JPS5915628B2 - Immobilization method for enzymes or microbial cells - Google Patents

Description

Detailed Description of the Invention The present invention involves irradiating an aqueous suspension of a hydrophilic photocurable resin and enzymes or microbial cells with active light in the presence of a photopolymerization initiator to inactivate the enzymes or microbial cells. A method of fixing,
In particular, the present invention relates to an immobilization method that improves the mechanical strength of immobilized materials.

Conventionally, when enzymes or microbial cells were immobilized by the entrapment method, the mechanical strength of the immobilized material was very weak, so it was necessary to reinforce the immobilized material by some means.

In order to achieve this reinforcement, for example, monomers were polymerized and immobilized on the inner wall surface of a certain tube, or the immobilized material was covered with a film.

However, such reinforcing means have the disadvantage that in the former case, the shape of the immobilized product is limited to a certain shape, and in the latter case, the degree of activation of the immobilized enzyme or microbial cells is further reduced. Ta.

Furthermore, when immobilizing these enzymes or microorganisms by the entrapment method, various reinforcing substrates are used for the immobilized product to increase its mechanical strength.

However, it is not uncommon for these enzymes or immobilized microorganisms to polymerize in a wet state and in a dry state, with the former being about 10 times as much as the latter, or even more, resulting in volume deformation due to swelling. Because it is extremely large, there is a large difference in the volumetric deformation of the reinforcing base material used for reinforcement purposes, and as a result, the adhesion between the reinforcing base material and the photocurable resin used for fixation is impaired. The photocurable resin used in
It breaks due to a single change from a hydrated state to a dry state,
In most cases, the reinforcing purpose could not be fully achieved due to peeling off from the reinforcing base material.

On the other hand, if one tries to obtain a small swellable immobilized material by photo-curing in order to avoid this difficulty, the diffusion of substrate molecules that should be brought into contact with enzymes or microbial cells becomes small.
This creates practical and efficiency problems.

It is also known to reinforce with gauze or silk cloth, but this causes similar problems.

The present inventors have completed the present invention through extensive research to solve the above-mentioned drawbacks.

That is, in the present invention, a photopolymerization initiator and a number average molecular weight of 300 to 30,000 are added to an aqueous suspension of enzymes or microorganisms.
In a method of uniformly mixing a hydrophilic photopolymerizable resin and irradiating it with actinic rays to immobilize enzymes or microbial cells,
The present invention relates to a method for immobilizing enzymes or microorganisms, which is characterized by reinforcing with a highly elastic reinforcing base material.

The highly stretchable reinforcing base material used in the present invention has 0.00% when dried at room temperature or lower humidity, assuming that one side of the immobilized material immediately after immobilization of enzymes or microorganisms is 1. When it contracts to 9 or less and reaches the maximum water absorption state, it expands and contracts to 1.3 or more.

If the reinforcing base material does not shrink by 0.9 times or less, or 1.
Unless it is more than 3 times larger, there is a large difference between the volume deformation of the photocurable resin immobilized with enzymes or microorganisms in the hydrated state and the dry state, and the difference in the volume deformation of the reinforcing base material under the same conditions. The main cause of this is that the adhesion of the immobilized resin to the reinforcing base material decreases, leading to problems such as peeling.

The highly elastic reinforcing base material may be made of natural or synthetic polymers, or may be made of metal, preferably synthetic polymers, and may be in the form of fibers, nets, woven fabrics, or non-woven fabrics. , knitted or woven, or sheet-like.

Among these, especially fibrous ones, even if the fiber alone shrinks by 0.9 times or more and expands and contracts by 1.3 times or less,
If it is made into a woven fabric, non-woven fabric, knitted fabric, etc. and exhibits a shrinkage of 0.9 times or less and an elongation of 1.3 times or more, it can be processed into the above-mentioned net shape or the like.

However, the fibrous structure alone has a shrinkage of 0.9 times or less and a shrinkage of 1.3
As long as it exhibits an elongation of more than double, it is sufficient to use a fibrous material in the center and immobilize enzymes or microorganisms on the outer periphery with a photocurable resin.

In addition, when using a sheet-like material as a reinforcing substrate, considering the adhesion of the hydrophilic photocurable resin that immobilizes enzymes or microorganisms to the sheet-like material, it is better to use a complete sheet-like material. It is also desirable to have a sheet-like material with at least a large number of holes.

Particularly desirable reinforcing base materials include nylon net, urethane fiber, cellulose net, cellulose acetate net, polyamide net, polyester net, polyvinyl net, polyolefin net, polycarbonate net, and polyacrylic net. Examples include nets that shrink by 0.9 times or less and expand by 1 to 3 times or more.

