JPH0154038B2 - - Google Patents

Description

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

Traditionally, enzyme reactions have been used to produce useful substances, but because the enzymes that act as catalysts are water-soluble, they are discarded after one use. However, enzymes are expensive, so we try to immobilize them, reuse them, and make the process continuous.
Manufacturing costs have been significantly reduced. In recent years, in addition to hydrolytic enzymes such as amylase and protease,
Synthetic enzymes and oxidoreductases that require coenzymes such as ATP and NAD (P) are also beginning to be applied in the field of medical analysis, and are gradually being used in the production of pharmaceuticals, industrial raw materials, etc. However, enzymes that require coenzymes are more expensive and less stable than conventional hydrolases or isomerases, so it is hoped that an immobilization method that is superior to conventional immobilization methods will emerge. ing.

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, since the enzyme interacts with the carrier through relatively weak bonds, desorption of the enzyme occurs during continuous reactions, which limits the conditions that can be used.

The second method is a covalent bonding method, in which the carrier and the enzyme are chemically covalently bonded.Since the carrier and the enzyme are bonded together with a strong bond, the detachment of the enzyme is extremely rare. Due to modification, deactivation is likely to occur. In particular, dehydrogenases and monooxygenases are often unstable and are greatly affected by their reactions.

The third method is an entrapment method, in which the enzyme is encapsulated in a gel matrix or microcapsules such as acrylamide or K-carrageenan. Unlike the covalent bond method, this comprehensive method does not directly chemically modify the enzyme molecule, so deactivation does not occur. In addition, by appropriately adjusting the gel mesh and the pore size of the microcapsule membrane, it is possible to capture and fix enzyme molecules within the gel and microcapsules while allowing only small substrate molecules to pass through.
Desorption of enzyme molecules can be prevented.

On the other hand, the enzymes that require the aforementioned coenzymes often form dimers or tetramers. The bonding force between each monomer is very weak, and if one part of the tetramer is covalently bonded to the carrier, 4
It is extremely difficult to maintain the same amount for a long time. In such cases, immobilization by gel entrapment is preferable. Furthermore, when simultaneously immobilizing two types of enzymes (malate dehydrogenase and formate dehydrogenase) in order to combine the regeneration reactions of NAD(P)H, conventional immobilization methods can only support microorganisms. The only method is to comprehensively immobilize the enzyme group within the capsule. Even with the gel entrapment method, it is extremely difficult to fit two types of enzymes into one compartment of the gel. Furthermore, the stability conditions for the enzyme and the optimal conditions for gelation do not necessarily match. However, existing microcapsules have fundamental deficiencies in practical use, such as deformation of the microcapsules, clogging, and high pressure loss when they are packed into columns for continuous production.

The present invention was made to solve the problems of the prior art, and includes enzymes contained in droplets surrounded by an organic solvent that is almost immiscible with water, and the droplets are dispersed in a solution containing a gel material. After that, a gelation reaction occurs, and after the gel is formed, the organic solvent is removed to prepare a droplet-immobilized enzyme gel that disperses and holds a large number of independent enzyme-containing droplets in a non-encapsulated state. Features.

In this method, the field where the gelation reaction occurs and the field where the enzyme exists are blocked by the organic solvent layer, so different conditions can be set, so the conditions are suitable for the enzyme to be stable and for gelation to be optimal. Stable immobilization can be achieved even under considerably different conditions. In addition, in the case of the usual gel entrapment method, when the enzyme content is increased,
Enzyme molecules may interfere with the formation of the gel matrix itself, resulting in a significant increase in enzyme leakage, but this has never happened with this method. In addition, especially when the conjugate reaction system is immobilized, it is all concentrated and arranged in countless independent droplets in the gel enclosing body.
The conjugation reaction is carried out efficiently. Furthermore, it is possible to easily perform the fixation of a coupling reaction between a cell fragment and an enzyme, which has not been carried out very often in the past. With the exception of temperature conditions, it is possible to select the optimal conditions for gelation, regardless of the limiting conditions necessary for stabilizing enzymes, bacterial cells, crushed bacterial cells, animal and plant cells, etc.
A hard and elastic gel can be made. Furthermore, it is also possible to obtain a droplet-entrained immobilized enzyme gel suitable for continuous use in a column.

