JPS6156076A - Immobilized enzyme and its preparation - Google Patents

Abstract

PURPOSE:To prepare an immobilized enzyme having high immobilization ratio, low escape rate, and high activity, by encapsulating an enzyme in a microcapsule, and dispersing the capsule in a polymer gel matrix. CONSTITUTION:An aqueous liquid containing an enzyme is mixed and emulsified with an organic solvent solution of a hydrophobic polymer to obtain the primary emulsion liquid containing enzyme-containing aqueous droplets dispersed in an organic solvent solution of the hydrophobic polymer. The emulsion liquid is added to an aqueous medium containing a hydrophilic polymer under agitation to obtain the secondary emulsion liquid composed of the droplets of the primary emulsion liquid dispersed in an aqueous medium containing the hydrophilic polymer. The organic solvent in the primary emulsion liquid is evaporated and removed through the aqueous medium to form a micro-encapsulated enzyme in the aqueous medium containing the hydrophilic polymer. The hydrophilic polymer is gelatinized to obtain the objective immobilized enzyme.

Description

DETAILED DESCRIPTION OF THE INVENTION [Technical Field] The present invention relates to an immobilized enzyme and a method for producing the same.

[Prior art]

In recent years, in addition to hydrolytic enzymes such as amylase and protease, A has been used as a catalyst for various enzymatic reactions.
Synthetic enzymes and oxidoreductases that require coenzymes such as TP, NAD, and NADP are beginning to be applied in the field of medical analysis, and are also gradually being used as catalysts for the production of pharmaceuticals and chemicals. .

However, enzymes that require coenzymes are generally more expensive and less stable than conventional hydrolases or isomerases; However, there is a need for the development of an immobilization method that is even better than conventional immobilization methods.

Enzyme immobilization methods can be broadly classified into three methods. The first method is to physically adsorb the enzyme onto an ion exchange resin, activated carbon, or the like. Although this method is easy to operate, it has the disadvantage that since the enzyme interacts with the carrier through relatively weak bonds, desorption of the enzyme occurs during continuous reactions, which limits the conditions under which it can be applied. The second method is a covalent bonding method, in which the carrier and the enzyme are chemically covalently bonded.

Because the carrier and the enzyme are bonded together with a strong bond, there is very little enzyme desorption, but there is a drawback that deactivation is likely to occur because the enzyme molecule is chemically modified. In the case of unstable enzymes, their deactivation is a major problem. The third method is an entrapment method, in which the enzyme is encapsulated in a gel matrix or microcapsules such as acrylamide or carrageenan. This comprehensive law.

Unlike the covalent bonding method, the enzyme molecule is not directly chemically modified, so it has the advantage of not causing deactivation. It is possible to allow only small substrate molecules to pass through while being captured and fixed in gels and microcapsules, and there is also the advantage that detachment of 18-element molecules can be prevented. This comprehensive method is preferably applied to the immobilization of enzymes that require the aforementioned coenzymes. That is.

In the case of such enzymes, the rJI element often forms dimers or tetramers, but in this case, the binding strength between the monomers is very weak. When one site is covalently bonded to a carrier, it is extremely difficult to maintain it as a tetramer for a long period of time. Therefore, in the case of such enzymes, immobilization by gel entrapment method is preferably applied. Also, when two types of enzymes are immobilized at the same time.

The only immobilization methods available up to now are methods that comprehensively immobilize enzyme groups within microcapsules, and even with the gel entrapment method, it is extremely difficult to fit two types of enzymes into one compartment of the gel. be. However, even with the conventional microcapsule method, there is almost no enzyme leakage, but when packed in a column for continuous production, the microcapsules deform and become clogged.
It had a fundamental practical defect in that the pressure loss was high.

〔the purpose〕

The present invention aims to overcome the above-mentioned drawbacks found in conventional immobilized enzymes and methods for their production.

〔composition〕

According to the present invention, as a first invention, an immobilized enzyme having a structure in which microencapsulated enzymes are dispersed and held in a polymer gel matrix is provided, and as a second invention, this immobilized enzyme is manufactured. In order to do this, a primary emulsion in which aqueous droplets containing an enzyme are dispersed in an organic solvent solution of a hydrophobic polymer is dispersed in an aqueous medium containing a hydrophilic polymer to form a secondary emulsion. , evaporating off the organic solvent,
A method for producing an immobilized enzyme is provided, which comprises producing a microencapsulated enzyme in an aqueous medium containing a hydrophilic polymer, and then gelling the hydrophilic polymer.