On the other hand, the photocurable resin used in the present invention is a hydrophilic resin and has a photopolymerizable unsaturated bond in the resin.

Examples of such resins include any commonly used photocurable resins that have hydrophilic groups and are water-soluble or water-dispersible. For example, these hydrophilic photopolymerizable resins include unsaturated polyester, unsaturated acrylic-urethane resin, unsaturated acrylic resin, unsaturated epoxy resin, unsaturated polyester-urethane resin, and unsaturated urethane resin. The resin has 0.5 to 3 double bonds per 1oooy of the resin, and has an ionic or nonionic hydrophilic group introduced into the resin.

Among these resins, the number of unsaturated groups is 0.5 per 1001 resin.
If the number is less than 3, the crosslinking density of the immobilized product becomes too high and the immobilized product becomes brittle, whereas if the number is more than 3, the crosslinking density of the immobilized product becomes too small, resulting in the disadvantage that it is difficult to develop the strength of the immobilized product. .

Therefore, the photopolymerizable double bond of the hydrophilic photocurable resin is resin 1
It is necessary that 0.5 to 3 pieces exist per 001.

Further, the photocurable resin used in the present invention is hydrophilic.

The hydrophilic group may be ionic or nonionic, and these hydrophilic groups may be present in the resin from the beginning or may be introduced later.

These hydrophilic groups are, for example, a carboxyl group, a hydroxyl group, an alkoxy group, an amide group, or an ether bond.

This hydrophilic photocurable resin can be mixed with an aqueous suspension of enzymes or microorganisms at any ratio, but usually 20 to 1
The mixed solution of the photocurable resin and the aqueous suspension of enzymes or microorganisms used in this method contains a known photopolymerization initiator, such as benzoin, to carry out the photopolymerization reaction. , α-7 carbonyl alcohols such as acetoin, acyloin ethers such as benzoin methyl ether, benzoin ethyl ether, benzoin propyl ether, anisoin ethyl ether, pivaloin ethyl ether, polycyclic aromatics such as naphthol, hydroxyanthracene, etc. compounds, α-monosubstituted acyloins such as methylbenzoin and α-methoxybenzoin, azoamide compounds such as 2-cyano-2-butylazoformamide, metals such as launyl nitrate and ferric chloride, mercaptans, and disulfides. , halokene compounds, and dyes.

These photopolymerization initiators are usually 0.01 to 10 PHR (pe
used at a rate of 10% (rhundred resiu).

In addition, as a light source of actinic rays used for irradiation, wavelength 22
Any device that emits light of 0 to 700 mμ, preferably 250 to 600 mμ can be used, such as low-pressure mercury lamps, high-pressure mercury lamps, fluorescent lamps, xenon lamps, carbon arc lamps, sunlight, and the like.

Irradiation time is usually in the range of 1 to 10 minutes.

Performing irradiation in an inert gas is effective for shortening irradiation time.

Examples of suitable enzymes to be immobilized in the method of the invention include urease, glucose oxidase, glucoamylase, glucose isomerase, glucose-6-phosphate dehydrogenase, catalase, invertase, glucose oxidase-catalase, lactase, D-amino acid oxidase, α-galactosidase, aminoacylase-aspartase, penicillin amidase, etc. Examples of microbial cells include Lactobacillus bulgaricus, Aerobacter aerokenes, Bacillus subtilis, Azotobacter vinellandii, Proteus vulgaris, Chloerachella, etc. Yeast, etc. can be mentioned.

In the method of the present invention, the method of immobilizing enzymes or microbial cells with a hydrophilic photocurable resin by entrapping method involves first adding a photopolymerization initiator and a hydrophilic photocurable resin to an aqueous suspension of enzymes or microbial cells. After uniformly mixing the reinforcing base material with the polymeric resin and immersing the reinforcing base material in this mixed solution, or after casting (spreading) the mixed solution on the reinforcing base material, or after reinforcing the reinforcing base material in the mixed solution. Enzymes or microorganisms may be immobilized by irradiating actinic rays after or while pulling up the substrate after immersing it.

In particular, when irradiating actinic rays, it is desirable to irradiate the coated object while cooling it so that the object to be exposed does not go to the edge.

According to the method of the present invention, unlike conventional methods for immobilizing enzymes or microorganisms, the temperature is at room temperature or below room temperature, so the activity of enzymes or microorganisms is not reduced. Since the use of monomers is not particularly essential during immobilization, there is no problem of toxicity of the immobilized product after immobilization.