The immobilized enzyme obtained by the present invention is novel in that it has a structure in which droplets containing the enzyme are dispersed and held in a non-microencapsulated state in a polymer gel matrix.

Basically, the method for preparing this immobilized enzyme is as follows.

A buffer or aqueous solution containing an enzyme and, if necessary, a stabilizer ( _W layer) is stirred and dispersed in an organic solvent that contains a surfactant and is immiscible with water (O layer) to form water-in-oil droplets. Make a primary emulsion. This primary emulsion is further stirred and dispersed in a buffer or aqueous solution (W ₂ layer) containing a gel material to obtain a (W ₁ /O)/W ₂ type composite emulsion (secondary emulsion). By adding, for example, a polymerization initiator to this secondary emulsion, it is immediately gelled to obtain a gel-enclosed body containing countless droplets. By processing this gel into an appropriate size by crushing, cutting, etc., and washing it with a buffer solution, the organic solvent in the O layer is gradually dissolved in water, and the organic solvent is removed from the washing solution surface. The immobilized enzyme containing enzyme-containing droplets is prepared by evaporating the solvent and removing the O layer. In this way, a droplet-enclosed immobilized enzyme gel is obtained which does not allow enzyme leakage and is suitable for a packed column reactor. By using this immobilization method,
A plurality of coupled reaction systems can be immobilized, and organelles, crushed bacterial cells, dried bacterial cells, live bacterial cells, animal and plant cells, etc. can be encapsulated and immobilized in droplets as they are. Such a new immobilization method has not been reported to date, and the effect of contributing to the development of enzyme utilization technology is extremely large.

The present invention will be specifically explained in detail below.

Since the enzyme used in the present invention does not require severe reaction conditions, it can be used unless the enzyme is particularly unstable. Specific enzyme names include alcohol dehydrogenase, aldehyde dehydrogenase, glutamate dehydrogenase, isoleucine dehydrogenase, formate dehydrogenase, lactate dehydrogenase, malate dehydrogenase, and glucose-6-phosphate dehydrogenase. dehydrogenases such as glyceraldehyde-3-phosphate dehydrogenase, monooxygenases such as orcinol-2-monooxygenase and phenol-2-monooxygenase, acetate kinase, hexokinase,
In addition to enzymes that require coenzymes such as kinases such as glycerol kinase, hydrolytic enzymes such as β-amylase, glucoamylase, β-glucosidase, lipase, phosphatase, protease, and esterase, glucose oxidase, and peroxidase Examples include oxygenase. Furthermore, the enzyme does not need to be purified. Therefore, it may be a culture solution, culture filtrate, live bacterial cells, dried bacterial cells, crushed bacterial cells, organelles, protoplasts, animal/plant cells, or crushed cells that have the necessary enzyme activity. However, if they are solid, they need to be so fine that they can fit into the droplets. Furthermore, these enzymes may be used as a mixture. An enzyme system that performs a coupling reaction mediated by a coenzyme is more convenient. This is because enzymes exist free even though they are confined in droplets.
Unlike the covalent bonding method, there is no reaction inhibition due to physical or steric hindrance, and because the enzyme is concentrated in the limited space of droplets in the gel, the reaction occurs quickly. This is for the purpose of

However, since the enzyme is contained in a polymer gel, this immobilization method is not effective for enzymes that use polymers as substrates, hydrolytic enzymes such as α-amylase, chitinase, and cellulase. However, these enzymes are effective when decomposing low molecular weight substrates.

