CA1072468A - Enzyme membrane - Google Patents

Abstract

ABSTRACT OF THE DISCLOSURE

The invention is an enzyme membrane which can be used as a chemical or electrochemical transducer. The enzyme membrane is permeable and consists of an organic high molecular weight polymer. The membrane is also characterized in that it is microporous, i.e., it preferable has pores having an average diameter of 10 to 10,000 A.
By using a microporous organic high polymer for the membrane, the disadvantages of prior art enzyme membranes can be avoided.

Description

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This invention relates to an immobi~ized enzyme and more particularly relates to an enzyme immobilized in membrane form used, for example, for an enzyme elec-trode which functions as a miniature electrochemical -transducer by means of com-bining enzymatic activity wi-th an electrochemical procedure for analysis.
Enzymes are indeed useful, by virtue of their substrate specificity and catalytic activity, for analysis, purification and reaction of various substances such as sugars, cholesterol etc., contained in living bodies or dissolved in liquids. However, the use of enzymes for such purposes is disadvantageous because of the usual instability of enzymes, the usual large consumption of expensive enzymes for each run of analysis and the requirement for long periods of time for analysis.
Attempts have been made to overcome these dis-advantages by providing enzyme-membranes, i.e., enzymes immobilized in, or supported by, plastic membranes which are permeable to, for example, oxygen,-ions, or substrates to be treated. The membranes thus obtained have the advantages of strength, stability and uniformity.
It has been also proposed to combine such enzyme-membranes with an electrochemical procedure for analysis, e.g., by providing the membrane at the head of an electrode of a cell used for analytical purposes in order to obtain the following advantages: -a) A determination can be carried out in a short period of time with good reproducibility and stability and the results can be obtained speedily;
b) The electrode is easily washable after each ~ 1 -- ~

1(37~

determination;
(c) The electrode can be used many times for analysis, because the consumption of enzyme per run of analysis is quite small.
The known types of enzyme-membranes or enzyme electrodes are exemplified as follows.
Type 1: An enzyme solution is sealed in a semi-permeable membrane which is directly contacted with the head of an electrode by means of o-rings; reported in Analytical Chemistry, 42, (1), 118-121 (1970). This type requires a long period of time to remove the waste resulting from the preceding operation, and the enzyme is unstable and non-uniform.
Type 2: A solution containing an enzyme, acrylamide, N,N'-methylenebisacrylamide and a polymerizing agent is subjected to photo-polymerization on a plastic membrane which is directly contacted with an electrode to entrap the enzyme in the matrix of acrylamide gel;
reported in Nature, 214, 986-988 (1967). This type may immobilize a large amount of an enzyme or a complex enzyme system of two or more enzymes, but requires a very com-plicated gelation reaction on the electrode for the preparation of the desired enzyme electrode. Furthermore, this type can be applied to high molecular substrates only with great diffuculty because of the excessively high density of the matrix structure of the acrylamide gel and because the resulting electrode is defective in reproducibility.
Type 3: An enzyme is mixed with an inactlvated protein and subjected to a cross-linking reaction to give a precipitate of the cross-linked pro~eins which is then put 7~

on a plastlc me~brane placed in direc-t contac-t with an electrode and supported by a net made of a plastic resin;
reported in Pathology Biology, 22, (6), 497-502 (1974).
This type has a similar advantage to that of Type 2, but the enzyme is unstable and non-uniform owing to the paste form of the cross-linked proteins.
Type 4: Amino groups of an organic polymer in powder form and an enzyme are coupled with each other to give a paste of an immobilized enzyme; reported ln 10 Analytical Letter, 6, (4), 301-312 (1973). This type has similar defects to those of Type 3 because of the paste form.
Type 5: A solution containing collagen fibrins and an enzyme is electrolysed with a constant electric current in a cell under acidic conditions to give an enzyme-collagen membrane around the cathode. The membrane is washed, dried and used as a part of a double membrane which also includes another membrane consisting of an oxygen-permeable membrane made of a plastic resin such ``` 20 as Teflon. This double membrane is directly contacted with . .
an electrode and tightly secured to the electrode with rubber rings; reported in Analytica Chimica Acta, 69, 431 (1974). This type has the disadvantage of a slow dispersion of the substrate and it is therefore time-consuming to use.
Even if this disadvantage can be avoided by providing a thin membrane, further disadvantages, such as poor mechanical property and difficulty in handling then result.
Also, this type cannot be used with some enzymes which may be inactivated under acidic conditions.
As described above, the difficulties in im mobilizing the enzyme are inherent to the known types of 1~i7'~;8 enzyme membranes or enzyrne electrodes. It has now been discovered that an enzyme-membrane not having the above-mentioned disadvantages can be prepared by using a porous membrane made of an organic high polymer.
An object of this invention is to provide an enzyme-membrane which may with advantage be used either solely for a chemical procedure or in combination with an electrode of a cell for an electrochemical procedure.
According to the invention there is provided an enzyme membrane having an enzyme supported by a plastic membrane wherein said membrane is microporous and permeable and made of an organic high molecular weight selected from the group consisting of (1) a copolymer of a major portion of acrylo-nitrile with a minor portion of methylacrylates, and (2~
polypropylene, the average diameter of the pores of said membrane ranging from 50 to5000A and the pores being unevenly distributed on the surface of the membrane, said pores being interconnected through tortuous paths which mav extend from one exterior surface to another.
The enzyme-membrane of the present invention can be used for chemical or electrochemical procedures such as analysis, purification, reaction and the like. The enzyme-membrane is generally used as an upper membrane of a double membrane, of which the lower membrane is an oxygen and ion-permeable plastic film. When a double membrane of the known type, such as a Teflon~ membrane or a nylon membrane, ' .

