JPS6239998B2 - - Google Patents

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]

When performing various enzymatic reactions, the enzymes used as catalysts are water-soluble and can only be used once. In order to overcome these drawbacks, techniques for immobilizing enzymes have been developed and are widely used.

In recent years, in addition to hydrolyzed oxygen such as amylase and protease, synthetic enzymes that do not require coenzymes such as ATP, NAD, and NADP, and redox have begun to be applied in the field of medical analysis, as well as pharmaceuticals, It is also gradually being used as a catalyst for the production of chemical products.

However, enzymes that require coenzymes are generally more expensive and less stable than conventional hydrolases or isomerases.
Conventional enzyme immobilization methods do not yield satisfactory immobilized enzymes, and 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 oxygen 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 a relatively weak bond, 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 enzyme are chemically covalently bonded.
Desorption of enzymes is extremely rare, but there is a disadvantage that deactivation etc. easily occur due to chemical modification of enzyme molecules.Especially in the case of unstable enzymes such as dehydrogenases and monooxygenases, the problem of deactivation is a problem. is big.
The third method is encapsulation, in which the enzyme is encapsulated in a gel matrix or microcapsules such as acrylamide or x-carrageenan. Unlike the covalent bonding method, this inclusion method does not directly chemically modify the enzyme molecule, so it has the advantage of not causing deactivation, and it also allows the mesh of the gel and the pore size of the microcapsule membrane to be adjusted appropriately. By. It is possible to capture and fix enzyme molecules within gels and microcapsules while allowing only small substrate molecules to pass through, and there is also the advantage that detachment of enzyme molecules can be prevented. This comprehensive method is preferably applied to the immobilization of enzymes that require the aforementioned coenzymes. In other words, in the case of such enzymes, the enzyme often forms dimers or tetramers, but
In that case, the bonding force between each monomer is very weak,
For example, when one part of a tetramer is covalently bonded to a carrier, it is extremely difficult to maintain the tetramer state for a long period of time. Therefore, in the case of such enzymes, immobilization by gel entrapment method is preferably applied. In addition, when two types of enzymes are immobilized at the same time, the only immobilization method that can be used up to now is the entrapping immobilization of enzyme groups in microcapsules. Fitting two types of enzymes into one compartment is extremely difficult. However, 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, causing pressure It had a fundamental practical flaw in that it resulted in high losses.

〔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 a microencapsulated enzyme is dispersed and held in a polymer gel matrix is provided, and as a second invention, an immobilized enzyme is provided that has a structure in which a microencapsulated enzyme is dispersed and retained in a polymer gel matrix. To produce a primary emulsion in which aqueous droplets containing enzymes are dispersed in a hydrophobic polymeric organic solvent solution,
Dispersing in an aqueous medium containing a hydrophilic polymer to form a secondary emulsion, removing the organic solvent by evaporation,
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, formate dehydrogenase, lactate dehydrogenase, malate dehydrogenase, glucose - Dehydrogenases such as 6-phosphate dehydrogenase and glyceraldehyde-3-phosphate dehydrogenase;
In addition to enzymes that require various coenzymes, such as monooxygenases such as orcinol-2-monooxygenase and phenol-2-monooxygenase; kinases such as acetate kinase, hexokinase, and glycerol xanase; Examples include hydrolytic enzymes such as amylase, β-glucosidase, lipase, phosphatase, protease, and esterase, and oxygenases such as glucose oxidase and peroxidase. Further, the enzyme may be a purified product or a crude product. Therefore, culture solution, culture filtrate, pre-viable cells, dried cells, crushed cells,
Animal and plant cells, cell fragments, and cell organelles may also be used. However, if they are solid, they need to be of a fine size so that they can 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 oxygen is concentrated in the limited space of microcapsules in the polymer gel, the conjugation reaction is carried out quickly, yielding excellent immobilized enzymes. However, since the enzyme is contained in a polymer gel, the enzyme activity is insufficient for enzymes that use polymers as substrates and hydrolytic enzymes such as α-amylase, chitinase, and cellulase. 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 an organic solvent solution of a hydrophobic polymer, and an aqueous solution containing the enzyme is mixed and emulsified in an organic solvent solution of a hydrophobic polymer. A primary emulsion with dispersed droplets is prepared. In this case, the enzyme contained in the aqueous liquid is
It may be in a suspended state as well as a dissolved state. Also,
In addition to the enzyme, the aqueous solution may contain conventional auxiliary additives such as stabilizers, PH buffers, and the like. When preparing the primary emulsion, a commonly used surfactant is added to the hydrophobic polymer organic solvent solution in advance in order to promote dispersion of the aqueous droplets into the hydrophobic polymer organic solvent solution. is advantageous.

