁₊ P _{2 +} 2H ₂ O

b) 2S ₁ + O ₂ -ÜLl_ ^ 2P ₂ + 2? ^r ₂ 0

(a: only secondary substrate oxidation)

The linking of E, and E ₂ via a quinone is achieved by two successive 1,4-addition reactions. For this purpose, either the oxidase or hydrogen peroxide-consuming enzyme in a buffer in which it is sufficiently stable, and dissolved with a solution of an excess of a quinone, preferably p-benzoquinone, 1,2-naphthoquinone or anthraquinones in a weakly alkaline buffer, preferably from pH 8 to 9, added. The absolute concentration should be as high as possible, it is usually determined by the solubility of the reactants.

The amount of quinone per se is not critical, but must at least be at least twice that of the enzyme to effect oxidation of the enzyme-substituted hydroquinone to the enzyme-substituted quinone. The quinone must contain at least two unsubstituted positions capable of 1,4-addition (Equation 3). If the solubility of the quinone in aqueous buffer is too low, it is advisable to add up to 30% of a water-miscible solvent, preferably dimethylformamide, alcohols or glycols.

Equation 3:

KtCi

OH

OH

OH

The formation of bis addition products which contain two molecules E ₁ and E ₂ linked to quinone takes place only after a reaction time of more than 10 hours in a larger amount, a reaction time beyond 20 hours is therefore not recommended. Oas Enzyme quinone adduct (II) is then freed in a conventional manner, for example by centrifugation, ultrafiltration or gel chromatography from the excess quinone and formed hydroquinone.

Subsequently, the procedure is repeated analogously. The enzyme-quinone adduct (II) is used either with an equimolar amount of hydrogen peroxide-consuming enzyme in the most concentrated solution of pH 7 to 9, preferably 8 (when the adduct [II] was prepared with the hydrogen peroxide generating enzyme), or otherwise with a equimolar amount of the hydrogen peroxide generating enzyme is added to form the reduced form of the novel oxidase prepared according to the invention (equation 4).

Ji

Since the reduced form (III) is easily oxidized by still unreacted adduct II, it is advisable, in order to avoid yield reductions, to convert (III) into the oxidized form (I) by means of small amounts of substutrate and atmospheric oxygen. (I) is then isolated or purified as before (II). Accordingly, the novel oxidases prepared according to the invention can in principle be used in the same way as the mixtures of E ₁ and E ₂ , without, however, having the disadvantages already described. Furthermore, they have advantageous properties which are intrinsic in general crosslinked or immobilized enzymes, for example, a higher functional stability, increased thermal stability and a higher storage stability compared to the starting components.

Ausführangsbeispiele

example 1

Production of a specific glucose, chromogen oxidase (linkage product of glucose oxidase (GOD) and peroxidase (PODl)

1. Preparation of the GOD-substituted quinone

0.8 ml of a 2 · 10 ^-5 molarenGOD solution prepared from 42 mg enriched GOD / ml 0.1 M Na-acetate buffer pH 5.0, are with 1.6m! a 0.1 M quinone solution pH 8.0 and 0.8 ml of 0.05M Na phosphate buffer pH 8.0 0.5 h at 25 ° C shaken gently. Then this solution is chromatographed on Molselect G 25 with 0.05 M phosphate buffer pH 8.0. The first brownish colored eluate contains the GOD-substituted quinone.

2. Coupling of POD to the GOD-substituted quinone

2.5ml D9R GOD substituted quinone solution are shaken with 0.25 ml of a 2 · 10 ^-5 molar POD solution prepared from 18 mg enriched POD / ml 0.1 M Na-quinone buffer pH 5,0,5 hours at 25 ° C.

3. Characterization of the enzymatic activity of the coupling product

The enzymatic activity of the resulting GOD / POD complex is determined as follows: Reagents: citric acid / phosphate buffer pH 5.5

(McIlvaine buffer)

o-dianisidine solution (0.42 mg o-dianisidine-HCl / ml Mcllvaine buffer) glucose solution (1 M in Mcllvaine buffer)

Gelatin solution (3% in Mcllvaine buffer)

GOD / POD linkage product (dilution with Mcllvaine buffer)

Execution: cuvette Reference cell 1.0ml 1.0 ml o-Dianisidinlösung 0.5ml 0.5ml glucose solution 0.1 ml 0.1 ml gelatin solution 1,35ml 1.4 ml buffer 0.05 ml _ GOD / POD

The start of the reaction takes place with the Zi gäbe the GOD / POD complex.

The reaction is followed spectrophotometrically at 436 μm and the activity is calculated from the initial rate.

A linkage product is obtained in which the activities of the enzyme components correspond to the starting activity before the linkage.

The kinetics of the enzymatic reaction of the linkage product differs in the start phase due to the lack of lag time of the reaction of the uncoupled individual components.

Example 2

Production of a specific uric acid, chromogenic oxidase (uricase-POD linkage product)

1. Preparation of uricase-substituted quinone

1 ml of a 2 x 10 ^"5 molar uricase solution prepared from 40 mg enriched uricase / ml 0.02 M Na-borate / HCl buffer pH 8.0, is treated with 2 ml of a 0.1 M solution pH 8.0 Chino and 1 ml of 0.05 M Na phosphate buffer pH 8.0 shaken gently at 25 ⁰ C for 2 h and further treated as described in Example 1.