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is used for an electrochemical procedure, the double membrane is directly and tightly placed in contact with an electrode of a cell, for example by using O-rings. The thickness of the plastic resin film is preferably from 1 to 500~.
The permeability required for the plastic membranes which may be used for the purpose of this invention is known in the art, and may vary, depending upon how the finished membrane is to be used. For example, permeability to glucose and oxygen is required for the plastic membrane used for a glucose oxidase oxygen electrode and permeability to the reaction product is required for the plastic membrane used for enzymatic reactions. Good results may be obtained by using ~, - 5 -;24~

an open-cell type of film. The microporous films used in the following examples may be prepared, for example, by the processes disclosed in United States Paten-t Specification No. 3,679,538 and Japanese laid-open specification Nos.
78,576/75, 90,579/75 and 43,878/74.
It is possible to immobilize the enzyme in the membrane by the single or combined application of the following methods.
- a chemical method for the covalent bonding of the enzyme with the functional groups of a high molecular plastic membrane (e.g., a methylester group and a nitrile group) - cross-linking with a matrix by the use of a cross-linking agent - physical adsorption.
As porous resin membranes are generally hydrophobic, it is preferable to immobilize the enzyme by contacting it with the porous membrane after treatment. For example, the membrane can be immersed in an organic solven-t, such `as, e.g., alcohol or acetone, followed by immersion in water.
The reaction (contact) of the enzyme with the membrane can be effected at a suitable temperature below the temperature at which the enzyme is inactivated (preferably from 0 to 40C). The contacting time need not be limited, but is preferably from 1 to 24 hours. The chemical treatment of the membrane can be effected with or without concurrent adsorption of the enzyme at a suitable tempera-ture for a suitable time. It is also possible to treat the membrane at an elevated temperature for a shorter period of time before combining it with the enzyme.

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l'he erlzymes which rnay be used for the invention areexemplified by oxidases such as glucose oxidase, amino acid oxidase, alcohol oxidase, uricase, choles-terol oxidase and like; various amino acid decarboxylases; urease, glutaminasei ~-glucosidase; penicillinase; choline esterase; cholesterol esterase; catalase; invertase; asparaginase; mutarotase and the like. It is possible to use the enzymes singly or in any combination of two or more enzymes. For example, complex enzyme systems are exemplified by the combinations of oxidase and catalase, cholesterol esterase and choles-terol oxidase, invertase and glucose oxidase, and invertase and mutarotase.
The invention may be applied to various selective electrodes for electrochemical and quantitative deter-minations, e.g., the following electrodes: Clarke or Galvani type oxygen electrodes; pH electrodes, cyanate electrodes, ammonium electrodes, carbonate electrodes, iodine ion electrodes and the like.
Enzyme electrodes using the enzyme-membrane of this invention can be used in any and all liquids provided the enzymatic reaction can be carried out without the inactivation of the enzyme and provided, of course, the liquid does not attack the structure of the membrane.
However, it is preferred to use the electrode in a liquid having a pH of 3 to 9 and a temperature of from O to 50 C.
The conditions may vary, depending upon the type o~ the electrode and the medium used for the enzymatic reaction.
For example, when the enzyme-membrane is with an oxygen electrode, the temperature is preferably kept below 50 C, and in the case of use with an ammonium ion electrode, good results can be obtained at a relatively higher pH.