Since the immobilization conditions for the enzyme used in the present invention are not strict, any enzyme can be used unless it is particularly unstable. Specific examples of such enzymes include alcohol dehydrogenase, aldehyde dehydrogenase, glutamate dehydrogenase, isoleucine dehydrogenase i4 enzyme, formate dehydrogenase, lactate-2-monooxygenase, and phenol-2-monooxygenase. monooxygenases such as;
Kinases such as acetate kinase, hexokinase, and glycerol kinase; as well as enzymes that require various coenzymes, such as β-amylase, glucoamylase, β-glucosidase, lipase, and phosphatase.

Examples include hydrolytic enzymes such as putase and esterase, and oxygenases such as glucose oxidase and peroxidase. Further, the enzyme may be a crude product as well as a purified product.

Culture solution, culture filtrate, and viable bacterial cells with the necessary enzyme activity.

Dried bacterial cells, crushed bacterial cells, animal and plant cells, crushed cells, and cell organelles may also be used, provided, however, that if they are solid,
It needs to be small enough to fit into microcapsules. These enzymes can be used alone or in mixtures. Enzyme systems that perform conjugation reactions such as those mediated by coenzymes are suitable raw materials for the present invention. That is, in the case of the present invention, although the enzyme is confined in microcapsules, it exists in a free form, so there is no reaction inhibition due to physical or steric hindrances as seen in covalent bonding methods. Furthermore, since the enzyme is concentrated in the limited space of microcapsules in the polymer gel, the conjugation reaction is carried out quickly and provides an excellent immobilized enzyme. However, since enzymes are contained in polymer gels, enzymes that use polymers as substrates, and hydrolytic enzymes such as α-7 mylase, chitinase, and cellulase, have poor enzymatic activity. However, it exhibits sufficient activity for decomposing low-molecular substrates.

To immobilize an enzyme according to the present invention, first, an aqueous solution containing the enzyme is mixed and emulsified with a solution of a hydrophobic polymer in an organic solvent, and aqueous droplets containing the enzyme are formed in the organic solvent solution of the hydrophobic polymer. Prepare a primary emulsion in which are dispersed. In this case, the enzyme contained in the aqueous liquid is.

In addition to the dissolved state, it may be in a suspended state.In addition to the enzyme, one commonly used auxiliary additive may be added to the aqueous liquid. It is advantageous to add surfactants.

The stabilizer added to the aqueous solution containing the enzyme is a drug that stabilizes the enzyme, and various conventionally known stabilizers are used. Proteins such as albumin; polyl'fA such as gum arabic and starch; water-soluble synthetic polymers such as polyethylene glycol and polyvinyl alcohol;
Coenzymes like NAD, NADP, ATP; EDT
Chelating agents such as A; in special cases, various metal salts,
Antioxidants such as glutathione are included and are appropriately selected depending on the enzyme used. Such stabilizers are usually
It is contained in commercially available enzyme preparations and is useful for maintaining long-term enzyme activity.It is not particularly needed in the case of highly stable enzymes by itself, and it is also useful for stabilizing small molecules. When using agents, it is not preferable to use agents that inhibit gelation (crosslinking reaction) of polymers.

Further, the pH buffering agent to be added to the aqueous solution containing the enzyme may be any one that does not inhibit the crosslinking reaction or enzyme activity, and the pH thereof may be in a range where the enzyme used can stably exist. In addition, even if the enzyme used is a buffer that provides a PH region that is not stable for a long time, VPli4
Is the time required for immobilization short? (Since it is removed by a washing process after immobilization, there is no problem when using it during enzyme immobilization. It is better to use a pH & l buffer at a lower concentration, but 0. It only needs to be 5M or less, preferably 0.1M
The following are good, and the aqueous liquid used when preparing the secondary emulsion and the secondary emulsion l! It is preferable to use agents with the same or equivalent ionic strength as additives to be added to the aqueous liquid containing the hydrophilic polymer used during gIm. This means that during microencapsulation, the concentration inside and outside the membrane is This is because the microcapsules may be deformed or destroyed due to water flowing in or out from the gap.

