JPS6013671B2 - Immobilization method for enzymes or microbial cells - Google Patents

Description

Detailed Description of the Invention The present invention has a hydrophilic side chain capable of introducing unsaturated groups in an aqueous solution, and has two or more photopolymerizable ethylenically unsaturated groups introduced into one molecule in an aqueous solution. Number average molecular weight: 30,000~
The present invention relates to a method for immobilizing enzymes or microbial cells, in which an aqueous suspension of enzymes or microbial cells is added to an aqueous solution of a hydrophilic photocurable resin having a molecular weight of 500,000 and irradiated with light.

Among the methods of immobilizing enzymes or microorganisms, the entrapment method of enzymes or microorganisms is a method of immobilizing enzymes or microorganisms into a fine lattice of gel without forming a strong bond with the enzyme itself. It is an extremely useful method for immobilizing enzymes, etc., as it has the potential to be universally used for many types of enzymes and microbial cells, and the activity of immobilized enzymes, etc. is high. There is a need for an immobilizing substance with excellent permselectivity that envelops enzymes or microbial cells and prevents them from melting, and has good substrate permeability.

As one of the immobilization methods using such a comprehensive method, an immobilization method has been attempted in which an aqueous suspension obtained by uniformly mixing enzymes or microorganisms in an aqueous solution of a polyfunctional resin is irradiated with active light. There is. In this method, enzymes and other substances are confined in a lattice made of polymer molecules in advance, and the molecular weight between photocurable ethylenically unsaturated groups in the resin is adjusted so that the lattice size is large enough to prevent melt scales and allow the substrate to easily pass through. A pre-conditioned water-repellent photocurable resin is used. This method provides better permselectivity than the conventional enzyme immobilization method that involves polymerization of hydrophilic monomers such as acrylamide and hydroxyalkyl (meth)acrylates, and also allows the film to be filmed simultaneously with light irradiation. It is possible to mold it into any shape such as shape and harden it.
In the photocurable resin used in this method, photocurable ethylenically unsaturated groups are introduced by adjusting the molecular weight between the unsaturated groups in advance, but the unsaturated group introduction reaction can be carried out quantitatively and efficiently. Therefore, the reaction is carried out in a reaction system with a high resin concentration, either without using any solvent or with only a small amount of solvent. This also has the purpose of minimizing the contamination of solvents harmful to the enzyme reaction after enzyme immobilization and side reaction impurities.
However, in such a reaction without a solvent or in a highly concentrated solution, there are significant limitations on the resin structure, molecular weight, and degree of unsaturation (number of moles of unsaturated groups per unit resin). That is,
The unsaturated group introduction reaction is usually carried out at the lowest possible temperature and low viscosity to prevent gelation due to reactions between unsaturated groups. It is limited to those with low molecular weight so that There is also a certain limit to increasing the degree of unsaturation because gelation tends to occur more easily. In this way, in the unsaturated group introduction reaction without a solvent or in a highly concentrated solution, the degree of unsaturation and molecular weight are increased in order to improve the photocurability of the resin and the mechanical strength of the film obtained by photocuring. It was extremely difficult to do so. Therefore, when particularly mechanical strength is required, it is necessary to mix other resins or use synthetic or natural materials as the base material, but in this case too, it is necessary to check the compatibility with other resins and the There was a drawback that adhesion was not good. In order to improve the mechanical strength of photocurable resins, an immobilization method has also been attempted in which an aqueous emulsion of a high molecular weight unsaturated resin is synthesized, and an aqueous suspension of enzymes or microbial cells is added to the emulsion and photocured. However, in order to maintain the stability of the emulsion, emulsifiers and stabilizers are required, which is unfavorable in terms of enzyme activity.

Furthermore, if the film is diluted too much, photo-crosslinking between particles will not proceed smoothly, and it will be difficult to photocure the cured film by incorporating enough water to cause an enzyme reaction. The present invention was completed as a result of intensive research to improve these drawbacks.

That is, the present invention has a hydrophilic side chain capable of introducing an unsaturated group in an aqueous solution, and has a number average molecular weight of 3 having two or more photopolymerizable ethylenically unsaturated groups introduced into one molecule in an aqueous solution.
0000-500000, preferably 35000-20
The present invention relates to a method for immobilizing enzymes or microbial cells, in which an aqueous suspension of enzymes or microbial cells is added to an aqueous solution (or aqueous dispersion) of a hydrophilic photocurable resin having a molecular weight of 0,000 and irradiated with light. The present invention involves enclosing enzymes or microbial cells in a photocurable resin and immobilizing them by irradiation with light. This method is characterized by using a photocurable resin into which an unsaturated group has been introduced.