The localizing agent added to the aqueous solution containing the enzyme is a drug that stabilizes the enzyme, and various conventionally known localizing agents are used, such as hemoglobin, gelatin, serum albumin, etc. Proteins such as: polysaccharides such as gum arabic and starch; water-soluble synthetic polymers such as polyethylene glycol and polyvinyl alcohol; NAD,
Coenzymes such as NADP and ATP; chelating agents such as EDTA; and in special cases, antioxidants such as various metal salts and glutathione, which are appropriately selected depending on the enzyme used. Such stabilizers are
It is usually included in commercially available oxygen samples, and is useful for maintaining oxygen activity over a long period of time, and is not particularly required in the case of enzymes that are highly stable by themselves. Furthermore, when using a low-molecular stabilizer, it is not preferable to use one that inhibits gelation (cross-polarization reaction) of the polymer. Further, the pH buffer added to the aqueous solution containing the enzyme is arbitrary as long as it does not inhibit the crosslinking reaction or enzyme activity, and the pH 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 range that does not stabilize it for a long time, the time required for enzyme immobilization is short and it can be removed by washing after immobilization. There is no problem in using it. The concentration of the PH buffer should be less than 0.5M, preferably 0.1M.
The following is good. In addition, it is recommended that the buffers added to the aqueous liquid used in the preparation of the primary emulsion and the aqueous liquid containing the hydrophilic polymer used in the preparation of the secondary emulsion be of the same or equivalent ionic strength. preferable.
This is because when forming microcapsules, water may flow in or out due to the concentration difference between the inside and outside of the membrane, which may cause the microcapsules to be deformed and destroyed.

As the hydrophobic polymer to be dissolved in the organic solvent that serves as a dispersion medium for the aqueous droplets containing the enzyme, almost all hydrophobic polymers that are soluble in organic solvents can be used. Specific examples of such substances include, for example, soluble addition polymers, such as homopolymers and copolymers thereof such as vinyl esters, vinyl ethers, acrylic esters, methacrylic esters, acrylonitrile, and soluble polycondensates, such as , polycarbonate, polyester, polysulfonate, polyurethane, polyamide, etc., as well as cellulose derivatives such as ethyl cellulose and cellulose acetate, and chlorinated rubber. It is also possible to use polymers that are soluble in organic solvents and whose solubility changes with changes in pH. For example, in addition to acrylic acid polymers and methacrylic acid polymers, examples of alkali-soluble and acid-insoluble polymers include polymers such as crotonic acid, maleic acid, itaconic acid, citraconic acid, and vinylbenzoic acid, or polymers that have vinyl monomers and acid groups. Examples include copolymers with monomers, cellulose derivatives, phthalic acid, succinic acid, or maleic acid derivatives of starch, and phthalic acid derivatives of cellulose. Furthermore, acid-soluble and alkali-insoluble basic polymers include polyvinylpyridine, polyvinylimidazole, polyvinylamine, polyvinylaniline, and the like. Furthermore, the organic solvent used as a dispersion medium for the hydrogen solution 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, Examples include carbon chloride, 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 [(W ₁ /
O)/W type ₂ composite emulsion] is prepared. In this secondary emulsion, the organic solvent contained in the primary emulsion is removed by evaporation through an aqueous medium containing a water-soluble polymer, thereby removing the hydrophobic polymer dissolved in the organic solvent. is precipitated around the aqueous droplets containing the enzyme to form an ultrafiltration membrane and provide microencapsulated enzyme. The rate of evaporative removal of the organic solvent 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 microcepherization enzyme is dispersed gelates (crosslinks) the polymer. In this case, gelation of the polymer can be performed by a conventionally known method, using an appropriate gelation method depending on the polymer.