As a hydrophobic polymer to be dissolved in an organic solvent that serves as a dispersion medium for the aqueous droplets containing the enzyme, 1 is a hydrophobic polymer,
Almost all organic solvent soluble compounds are used, and specific examples of such compounds include soluble addition polymers such as vinyl esters, vinyl ethers, Acrylic acid esters, Homopolymers and copolymers thereof such as methacrylic acid esters and acrylonitrile, soluble polycondensates such as polycarbonate, BoIJ ester, polysulfonate, polyurethane, polyamide, etc., as well as ethyl cellulose, cellulose acetate, etc. Examples include inducing fiber retention and chlorinated rubber.

In addition, polymers that are soluble in organic solvents and whose solubility changes due to pH changes can also be used. For example, alkaline soluble,
In addition to acrylic acid polymers and methacrylic acid polymers, examples of acid-insoluble polymers include polymers such as crotonic acid, maleic acid, itaconic acid, citraconic acid, and vinylbenzoic acid; Polymers, cellulose derivatives, starch phthalic acid, secondary acid or maleic acid derivatives, and cellulose phthalic acid! t2
There are derivatives etc. Furthermore, acid-soluble and alkali-insoluble basic polymers include polyvinylpyridine, polyvinylimidazole, polyvinylamine, polyvinylaniline, and the like. Further, the organic solvent used as a dispersion medium for the aqueous liquid containing the enzyme may be one having a boiling point of 100°C or lower and immiscible with water, such as benzene, cyclohexane, ethyl ether, ethyl acetate, Carbon tetrachloride.

Examples include chloroform and halogenated ethylene.

Next, the primary emulsion obtained as described above is added to an aqueous medium containing a water-soluble polymer while stirring, and primary emulsion droplets are dispersed in the aqueous medium containing a water-soluble polymer. Secondary emulsion ((V 110) lv2 type composite emulsion)
1 Ilys. In this secondary emulsion, the organic solvent contained in the secondary emulsion is removed by evaporation through an aqueous medium containing a water-soluble polymer, thereby removing the hydrophobic polymer dissolved in the organic solvent. It precipitates on the periphery of the aqueous droplet containing the enzyme to form an ultrafiltration membrane, giving microencapsulated element 8I. The rate of evaporative removal of this organic solvent is
It can be adjusted by adjusting the environmental temperature and pressure, or by bubbling gas such as nitrogen or air into the secondary emulsion. The thus obtained aqueous medium containing the hydrophilic polymer in which the microencapsulated enzyme is dispersed gels (crosslinks) the polymer. An appropriate gelation method is adopted depending on the situation, and the gelation can be carried out by conventionally known methods.

In the case of the present invention, the hydrophilic polymer dissolved in water used as a dispersion medium for the primary emulsified droplets acts as a protective colloid during microcapsule formation, and further acts as a gel matrix for the microencapsulated enzyme after microencapsulation. As such a hydrophilic polymer that plays the role of Proteins such as gelatin, collagen, albumin, and casein; Synthetic polymers having vinyl groups such as polyethylene glycol diacrylate; Polyvinyl alcohol; Polyethyleneimine; Polyacrylamide; Polyvinylpyrrolidone.

In the present invention, as described above, the secondary emulsion is subjected to a gelling treatment after microencapsulation is completed to gel the hydrophilic polymer contained in the aqueous medium as a dispersion medium. The gelation method in this case is appropriately selected depending on the polymer. Specifically, the gelation method in this case is shown in relation to the polymer used: 1. For example, alginic acid is crosslinked by polyvalent cations; polysaccharides such as agar and carrageenan are gelled by cooling; gelatin, Proteins such as collagen, albumin, and casein are crosslinked using glutaraldehyde and Schiff bases; synthetic polymers with vinyl groups such as polyethylene glycol diacrylate are crosslinked by radical polymerization of the vinyl groups; polyvinyl alcohol is crosslinked with glutaraldehyde; Crosslinking with aldehyde or epichlorohydrin, radiation crosslinking, and photocrosslinking with sodium benzoate and UV irradiation; for polyethyleneimine, crosslinking with epichlorohydrin; for polyacrylamide and polyvinylpyrrolidone, radiation crosslinking, etc. are applied.