This resin has a lower degree of unsaturation and molecular weight compared to photocurable resins obtained by conventional unsaturated group introduction reactions without solvent or in high concentration solutions because the viscosity of the reaction etc. can be adjusted arbitrarily with water during the introduction of unsaturated groups. Therefore, the photocurability of the resin and the mechanical strength of the film obtained by photocuring can be improved. Furthermore, there is no risk of contamination with solvents or side reaction impurities that are harmful to enzyme activity. In addition, it improves workability during photocuring, and it is easy to photocure by adding enough moisture to cause an enzyme reaction in the cured film. There is no fear that your sexuality will deteriorate. The number average molecular weight of the photocurable resin is in the range of 30,000 to 500,000, preferably in the range of 35,000 to 200,000.

When the number average molecular weight is less than 30,000, the mechanical strength of the resin alone is insufficient, so if particularly mechanical strength is required, it is necessary to mix it with other resins or to use it by coating it on a base material. When the number average molecular weight is 500,000 or more, entanglement, aggregation, crystallization, etc. between molecules tend to occur, making it difficult to dissolve in water. In addition, since photocurable resins can cause crosslinking between resins by light irradiation,
It is necessary that each resin molecule has at least two photopolymerizable ethylenically unsaturated groups.

In order to ensure crosslinking between the resins, there is no problem in using a monofunctional unsaturated resin or a polyfunctional unsaturated resin having a number average molecular weight of 30,000 or less in combination. Furthermore, in order to increase enzyme activity, a saturated resin that has a strong affinity for enzymes etc. can be added. A photocurable resin has a hydrophilic functional group on its side iron that can introduce an unsaturated group in an aqueous solution, and the hydrophilic functional group that introduces an unsaturated group and the hydrophilic functional group that provides water solubility have the same structure. It may be of a different structure. The hydrophilic group may be either nonionic or ionic. In the case of ionic hydrophilic groups, p of the aqueous solution
If H is out of the optimum pH range of 4 to 8 for enzymatic reactions, it may be adjusted to the optimum f1 day range by neutralization after introduction of the unsaturated group. Also, regardless of whether it is non-ionic or ionic,
The same applies when the pH deviates from the optimum range for the enzymatic reaction due to the reaction conditions up to the introduction of the unsaturated group. Since active light is used for enzyme immobilization, the temperature does not rise during curing, and the activity of the immobilized enzyme or microorganism is not impaired. In addition, the ability to contain enough water to carry out an enzyme reaction in the cured film to form a transparent coating is also effective in preventing temperature rise during curing. Since the resin used is water-soluble, it has good hydrophilicity especially with aqueous suspensions of enzymes or bacterial cells, and has the advantage of less lysis and detachment after immobilization. In addition, the number average molecular weight of the photocurable resin is within a range that allows it to be used as fibers and plastics, so the resin alone can provide sufficient strength to withstand use, but it can also be used in combination with other resins or reinforced with a base material. , due to the high molecular weight of the resin, entanglement and
Excellent results can be obtained due to the good affinity due to the hydrophilic groups. The water-soluble photocurable unsaturated resin into which an unsaturated group has been introduced in an aqueous solution used in the present invention may be any resin as long as it falls within the above limitations, and the following specific examples are merely examples. .

Polyvinyl alcohol (PVA) with a number average molecular weight of 50,000 to 100,000 (saponification degree of 85 to 90%),
In an aqueous solution, 0.3 to 3 mol of N-methylolacrylamide (N-MAM) is added to 1 kg of PVA using an acidic catalyst (phosphoric acid, sulfuric acid, P-toluenesulfonic acid, etc.). Unsaturated PVA obtained by reacting the methylol group with the hydroxyl group of PVA.

{2) For polyvinyl alcohol (number average molecular weight 35,000 to 50,000) containing at least one type of water-soluble acrylic monomer such as acrylamide, methacrylamide, acrylic acid, and methacrylic acid as a copolymer component, N
- An unsaturated PVA copolymer obtained by reacting methylol acrylamide under the conditions described in {1}' above.

(3- Methyl cellulose, ethyl cellulose, hydroxyethyl cellulose, hydroxyph.