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The concentration oE the substrate used for erlzyma-tic reaction may vary, depen~ing upon -the properties of -the enzyme membrane, the type of the substrate and the type of electrode, e-tc., but 10 5 mol/l to 10 2 mol/l is preferable.
In use, the enzyme electrode is usually put into a solution which does not contain the substrate and loaded wi-th a constant electric current or voltage. After this, a small amount of the solution to be determined is added gradually to check the change of the current or voltage in a conven-tional manner. Even when the solution initially containsa small amount of the substrate, it is possible to obtain an equilibrium by allowing the solution to s-tand for a suitable period of time.
According to this invention, it is usually possible to obtain an enzyme-membrane which does not have the aforementioned disadvantages of the prior art membranes and which usually has the following advantageous results.
a) Many types of enzymes can be immobilized to form the enzyme membrane and it is possible to use various enzymes which would be inactivated at the pH which would be used with collagen-membranes of the known type.
b) The enzyme immobilized in the enzyme-membrane is stable and the membrane thus obtained is uniform. For example, when the enzyme membrane described in Example 1 below was used 100 times in a stationary electric current procedure for the determination of glucose concentration (16 mg/dl), the result obtained in the final determination was within 98% of the initially obtained result.
Various disadvantages of the known enzyme electrodes with respect to stability, constant reproduci-bility, washability and operation time are l~sually completely ~07;~4~3 overcome by this invention and a wide ap~licability to various enzymes is affor~ed by th,is invention.
The accompanying drawings are repxoduced from Figures 1-3 on page ~87 of .~ature, Vol. 214 (1957) to illustrate the construction and func-tion of an enzyme , electrode used in Example 1 given below.
In the drawings, Figure 1 is a diagram showing the principle of an enzyme electrode;
Figure 2 is a diagram showing a dual cathode enzyme electrode; and F,igure 3 is a circuit diagram showing the circuit used for the measurement of the oxygen electrode current response.
The invention is illustrated by the following non-limitative Examples, in which all procedures were carried out at room temperature unless otherwise specified, and in which the plastic membranes used in the preparation of the enzyme-membranes were prepared as follows.
Prepara-tion of the membranes used in Examples 1-4 and 6~12.
~crylonitrile (95 parts by weight) and methyl-acrylate (5 parts by weight) were copolymerized in a conventional manner as disclosed in JA-OS 90579/75 and FR-PS 2254355. The resulting copolymer was dissolved in dimethylformaldehyde to give a solution having a copolymer concentration of 20~ by weight. The solution was cast onto a glass plate, followed by immersion in water at 20 C for 10 minutes to give a semi-permeable membrane. This membrane was washed with water and then subjected to thermal treatment in water at 80C for 10 minutes under substantially g ~7Z~

no tension. Irhe membrclne thus obtained was microporous and permeabie to the subst:rate, oxygen and ions to be treated. The pores were distributed closely over the entire exterior surfaces and were microscopic having an average diameter of from 50 to 1, OOOA.
Prepara-tion of the membrane used in Example 13 ... . . . ~ .
A plastic membrane was prepared in a similar manner to that described in U.S. Patent 3,679,538 as fol~.ows:

7 ~
l'oly[>ropylerle having a ~lle1L inclex of (~.7 to S.~ and a density of (~.9() to 0.9~ w.ls melt extru~ed at 200 to 250C
through a T-die ~n~ cont<,lc~ecl ~iLh cl rotating casting roller maintalned at 50C~ 'I'he pro~uced melnbrane was oven annealed in air with a s1ig~ tension at 125-140C for 30 to 60 minutes.
The mernbralle was subjected to, ~or example~ 100~Z ex~ension lengthwise at room temperature and then heat-set under tension at 100-150C for S minutes to obtain a microporous membrane having, for example, about 1.7~ g/cm3 of bulk density and about 0.17 cm3/g o cubic densi~y.
E~ample 1:
A porous membrane (thickness - 75~9 space ratio - 53~o;
average pore diame~er- 90A) macle of a copolymer of acrylonitrile (95 parts by weight) and methylacrylate (5 parts by weight) was put in a 25'~o methanol solution of hydrazine hydrate. 'rhe aMount of the used hydrazine solu~ion was 1 ml per 1 cm2 of the starting membrane. The membrane was allowed to stand at room temperature for 3 hours in the m~thanol solution and was thell put in a 10%
glutaraldehyde solution in 0.1 mol phosphate buffer solution "~ (p~l 8) for 10 minutes. l'he amount o~ ~he glutaraldehyde solution was 1 ml per 1 cm of the starting membrane. An enzyme solution containing 50 mg/ml of glucose oxidase (specific acti~ity -14 IU/mg; a commercial product of Kyowa Hakko Kogyo K.l~., Japan) was prepared by dissolving the enzyme in 0.1 mol phosphate buffer solution (pH 6) and was applied to the membrane in an amount of 0.2 ml per l cm of the starting membrane to immobilize the enzyme. The thus-treated membrane having an enzymatic activity of about 0.2 IU/cm2 was closely attached to Teflon film (thickness -2011) at the head of a Clarke type oxygen electrode by using an 0-ring consisting o~' rubber. The electrode was = _~.............. . . _ _ ., __ 1~7'~
~r~ar~cl i~ si~ r fast~io~ t h~l~ reported in Na~;ure, 2l4, 9~6-98~ (1967). Whetl ~his elec~rode was tes~e(l in a phosphate buffer sol~ion (p~l 6) as an oxygerl electrode without using the enzyme me~nl)rane and the stationary electric currency was adjusted to 0.65 ~A, it was obserbed Llle amount of oxygen dissolved in the buffer sol~tion being 2.2 x 10 4 mol/l. The electrode was used togeLher with the enzyme rnetnbrane and the -stationary electric current was adjusted to 0.70 ~A.
The electrode obt~ined was pu-t in a cell. A given amount of ~ test solution containing glucose was added to -the cell. I'he electric curren~ of the electrode became stationary after about l minute. The glucose concentration in the test solution was determined by the difference between the initial and stationary electric curren~s to give the results sho~n in Table l. The decrease rate of oxyger. concentration at the head of the electrode ~as proportional to the glucose concentration.
It took about l to 2 minutes Lo carry out the stationary con-dition method above-described, while only abou~ 15 seconds were enough to carry out the rate metho~.
Table L

.. . _ . .. . . ~ _ ~tationary Method Glucose (Current l~ate (.oncentration Differentia1) Method (mg/dl) (~A~ in) ~5 - 4 0.037 0.070 8 0.(~7~ 0.142 12 0.106 0.208 1~ 0.1~4 0.275 ~729 f~;xarn~l~ 2:
~ simillr acry1Onitri1e-Lype membrane to that used in Example l was put in a 0.2 ml of e~zyme svlution per l cm of the starting membrane at 5C for 16 hours. The enzyme solution contained 50 mg/ml of glucose oxidase (specific activity -14 IU/mg; commercial product of Kyowa l-lakko Kogyo K.K., Japan) was prepared by dissolving the enzyme in a O.l mol phosphate buffer solution (p~l 6). After this, the membrane was slightly washed with 0.1 mol p~losphate buffer so].ution (pH 6) and put l~ at room temperature for 20 minutes in 10% glutaraldehyde solu-tion prepared by dissolving glutaraldehyde in O.l mol phosphate buffer solution (pH 8). In this manner9 the enzyme adsorbed onto the porous membrane was immobilized by the cross-linking reaction. The thus-obtained metnbrane combined with the enzyme had an enzymatic activity of 0.2 lU/cm2 of the starting membrane and was used to prepare a simi:lar e~zyme electrode to that described in Example l. In a similar manner to that described in Example l glucose concentration was determined quantitatively and the results obtained are shown in Table 2.
~0 Table 2 . ~
Glucvse Current Concentration Dif~erential Rate (mg/dl) (~)A) (~A/min) 4 0.036 0~076 ;j 8 0.072 0.141 12 0.107 ~).209 16 ~.144 0.275 . _ ~