When producing an immobilized enzyme according to the present invention, the amount of hydrophilic polymer contained in the aqueous medium used as a dispersion medium for the secondary emulsion is generally 0.01 to 50%, preferably 0.01 to 50% by weight. The weight ratio of the secondary emulsion to the aqueous medium containing the crude aqueous polymer for dispersing it is generally 5 to 30%.

l:2-100, preferably l:5-20. Furthermore, the weight ratio of the aqueous liquid containing the enzyme in the secondary emulsion to the organic solvent solution of the hydrophobic polymer for dispersing this is generally 1:1 to 20. Preferably it is 1:1-5.

The polymer gel containing the microencapsulated enzyme obtained as described above is finely divided into appropriate sizes suitable for column packing (for example, particle size of about 0.01 to 5 m+s) and used for enzyme reaction. Used as an immobilized enzyme. In this case, the microencapsulated enzyme has a size of several micrometers to several hundred micrometers, and will not be detached from the polymer gel that acts as its matrix.

[Effects] As described above, the method of the present invention is a method in which enzymes are confined in microcapsules and these microcapsules are dispersed and held in a polymer gel matrix, so the enzyme immobilization rate is high. Moreover, it has a characteristic that the enzyme leakage rate is low.

According to the present invention, the enzyme is a crushed bacterial cell product or a dried bacterial cell product.

It is possible to immobilize it directly in the form of live bacterial cells, etc.
Furthermore, unstable enzymes or multiple types of enzymes, for example enzymes for multiple conjugation reaction systems, can be effectively immobilized.

When using ordinary microencapsulated enzymes, when filling a column, fine microencapsulated enzymes that cause clogging are often removed, and the enzyme immobilization rate decreases accordingly. In contrast, in the case of the present invention, the immobilized enzyme has a structure in which the microencapsulated enzyme is dispersed and held in a polymer gel matrix.

Even if the microencapsulated enzyme is minute, the problem of clogging does not occur; on the contrary, immobilized enzymes made of polymer gel in which mf and ID encapsulated enzymes are uniformly dispersed are more expensive. This is advantageous because an active yield can be obtained.

〔Example〕

Next, the present invention will be explained in more detail with reference to Examples.

Example 1 Heat-resistant malic acid dehydrogenation cable (manufactured by Seikagaku Corporation, part name:
Thermostable malate dehydrogenase, hereinafter abbreviated as rMDHJ. ) 350 units (u), formate dehydrogenase (manufactured by Boehringer, trade name, "formate dehydrogenase".

Hereinafter, it will be abbreviated as rFDHJ. ) 50 units (υ), hemoglobin 30 mg, NAD containing 1.3 mg cotin adenine dinucleotide) 1 mQ of 0.0 g M pyrophosphate buffer (P) 18.5), surfactant (Spa
n85) One drop was added to a benzene solution containing 3% polystyrene to which two drops were added while stirring vigorously. The resulting emulsion (-secondary emulsion).

Polyethylene glycol diacrylate (molecular weight approximately 40
0.00) containing 2g, methylenebisacrylamide 100g. OIM birophosphate buffer (p!(8,5) l
ow m with vigorous stirring. The resulting emulsion (secondary emulsion) was continued to be stirred while blowing nitrogen gas to remove benzene by evaporation. When the benzene odor disappeared, stirring was stopped, and nitrogen gas was bubbled through the emulsion for 5 minutes. After confirming the formation of microcapsules with a microscope, 500 μQ of a 6% aqueous potassium persulfate solution and 100 μA of dimethylaminopropionitrile were added, and after shaking and mixing thoroughly, the mixture was allowed to stand while being blown with nitrogen gas. Gelation occurred in about 3 minutes and was then allowed to proceed for 15 minutes at 30°C. The generated gel (immobilized enzyme) was ground with a Waring blender and mixed with 100 mA of 0.01M phosphate buffer (
Wash with pH 7.5).

Immobilized enzyme (13.34 g) was obtained.

