DE2634562A1 - ENZYME MEMBRANE - Google Patents

Description

Andrejewski, Honke, Gesthuysen & Masch patent attorneys Physicist Dr. Walter Andrejewski Graduate engineer Dr.-lng. Manfred Honke Graduate engineer Hans Dieter Gesthuysen Physicist Dr. Karl Gerhard Masch Lawyer file:

459 / _{yes +}

43 Essen 1, Theaterplatz 3, Postf. 789

July 29, 1976

Patent application KYOWA HAKKO KOGYO KABUSHIKI KAISHA No. 6-1, 1-chome, Chiyoda-ku, Tokyo-to, Japan

Enzyme membrane

This invention relates to an immobilized enzyme and, more particularly, relates to an enzyme - immobilized on a membrane z. B. is used for an enzyme electrode, which by combining an enzymatic Activity using an electrochemical analysis method works as a small electrochemical transducer.

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Enzymes are by virtue of their substrate specificity and catalytic Activity for analyzing, purifying and reacting various substances such as sugar, cholesterol and the like., contained in living bodies or dissolved in liquids are indeed useful.

However, the use of an enzyme for such purposes is because of the instability of the enzymes, great consumption of expensive enzymes useful for any course of the analysis and the requirement of a long period of time for the analysis.

With a view to overcoming these disadvantages, attempts have been made to create an enzyme membrane, e.g. an enzyme that is immobilized in it or carried by a plastic membrane, which e.g. for oxygen, Ions, substrates and the like. To be treated, is permeable. A membrane obtained in this way has the advantages of Strength, stability and evenness.

It has also been proposed to combine such an enzyme membrane with an electrotechnical process Attaching the membrane to the head of an electrode of a cell, which is used for analytical purposes, in order to achieve the following advantages to obtain:

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a) The determination is made in a short period of time with good reproducibility and stability executed.

b) The electrode can be easily washed off after the previous operations have been completed (e.g. easy to reprocess).

c) The electrode can be used several times for analysis because of the consumption of enzyme per analysis run is very low.

The enzyme membrane or enzyme electrodes of the well-known Types are illustrated as follows.

Type 1 : An enzyme solution is enclosed in a semipermeable membrane which is in direct contact with the tip of an electrode using O-rings, reported in Analytischen Chemie, £ 2 (1), 118-121 (1970). This type requires a long period of time to remove the wastes from the previous operation, and the enzyme is inconsistent and non-uniform.

Type 2 : a solution containing an enzyme, acrylamide; Ν, Ν-methylenebisacrylamide and a polymerizing agent is subjected to photo-polymerization on a plastic membrane which is in direct contact with an electrode

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stands to include the enzyme in the structure of the acrylamide gel - reported in Nature, 214 , 986-988 (1967). This type can fix a large amount of an enzyme or an enzyme complex system of two or more enzymes, but it requires a very complicated gelation reaction on the electrode for obtaining the desired enzyme electrode. Further, this type can be troublesome to apply to high molecular substrates because of an excessively high density of the structural structure of the acrylamide gel, and it is also poor in reproducibility.

Type 3 : An enzyme is mixed with an inactive protein and subjected to the crosslinking reaction to give excretions of crosslinked proteins, which are then applied to a plastic membrane placed in direct contact with an electrode and through a mesh, made of plastic resin - reported in Pathology Biology, ^ 2 (6), 497-502 (1974). This type has an advantage similar to that of type 2, but the enzyme is unstable and uneven due to the paste form of the crosslinked proteins.

Type 4 : Amino groups of an organic polymer in powder form and an enzyme are coupled to one another in order to produce a paste from an immobilized enzyme - reported in Analytical Letter, j [(4), 301-312 (1973). That guy

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because of the paste form has similar defects as that of the Type 3.

Type 5 ; A solution containing collagen fibers and an enzyme is introduced into a cell with a constant electric current under an acidic condition to give an enzyme-collagen membrane around the cathode. The membrane is washed, dried and used as part of a double membrane which has yet another membrane consisting of an oxygen permeable membrane made of a plastic resin, e.g. B. Teflon. This double membrane is brought into direct contact with an electrode and tightly attached to the electrode with rubber rings - reported in Analytica Chimica Acta, 69 , 431 (1974). This type suffers from poor substrate dispersion and time consumption. Even if these shortcomings can be avoided by using a thin membrane, there are still disadvantages of poor mechanical property and difficulty in handling. This type can also hardly be applicable to some enzymes which can be inactivated under acidic conditions.