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:xa~ e ~ similar polyacrylonitrjle type membralle to that used in ~xample 1 w~s puL~ at 5C Lor 16 llours, in a 0.2 ml of mixed enzyme solution containing 300 mg/ml of invertase (specific activity - 23 IU/mg; commercial product of Kyowa Hakko Kogyo K.K., Japan), 50 mg/ml of glucose oxidase (specific activity - 14 IU/mg; commercial product of Kyowa Hakko Kogyo K.K.9 Japan) and 2.5 mg~ml of mutarotase (specific activity - 6,000 IU/mg; commercial product of Boehr]nger Mannheim GmbH, Germany) per 1 cm of the membrane. The enzyme solution was prepared by dissolving ~he enzymes in 0.1 mol phosphate buffer solution (pE~ 6.5). The membrane was slightly washed with 0.1 mol phosphate buffer solution (pH 6.5) and put, at room temperature for 20 minutes, in 10% glutaraldehyde solution prepared by dissolving glutaraldehyde in 0.1 mol phosphate buffer solution (p~l 8) to combine at once invertase, glucose oxidase and mutarotase with the membrane which was used to prepare a similar enzyme electrode to that used in Example 1~ 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 each other.
Table 3 ~ ____ >5 of Glucose & Gurrent Differential (~A) Sucrose (~Imoltml) Sucrose Glucose .. .. . _ .. .. .
0.3 0.022 0.024 0.6 0.045 0.047 1.2 0.089 0.095 1.8 0.125 0.140

2.4 0.172 0.185 . _ . _ ... . .. . .. _ _ ~ ~7 l;xcn~ JJ.e 4:
~ similar m~mbrall~ of ~oJ.yacrylonitri.le ~ype to that use~ in ~xampl~ 1 was treaL~d in a similar manncr to that described in ~xamp.le .I to introduce the aldehyde group and was then put in 0.2 ml of an en~yme solution containing catalase per 1 cm of the membrane at 5C ~or 16 hours. The enzyme solution was pr~pared by dissolving catal.ase (cow liver enzyme ~ available from Kyowa Hakko Kogyo K.K., Japan) in 0.1 mol phos-phate buffer solution (p~l 7) ancl had an activity of 110,000 lU IU/rnl. The membrane combine~ with the enzyme had a catalase activity of 1.1 IU/cm2 of the s~arting membrane and was used to prepare a similar enzyme electrode to that used in Example 1.
The s,ubstrate (hydrogen peroxide) was decomposed by catalase to give wa~er and oxygen. 'l`he incr~ase rate of oxygen concentrati at the head of the electrode was proportional to ~he hydrogen peroxide concentration. ~ccordingly the level of oxygen at the electrode elevated in the presence of the substrate, and hydro-gen peroxide was determined in reliance with the level and speed of the oxygen rising. The quantitative determination of hydrogen peroxide concentration was performed at 2C at a pH of 7.4. The results obtained are shown in Table 4.
Table 4 _ _ _ Concentration Stationary Condition ~Iydro~en Peroxide Method (CurrentKate ~eLhod ~g/ml) Differentia]. - ~A)(Rate-~lA/min) --. .. _ __ 0.08~ 0.10 37.5 0.226 0.26$
0.443 0.503 112.5 0.679 0.770 150 0.890 0.9~4 187.5 1.144 1.240 225 1.276 1.420 .. . .. .