The stabilizer in the present invention refers to a drug that stabilizes enzymes, including proteins such as hemoglobin, gelatin, and serum albumin, polysaccharides such as gum arabic and starch, and water-soluble substances such as polyethylene glycol and polyvinyl alcohol. synthetic polymer,
Coenzymes such as NAD(P) and ATP, chelating agents such as EDTA, and in special cases, metal salts, antioxidants such as glutathione, etc. The combination of these substances depends on the enzyme used. It depends. These substances are included in commercially available enzyme preparations and are useful for maintaining long-term enzyme activity. They are not necessary for enzymes that are naturally highly stable, and are used in this immobilized enzyme preparation. There is no need to add it at any time.

Furthermore, since the W ₁ layer and W ₂ layer are separated by the O layer during gelation, even substances that inherently inhibit the gel crosslinking reaction can be used as a stabilizer.

The buffer as used in the present invention may be any buffer as long as it is suitable for cross-linking reactions and stabilizing enzyme activity.
Since the W ₁ layer and the W ₂ layer are separated by the O layer during gelation, they may be completely different, so it is possible to select the one that is most suitable for stabilizing the crosslinking reaction and enzyme activity.

The organic solvent used in the present invention may be one having a boiling point of 100°C or less and being almost immiscible with water, such as benzene, cyclohexane, ethyl ether, ethyl acetate, carbon tetrachloride, chloroform, halogenated ethylene, etc. can be mentioned.

The gel material used in the present invention is contained in the W ₂ layer,
Any material that can supply a gel matrix of immobilized enzymes may be used, and since the droplets produced have a size of several μm to several hundred μm, they will not fall off from most gels. For example, there are polymerization methods using commonly used acrylamide, copolymerization methods with copolymers such as methacrylamide, acrylic acid, and hydroxyethyl methacrylate, and methods using photocrosslinkable monomers, photocrosslinkable prepolymers, urethane prepolymers, and the like. There is also a gelation method using a hydrophilic polymer combined with crosslinking as shown below.

Alginic acid (crosslinked with polyvalent cations); agar,
Polysaccharides such as carrageenan (gelling upon cooling); proteins such as gelatin, collagen, albumin, casein (crosslinking with glutaraldehyde etc. via Schiff base); polyethylene glycol diacrylate (radical crosslinking polymerization of double bonds)
Synthetic polymers with vinyl groups such as; polyvinyl alcohol (PVA) (crosslinked with glutaraldehyde, etc.); PVA, polyethyleneimine (crosslinked with epichlorohydrin); PVA, polyacrylamide, polyvinylpyrrolidone (crosslinked with radiation);
PVA (photocrosslinking by UV irradiation with sodium benzoate), etc.

The present invention will be explained below using examples. The present invention is not limited to these examples.

Example 1 Thermostable malate dehydrogenase (hereinafter abbreviated as MDH, derived from Thermus thermophilus, manufactured by Seikagaku Corporation) 150U (international standard unit), formate dehydrogenase (hereinafter abbreviated as FDH,
Add 1 ml of 0.01 M pyrophosphate buffer (PH8.5) containing 45 U (derived from Candida boidinii, manufactured by Boehringer) to 1 ml of benzene containing 4 drops of surfactant (Span 85) while stirring vigorously. dripped. The resulting primary emulsion was mixed with polyethylene glycol diacrylate (molecular weight approx.
4000) 2g, methylenebisacrylamide 100mg
The mixture was added dropwise to 10 ml of 0.01M pyrophosphate buffer (PH8.5) with vigorous stirring. Immediately add 500μ of 6% potassium persulfate aqueous solution and 100μ of dimethylaminopropionitrile to the generated secondary emulsion,
After stirring well, while blowing nitrogen gas,
It was left standing at 30°C. Gelation occurred in about 5 minutes, and then kept at 30°C for 2 hours. The resulting gel was ground with a Waring blender, kept in a beaker at 30°C with 100ml of 0.1M Tris-HCl buffer, and benzene was removed while shaking. After shaking for 2 hours, the mixture was filtered with suction to obtain an immobilized enzyme preparation (14.5 g).

