JPS6371176A - immobilized enzyme - Google Patents

Abstract

PURPOSE:To provide an immobilized enzyme composed of an ionic polymer, an organic compound having straight-chain hydrophobic group and an enzyme, having excellent moldability even in the state containing immobilized enzyme and keeping excellent enzymatic activity even after molding. CONSTITUTION:The objective immobilized enzyme can be produced by dissolving or dispersing an ionic polymer, an organic compound having straight-chain hydrophobic group and an enzyme in the same or different reagents at respective specific amount, mixing the solutions or dispersions and collecting the produced precipitate. The ionic polymer is e.g. the polymer of formula I (R is H, alkyl or carboxymethyl; X is methylene, etc.; M is H, metallic atom or lower ammonium; c is 0-2; a and b are 0 or 1) and the organic compound having straight- chain hydrophobic group is e.g. the compound of formula II [R<1> and R<2> are 6-30C straight-chain alkyl or its halogen-substituted group; R<3> and R<4> are 1-4C (substituted) alkyl; X is halogen or OH].

Description

DETAILED DESCRIPTION OF THE INVENTION (Industrial Application Field) The present invention relates to a novel immobilized enzyme comprising a specific polymer, a specific organic compound, and an enzyme.

(Prior art and problems to be solved by the invention) Enzymes are in-vivo catalysts made of proteins that allow reactions to proceed efficiently under mild conditions of room temperature and normal pressure, and have extremely high specificity of action. It has the following characteristics. Hitherto, various so-called immobilized enzymes immobilized on enzyme carriers have been provided in order to industrially utilize the action of g element.

Among the uses of such immobilized enzymes, in applications as biosensors, it has become necessary to make the immobilized enzyme thinner in order to increase the response speed of the sensor or to make it thinner.

On the other hand, immobilized enzymes are roughly divided into three types obtained by carrier binding method, crosslinking method, and entrapment method, but in applications that require fine molding such as the above-mentioned U film formation, the following problems arise. have. That is, in the carrier binding method, fine molding is possible because the carrier is molded in advance, but there are problems in that it is difficult to immobilize the enzyme and the binding force of the enzyme is weak. In addition, the crosslinking method and entrapment method have a strong enzyme binding force and can produce products with high enzyme activity, but they have the problem that fine molding is difficult because crosslinking or entrapment is performed at the same time as molding.

(Means for Solving the Problems) The present inventors have conducted repeated research in order to solve the above problems. As a result, it was found that an immobilized enzyme made of a specific polymer, a specific organic compound, and an enzyme has excellent moldability in the state in which the enzyme is immobilized, and also has excellent activity even after molding. This discovery led to the completion of the present invention.

That is, the present invention provides +1) a polymer having an ionic group (hereinafter abbreviated as an ionic polymer); (2) (a) a polymer having two or three linear hydrophobic groups or a rigid portion in the chain; (3) An immobilized enzyme consisting of an organic compound (hereinafter abbreviated as a linear organic compound) having one linear hydrophobic group and (ii) an ionic group.

In the present invention, an ionic group is defined as a general term for an acidic group or a basic group. Here, acidic or basic means Brønsted acid or Brønsted base, and the acidic group generally includes a sulfone group or a carboxyl group.

Phosphoric acid groups, phenolic hydroxyl groups, and their salts; as basic groups, generally, 7-mino groups, substituted 7-mino groups, quaternary ammonium groups, and salts of these are preferably used. Ru. Furthermore, the term "linear hydrophobic group" as used in the present invention is used to include not only completely linear groups but also branched groups having up to 2 carbon atoms. Such a straight chain hydrophobic group is typically a straight chain alkyl group having 4 to 30 carbon atoms or a hapgen-substituted product thereof.

Furthermore, in the present invention, the rigid moiety refers to groups shown in the following (1), (2), and (2).

■A divalent group consisting of at least two aromatic rings connected directly or through a carbon-carbon multiple bond, carbon-nitrogen multiple bond, nitrogen-nitrogen multiple bond, ester bond, 7-mid bond, etc. If such a group is specifically shown, for example, o
Examples include 2f5 groups such as O and the like.

0 A divalent group in which the number of bonds between two aromatic rings is multiple or a single bond between multiple atoms, and the rotation of which is energetically constrained. Specific examples of such groups include, for example: , CM.

Examples include divalent groups such as.

■ A divalent group in which the aromatic rings form a fused ring, and when this fused ring is stacked between multiple molecules, the rotation is sterically constrained to each other. Examples include hexavalent groups such as the following.