~L~7Z~6i 3 Example 5: ~
Juragard 2400 (tr~de ~ o~ porous membrane made of polypropylene available from l'olyplastics Corporation, Japan;
thickness -25~; average pore diameters - l,OOOA (major axis) and 200A (minor axis)_7 was put in acetone to afford an affinity to water by subjecting to water replacement and was put in 0.2 ml of an enzyme solution containing 50 mg/ml of glucose oxidase `(specific activity - 141U/mg; commercial product of Kyowa Hakko Kogyo K.K., Japan) per 1 cm2 of the starting membraneO The en~yme solution was prepared by dissolving the enzyme in 0.1 mol phosphate buffer solution ~pH 5.6). The thus obtained porous membrane was slightly washed with 0.1 mol phosphate buffer solution (pH 5.6) and put at room temperature for 20 minutes, in 10% glutaraldehyde solution prepared by dissolving glutaraldehyde in 0.1 mol phosphate buffer solution (p~l 8) to combine glucose oxidase with ~he matrix of polypropylene.
The membrane combined with the enzyme and having an activity of 0.1 IU/cm2 of the starting membrane was used to prepare a similar enzyme electrode to that used in xample 1~ According to a similar procedure to that described in Example 1, glucose concentration in the test solution was determined at 37C at a pH of 7.4 by using the electrode. The results obtained are shown in Table 5.
Table 5 _ ~ ~ - . . _ . . ,._. _ Glucose Concentration (m~/dl) .. . .. _ . _ . _ . _ .. . ..... .
CD(~A) 0.063 0.125 00180 0.242 0.302 0.362 0.415 0.463 _ _ .... . . . .. . . . _ _ . .. _ _ Note: CD - Current differential ~0 =~ - -- ----------- -- .. _ _ .. ... ~ . _ . ,_.. __ , ,_ _ 1~97;~ 8 ~x~mp I e 6 A similar porous rn~m~rane of polyacrylonitrile-type to that useci in E,xa[nple 1 exc~pt a thickness o~ 1O0~L~ space ratio of 58% and avarage pore-diameter of 120A was subjected to a similar treatmenh with hydrazine and glutaraldehyde to that applied in Example l to introduce the aldehyde group into the membrane. The membrane was put, at 5C for 16 hours, iTI O . 2 ml of a mixed enzyme solution per l cm2 of the starting membrane to in~obilize ~he enzymes. This enzyme solution contained S0 mg/ml of cholesterol esterase (specific activit~ 2 IU/mg) and 10 mg/ml of cholesterol oxidase (spe-cific activity - 10 IU/mg) (both enzymes being commercially available from Kyowa Hakko Kogyo K.K., Japan) and was prepared by dissolving these enzymes in 0.1 mol phosphate buffer solution (pH 7.4). The membrane was used to prepare a similar enzyme electrode to that described in ~xample 1. According to a similar procedure to that described in Example 1~ cholesterol .
concentrations(free and ester forms) in the test solution were determined at 37C at a pH of 7.4 by using the electrode to give the results sh~n in Tables 6-l and 6-2. Cholesterol linolate was used as one in ester form~ and a solution con-taining 5% ethanol and 1% Triton-X-100 (surfactant commercially available from Nakarai Kagaku Yakuhin K,K., Japan) was used as a substrate solution.
~5 ( ~:s ter f orm) Cholesterol C.oncentration Current l)iff~rential (mg/dl) (1~
. _ .. . _ . _ _ . . . . ..

0 . ()0~
0 . ()13 0.017 . _ . . . _ . _ .

Tablo 6-2 (Free ~`orm) -- - . .. ...
Cholesterol Concentration Current Differential (mg/dl) (~A) _ _ . . _ . _ 0.010 0. 020 0 . 031 - ---Example 7:
A similar enzyme electrode to that used in Example 6 was used to determine blood sugar and was compared with another analytical method (Somogi-Nelson~s method) or the determination of glucose concentration. The results are shown in Table 7.
Table 7 . . ~ . . .
Somogi-Nelsonls Sample Method Present Process 1 81 mg/dl 82.7 mg/dl 2 155 mg/dl 152 mg/dl ~ . . _ .. . . _ ._ __. _ _ _ . ,. . .. . .. , .... _~ ____ .
I

3 ~7Z~68 I~X~ P I ~
A po1yacry1OI~i.triLe ~y1~e membrane ~hi.ch WC1S s.imi lar to ~ha~ u~ed in ~xarnple l was immersed, at 5~(, for l6 llours, in 0. 2 ml of an enzyme solution containing 25 mg of ~-amino rO acid oxidase per 1 cm of th~ s~arting membrane- The enzyme solution was prepared by dissolving the enzyme (specific activity - lS IU/mg; commercial produc~ of Bo~inger Mannheim GmbH9 West Germany) in 0.1 mol phosphate buffer solution (p~l 8~. The membrane was then slightly washed with O.l mol phosphate buffer solution (pH 8) and put in O.l mol phosphate buffer solution containing lO~o glutaraldehyde (pH 8) for 20 minutes to obtain an enzyme-membrane. D-alanine concentration in the test solution was quantitatively determined by using the enzyme membrane in a similar manner to that described in Example l. The results obtained are shown in Table 8.
'la le 8 Concentration of D-alanine(mg/dl) 1.25 2.50 3.75 5.~0 : Current D fferential 0.041 0.084 0.123 0.159 . .