When we measured the activity of this immobilized enzyme preparation and the washing solution, we found that the washing solution contained 19% of the input MDH activity.
0.5% of the FDH activity was measured, and the immobilized enzyme preparation contained 19% of the input MDH activity and 19% of the input FDH activity.
10% was measured. The activity of MDH was measured using a 0.1M phosphate buffer (PH7.5) containing 0.52mM oxaloacetate and 0.2mM NADH at 30°C.
It was determined from the initial rate of decrease in absorbance at 340 nm (1 unit is the oxygen activity required to reduce 1 μmole of oxaloacetic acid per minute at pH 7.5 and 30°C).
In addition, FDH activity measurement was performed using 33mM formic acid and
0.1M phosphate buffer (PH
7.5), FDH activity was determined from the initial rate of increase in absorbance at 340 nm at 30°C (1 unit is PH
7.5, the enzyme activity required to oxidize 1 μmole of formic acid per minute at 30°C).

Example 2 600 mg of the gel prepared in Example 1 was mixed with 300 mg of chitin flakes (wet weight) into a container with an inner diameter of 5 mm x total length.
It was packed into a 100 mm column, and both ends were further sealed with glass wool. Next, this column was submerged in a constant temperature water bath at 30° C., and the substrate solution was fed at a flow rate of 15 ml/hour to carry out a conjugation reaction. In addition, as a substrate solution, oxaloacetic acid 15mM, formic acid 100mM, NAD 0.3mM
Contains 1mM cysteine as a stabilizer,
0.1M Tris-HCl buffer (PH7.5) containing 0.1mM EDTA and 0.01% potassium sorbate as preservative
was used. The substrate solution was replaced every day, and the conjugation reaction was carried out for 2 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 10 units/ml of malate dehydrogenase (derived from pig heart, manufactured by Boehringer) at 30℃. The reaction was carried out, and the amount of NADH produced was determined from the difference in absorbance at 340 nm between the initial reaction and the end of the reaction, and the amount of malic acid in the sample was calculated.

As a result, an increase in conjugated activity was observed in the early stage (days 2 to 4), and this activity was maintained over two weeks, indicating stable production of malic acid. In this case, the substrate conversion rate at about day 12 is about 3.5%
It was hot.

Example 3 0.1M Tris-HCl buffer (PH
7.5) 1 ml of benzene with 4 drops of Span85 added to 1 ml
was added dropwise to the solution while stirring vigorously. The resulting primary emulsion was added dropwise to 10 ml of 0.01M pyrophosphate buffer (PH8.5) containing 1.9 g of acrylamide and 100 mg of methylenebisacrylamide with vigorous stirring. Immediately, 0.6% potassium persulfate and 10μ of dimethylaminopropionitrile were added to the resulting secondary emulsion, and the mixture was stirred and left at 30°C while blowing nitrogen gas. It gelated in about 3 minutes and was then kept at 30°C for 2 hours. The resulting gel was pulverized using a Waring blender and placed in a beaker with 100 ml of Tris-HCl buffer (PH7.5), kept at 30°C and shaken to remove benzene. After shaking for 2 hours,
The immobilized enzyme preparation (14.6 g) was obtained by suction filtration.

As a result, 14% of the input MDH activity was detected in the gel washing solution. In addition, the total enzyme activity of the gel is
5% of input MDH activity was measured and it was found that acrylamide could be used similarly to polyethylene glycol diacrylate.

Example 4 Candida tropicalis
tropicalis, IAM12202), potassium dihydrogen phosphate 0.1%, magnesium sulfate heptahydrate 0.05%, calcium chloride dihydrate 0.01%, salt 0.02%, casamino acid 0.4%, yeast extract 0.1%, D-xylose 8%. Inoculate into a pH5.0 culture solution containing 30
The cells were cultured with shaking at ℃ for 48 hours. The bacterial cells were collected by centrifugation, washed, and then freeze-dried to obtain a cell lysate with aldose reductase activity.