One of the constituent elements of the immobilized enzyme of the present invention is an ionic polymer. As such ionic polymers, known ones having the above-mentioned ionic groups may be used without particular limitation. Among them, in consideration of the strength and stability of the resulting immobilized enzyme, it is generally desirable to use one with a molecular weight of s, ooo or more. In addition, the amount of ionic groups contained in the polymer varies depending on the type thereof, the linear organic compound described later, etc., and cannot be absolutely limited, but it is generally o, 1 maq /
79 or more, preferably 0 meq/9 or more is desirable.

The general formula of the ionic polymer generally suitably used in the present invention is as follows.

(2) Polymers having carboxyl groups, where R is a hydrogen atom, a furkyl group or a carboxymethyl group, and X is a 10H,- group.

R' 0 alkyl group or 7-aryl group), and M is a hydrogen atom, a metal atom or a lower ammonium. C is an integer of 0 to 2, and a and b are O or 1. Here, a is 0
When 0, b = 1, c = Q, R is a hydrogen atom, and when a is 1, b = o, c-0~2, R
is a hydrogen atom, an alkyl group or a carboxymethyl group.

■Polymer with sulfonic acid group "-"Hl (1:1 d So, M However, Ra is a hydrogen atom or an alkyl group, OCI (8 10- or (-CH, +. However, e is a positive ), M is a hydrogen atom, a metal atom or a lower ammonium, and d is 0 or 1.

■Polymer R″P(OM)B having a phosphoric acid group, where R■ is a hydrogen atom or a furkyl group, Z
i! -C-0+CHa+gO (where I is a positive integer), M is a hydrogen atom, a gold scrap atom, or lower ammonium, and f is O or 1.

■Polymer R'' having ammonium base (However, R1) + is a hydrogen atom or a furkyl group, 2 is -0- or -NH- group, and Y has 1 to 1 carbon atoms.
2, methylene chains having 1 to 8 carbon atoms are particularly preferred, and halogen-substituted or hydroxyl-substituted methylene chains are also preferred. It is a substituted product by an atom and/or a hydroxyl group, and X is a halogen, hydroxy acid, nitric acid, perchloric acid, or thiocyanic acid.

It is 7 onions of vinegar. ) The above general formula (1), (I[), (■) and (f
In f), the furkyl group represented by R, R', R'', R'', and R open is not limited to the number of carbon atoms, and any group can be used, but in general, a group having 1 to 4 carbon atoms can be used. Preferably. In addition, the above general formula (1) and [I]
In the above, C and I may be positive integers, but are preferably integers from 1 to 4 from the viewpoint of ease of handling raw materials.

As the polymer having an ionic group according to the present invention,
In addition to the above-mentioned polymers, natural polymers having ionic groups are also preferably used.

Examples of natural polymers having ionic groups that are generally preferably used in the present invention include alginic acid, sodium alginate, carboxymethyl cellulose, heparin,
Examples include ConFE itin sulfate and derivatives thereof.

In addition, in the present invention, the linear organic compound has two or three linear hydrophobic groups or one linear hydrophobic group containing a rigid part in the chain, and any known ionic group can be used. It can be used without particular limitation. Typical examples that are generally suitably used include those shown in the following general formula.

(However, R and R are the same or different straight chain fulkyl groups having 6 to 30 carbon atoms or halogen-substituted products thereof, and R,
R is the same or different alkyl group having 1 to 4 carbon atoms, or a substituent thereof with a halogen atom and/or a hydrogen group, and X is a halogen atom or an OH atomic group. ) (However, R, R, and X are the same as above,
j is O or 1, and k is a positive integer. ), and h and l are positive integers. R, R, and R are the same as the description of R and R above. ) +1 (However, R, R, R, R, R, A and X are the same as above, t is 1 or 2, m is O or 1.) (However, R1, RN, R8, R4 , Hli and X
is the same as above, and n is a positive integer. )R'-D
-E-N''-R' XeCE)S (However, R, R, R and X are the same as above, and R6
is a furkyl group having 4 to 30 carbon atoms.

A fulkyloxy group, a fulkyloxycarbonyl group, or a norogen-substituted product thereof (F is -N
=CH-, -N=N-.

Q　　　　　　　　　0-C-.

H8 -NHCH,-, -CH=CHC-.

E is 4CH, +9 or -〇-(CHfi+,
It is. (However, q and r are positive integers.) In the above general formulas (B), CD) and (E), k and n.