N-M for the hydroxyl group of water-soluble cellulose (number average molecular weight 100,000 to 200,000) such as lopil cellulose
Unsaturated cellulose obtained by reacting AM under the conditions described in {1}' above. {4) Hydroxyethyl acrylate (HEA), hydroxyethyl methacrylate (HE
MA), hydroxypropyl acrylate (HPA),
Hide.

A homopolymer of hydroxyalkyl (meth)acrylate such as oxypropyl methacrylate (HPMA),
Contains at least 5% of at least one of these, and nonionic hydrophilic monomers such as acrylamide and methacrylamide, anionic hydrophilic monomers such as acrylic acid and methacrylic acid, dimethylaminoethyl acrylate, dimethylaminoethyl methacrylate, etc. A water-soluble acrylic copolymer (number average molecular weight 100,000) containing at least 50% of at least one cationic hydrophilic monomer of N-MA
An unsaturated acrylic resin obtained by reacting M. '6l
A homopolymer of N-methylol acrylamide, or a nonionic hydrophilic monomer such as acrylamide or methacrylamide, an anionic hydrophilic monomer such as acrylic acid or methacrylic acid, containing at least 5% of this homopolymer,
An unsaturated acrylic resin (number average molecular weight 50,000).

The method of the present invention can be applied to a wide variety of enzymes and microbial cells, and can impart the effect of immobilization or insolubilization to all of them while preserving their activity.

Enzymes to which the method of the present invention can be applied are shown below, but this is just one example. Examples of suitable enzymes include urease, glycose oxidase, glucoamylase, glucose isomerase, catalase, invertase, lactase, D-amino acid oxidase, Q-galactosidase, aminoacylase, as'glutase, penicillin acidase, and the like. Examples of microbial cells include Lactopatillus vulgaris, Aerobacter aerogenes, Pachyrus subtilis, Azotobacter pinellandii, and Proteus vulgaris. A known photosensitizer can be added to the mixture of the aqueous photocurable resin solution and the aqueous suspension of enzymes or microbial cells used in this method for the purpose of promoting the photopolymerization reaction.

For example, Q-carbonyl alcohols such as benzoin and acetoin; penzoin methyl ether, benzoin ethyl ether, benzoin isopropyl ether,
Acilloin ethers such as anisoin ethyl ether and pivaloin ethyl ether; Q-methylbenzoin,
Q-Methoxybenzoin and other fusuma acyloins:
Preferred are polycyclic aromatic compounds such as naphthol and hydroxyanthracene; azoamide compounds such as 2-cyano-2-butylazoformamide; metals such as uranyl nitrate and ferric chloride;
Disulfides, halogen compounds, dyes, etc. can also be used.The mixed solution of a photocurable resin aqueous solution and an aqueous suspension of enzymes or microorganisms used in this method is molded into a shape suitable for the purpose of use. There are no restrictions on the thickness, presence or absence of vessel walls, etc., as long as the shape does not interfere with the transmission of the active rays. It is optional, such as filling it in a permeable container, impregnating it with a needle-shaped object, or allowing it to flow freely through the space during irradiation.The objects here include inorganic and organic natural products, synthetic materials, and metals. As the light source of the actinic rays used for irradiation, any source that emits light with a wavelength of 250 to 600 can be used, such as a low-pressure mercury lamp, a high-pressure mercury lamp, a poppy lamp, a light lamp, a xenon lamp, Carbon arc lamps, sunlight, etc. are used.

Irradiation time is usually in the range of 1 to 10 minutes. Irradiation in an inert gas is effective in shortening the irradiation time. Example 1 5 parts of polyvinyl alcohol (hereinafter abbreviated as PVA) having a degree of saponification of 90% and a number average molecular weight of 75,000 were dissolved in 150 parts of water, and while maintaining the temperature at 60 oo, N-methylolacrylamide (hereinafter abbreviated as N-MAM) was dissolved. ) was dissolved in 5 mounds of water, added thereto, added 1 part of 85% phosphoric acid, and reacted at 60°C for 5 hours to obtain unsaturated PVA with an unsaturation degree of 0.1 mol/k9.
An aqueous solution was obtained.