As described above, the difficulties in immobilizing are of the enzyme inextricably linked with the enzyme membranes or enzyme electrodes of the known types. One has however, now discovered that an enzyme membrane without that previously

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mentioned disadvantages due to the use of a porous membrane made of an organic high polymer, can be prepared.

An object of the invention is to provide an enzyme membrane, which with advantage either alone for chemical Process or to be used in combination with an electrode of a cell for electrochemical processes.

This invention relates to an enzyme membrane for chemical or electrotechnical processes such as analysis, purification, reaction and the like. The enzyme membrane in general A double membrane is used as an upper membrane, of which the lower membrane is an oxygen or ion permeable Plasticfilm is. When a double membrane of the known types, e.g. Teflon membrane or nylon membrane is used When an electrochemical process is used, the double membrane is directly and tightly in contact with one Electrode attached to a cell e.g. by using O-rings.

This invention teaches an enzyme membrane which comprises an enzyme trapped in a porous membrane which is permeable and which is made of an organic high molecular polymer.

The plastic membrane used for the purpose of the invention has a microporous structure, the pores, the

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have an average diameter of 10 to 10 000 8 (preferably 50 to 5000 R) , are dense and unevenly distributed over the entire surface. The thickness of the plastic resin film is between 1 and 500, u. Such films are known in the art and are used, for example, as membrane filters. All and any plastic resin films that are used for the production of the known enzyme membranes can be used for the purpose of the invention, although it is also possible to use films made of polyacrylonitrile, polyurethane, polystyrene, polyamide, polyurea resin, Polyester, polyethylene, polypropylene and the like. Are made. Good results can be obtained by using a resin film made from a copolymer of acrylonitrile with either vinyl acetate, acrylamide, methyl acrylate or the like. Polypropylene films, which have an average pore diameter of 50 to 5000 8, are also preferable.

Porous resin films of the known types are of two types classified. One is equipped with pores which are not connected to one another, e.g. B. a closed one Zeil type, and another is equipped with pores, which are connected to each other by winding paths, which can expand from one outer surface to another, e.g., as an open cell type. Both types of film

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can be used for the purpose of the invention when the films are of a microporous structure, which is a high affinity and good adhesion of the enzyme. grant the film. The ones that can be used within the scope of the invention Plastic membranes required permeability is known in the art and can be according to the The type of use of the finished enzyme membrane will vary. For example, there is a permeability for glucose if the plastic membrane for a glucose oxidase oxygen electrode is used and a permeability for the reaction product if the plastic membrane for the enzymatic Reaction is used. Good results can be obtained by using an open cell type. Microporous Films as used in the following examples can e.g. B. produced by process as disclosed in U.S. Patent 3,679,538, Japanese Patent Laid-Open Nos. 78576/75, 90579/75 and 43878/74 are disclosed.

It is possible to keep the enzyme in the membrane by a simple or combined application of the following methods to be determined.

- chemical methods for covalent binding of the enzyme with the active groups of the high molecular weight plastic membrane (e.g. methyl ester group and nitrile group) -

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- Meshing in a die by using a NETWORKING MEANS -

- physical adsorption.

Since the porous resin membrane is generally hydrophobic, it is advantageous to coat the enzyme by painting the porous Set membrane after treatment. For example, the membrane is immersed in an organic solvent such as alcohol or acetone, immersed with subsequent immersion in water.

The reaction (contact) of the enzyme with the membrane is carried out at a suitable temperature which is below the temperature at which the enzyme is inactivated (preferably at 0 to 40 ° e). The contact time is not limited, but it is preferably between 1 and 24 hours. The chemical treatment of the membrane is done with or without the adsorbed enzyme for a suitable time at a suitable temperature. It is also possible to remove the membrane prior to combining with the enzyme at elevated temperature and over a shorter period of time to treat.