Although q and r may be positive integers, they are generally preferably 1 to 16 from the viewpoint of ease of handling raw materials. Also,
In the above general formula CB), h and I can be positive integers without any restriction, but generally from 1 to 1 due to the ease of handling the raw materials.
It is preferable that it is 4.

(However, R and R are the same as the explanations for organic compounds (A) to (D), and M is not particularly limited and is generally an atom or atomic group that becomes an organic or inorganic cation, such as hydrogen, alkali metal, Alkaline earth metal atoms and ammonium atomic groups are preferable) ■ 0 (However, R, R and M are the same as above.) In addition, the linear fulkyl group as used in the present invention refers to completely linear fulkyl groups. In addition to the above, it is also used to include branched forms having up to 2 carbon atoms. Also, the above general formula (A)
, CB).

(C) , (D) , OE) , CF) and (G
), examples of the halogen atom of the halogen-substituted alkyl group represented by R1, .R, R, R, R, and R include fluorine, chlorine, bromine, and iodine atoms.

In the present invention, the enzyme is not particularly limited and refers to any known enzyme, but generally refers to a protein molecule with a molecular weight of 10,000 to 1,000,000 and exhibits catalytic activity. Representative enzymes that can be used in the present invention include alcohol dehydrogenase, glycerol dehydrogenase, lactate dehydrogenase, malate dehydrogenase, glutamate dehydrogenase, D-amino acid oxidase,
Oxidoreductases such as heptomine oxidase and tipsinase; transferases such as aspartate aminotransferase, catefyl methyltransferase, phospholytase, and creazahn kinase; lipase.

Chymotrypsin, alkaline phosphatase.

trypsin, amino-7 sylase, papain.

Hydrolases such as pepsin, lysozyme, and urease; Lyases such as glutamate decarboxylase, fumarase, and carbonate dehydratase; Immerases such as glucose phosphorylase, UDP-glucose 4-epimerase; Gluecases such as formyltetrahydrofolate synthetase, etc. can be mentioned.

The immobilized enzyme of the present invention, which is composed of an ionic polymer, a linear organic compound, and an enzyme, is not simply a mixture at °C. Since it exists as an integrated substance due to physical adsorption due to the hydrophobicity of the chain organic compound, it can generally be handled as a single substance. In this way, as long as the ionic polymer, the linear organic compound, and the enzyme can be handled as an integrated substance, their network ratio is not particularly limited and they can be blended. In general, considering the stability in water due to manufacturing or handling,
0.2 to 5 times 1L for the ionic group of the ionic polymer
Preferably, 0.5 to 3 equivalents of the linear organic compound are blended. Furthermore, the enzyme expresses its enzymatic function,
It can be blended without any particular limitation as long as it is in a sufficient amount for use. Generally speaking, taking into account manufacturing and industrial manpower constraints, O,OO is generally 1 to 10% by weight based on the total weight.
It is suitable to incorporate an enzyme in an amount of preferably o, oi to 5 gL.

Further, the ionic polymer and the linear organic compound are preferably used in combination so that the ionic groups have opposite charges to each other (8).

The method for producing an immobilized enzyme comprising an ionic polymer, a linear organic compound, and an enzyme according to the present invention is not limited to a specific method, and any method may be used as long as these components are finally mixed. You can. Generally preferred manufacturing methods include the following methods. That is, ←a) A method in which predetermined amounts of an ionic polymer, a linear organic compound, and an enzyme are dissolved or dried in the same or different solvents, and the precipitate produced by mixing these is collected. The mixing order in this case is not particularly limited, but in general, a method of adding a linear organic compound to an ionic polymer and an rII element, or a method of adding an ionic polymer to a linear organic compound and an enzyme is preferably used. .

Alternatively, (b) a method in which a composition consisting of an ionic polymer and a linear organic compound is prepared in advance, and the composition is mixed with or brought into contact with an enzyme. In this case, the method for producing the composition is the method shown in JP-A No. 60-228564, for example,
A suitable method is to dissolve or suspend predetermined amounts of an ionic polymer and a linear organic compound in the same or different solvents, mix them, and collect the resulting precipitate.

When the solvent used here is the same solvent, it may be water or a mixed solvent with an organic solvent that is compatible with water, such as water/methanol mixed solvent, water/ethanol mixed solvent, water/7cetone mixed solvent, etc. generally preferred. When different solvents are used for the polymer and the linear organic compound, water is generally preferred as the solvent for the polymer. Water, methanol, and ethanol are used as solvents for linear organic compounds.