Next, the activities of unimmobilized MDH and FDH, the activities of unimmobilized MDH and FD)I in the gel washing solution, and the activities of immobilized MDH
and FD) I activity was measured as follows.

In addition, in D)l, one unit (u) is p
) I7.5. It exhibits the enzyme activity necessary to reduce 1 g mole of oxaloacetate in 1 minute at 30°C, and in FD) I, one unit (u) of it is P)17.5.3
It shows the enzyme activity required to oxidize 1 g mole of formic acid in 1 minute at 0'C.

In the activity measurement of unimmobilized HDH, oxaloacetic acid 0.52
11N and NADHo, using 0.1M phosphate buffer (P) 17.5) with a concentration of 2mM, 34On at 30℃.
MDI+ activity was determined from the initial rate of decrease in absorbance of +i. In addition, the immobilized MDI activity was determined by taking into account diffusion inhibition of polymer gel.

Oxaloacetic acid 1.04mM and NADHo, 4mM 0.1M phosphoric acid 1118? It was determined in the same manner using a solution (pH 7.5).

For activity measurement of unimmobilized FD), formic acid 33d and NA
D) Phosphorus buffer (P) 1 to 0.1 of the concentration condition of l ld
7.5), FDH activity was determined from the initial rate of increase in absorbance of 340na+ at 30°C. Immobilized FDII
Activity was determined by formic acid 66mM, taking into account polymer gel diffusion inhibition.
It was determined in the same manner using O, 1M phosphate buffer (p17.5) with a concentration of 2mM of NAD and 2mM of NAD.

As a result of the above activity measurement, it was found that the gel washing solution contained MD
4% of the I activity and 0.2% of the input FDI+ activity were detected, indicating that the enzyme activity was immobilized very efficiently. The total dox activity of the immobilized enzyme is 1 of the input MDH activity.
5% and 35% of the input FDH activity was measured.

This apparent decrease in activity is presumed to be due to the internal diffusion resistance of the gel, and by selecting a polymer gel material,
It seems that even higher activity can be obtained.

Next, 600 m5 of immobilized enzyme was added to 500 m5 of chitin flakes.
mg (wet weight 5), inner diameter 5m+i, total length 100m
m column, and both ends were further sealed with glass wool. The column was then submerged in a constant temperature water bath at 30°C, and the substrate solution was fed at a flow rate of 15o+Q for 1 hour to carry out a conjugation reaction. In addition, the substrate solution is oxaloacetate 15II
IM, 100++M formic acid, 100μM NAD.
A 1M phosphate buffer (PH 7,5) was used to saturate chloroform as a preservative.

The substrate solution was replaced every day, and the conjugation reaction was carried out for 2 weeks. The eluate was collected in 7.5 mfl portions using a fraction collector, and the amount of malic acid produced was measured.

The amount of malic acid was measured using a 0.2M hydrazine hydrochloride buffer (pH 9.3) with a concentration of 2mM NAD and 10u/m Q of malic acid dehydrogenation resistance (derived from pig heart).
The reaction was carried out at 30"C, and the temperature was 340" at the time of the initial reaction and at the end of the reaction.
The amount of NADH produced was determined from the difference in absorbance in nm, and the amount of malic acid in the sample was calculated.

As a result of the above measurement, it was determined that the reaction-completed solution contained a large amount of malic acid, and from this, MO)
It was confirmed that 1 and FD) I were conjugated and reacted.

During two weeks of continuous operation, the initial substrate conversion rate was 100%.
%, the substrate conversion rate after 2 weeks was 55%.

Example 2 MDH170u, 0.0 containing 30mg hemoglobin
1 mQ of IM pyrophosphate a solution (pH 8,5) was added dropwise to 1fflQ of a benzene solution containing 3% polymethyl methacrylate to which 4 drops of a surfactant (Span 85) had been added, with vigorous stirring.