The enzymes which can be used for the invention are exemplified by oxidases such as glucose oxidase, Amino acid oxidase, alcohol oxidase, urate oxidase, Cholesterol oxidase and the like; different amino acids

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Decarboxylases, urease, glutaminase, p-glucosidase, Penicillinase, cholinesterase, cholesterol esterase, catalase, invertase, asparaginase, mutarotase and the like. It is possible to use the enzymes alone or in combination of at least two of them. The complex enzyme systems are illustrated by the combination of oxidase and catalase, and cholesterol esterase Cholesterol oxidase, invertase and glucose oxidase, and Invertase and mutarotase.

The invention can be applied to various selective electrodes for electrochemical and quantitative determination e.g. The following electrodes can be used.

Clarke or Galvani-type oxygen electrode; pH electrode, Cyanate electrode; Ammonium electrode; Carbonate electrode; Iodine electrode and the like.

The enzyme electrode equipped with the one according to the invention Enzyme membrane is used in all liquids as long as the enzymatic reaction is carried out without inactivating the one used Enzyme can be run. However, it is preferred to keep them in a liquid at pH 3 to 9 and at at a temperature of 0 to 50 ° C. The conditions can vary depending on the types of electrodes and the used enzymatic reaction medium used vary. if

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For example, if the enzyme membrane is used for an oxygen electrode, the temperature is preferably below 50 ^° C., and in the case of an ammonium ion electrode, good results can be obtained at a relatively higher pH. The concentration of the substrate used for an enzymatic reaction may vary depending on the properties of the enzyme membrane used, the types of substrates, the electrode, and the like, but 10 ~ mol / to 10 mol / is preferable. During use, the enzyme electrode is placed in a solution which does not contain the substrate and which is loaded with a constant electric current and full voltage. Then a small amount of the solution to be determined is gradually added to control the change in current or full voltage in the usual way. Even if the solution contains a small amount of the substrate at first, it is possible to equilibrate by allowing the solution to stand for an appropriate period of time.

According to the invention it is possible to use the enzyme membrane without the However, the aforementioned disadvantages can be obtained with the following advantageous results.

a) Many types of enzymes can be immobilized, and it is also possible to use different enzymes which one can be inactivated at a pH which is not applicable to the collagen membrane of the known type.

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b) The enzyme fixed on the enzyme membrane is stable and the membrane thus obtained is uniform. If e.g. the same enzyme membrane - as that used in Example 1 - 100 times in the stationary, electrical circulation process is used to determine the glucose concentration (16 mg / dl), the result obtained is indicated by the Final determination in the range of 98% of the result initially obtained.

Various disadvantages of the known enzyme electrodes with regard to stability, constant repeatability, processability and time of action are overcome by this invention, and this invention has wide applicability supplied to a wide variety of enzymes.

The accompanying drawings are reproduced in Figures 1-3 at p. 987 of Nature, Volume 214 (1967) to explain the construction and function of an enzyme electrode as used in Example 1 below.

The invention is illustrated by, but not limited to, the following Examples are explained in which all process steps are carried out at room temperature - otherwise specified individually and in which the plastic membranes in which the enzymes are fixed are made as follows can be.

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Manufacture of the membranes used in Examples 1-4 and 6-12 .

Acrylonitrile (95 parts by weight) and methyl acrylate (5 parts by weight) are conventionally disclosed as copolymerized in French patent specification 2,254,355 to prepare a copolymer which is converted into dimethylformaldehyde was dissolved to give a solution with a copolymer concentration of 20 wt .-%. the Solution was poured onto a glass plate, followed by immersion in water at 20 ° C. for 10 minutes to obtain a to result in semipermeable membrane. This membrane was washed with water and then a thermal one for 10 minutes Treatment in hot water at 80 C under halfway no pressure. The membrane obtained was microporous and permeable to the substrates to be treated, oxygen and ions. The dense on the entire outer surface distributed pores were microscopic and had an average diameter of 50 to 1000 A.

Production of the membrane used in Example 13 .