2-propertool, 7setone, ethyl acetate.

Ethyl/reether, benzene, chloroform.

Methylene chloride, tetrahydrofuran, dimethylform7
Mido, dimethylacetamide, 7cetonitrile, etc. are preferably used. In the case of solvents that are not miscible with each other, it is generally effective to form an emulsion by stirring vigorously during mixing to obtain a precipitate. Generally, the solvent resistance of the composition obtained by the above operation is improved.

The immobilized enzyme of the present invention can be molded in a state containing the enzyme. For example, (1) melt moldability, (2) solvent evaporation method, etc. are suitable. The melt molding method (2) is a known melt molding method, such as injection molding, at a temperature that does not cause loss of enzyme activity.

This can be done by extrusion molding, press molding, etc. In this case, the softening temperature of the immobilized enzyme can be adjusted from -25 to -25 by adjusting the composition ratio of the ionic polymer and the linear organic compound.
The temperature can be changed up to about 100°C. Therefore, the composition ratio may be determined by taking into account the above-mentioned molding temperature, use environment, etc. In addition, in the solvent evaporation method (2), the immobilized enzyme is dissolved in a solvent that can dissolve the immobilized enzyme without deactivating the enzyme, and this is cast, for example, on a substrate having an appropriate plane. This is a method for removing the solvent.

According to this method, a thin film having a thickness of 20 μm or less can be obtained. Examples of the above solvents include methanol, ethanol, and 2-fuc=panol.

Ethyl acetate, acetone, benzene, chloroform, methylene chloride, tetrahydrofuran.

Examples include ethyl ether, acetonitrile, dimethylformamide, dimethylformamide, and the like. In general, the solubility of the immobilized enzyme in organic solvents varies greatly depending on the type and combination of the ionic polymer, linear organic compound, and enzyme that are its components, so it is necessary to test the solubility beforehand. desirable. Generally, air drying, reduced pressure drying, etc. are preferably used to remove the above solvent.

(Effects) As explained above, the immobilized enzyme of the present invention has excellent moldability in a state in which the enzyme is immobilized, and can maintain sufficient enzyme activity even after molding. Therefore, it is suitably used not only as a catalyst for a bioreactor or a carrier for chromatography, but also as a biosensor, a microsensor, etc. that requires microfabrication. Note that the enzyme activity is confirmed by measuring the enzyme reaction, and any known method can be used without particular limitation. In general, suitable measurement methods include (a) spectroscopic measurement, which measures changes in absorbance of ultraviolet to visible light, and (b) spectroscopy, which measures changes in fluorescence intensity.
Fluorescent probes can also be used, and generally high-sensitivity measurements are possible.
Examples include measuring changes in sensory concentration and pH using Ht electrodes.

(Function) Although the mechanism of action by which the immobilized enzyme of the present invention exhibits good molding processability and expresses enzyme function is not necessarily clear, the composition comprising the ionic polymer of the present invention and the linear organic compound Object exhibits liquid crystallinity (Japanese Patent Application Laid-Open No. 60-2285
64) is believed to be involved in some way. That is, it is presumed that the liquid crystallinity of the composition increases the bonding force with the enzyme, which not only facilitates the expression of its function as an immobilized enzyme, but also reduces the deactivation of the enzyme during molding. This is presumed to be related to the fact that the linear organic compound of the present invention forms a bilayer structure similar to a lipid bilayer membrane in water. That is, it is inferred that the orientation of the linear organic compound contributes to the stabilization of the enzyme.

(Examples) Examples are given below to explain the present invention more specifically, but the present invention is not limited to these Examples.

Example 1 Compound 19 was ultrasonically dispersed in water 501 to obtain a soap-like liquid,
This is combined with 7 sylase (fromAa-pergillus
genus, Tokyo Kasei Kogyo) 50mg, and
While stirring vigorously at 0°C, add sodium polystyrene sulfonate (viscosity average molecular weight: 1,060,000) 0.4F! A solution prepared by dissolving the above in water was added. The resulting precipitate was stirred at 40° C. for 30 minutes, filtered, washed thoroughly with water, lightly washed with 50% methanol, and dried under reduced pressure. Pale and colored solids, 0
I got g.

0.1 F of this solid was dissolved in chloroform 51) 17 and poured into a Teflon petri dish with a diameter of 5 cIR, and the solvent was evaporated to obtain a film with a thickness of 35 μm.