After taking 10 parts of this unsaturated PVA aqueous solution and adding 0.1 part of penzoin ethyl ether, the phosphoric acid was neutralized with a caustic soda aqueous solution and the pH was adjusted to 5. Mix one part of Proteus vulgaris (bacterial cells) dispersion suspended in 0.1M phosphate buffer to this, and place it on a horizontally arranged glass plate with a thickness of 1 side. A square with an inner frame of 5 x 5 was made and poured in. A polyester film with a thickness of 0.2 mm was tightly attached to the top of the square, and the top of the film was irradiated with a low-pressure mercury lamp for 5 minutes to create an immobilized bacterial cell film. Example 2 Number average molecular weight 3500 containing 10% acrylamide component
0 parts of N-MAMIO to 50 parts of PVA copolymer and 15 parts of water.
Dissolve and add 2 parts of phosphoric acid and react under the same reaction conditions as in Example 1 to obtain an unsaturation degree of 2.0 mol/k.
g of an unsaturated PVA copolymer was obtained.

After adding 0.1 part of penzoin ethyl ether to this and neutralizing the phosphoric acid with aqueous ammonia and adjusting the pH to 6.5,
Add 1 part of 1% urease aqueous solution dissolved in 0.01M phosphate buffer and pour it into the same frame as in Example 1. A polyester plate with a thickness of 0.5 ribs was placed on top of the frame, and the upper surface 20 The skin was irradiated with light using a Fox W high-pressure mercury lamp for 5 minutes to obtain a molded film of immobilized enzyme. The formed membrane was washed 3 times with 200ml of distilled water, immersed in 100ml of 0.01M urea solution dissolved in 0.01M phosphate buffer, and heated to 30℃ for 30 minutes.
It was allowed to react for a minute. Take 5' of reaction solution and add 0.1N-HC1
5 skin was added and back titrated with 0.1N-NaOH to determine the specific activity relative to the activity of the unimmobilized enzyme.68
%showed that. Example 3 5 parts of N-MAM was dissolved in 200 parts of water and added to 50 parts of hydroxypropyl cellulose having a number average molecular weight of 150,000, and 1 part of 97% sulfuric acid was added and reacted under the same reaction conditions as in Example 1 to obtain unsaturated degree 1.0m
An unsaturated cellulose aqueous solution of ol/k9 was obtained.

After adjusting the pH to 7 with a caustic soda aqueous solution, add this resin aqueous solution to 1st floor B' this pensoin isopropyl ether 0.2
and 5 parts of 0.5% glucose oxidase (catalase-free) aqueous solution dissolved in 0.1M phosphate buffer, applied to a platinum electrode, and exposed around it using a high-pressure mercury lamp.
An immobilized enzyme electrode was prepared by irradiation for 1 minute. When electrical measurements were performed using a lead electrode as a counter electrode and immersing the immobilized electrode and the counter electrode in a 0.1% glucose solution, a response of the electrode due to glucose was observed. Example 4 A 10% aqueous solution of an acrylamide copolymer (number average molecular weight 50,000) containing 20% of hydroxyethyl methacrylate (HEMA) was prepared in the same manner as in Example 1 by adding 5 parts of N-methylolacrylamide and 1 part of phosphoric acid. An aqueous solution of unsaturated acrylamide copolymer was prepared under the following reaction conditions.

After adjusting the pH to 7 with an aqueous solution of caustic soda, 0.0% of this aqueous resin solution was added to the base of the aqueous resin solution.
5 parts of a composite enzyme solution of 0.1% invertase and 0.5% glucose oxidase dissolved in 0.1M phosphate buffer was mixed and applied to gauze to a thickness of about 0.5 willow. . A transparent polyester plate with a thickness of 0.3 skin was placed on top of this, and a high-pressure mercury lamp was applied to the top surface from 50 skin.
When the polyether plate was peeled off, a film of immobilized enzyme made of gauze was obtained. A hydroxyethyl acrylate female part and 1 part of phosphoric acid were added to 500 parts of a 10% aqueous solution, and an unsaturated acrylamide copolymer aqueous solution was prepared under the same reaction conditions as in Example 1.

Claims (1)

[Claims] 1 Has a hydrophilic side chain that can introduce an unsaturated group in an aqueous solution,
Two or more photopolymerizable ethylenically unsaturated groups are introduced into one molecule in an aqueous solution, and the number average molecular weight is 30,000 to 50.
Immobilization of enzymes or microbial cells, characterized by irradiating active light to a mixture of an aqueous solution (or aqueous dispersion) of a 0000 hydrophilic photocurable resin and an aqueous suspension of enzymes or microbial cells. Method.