The plastic membrane was made in a manner similar to that described in U.S. Patent 3,679,538 - as follows manufactured:

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Polypropylene having a melt index of 0.7 to 5.0, and a density from 0.90 to 0.925 was melted at 200 to 25O ° C extruded through a T-die and with a rotating casting roll kept at 50 ⁰ C, is brought into contact . The membrane obtained in this way was heat-treated openly at 125 to 140 ° C. for 30 to 60 minutes in a stream of air with a low voltage. The membrane was then subjected to, for example, 100% longitudinal elongation at room temperature and then heat-set for 5 minutes at 100 to 150 ° C. under pressure. A microporous membrane of, for example, about 1.78 g / cm bulk density and about 0.17 cm / g cubic density was obtained.

example 1

A porous membrane (thickness - 75 , u) ; Space ratio 53%; average pore diameter - 90 S, prepared from a copolymer of acrylonitrile (95 parts by weight) and methyl acrylate (5 parts by weight) was placed in a 25% methanol solution of hydrazine hydrate. The amount

ο the hydrazine solution used was 1 ml / 1 cm of the starting membrane. The membrane was left for 3 hours Room temperature in the methanol solution and then it was immersed in a 10% glutaraldehyde solution for 10 minutes placed in a 0.1 mol phosphate buffer solution (pH 8).

2 The amount of glutaraldehyde solution was 1 ml / 1 cm of the

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Exit membrane. An enzyme solution containing 50 mg / ml of glucose oxidase (specific activity: 14 IU / mg, a commercial product of Kyowa Hakko Kogyo KK, Japan) was prepared by dissolving the enzyme in a 0.1 molar phosphate buffer solution (pH 6) and applied to the membrane in an amount of 0.2 ml / 1 cm starting membrane in order to fix the enzyme. The membrane treated in this way, with an enzymatic activity of approx Rings made of rubber. The electrode was made in a similar manner as reported in Nature, 214 , 986-988 (1967). When this electrode was tested in a phosphate buffer solution (pH 6) as an oxygen electrode without using the enzyme membrane and stationary electrical circulation (currency), which was set to 0.65 / uA, the amount of oxygen was observed, which was present in the buffer solution, that it was 2.2 10 mol / l. The electrode was used together with the enzyme membrane and the steady state electric current was adjusted to 0.70 µA.

The obtained electrode was placed in a cell. A given amount of a glucose was given to the cell Test solution added. The electric current became stationary after about 1 minute. The glucose concentration in the Test solution was determined by the difference between the

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initial and steady state electric currents and gave the results shown in Table 1. The degree of waste the oxygen concentration at the tip of the electrode is proportional to the glucose concentration. He needed approx. 1 to 2 minutes to perform the stationary conditioning method as described above, while only 15 seconds were sufficient, to do the waste rate method.

Table 1

Glucose
concentration
(mg / dl) Stationary
method
(Current difference)
^ uA) Degree of waste (rate) -
method
(, uA / min.) 4th
8th
12th
16 0.037
0.072
0.106
0.144 0.070
0.142
0.208
0.275

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Example 2

A similar acrylonitrile type membrane - as in Example 1 used - was placed in 0.2 ml of an enzyme solution for 16 hours at 5 ° C per 1 cm of starting membrane. The enzyme solution containing 50 mg / ml glucose oxidase (specific activity - 14 IU / mg, commercial product of Kyowa Hakko Kogyo K.K., Japan) was obtained by dissolving the enzyme in a 0.1 molar phosphate buffer solution (pH 6). The membrane was then coated with a 0.1 molar phosphate buffer solution (pH 6) lightly washed and placed in a 10% glutaraldehyde solution for 20 minutes at room temperature, which was prepared by dissolving glutaraldehyde in a 0.1 molar phosphate buffer solution (pH 8). In this way the enzyme adsorbed on the porous membrane was fixed by the crosslinking reaction. The thus obtained with the membrane combined with the enzyme had an enzymatic activity

vity of 0.2 IU / cm of the starting membrane and was used to prepare an enzyme electrode similar to that described in Example 1. In the same way - like Described in Example 1 - the glucose concentration was determined quantitatively. The results obtained are in Table 2 shown.