Cut out the above film-like substance, Od, 97WM7 Cetyl L
-Phenylalanine solution (-<+=+-le buffer PH=
7.95) 0.2m, 25mM Cobalt Chloride 0.
Add 1 Go, Peronal buffer layer (PH = 7.95), 6M, and add water to 21 lil#! 38℃
The mixture was allowed to react for 4 hours. Then this reaction solution 0.5 R/
Take out and add 0.2M borate buffer (P)I=9.0)
was added, and further a 0.02% solution of fluorescamine in 7 setone (0.51) 7 was added, stirred vigorously, and this solution was analyzed using a spectrophotometer (Hitachi 204RW) at an excitation wavelength of 380 fM and a range of 450 to 500 yen. As a result of measuring the fluorescence intensity, it was confirmed that L-phenylalanine, which is an enzymatic reaction product of 7cetyl-L-phenylalanine, was produced at 1% (based on the substrate).

Example 2 Compound II was ultrasonically dispersed in 50s+j of water to obtain a soap-like liquid, to which 20mg of α-chymotrypsin (SrGMA) was added.
of poly(trimethylammonioethyl methacrylate) 0.41) while stirring vigorously at 40°C.
A solution prepared by dissolving 201% of water was added. 30 at 40℃
After stirring for a minute, the resulting precipitate was collected, thoroughly washed with water, lightly washed with 50% methanol, and then dried under reduced pressure. A white fixative, Ol, was obtained.

0.1E of this fixed material was dissolved in 50,000 ml of chloroform and cast in a Teflon Petri dish with a diameter of 53 mm, and the solvent was evaporated to obtain a film-like material with a thickness of about 40 μm.

Cut out 0d of the above film-like material, put it in a cell for absorbance measurement together with loMN-7 cetyl-L-tyrosine ethyl ester (1PT (=7.08)), and add N-7 cetyl-L-tyrosine. The difference in absorbance (at237U+) with N-7 cetyl-L-tyrosine, a hydrolysis product of ethyl ester, was measured using an ultraviolet-visible spectrophotometer (Hitachi 2
00fEi) to confirm the progress of # elementary reaction. As a result, after heating at 26°C for 60 minutes, a change in absorbance of 6×10 was observed.

Example 3 Compound 1y was ultrasonically dispersed in water 501 to obtain a soap-like liquid,
Add trypsin (SIGMA) 20 and 9 to this, and add 40
℃ [While stirring vigorously, add 0.4.9% of sodium polystyrene sulfone (viscosity average molecular weight = 1,060,000) to 22% of water.
A solution dissolved in 017 was added. After stirring for 30 minutes at 40°C, the resulting precipitate was filtered and thoroughly washed with water.
Washed with % methanol and dried under vacuum, white solid 0.9%
I got 1.

This solid 2CJ19 was dissolved in chloroform 1-, and 0.15- of this was cast on a quartz plate (0.9 x 2.5 crA), and the solvent was evaporated to form a film on the quartz plate. .

The above quartz plate was placed in a cell for measuring absorbance together with 2d of 5X10-'MN-benzoyl-L-fulginine ethyl ester (phosphate buffer pH = 8.02), and N-benzoyl-
Difference in absorbance between L-fulginine ethyl ester and the hydrolysis product N-benzoyl-L-fulginine (at253
1m) was measured using an ultraviolet-visible spectrophotometer (Hitachi 200m) to confirm the progress of the enzymatic reaction.

As a result, a change in absorbance of 5X10-B was observed after 30 minutes at 38°C.

Example 4 In the same manner as in Examples 1 to 3, a solid was obtained from 1 mmole of the linear organic compound shown in Table 1, 1 mmole of the ionic polymer equivalent to the ionic group, and 10 to 9 of the enzyme. .

Dissolve 0.19 of this solid in the solvent 51 shown in Table 1,
After spreading the mixture on a Teflon Petri dish with a diameter of 5 cm, one solvent was evaporated to obtain a film-like product.

The above film-like substance, 0- is cut out and the enzymatic reaction with the substrate is first carried out.
It was measured using the method shown in the table and confirmed to have a function as an immobilized enzyme. As a result, it was confirmed that all the immobilized enzymes shown in Table 1 had sufficient enzyme activity.

Claims (3)

[Claims] (1) Polymer with ionic group (2) (a) An organic compound having two or three straight-chain hydrophobic groups, or one straight-chain hydrophobic group containing a rigid part in the chain, and (b) an ionic group. (3) Enzymes Immobilized enzyme consisting of