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 furthermore, after microencapsulation, it acts as a gelatin for the microencapsulated enzyme. It serves as a trix. Any hydrophilic polymer can be used as long as it can be gelatinized (crosslinked), and examples include alginic acid; polysaccharides such as agar and carrageenan; gelatin, collagen, Examples include proteins such as 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. For example, in the case of alginic acid, crosslinking with polyvalent cations; in the case of polysaccharides such as agar and carrageenan, gelation by cooling; and in the case of gelatin, For proteins such as collagen, albumin, and casein, crosslinking using glutaraldehyde and Schiff's base; for synthetic polymers with vinyl groups such as polyethylene glycol diacrylate, radical polymerization crosslinking of the vinyl groups; for polyvinyl alcohol,
Crosslinking with glutaraldehyde and 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 primary emulsion is generally from 0.001 to
50% by weight, preferably 0.5 to 30% by weight, and the weight ratio of the primary emulsion to the aqueous medium containing the hydrophilic polymer for dispersing it is generally 1:2 to 100. Preferably it is 1:5-20. Furthermore, the weight ratio of the aqueous liquid containing the enzyme in the primary emulsion and the organic solvent solution of the hydrophobic polymer for dispersing this is generally 1:1 to 1.
20, preferably 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 mm) and immobilized for enzyme reaction. Used as a converting enzyme. In this case, the microencapsulated enzyme has a size of several micrometers to several hundred micrometers, and is not detached from the polymer gel that acts as its matrix.

〔effect〕

As mentioned 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 and enzyme leakage is achieved. It is characterized by a low production rate. According to the present invention, enzymes can be immobilized as they are in the form of crushed bacterial cells, dried bacterial cells, live bacterial cells, etc., and can also be immobilized as they are, such as unstable enzymes or multiple types of enzymes, such as multiple conjugated enzymes. Enzymes in the reaction system 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, so even if the microencapsulated enzyme is minute, No clogging problems occur. On the contrary, an immobilized enzyme made of a polymer gel in which a large amount of finely encapsulated enzyme is uniformly dispersed is more advantageous because a higher activity yield can be obtained.

〔Example〕

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

Example 1 Heat-stable malate dehydrogenase (manufactured by Seikagaku Corporation, trade name, "heat-stable malate dehydrogenase", hereinafter abbreviated as "MDH") 350 units (u), formate dehydrogenase (Boehringer) Contains 50 units (U), 30 mg of hemoglobin, and 1.3 mg of NAD (nicotine adenine dinucleotide).
1 ml of 0.01M pyrophosphate buffer (PH8.5) was added dropwise to a benzene solution containing 3% polystyrene and 2 drops of surfactant (Span 85) with vigorous stirring. The generated emulsion (primary emulsion) is mixed with polyethylene glycol diacrylate (molecular weight approx.
4000) 2g, methylenebisacrylamide 100g
The mixture was added dropwise to 10 ml of 0.01M pyrophosphate buffer (PH8.5) with vigorous stirring. Stirring was continued while blowing nitrogen gas onto the resulting emulsion (secondary emulsion) to evaporate and 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 using a microscope, add 500μ of a 6% potassium persulfate aqueous solution and dimethylaminopropionitrile.
After adding 100μ and shaking well, the mixture was allowed to stand while blowing nitrogen gas. Gelation occurred in about 3 minutes, and was then continued at 30°C for 15 minutes. The generated gel (immobilized enzyme) was ground in a Waring blender and added to 100 ml of 0.01M phosphate buffer (PH
7.5) to obtain immobilized enzyme (13.34 g).

Next, the activities of unimmobilized MDH and FDH, the activities of unimmobilized MDH and FDH in the gel washing solution, and the activities of immobilized MDH and FDH were measured as follows.

In addition, in MDH, one unit (u)
shows the enzyme activity necessary to reduce 1 μmol of oxaloacetate per minute at PH7.5 and 30°C, and
In FDH, one unit (u) is PH
7.5, indicating the enzyme activity required to oxidize 1 μmol of formic acid per minute at 30°C.

For activity measurement of unimmobilized MDH, oxaloacetate
MDH activity was determined from the initial rate of decrease in absorbance at 340 nm at 30°C using a 0.1M phosphate buffer (PH7.5) with concentrations of 0.52mM and 0.2mM NADH.
In addition, the immobilized MDH activity was determined by oxaloacetic acid 1.04mM and
It was determined in the same manner using 0.1M phosphate buffer (PH7.5) with a concentration of NADH of 0.4mM.

For activity measurement of unimmobilized FDH, 0.1M phosphorus buffer (PH) with a concentration of 33mM formic acid and 1mM NADH was used.
7.5), FDH activity was determined from the initial rate of increase in absorbance at 340 nm at 30°C. Immobilized FDH activity was determined by formic acid 66mM, taking into account polymer gel dispersion inhibition.
It was determined in the same manner using 0.1M phosphate buffer (PH7.5) with a concentration of 2mM and NAD.