The resulting emulsion was mixed with 2 g of polyethylene glycol diacrylate (molecular weight approximately 4000) and 0.01M pyrophosphoric acid containing 100B of methylene bisacrylamide! The mixture was added dropwise to 10 m of II buffer solution (pt+8.5) with vigorous stirring. Stirring was continued while blowing nitrogen gas to the resulting emulsion to remove benzene. Stirring was stopped when the benzene odor disappeared, and nitrogen gas was further bubbled through the emulsion for 5 minutes. After confirming the formation of microcapsules with a microscope, 500 μQ of 6% potassium persulfate aqueous solution and 100 μQ of dimethylaminopropionitrile were added, and after stirring,
It was left standing while blowing nitrogen gas. Gelation occurred in about 3 minutes, and gelation was then continued at 30°C for an additional 15 minutes. Grind the generated gel with a Waring blender,
0.018 phosphorus rs of 011IQ! ! Buffer solution (pH 7,
5) to obtain immobilized enzyme (13,49E).

Next, in the same manner as in Example 1, MDll in the gel washing solution was
and immobilized MDI (activity was measured).

As a result, 4% of the input MDH activity was detected in the gel washing solution, indicating that the enzyme activity was immobilized very efficiently. The total enzyme activity of the immobilized enzyme was determined by the input MD11
20% relative to activity was determined. From this, it was confirmed that polymethyl methacrylate can be used as a wall material for microcapsules in the same way as polystyrene.

Example 3 150 u of MDII, 1 water containing 30 mg of hemoglobin
mA.

It was added dropwise to a benzene solution containing 3% polystyrene to which 4 drops of a surfactant (Span 85) had been added while stirring vigorously. The resulting emulsion was treated with thorium alginate 15011.
The mixture was added dropwise to 1OIIIQ of water containing E with vigorous stirring. The resulting emulsion was then stirred while blowing nitrogen gas to remove the benzene. Stirring was stopped when the benzene odor disappeared, and the formation of microcapsules was confirmed using a microscope.

Pour the generated microcapsule solution into a syringe and inject 1.5
% calcium chloride solution to form a spherical gel. The gel was stored overnight at 4° C. in a 1.5% calcium chloride solution to allow gelation to proceed. The gel was washed with 100 mQ of 1.5% calcium chloride solution and immobilized enzyme (4
, 54E) were obtained.

This immobilized enzyme consists of a spherical gel with a diameter of 1 to 1
, 5 mm.

Next, MDI in the gel washing solution was measured in the same manner as in Example 1, and the activity of immobilized DI': c 31! I decided.

As a result, the gel washing solution contained 15% of the input MDI activity.
was detected. This seems to be due to part of the microcapsules being destroyed due to rapid changes in ionic strength during gelation and gel contraction, and can be prevented by increasing the ionic strength in the microcapsules. Fixed M
The DH activity was 14% of the input MDH activity.

Example 4 MDHlou, hemoglobin 30mg, NAD2+n
0.0'l+'l phosphorus II containing g! fi uiN liquid (
pH47,5) 1m Q, surfactant (Span85
) was added dropwise to 1 mff of a benzene solution containing 3% polymethyl methacrylate with vigorous stirring. The resulting emulsion was added dropwise to 10 mQ of 0.01M phosphate buffer (pH 7.5) containing 1 g of gelatin with vigorous stirring. Stirring was continued while blowing nitrogen gas to the resulting emulsion to remove benzene. After confirming the formation of microcapsules using a microscope, 200 μl of 25% glutaraldehyde solution was added.

Gelation occurred in about 10 minutes, and gelation was continued for an additional 2 hours at 4°C. The generated gel was pulverized with a Waring blender and mixed with 100 mQ of O, OIM phosphoric acid Dfl solution (pH
7,5) to obtain immobilized enzyme (TA, 40 g).

Next, in the same manner as in Example 1, the MDI activity and the immobilized MDH activity in the gel washing solution were measured. As a result, 0.4% of the input MDI activity was detected in the gel washing solution, indicating that the enzyme activity was immobilized very efficiently. In addition, the total enzyme activity of immobilized MDI (injected MDH
It was 26% of the activity.

Example 5 In order to demonstrate that the immobilization method of the present invention is applicable not only to Jingji dehydrogenation resistant chain but also to other kinase enzymes, AT
Experiments were also conducted on P-related kinases.

Hexokinase (manufactured by Boehringer, trade name).