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Table 2

Glucose
concentration
(mg / dl) Current difference
(/ UA) - Rate of waste
(, uA / min.) 4th
8th
12th
16 0.036
0.072
0.107
0.144 0.076
0.141
0.209
0.275

Example 3

A similar polyacrylonitrile-type membrane - like that used in Example 1 - was 16 hours at 5 ° C in 0.2 ml of a mixed enzyme solution containing 300 mg / ml invertase (specific activity - 23 IU / mg; commercial product of Kyowa Hakko Kogyo K.K., Japan), 50 mg / ml glucose oxidase (specific activity - 14 IU / mg; commercial product of Kyowa Hakko Kogyo K.K., Japan) and 2.5 mg / ml mutarotase (specific activity - 6,000 IU / mg; commercial product from Boehringer Mannheim GmbH, Germany) to 1 cm of the Membrane laid. The enzyme solution was by dissolving the

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Enzymes in a 0.1 molar phosphate buffer solution (pH 6.5) prepares. The membrane was washed lightly with a 0.1 molar phosphate buffer solution (pH 6.5) and left for 20 min. placed in a 10% glutaraldehyde solution at room temperature, which is obtained by dissolving glutaraldehyde in a 0.1 molar Phosphate buffer solution (pH 8) was prepared to instantly use invertase, glucose oxidase and mutarotase of the membrane used to make a similar enzyme electrode - as that used in Example 1 - to manufacture. The electrode was used to determine sucrose and glucose concentrations. The results obtained are shown in Table 3, from which it appears that the sensitivities of the electrode to both sugars are almost equal to each other.

Table 3

concentration
the glucose and
Sucrose
(/ U mol / ml) Current difference (/ UAJ Glucose 0.3
0.6
1.2
1.8
2.4 Sucrose 0.024
0.047
0.095
0.140
0.185 0.022
0.045
0.089
0.125
0.172

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Example 4

A similar membrane of the polyacrylonitrile type - like ■ that used in Example 1 - was treated in a similar manner - as that described in Example 1 - to to introduce the aldehyde group, and it was then dissolved in 0.2 ml

2 put an enzyme solution containing catalase per 1 cm membrane

contained. Duration of the insert 16 hours at 5 ° C. The enzyme solution was prepared by dissolving catalase (cow liver enzyme, available from Kyowa Hakko Kogyo K.K., Japan) in 0.1 molar phosphate buffer solution (pH 7), and it had an activity of 110,000 IU / ml. Those with the enzyme koitibi-

The second membrane had a catalase activity of 1.1 IU / cm the starting membrane and was used to create an enzyme electrode similar to that used in Example 1 create. The substrate (hydrogen peroxide) was through Catalase decomposes in water and oxygen. The rate of increase in oxygen concentration at the tip of the electrode was proportional to the hydrogen peroxide concentration. As a result, the level of oxygen at the increased Electrode in the presence of the substrate, and the hydrogen peroxide content was proportional to the level of oxygen and its rate of incline is determined. The quantitative determination of the hydrogen peroxide concentration was carried out at 2 C and a pH of 7.4. the Results obtained are shown in Table 4.

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Table 4

Focus on
Hydrogen peroxide
(/ ug / ml) Stationary conditions
method (electricity
difference - / UA) Degree of waste (rate) -
method
(Rate -, uA / min.) 15th 0.088 0.109 37.5 0.226 0.268 75 0.443 0.503 112.5 0.679 0.770 150 0.890 0.994 187.5 1,144 1,240 225 1,276 1,420

Example 5

Juragard 2400 (trade name for a membrane made of polypropylene, available from Polyplastics Corporation, Japan; Thickness - 25, u; average pore diameter was 100 8 (major axis) and 200 8 (minor axis)) placed in acetone to provide an affinity for water by hanging in water, and it was then dissolved in 0.2 ml placed an enzyme solution containing 50 mg / ml glucose oxidase (specific activity - 14 IU / mg; commercial product of

2 Kyowa Hakko Kogyo K.K., Japan) per 1 cm of the starting membrane

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contained. The enzyme solution was made by adding the Enzyme dissolved in a 0.1 molar phosphate buffer solution (pH 5.6). The porous membrane thus obtained was 0.1 mol a phosphate buffer solution (pH 5.6) and 20 min. At room temperature in a 10% glutaraldehyde solution which was prepared by dissolving glutaraldehyde in a 0.1 molar phosphate buffer solution (pH 8), to combine glucose oxidase with the polypropylene matrix. The membrane that was combined with the enzyme and

2
had an activity of 0.1 IU / cm of starting membrane was used to prepare an enzyme electrode similar to that used in Example 1. According to a method described in Example 1, the glucose concentration in the test solution was determined using the electrode at 37 ° C and a pH of 7.4. The results obtained are shown in Table 5.