Example 9:
A polyacrylonitrile type membrane which was similar to that used in Example l was treated in a similar manner to - that described in Example l to introduce the aldehyde group and was then put in 0.2 ml of an enzyme solution per l cm2 of the starting membrane at 5C for 16 hours to give an enzyme membrane. The enzyme solution was prepared by dissolving uricase (specific activity (Candida utilis) - 2.5 IU/mg;
commercial product of Sigma Corporation, U.S.A~) m O.l mol ~ ' ' , . . .__ _ 7 ~4 ~ ~
borate ~ur~er solu~ion (ptl 9.U) and contained 10 mg/ml of the en~yme. I`he obtaine~ enzyme meinbrane was used to determine uric acid concentration in the test solution in a similar manner t o that described in Ex~mple 1. The results obtained are shown in Table 9.
Table 9 .. ., ............... . . _ Concentration of Uric Acid (mg/dl) 2.5 5.0 10.0 15.0 Differencial(~A) 0.102 0.200 0.385 0.540 .. _ _ . . ..
Example 10:
A polyacrylonitrile type membrane which was similar to that used in Example 1 was treated in a similar m~nner to that described in Example 1 to introduce the al~ehyde group and was then immersed in 0~2 ml of an enzyme solution(per 1 cm2 of the starting membrane) containing 2 mg/ml of aspara-ginase at 5C for 16 hours to give an enzyme membrane. The enzyme solution was prepared by dissolving the enzyme (spe-cific activity- 110 IU/mg; commercial product of Kyowa Hakko Kogyo K.K. J Japan) in 0.1 mol phosphate buffer solution (pH 7).
The enzyme membrane was put on the head of an ammonium electrode (Type 7161, a commercial product of Denki Kagaku Keiki K.K. 9 Japan). The concentration of L-asperagine was determined by using the electrode. Table 10 shows the results obtained~

A 0.1 mol phosphate buffer solution was used in the determination (pH 8.5). The logarithm of ammonium concentration was proportional to the voltage.

~ 20 -~,~t~ 8 ~ ~

lable :lO
-Concentration o L-asparagine (mole/l) S x10-4 10-3 5x 10 3 ]o_2 _ _ _ ___ . ... _ _ D _ _. .. _ ~.. ~.. -- _ DViolfage i 1 ( ) 17 25 43 51 _ . .. __ .... . ... ~ _ .. . .

Example 11:
A polyacrylonitrile type membrane which was similar to that used in Example 1 was treated in a similar manner to that described in Example 1 to introduce the aldehyde group and was then immersed, at 5C for 16 hours, in 0.2 ml of an enzyme solution containing urease (10 mg/ml) to give an enzyme membrane. The enzyme solution was prepared by dis-solving the enzyme (specific activity - 700 IU/mg; a commercial product of Sigma Corporation, U.S.A.) in 0.1 mol phosphate buffer solution (p~l 7), The enzyme membrane was used in a similar manner to that described in l`xamplelO to determine quantita-tively urea concent~tion in the test solution. The results obtained are shown in Table 11.
."~
Tab].e 11 .
_ _ _ . _ . _ _ . . . . . . . ~ . ..
Concentration of Urea (mo].e/l) 5xlO 4 10-3 5xlO 3 1o~2 5xlO 2 . . _ ~ . ~ . . _ . .

Dioffage i 1( ) 10 22 48 59 80 Example 12:
A similar membrane of polyacrylonitrile-type to that used in Example 1 was ~ubjected to a similar treatment with hydrazine .. ~

~7'~
anc~ g~ ar~:ll(lellycle Lo t:.h~ applied in .xampl~ 1 Lo introduce ~h~ al~lehyde group in~o the me~nbrane. The membrane was irnmersed in 0.2 ml of arl enzyme solution per 1 cm2 of ~he starting membrane at 5C for 16 hours to i~MIobilize the enzyme. The enzyme solution contained 50 mg/rnl of lactase-Y (commer~ial product of Kyowa ~lakko Kogyo K.K., .Japan; specific activity -~6 IU/ml ) and was prepared by dissolving the en~yme in 0.1 mol phosphate buffer solution (pll 7.0). ~he membrane combined with the enzyme had an enzymatic activity of 2.8 IU/cm2 of the lo starting membrane. DIAFLO-C~LL Type 402 (an apparatus for ul~ra-filtration commercial]y available from Amicon Corporation, U.S.A.) provided with this membrane was used to treat about 200 ml of 8~/o cheese whey solution under pressure of nitrogen gas (3 Kg/cm of the membrarle) to ~ive a filtra~e (20 ml/hour).
By concentrating the filtrate to dryness under reduced pressure, there was obtained desired product, of which contents are shown in Table 12.
Iable L2 ~heese Whey Product . . . _ ~ .
Lactose 64. 2~o 12V/o Glucose 0 31%
Galactose 0 30V/o Protein 6.7V/o 0 ~5 Example_13:
.
A porous polypropylene membrane (thickness -30~;
space ratio- 38~/~; average pore diameter- l,SOOA in major axis and 400A in minor axis) prepared in a similar manner to that described in U.S. Patent 3,679,538 was used. A similar procedure !