"Hexokinase", hereinafter abbreviated as "H". )1
50u, glucose-6-phosphate hydrogenase (manufactured by Boehringer, trade name "glucose-6-phosphate dehydrogenase", hereinafter abbreviated as rGSPD), 120u, hemoglobin 30rag, ATP (azodinone-5'-3) phosphoric acid) 2m3. Cotin adenine dinucleotide phosphorus lj7) 2mg in NADP.

Add 1 m of 0.05M )-lith hydrochloric acid buffer (pH 8,5) containing 20+ag of magnesium chloride to 1 ml of benzene solution containing 3% polystyrene and 4 drops of surfactant (Span 85) with vigorous stirring. did. The resulting emulsion was treated with polyethylene glycol diacrylate (molecular weight approximately 4000)2. The mixture was added dropwise to 10 m of 0.05I Lis-HCl buffer (pH 8,5) containing 100 m of methylene bisacrylamide and 00 mg of magnesium chloride with vigorous stirring.

Stirring was continued while blowing nitrogen gas to the resulting emulsion to remove benzene. Stirring was stopped when the benzene odor disappeared, and nitrogen gas was bubbled through the emulsion for 5 minutes. After confirming the formation of microcapsules with a microscope, 500 μQ of a 6% potassium persulfate aqueous solution and 100 μQ of dimethylaminopropionitrile were added, shaken thoroughly, and left still while blowing nitrogen gas. Gelation occurred in about 3 minutes, and was then allowed to stand at room temperature for an additional 20 minutes to advance gelation.

The generated gel was crushed with a Waring blender and lo
om Q's 0.01M Tris-HCl aFJf solution (P11
7.5) to obtain immobilized enzyme (13,22F!).

Next, unimmobilized G6PD)l and )IK, unimmobilized G6PDH and H in the gel washing solution, and further immobilized G6PDI
+ and H activities were measured as follows.

In addition, in G 6 P D H, one unit (
u) exhibits the enzyme activity required to oxidize 1 μmol of glucose-6-phosphate at pH 7, 6, 30 °C for 11 minutes, and in )iK, its 1 unit (u) is p) 17.6. Demonstrates the enzyme activity necessary to phosphorylate 1 μmol of glucose in 1 minute at 30°C, II
The product of K is glycose-6-phosphate, and the excess G6PDH converts its amount into the amount of NADH and is measured.

In the activity measurement of unimmobilized G6PDH, glucose-6-
86.3mM I with concentration conditions of 1.2mM phosphoric acid, 0.37mM NADP, 6 and 7mM magnesium chloride
~ using reethanolamine buffer (p) 17.6),
G6P leaf activity was determined from the initial velocity bottom of the increase in absorbance of 340nII+ at 30°C. The activity of immobilized G6PDH was also determined using the same measurement method as described above. 11, glucose 222mM, ATP 2.7mM, NADP 0.
73mM, magnesium chloride 6.7mM, G6PDII
Using 82.31 triethanolamine grade buffer solution (pH 7,6) with a concentration of 0.5 u/nu,
The activity of H was determined from the initial rate of increase in absorbance at 0 nm. The same measuring method as above was used for the activity on immobilized H.

The conjugation reaction between HK and G6PDH was carried out at 30°C using a reagent for measuring HK activity without G 6 PDH.
In this study, the production of NADPH, that is, the increase in absorbance at 340 nm, was investigated using only the immobilized enzyme.

As a result, in the gel washing solution, there are 2.
5% and 0.6% of the input G6PDH activity was detected, indicating that the enzyme activity was immobilized very efficiently.

For immobilized enzyme activity, input 1 (33% of the activity, input G
7% of 6PDH activity was measured. Furthermore, the coupled reaction between immobilized IK activity and G6PD) activity was investigated.

Claims (2)

[Claims] (1) An immobilized enzyme having a structure in which microencapsulated enzymes are dispersed and held in a polymer gel matrix. (2) A primary emulsion in which aqueous droplets containing an enzyme are dispersed in an organic solvent solution of a hydrophobic polymer is dispersed in an aqueous medium containing a hydrophilic polymer to form a secondary emulsion. , producing a microencapsulated enzyme in an aqueous medium containing a hydrophilic polymer by evaporating the organic solvent, and then gelling the hydrophilic polymer. Method.