Table 5

(/ UA) 10 Glucose concentration (mg / dl) 20th 30th 40 50 60 70 80 0.063 0.125 0.180 0.242 0.302 0.362 0.415 0.463 CD

Note: CD - current difference.

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Example 6

A similar polyacrylonitrile type porous membrane

- As that used in Example 1 - with the exception of a thickness of 100, u, a space ratio of 58% and one average pore diameter of 120 8 - was similar to treatment with hydrazine and glutaraldehyde applied in Example 1 - subjected to the introduction of the aldehyde group into the membrane. The membrane was 16 hrs.

long at 5 ° C in 0.2 ml of a mixed enzyme solution per

Inserted 1 cm of the initial membrane to fix the enzymes. This enzyme solution contained 50 mg / ml cholesterol esterase (specific activity - 2 IU / mg) and 10 mg / ml cholesterol oxidase (specific activity - 10 IU / mg)

- Both enzymes are commercially available from Kyowa Hakko Kogyo K.K., Japan - and it was prepared by these enzymes in a 0.1 molar phosphate buffer solution (pH 7.4) dissolved. The membrane was used by placing an enzyme electrode similar to that described in Example 1 - made. According to a similar procedure

- as described in Example 1 - were at 37 ° C and a pH of 7.4 using the electrode cholesterol concentrations (free and in ester forms) determined in the test solution. The results obtained are in the tables 6-1 and 6-2. Cholesterol linolate was used as one of the ester forms, and as a substrate solution, one became

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Solution containing 5% ethanol and 1% Triton-X-100 (a wetting agent, commercially available from Nakarai Kagaku Yakuhin K.K., Japan).

Table 6-1

(Ester form)

Cholesterol concentration
(mg / dl) Current difference
(/ UA) 20th
40
60
80 0.004
0.008
0.013
0.017

Table 6-2 (free form)

Cholesterol concentration
(mg / dl) Power Di fference
(/ UA) 20th
40
60
80 0.010
0.020
0.031
0.034

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Example 7

A similar enzyme electrode as that in Example 6 used - was used to determine blood sugar and was determined by another analytical method (Somogi-Nelson's Method) for determining the glucose concentration. The results are shown in Table 7.

Table 7

sample Somogi-Nelson's
method present proceedings 1
2 81 mg / dl
155 mg / dl 82.7 mg / dl
152 mg / dl

Example 8

A membrane of the polyacrylonitrile type, which is the im Example 1 used was similar to 16 hrs at 5 ° C

in 0.2 ml of an enzyme solution containing 25 mg of d-amino acid oxidase

2
contained per 1 cm of the starting membrane, immersed. The enzyme solution was prepared by adding the enzyme (specific activity - 15 IU / mg; commercial product of Boehringer Mannheim GmbH, Germany) _r l in a molar 0 phosphate buffer solution dissolved (pH 8). The membrane then became light

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washed with a 0.1 molar phosphate buffer solution (pH 8) and in a 0.1 molar phosphate buffer solution which 10% glutaraldehyde was placed for 20 minutes, and an enzyme membrane was obtained. The d-alanine concentration in the test solution was quantified by similarly using the enzyme membrane used in Example 1 The results obtained are shown in Table 8.

Table 8

Concentration of d-alanine (mg / dl) 1.25 2.50 3.75 5.00

Current difference

(, uA) 0.041 0.084 0.123 0.159

Example 9

A polyacrylonitrile type membrane - a similar one to used in Example 1 - was treated in the same manner as described in Example 1 - to remove the aldehyde group and then soaked it in 0.2 ml of an enzyme solution per 1 cm of the starting membrane at 5 C for 16 hours laid to give an enzyme membrane. The enzyme solution was prepared by using Uricase (specific Activity (Candida utilis) - 2.5 IU / mg; commercial

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Product of Sigma Corp., USA) in a 0.1 molar borate buffer solution (pH 9.0), it contained 10 mg / ml of the enzyme. The obtained enzyme membrane was used to to determine the uric acid in the test solution in the same manner as described in Example 1. The results obtained are shown in Table 9.