As a result of the above activity measurement, the gel washing solution contained
4% of the input MDH activity and 0.2% of the input FDH activity were detected, indicating that the enzyme activity was immobilized very efficiently. The total enzyme activity of the immobilized enzyme is
15% of input MDH activity and 35% of input FDH activity were measured. This apparent decrease in activity is presumed to be due to internal diffusion resistance of the gel, and it is thought that even higher activity can be obtained by selecting a polymer gel material.

Next, add 600 mg of immobilized enzyme and 500 mg of chitin flakes.
mg (wet weight) into a column with an inner diameter of 5 mm and a total length of 100 mm, and both ends were further sealed with glass wool. The column was then submerged in a 30°C water bath, and 15 ml of the substrate solution was fed at a flow rate of 1 hour to carry out a conjugation reaction. The substrate solution is oxaloacetic acid.
A 0.1M phosphate buffer (PH7.5) containing 15mM of formic acid, 100mM of formic acid, and 100μM of NAD was used to saturate chloroform as a preservative. Replace the substrate solution every day,
Conjugation reactions were performed for weeks. The eluate was collected in 7.5 ml portions using a fraction collector, and the amount of malic acid produced was measured.

The amount of malic acid was measured using 0.2M hydrazine hydrochloride buffer (PH9.3) with a concentration of 2mM NAD and malate dehydrogenase (derived from pig heart) 10u/ml at 30°C. time and at the end of the reaction
The amount of NADH produced was determined from the difference in absorbance at 340 nm, and the amount of malic acid in the sample was calculated.

As a result of the above measurement, it was determined that a large amount of malic acid was contained in the reaction-completed solution, and from this, it was confirmed that MDH and FDH were conjugated and reacted.

In continuous operation for 2 weeks, if the initial substrate conversion rate is 100%, the substrate conversion rate after 2 weeks is 55%.
It was hot.

Example 2 1 ml of 0.01M pyrophosphate buffer (PH8.5) containing MDH170u and 30 mg of hemoglobin was added to 1 ml of benzene solution containing 3% polymethyl polymetalate to which 4 drops of surfactant (Span85) was added while stirring vigorously. dripped. The resulting emulsion was added dropwise to 10 ml of 0.01M pyrophosphate buffer (PH8.5) containing 2 g of polyethylene glycol diacrylate (molecular weight approximately 4000) and 100 mg of methylenebisacrylamide while stirring vigorously. 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 using a microscope, 500μ of a 6% aqueous potassium persulfate solution and 100μ of dimethylaminopropionitrile were added, stirred, and allowed to stand while blowing nitrogen gas. Gelation occurred in about 3 minutes, and gelation was then continued for an additional 15 minutes at 30°C. The generated gel was crushed with a Waring blender and washed with 100 ml of 0.01M phosphate buffer (PH7.5) to obtain immobilized enzyme (13.49 g).

Next, in the same manner as in Example 1,
The activity of MDH and immobilized MDH 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 to be 20% of the input MDH activity. 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 1ml of water containing MHD150u, hemoglobin 30mg
was added dropwise with vigorous stirring to a benzene solution containing 3% polystyrene to which 4 drops of surfactant (Span 85) were added. The resulting emulsion was added dropwise to 10 ml of water containing 150 mg of sodium alginate with vigorous stirring. The resulting emulsion was continuously stirred while blowing nitrogen gas to remove benzene. Stirring was stopped when the benzene odor disappeared, and the formation of microcapsules was confirmed using a microscope. The produced microcapsule solution was placed in a syringe and dropped into 40 ml of 1.5% calcium chloride solution to produce a spherical gel. The gel was stored overnight at 4°C in a 1.5% calcium chloride solution to allow gelation to proceed. 1.5 for 100ml of gel
The immobilized enzyme (4.54 g) was obtained by washing with % calcium chloride solution. This immobilized enzyme, consisting of a spherical gel, had a diameter of 1 to 1.5 mm.

Next, in the same manner as in Example 1,
MDH was measured, and the activity of immobilized MDH was also measured. As a result, 15% of the input MDH activity was detected in the gel washing solution. 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. The immobilized MDH activity is
14% of the input MDH activity was achieved.