2. Coupling of the POD to the uricase-substituted quinone

To 2.5 ml of the resulting solution is added 0.25 ml of a 2 x 10 -6 molar POD solution prepared from 9 mg of enriched POD / ml 0.1 M Na citrate buffer pH 5.0 and added after the in Method described in Example 1.

3. Characterization of the enzymatic activity of the coupling product

The enzymatic activity of the uricase / POD complex is determined by the following methodology: Reagents: Na borate / HCl buffer, 0.02M pH 8.0

o-dianisidine solution (0.42 mg o-dianisidine HCl / ml Na borate / HCl buffer) uric acid solution, 0.012 M in Na borate / HCl buffer

Gelatin solution, 3% in Na borate / HCI buffer

Uricase / POD complex (diluted with Na borate / HCI buffer)

Execution: Meßküveite Reference cell 1.0ml 1.0ml o-dianisidine solution 0.2ml 0.2 ml Uric acid solution 0.1 ml 0.1 ml Gelatin solution 1,65ml 1.7ml buffer 0.05 ml _ Uricase / peroxidase complex

The reaction is started by addition of the enzyme complex and the course followed spectrophotometrically at 436 nm. The initial speed serves as the basis for calculating the activity.

The new oxidase from uricase and POD thus obtained differs in the starting phase of the reaction from the subsequent reaction of the two individual components. Due to the covalent coupling of the two enzymes, the elimination of the lag phase is achieved at a lower concentration of the second enzyme.

The activities of the individual enzymes of the complex still have their initial activities after the chemical modification.

Example 3

Preparation of Specific Xanthine, Chromogen Oxidase Linkage Product

1. Preparation of the XOD-substituted quinone

1 ml of an XOD solution prepared from 10 mg XOD dissolved in 1 ml 0.02 M phosphate buffer, 1 M (NH ₄ J ₂ SO ₄ ) - are mixed with 2 ml of a 0.1 M quinone solution pH 8.0 and 1 ml 0.05 M phosphate buffer pH 8.0 2h at 25 ⁰ C with gentle shaking reacted. The resulting coupling product is chromatographed with Molselect ΰ-25 at pH 8.0 to remove the excess quinone. The LEIuat-a brownish colored solution-contains the enzyme-substituted quinone.

2. Preparation of the complex XOD / POD

3 ml of XOD-substituted quinone fraction with 1 ml of a POD-Lösunq containing 5 mg enzyme / ml 5,0enthält 0.05 M Na-citrate buffer pH, again shaken 10 h at 25 ⁰ C.

After this reaction time, a product is formed in which POD and XOD are covalently coupled to each other.

3. Characterization of the XOD / POD Coupling Product

The enzymatic activity of the resulting enzyme complex can be demonstrated by product formation, in this case formation of a chromogen from its leuco form

 Reagents: 0.1 M Na phosphate buffer pH 7.0

2OmM ABTS (2,2'-azino-di- [3-ethyl-benzthiazoline-suifonate (6)] in phosphate buffer 10 mM hypoxanthine solution in phosphate buffer

1 mM EDTA in phosphate buffer

XOD / POD-Kornplex (dilution with phosphate buffer)

Execution: cuvette Referenzküvelte 1ml 1ml ABTS solution 1ml 1ml Hypoxanthine -'- dissolution 0.1 ml 0.1 ml EDTA solution 0.85 ml 0.9 ml buffer 0.05 ml - XOD / POD complex

By addition of the enzyme complex, the reaction is started and its course followed spectrophotometrically at 414 nm. The initial speed serves as the basis for calculating the specific activity.

The resulting linkage product is characterized by similar high activity (retention of at least 80% of the starting activities of the two enzyme partners) and similarly fast reaction kinetics. Despite a reduction in the amount of the sequence enzyme used, there is a loss of the lag phase. Furthermore, linking products produced in this way are distinguished by increased storage stability.

Claims (6)

1. A process for preparing new specific oxidases of general formula O. wherein A is a quinoid compound having 1-3 nuclei, E _{1 is} an oxygen-consuming enzyme and E _{2 is} an H ₂ O _2- consuming enzyme, characterized in that an aqueous 0-30 vol .-% of a water-miscible solvent containing solution of Enzyme E ₁ (or E ₂ ) is reacted with a 2-10000 molar excess of a quinoid compound having at least two positions capable of 1,4-addition in the range of pH 7-9 within 0.5-20 hours, the reaction product enriched by known methods and then reacted in an analogous manner with the enzyme E ₂ (or E ₁ ) to (I). 2. The method according to item 1, characterized in that as the quinoid compound A preferably p-benzoquinone, 1,2-naphthoquinone or anthraquinone is used. 3. The method according to item 1 and 2, characterized in that as oxygen-consuming enzyme E ₁ z. As alcohol oxidase, cholesterol oxidase, choline oxidase, glucose oxidase, glutamate, uricase or xanthine oxidase is used. 4. The method according to item 1-3, characterized in that as H ₂ O _2- consuming enzyme E ₂ , z. As lactoperoxidase, horseradish peroxidase, hemoglobin or catalase is used. 5. The method according to item 1- /, characterized in that is used as the water-miscible solvent, in particular dimethylformamide, alcohols or glycol. 6. The method according to item 1-5, characterized in that the enrichment of the reaction product by ultra or gel filtration, by centrifugation or by chromatographic methods.