Table 9

Uric acid concentration (mg / dl) 2.5 5.0 10.0 15.0

Current difference (, uA) 0.102 0.200 0.385 0.540

Example 10

A polyacrylonitrile type membrane - a similar one to used in Example 1 - was treated in the same manner as described in Example 1 - to remove the aldehyde group and then it was immersed in 0.2 ml of an enzyme solution (per 1 cm of the starting membrane) for 16 hours at 5 C containing 2 mg / ml asparaginase was laid to give an enzyme membrane. The enzyme solution was prepared by adding the enzyme (specific activity - 110 IU / mg; commercial product of Kyowa Hakko Kogyo K.K., Japan)

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dissolved in a 0.1 molar phosphate buffer solution (pH 7). The enzyme membrane was placed on the tip of an ammonium electrode (type 7161, commercial product from Denki Kagaku. Keiki K.K., Japan). The concentration of 1-asparagine was determined by using the electrode. Table shows the results obtained. In determining it was a 0.1 molar phosphate buffer solution is used (pH 8.5). The logarithm of the ammonium concentration is proportional the tension.

Table 10

Concentration ml-asparagine (mol / l)

5 χ 10 ~ ⁴ 10 " ³ 5 χ 10 ~ ³ 10" ²

Voltage difference (mV) 17 25 43 51

Example 11

A polyacrylonitrile type membrane - a similar one to used in Example 1 - was treated in the same way - as described in Example 1 - to remove the aldehyde group and then soaked it in 0.2 ml of an enzyme solution containing urease at 5 ° C for 16 hours (Io mg / ml) immersed to give an enzyme membrane. the

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Enzyme solution was prepared by adding the enzyme (specific activity - 700 IU / mg; commercial product of Sigma Corp., USA) in a 0.1 molar phosphate buffer solution (pH 7). The enzyme membrane was on the same The manner described in Example 10 was used to quantitatively determine the urea concentration in the test solution. The results obtained are shown in Table 11.

Table 11

Concentration of urea (mol / l) 5 x 10 " ⁴ 10 ~ ³ 5 χ 10" ³ 10 " ² 5xlO ~ ²

Voltage difference (mV) 10 22 48 59

Example 12

A polyacrylonitrile type membrane similar to that used in Example 1 was subjected to the same treatment with hydrazine and glutaraldehyde - as applied in Example 1 - subjected to the aldehyde group in the membrane

to introduce. The membrane was in

0.2 ml of an enzyme solution immersed per 1 cm of the output membrane, to set the enzyme. The enzyme solution contained 50 mg / ml lactase-Y (a commercial product from Kyowa Hakko Kogyo K.K., Japan; specific activity - 26 IU / ml).

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It was prepared by dissolving the enzyme in a 0.1 molar phosphate buffer solution (pH 7). The one with the Enzyme combined membrane had enzymatic activity of 2.8 IU / cm of the exit membrane. It became a Diaflo cell (Type 402) (an apparatus for ultra-filtration, commercially available from Amicon Corp., USA), the was equipped with this membrane, used to treat about 200 ml of an 8% cheese whey, under pressure

2
with a nitrogen gas (3 kg / cm of membrane) to give a filtrate (20 ml / hour). By concentrating the filtrate under reduced pressure to dryness, the desired product, the content of which is shown in Table 12, was obtained.

Table 12

Cheese whey product

Lactose 64.2% 12%

Glucose 0 31%

Galactose 0 30%

Protein 6.7% 0

Example 13

A porous polypropylene membrane (thickness - 30, u; space-

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Andrejewski, Honke, Gesthuysen & Masch, patent attorneys in Essen

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Ratio - 38%; Average pore diameter 1500 8 in the larger axis and 400 8 in the smaller one Axle) made in the same way as described in US Pat. No. 3,679,538 used. The same procedure as described in Example 5 was carried out, but using one Enzyme membrane (enzymatic activity - 0.15 IU / cm of the Output membrane), which was prepared by using 60 mg / ml glucose oxidase solution. The glucose concentration in the test solution was determined at 37 ° C. and a pH of 7.4. The results obtained are in Table 13 shown.