Example 4 1 ml of 0.01M phosphate buffer (PH7.5) containing 100 u of MDH, 30 mg of hemoglobin, and 2 mg of NAD was vigorously stirred into 1 ml of benzene solution containing 3% polymethyl methacrylate to which 4 drops of surfactant (Span 85) were added. while dripping. The resulting emulsion was mixed with 10 ml of 0.01 M phosphate buffer (PH7.5) containing 1 g of gelatin).
was added dropwise to the solution while stirring vigorously. Stirring was continued while blowing nitrogen gas to the resulting emulsion to remove benzene. After confirming the formation of microcapsules using a microscope, a 25% glutaraldehyde solution was added.
Added 200μ. Gelation occurs in about 10 minutes, and then
Gelation was allowed to proceed at 4°C for an hour. The generated gel was crushed with a Waring blender and washed with 100 ml of 0.01M phosphate buffer (PH7.5) to obtain immobilized enzyme (18.40 g).

Next, in the same manner as in Example 1,
MDH activity and the activity of immobilized MDH were measured. As a result, the gel washing solution contains the input MDH activity.
0.4% was detected, indicating that oxygen was immobilized very efficiently. Furthermore, the total oxygen activity of immobilized MDH was 26% of the input MDH activity.

Example 5 In order to demonstrate that the immobilization method of the present invention is applicable not only to the above-mentioned dehydrogenases but also to other kinase enzymes, experiments were also conducted on ATP-related kinases.

Hexokinase (manufactured by Boehringer, trade name,
"Hexokinase", hereinafter abbreviated as "HK". )
150u, Glucose-6-phosphate dehydrogenase (manufactured by Boehringer, trade name: "Glucose-6-phosphate dehydrogenase", hereinafter abbreviated as "G6PDH")
120u, hemoglobin 30mg, ATP (adecynon-
0.05 M Tris-HCl buffer (PH
8.5) 1 ml was added dropwise to 1 ml of a benzene solution containing 3% polystyrene to which 4 drops of surfactant (Span 85) were added with vigorous stirring. The emulsion produced is
2 g of polyethylene glycol diacrylate (molecular weight approx. 4000), 100 methylene bisacrylamide
It was added dropwise to 10 ml of 0.05 M Tris-HCl buffer (PH8.5) containing 200 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, 500μ of a 6% aqueous potassium persulfate solution and 100μ of dimethylaminopropionitrile were added, shaken thoroughly, and allowed to stand while blowing nitrogen gas. Gelation occurred in about 3 minutes, and then the mixture was allowed to stand at room temperature for an additional 20 minutes to advance gelation. The generated gel was crushed with a Waring blender and washed with 100 ml of 0.01M Tris-HCl buffer (PH7.5) to obtain immobilized enzyme (13.22 g).

Next, unimmobilized G6PDH and HK, unimmobilized G6PDH and HK in the gel washing solution, and further immobilized G6PDH
and HK activity was measured as follows.

In addition, one unit (u) of G6PDH exhibits the enzyme activity necessary to oxidize 1 μmol of glucose-6-phosphate in 1 minute at PH7.6 and 30°C. One unit (u) represents the enzyme activity necessary to phosphorylate 1 μmol of glucose in 1 minute at 30°C and a pH of 7.6. The product of HK is glycose-6-phosphate, and the excess G6PDH converts its amount into the amount of NADH and is measured.

To measure the activity of unimmobilized G6PDH, 86.3mM triethanolamine buffer (PH7.6) with a concentration of 1.2mM glucose-6-phosphate, 0.37mM NADP, and 6.7mM magnesium chloride was used at 30°C.
G6PDH activity was determined from the initial rate of increase in absorbance at 340 nm. The activity of immobilized G6PDH was also determined using the same measurement method as described above. For HK activity measurement, 82.3mM triethanolamine buffer (PH
7.6), HK activity was determined from the initial rate of increase in absorbance at 340 nm at 30°C. The same method as above was used to measure the activity of immobilized HK.

For the coupling reaction of HK and G6PDH, the production of NADPH, that is, the increase in absorbance at 340 nm, was investigated using only the immobilized enzyme at 30° C. using a reagent for measuring HK activity without G6PDH.

As a result, 2.5% of the input HK activity and 0.6% of the input G6PDH activity were detected in the gel washing solution, indicating that the enzyme activity was immobilized very efficiently. For immobilized enzyme activity, 33 of input HK activity
%, 7% of the input G6PDH activity was measured. Furthermore, when the coupled reaction between the immobilized KH activity and G6PDH activity was investigated, it was confirmed that the enzyme had sufficient activity, and it was confirmed that the amount of glucose could be measured using this immobilized enzyme.

Claims (1)

[Scope of 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, and the A method for producing an immobilized enzyme, which comprises evaporating an organic solvent to produce a microencapsulated enzyme in an aqueous medium containing a hydrophilic polymer, and then gelling the hydrophilic polymer.