110 mg of freeze-dried bacterial cells were suspended in 0.4 ml of 0.1 M Tris-HCl buffer (PH7.5) and added dropwise to 0.5 ml of benzene containing 2 drops of Span 85 with vigorous stirring to effect emulsification. This primary emulsion was 0.01M containing 1.2g of polyethylene glycol diacrylate (molecular weight approximately 4000) and 50mg of methylene bisacrylamide.
The mixture was added dropwise to 5 ml of pyrophosphate buffer (PH8.5) with vigorous stirring to obtain a secondary emulsion. 250μ of 6% potassium persulfate and 50μ of dimethylaminopropionitrile were added to the secondary emulsion, mixed well, and then allowed to stand at 30°C while blowing nitrogen gas. Approximately 5
Gelation occurred within minutes and then kept at 25°C for 3 hours.
Grind the generated gel with a Waring blender,
The mixture was kept at 30°C in a beaker with 50 ml of Tris-HCl buffer (PH7.5), and benzene was removed while shaking. After shaking for 1 hour, filter with suction, 7.7
A fixed cell lysate of g was obtained.

This fixed cell lysate and washing solution are
NADP 2mM, glucose 1M, glucose-6
-Phosphoric acid 0.3M, glucose-6-phosphate dehydrogenase (yeast derived, manufactured by Boehringer) 5 units/ml
with 0.1M Tris-HCl buffer (PH7.5) containing
React at 30℃ for 5 hours, take a portion of the reaction solution,
After removing solids, HPLC using ion exchange resin
The amount of sorbitol produced was measured.

As a result, no production of sorbitol was observed in the washing solution, and 35% of the input aldose reductase activity was measured in the fixed cell lysate gel. Immobilized aldose reductase can also be obtained by such a method, and it is considered to be extremely useful from a practical standpoint.

Example 5 Saccharomyces cerevisiae (Haken No. 1) was inoculated into a medium containing 1% glucose, 0.3% yeast extract, 0.5% peptone, and 0.3% malt extract, and cultured with shaking at 30°C for 2 days. Ivy. After collecting the bacterial cells by centrifugation and washing, take 300 mg (wet weight) of the bacterial cells and add them to 700μ of 0.1M phosphate buffer (PH
6.0). Drop the bacterial cell suspension into 1 ml of benzene containing 4 drops of Span85 while stirring vigorously.
A second emulsion was obtained. This emulsion was added dropwise to a 3% aqueous Katsupar carrageenan solution at about 60° C. with vigorous stirring to obtain a secondary emulsion. Spread this secondary emulsion on a glass plate to a thickness of 3 mm and gel it.
It was kept at ℃ for 1 hour. This gel was cut into 5 mm x 5 mm pieces and shaken in 50 ml of 1.2% KCl solution at 30°C to strengthen the gel and remove benzene. After shaking for 2 hours, suction filtration was performed to obtain 8 g of an immobilized microbial specimen.

2 g of this sample was put into a culture solution containing 1% glucose, 0.3% yeast extract, 0.5% peptone, 0.3% malt extract, and 1.2% potassium chloride, and left at 30° C. for 2 days. As a result, microbial cell growth was observed in the gel under a microscope, and significant ethanol accumulation was observed in the culture filtrate.

Claims (1)

[Scope of Claims] 1. An immobilized enzyme having a structure in which a large number of independent droplets containing the enzyme are dispersed and held in a non-microencapsulated state in a polymer gel matrix. 2 A primary emulsion in which aqueous droplets containing an enzyme are dispersed in an organic solvent solution is dispersed in an aqueous medium containing a gel material to form a secondary emulsion, and the gel material is gelled. A method for producing an immobilized enzyme, which comprises subsequently removing the organic solvent and entrappingly immobilizing the droplets.