Table 13

Glucose concentration (.ug / ml) 10 20 30 40 50 60 70 80

A-O.061 0.120 0.178 0.238 0.290 0.350 0.400 0.445

Example 14

The pH resistance, heat resistance and durability a membrane made according to Example 1 was compared with a membrane made in an acrylamide gel and contained free enzyme, and gave the results shown in Tables 14, 15 and 16.

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Andrejewski, Honke, Gesthuysen & Masch, patent attorneys in Essen

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A membrane enclosed in an acrylamide gel was made as follows.

95 mg of acrylamide, 5 mg of NN-methylenebisacrylamide and 50 mg of glucose oxidase (a product similar to that used in Example 1) were dissolved in 0.8 ml of 0.1 μm phosphate buffer solution. 5 mg Ν, Ν, Ν ¹ , N ¹ -Tetramethyläthylendiamxn and 1 mg ammonium persulfate were dissolved in 0.2 ml 0.1 η phosphate buffer solution. Immediately afterwards the two solutions were

mixed and a nylon mesh (50 cm) was covered with the combined solution. The coated nylon net was left Stand at room temperature in nitrogen atmosphere until the membrane is enclosed in acrylamide gel became.

Table 14

pH stability

Sample pH

5.6

6.5

7.5

A. 90 92 91 88 80 78 85 B. 90 90 90 80 64 52 40 C. 91 91 90 75 54 45 5

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Andrejewski, Honke, Gesthuysen & Masch, patent attorneys in Essen

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Note: A - membrane according to Example 1 B - membrane enclosed in acrylamide gel C - free enzyme

The results shown in Table 14 characterize the enzymatic activities of the samples, determined from the displayed pH values (initial activity - 100). The preservation was carried out ^{at 60 ° C. for 1 hour.}

Table 15

100 100 98 92 72 100 100 95 90 48 100 100 92 91 13th

Sample temperature 30 ⁰ C 40 ° C 50 ° C 60 ° C 70 ⁰ C

Note: A - membrane according to Example 1 B - membrane enclosed in acrylamide gel C - free enzyme

The results shown in Table 15 characterize the enzymatic activities of the samples, which in a 0.1 molar phosphate buffer solution and which at the indicated temperatures for one hour (initial activity - 100) have been preserved.

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Andrejewski, Honke, Gesthuysen & Masch, patent attorneys in Essen

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Table 16

Holding

Sample 1 week

Shelf life period 2 weeks "3 weeks

4 weeks 5 weeks

A. 92 91 90 90 90 B. 85 75 70 65 63 C. 69 50 42 25th 12th

Note: A - membrane According to example 1

B - membrane enclosed in acrylamide gel C - free enzyme

The results shown in Table 16 indicate the enzymatic activities of the sample, which in a 0.1 molar phosphate buffer solution (pH 5.6) were dissolved while being stored at 30 ° C.

As can be seen from Tables 14-16, the pH stability, the heat resistance and the durability are of the membranes that have bonded with the specified enzyme according to the method of the invention, both those of the established enzymes of the known types as well

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Andrejewski, Honke, Gesthuysen & Masch, patent attorneys j & Jfs & k _o

superior to free enzymes. The specified enzymes of the invention undoubtedly provide an advantage in the Technology and provide an excellent enzyme electrode.

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Claims (6)

Andreiewslei, Honke, Gesthuysen & Masch, patent attorneys in Essen - 36 - Patent claims: 1y enzyme membrane, used as a chemical or electrochemical Transducer, whereby the enzyme membrane is permeable and made of an organic high molecular weight Polymer consists, characterized in that the membrane is microporous. 2. Enzyme membrane according to claim 1, characterized in that that the pores are distributed over the entire surface of their exterior. 3. enzyme membrane according to claims 1 and 2, characterized characterized in that the average diameter of the pores is between 10 and 10,000 S. 4. Enzyme membrane according to claims 1-3, characterized in that that the plastic membrane is of the open cell type. 5. double membrane including an enzyme membrane according to claim 1 as a top membrane. 6. Enzyme electrode including an enzyme membrane according to claim 1. 709807/081 1