4~
1 to th~t clescribc(l irl L~x~lnple 5 was carried out however by using an enzyme membralle (enzyr~ic ~ctivity - O.lS lU/cm2 ~f the starting membrane) prepared by using 60 mg/ml of glucose oxidase solution. ~he glucose concentration in the test solution was determined at 37C at p~l 7.4. The results obtained are shown in Table 13~
Table 13 Glucose Concentration (~g/ml) 30 40 ~0 ~0 70 80 . . . _ _ _ . . ~
A -0.061 0.120 0.178 0.238 0.290 0.350 0.400 0.445 ... . _ . _ _ _ Note: A - Current differential (~A) Exa~le 14:
The p~l stability, heat s~abili~y and preservability of lS a membrane prepared by Example 1 was compared with a membrane entrapped in ~crylamide gel and free enzyme to give the results shown in Tables 14, 15 and 16.
Membrane entrapped in acry-lamide gel was prepared as f~llows.
95 mg of acrylamide, 5 m~ of N,N-methylenebisacrylamide and 50 mg of glucose oxidase (a simil~ product to that used in Example 1) were dissolved in 0.8 ml of O.lN phosphate buffer solution. 5 mg of N,N,N',N'-tetramethylethylenediamine and 1 mg o~ ammonium-persulfate were dissolved in 0.2 ml of a O.lN
~5 phosphate buffer solution. Immediately after this, two solution~
were mixed and the combined solution was coated on a nylon net(SOcm2 The coated nylon net was allowed to stand at room temperature in nitrogen atmosphere un~il the membrane entrapped in acrylamide gel w~ obtained.

I

~724~i~

SampI~e~l 5 5~6 6 6.5 7 7.5 8 B 90 gO 90 80 64 52 40 , _ . . _ . _ . . . _ Note: A - mernbrane according to Example 1 B - membrane entrapped in acrylamide gel C - Free enzyme The results shown in Table 14 denote the enzymatic activities of the samples determined at the indicated pHs (initial activity = lOO). 'lhe preservation was carried out at 60C for 1 hour.

~leat--- ........ , .. _ Sam iè~ T.~emP 30C 40C 50C 60C 70C

C 100 1~0 92 91 13 ~ . . _ _ . .. . _ . _ , Note: A - membrane according to Example 1 B - membrane entrapped in acrylamide gel C - free enzyme The results shown in Table 15 denote the enzymatic activities of the samples which were dissolved in Ool mol phos-phate buffer solution and which were preserved at the indicated temperatures for one hour (initial activity = 100).

{ -- . . . . ....

~7~46i8 Tc~ l6 ~'reserv~lbility Preservation Perio~l Sample 1 ~eek 2 weeks 3 weeks 4 weeks S weeks _ _ _ _ _ .. ~ . . . . . .. . . _ _ _ Note: A - Membrane according to Example 1 B - Membrane entrapped in acryLamide gel C - Free enzyme The results shown in Table 16 denote the enzymatic activities of the samples dissolved in 0.1 mol phosphate bur'fer solution (p~l 5.6~ when preserved at 30C.-As apparent ~rom Tables 14-16, the pH stability, heat stability and preservability of the membrane bonded with the immobilized enzyme according to the process of the inventio are superior to those of the immobilized enzyme of the known type as well as of the free enzyme~ The immobilized enzyme of the invention no doubt represeTIts an advance in the art and provides an exce~llent enzyme electrode.

j==_... __.... _.... .. ____ ., . . ......... . . .__ _ _ _ . .

Claims (4)

The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:

1. An enzyme membrane having an enzyme supported by a plastic membrane wherein said membrane is microporous and permeable and made of an organic high molecular weight selected from the group consisting of (1) a copolymer of a major portion of acrylonitrile with a minor portion of methlyacrylates, and (2) polypropylene, the average diameter of the pores of said membrane ranging from 50 to 5000A and the pores being unevenly distributed on the surface of the membrane, said pores being interconnected through tortuous paths which may extend from one exterior surface to another.

2. An enzyme membrane according to claim 1 wherein the membrane has a thickness of 1 to 500µ.

3. A double membrane comprising an enzyme membrane as claimed in claim 1 and a second membrane contacting said enzyme membrane.

4. A double membrane according to claim 3 wherein said enzyme membrane forms an upper layer of the double membrane and said second membrane forms a lower layer, said second membrane being an oxygen and ion-permeable plastic film.