Furthermore, by using a highly elastic reinforcing base material when immobilizing enzymes or microbial cells using a hydrophilic photocurable resin, the mechanical strength of the immobilized product is significantly increased, and at the same time, the shrinkage of the immobilized product is reduced. Oaks, which conventionally used to crack, are no longer cracked by the present invention.

Therefore, whereas conventionally immobilized materials had to be stored and transported by being constantly immersed in an aqueous dispersion system, it is now possible to store and transport them in a dry state.

Regarding storage stability, in the case of immobilized products containing conventional reinforcing substrates, enzymes and microbial cells are stored in an aqueous dispersion system, which may lead to denaturation of enzymes or microorganisms. Long-term storage was impossible due to the growth of different types of microorganisms such as molds, which lowered the activity of the target enzyme or microbial cells.However, according to the method of the present invention, the immobilized product can be stored in a dry state. It has many features, such as being able to preserve the activity of enzymes or microbial cells over a long period of time.

The immobilized enzyme or microbial cell obtained by the present invention can be effectively used in a wide range of industrial fields such as pharmaceuticals, fermentation, catalysts, foods, clinical practice, analysis, and purification.

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

In the Examples, parts and weights refer to parts by weight and percent by weight, respectively.

Example 1 1 mole of xylylene diisocyanate and a molecular weight of about 1500
85 parts of a photocurable resin (number average molecular weight: 2160) consisting of 750 g of polyethylene glycol and 1.1 moles of 2-hydroxyethyl meccrylate were suspended in a 0.1 mole phosphoric acid buffer solution of H6.0. 1% invertase solution 1
A mixed solution was prepared by uniformly mixing 20 parts and 1 part of benzoin methyl ether.

A 0.3 mm thick spacer inner frame 5 crrL x 0.1 mm thick polypropylene sheet placed horizontally
A 5cIrL square was made, a nylon net was placed in the inner frame, and the above mixture was poured into the square.

A polypropylene sheet with a thickness of 0.1 mm was adhered thereon and irradiated with a 2 kW high-pressure mercury lamp for 2 minutes from a distance of 10 c/rL from the upper surface of the sheet to obtain a membrane-like immobilized enzyme reinforced with a nylon net.

When this membrane-like immobilized enzyme was left at room temperature for 3 days, it shrunk and the length of one side of the immobilized product was approximately 0.
．． It was 84 times larger.

When this thing is immersed in water, the size when immobilized is 1.7
Despite swelling four times, the membrane did not crack.

Example 2 A membrane-like immobilized enzyme prepared in the same manner as in Example 1 was dried under reduced pressure in a vacuum dryer, stored in a warehouse for 30 days, and then processed into IC1r.
It was cut into a size of L×ICrrL.

Put 5 of these cut membranes into a test tube, and add 20 ml of 50 mmol sucrose solution (0.1 molar phosphate buffer of pn6.o) to the test tube.
was added and reacted with stirring at 40° C. for 10 minutes, and the activity was determined by quantifying glucose with Glucoscut (manufactured by Fujisawa Medical Supply) solution.

When the specific activity was measured relative to the activity before storage in the refrigerator, the specific activity was 86%.

Example 3 NK ester 23G (polyethylene glycol dimethacrylate with a molecular weight of 1000, manufactured by Shin Nakamura Chemical Industry) 90
part, dissolved in 0.1 M acetate buffer at pH 5,6.
, 100 parts of a 5% glycose oxidase (including catalase) aqueous solution, and 1 part of benzoin ethyl ether were uniformly mixed to prepare a mixed solution.

A nylon net was immersed in this mixed solution, pulled up in a nitrogen gas atmosphere, and immediately irradiated with a 2 kW low-pressure water lamp from both sides for 3 minutes to obtain a membrane-like immobilized enzyme reinforced with the nylon net.

This membrane-like immobilized enzyme did not cause any cracks in the membrane even after shrinking (085 times as much as when immobilized) and swelling (1.57 times as much as when being immobilized) 10 times.

Example 4 1 mole of Epicoat 1001 resin (manufactured by Shell Chemical Company, trade name) was reacted with 1.5 moles of adipic acid, then esterified with 4.5 moles of succinic anhydride, and then reacted with 2.75 moles of glycidyl methacrylate. Oxidation 75 generated by
photocurable resin (number average molecular weight approximately 4100) 85 parts, 0
．． 5 parts of 2% NaOH solution and 1.1% glucose isomerase dissolved in 0.1M phosphate buffer, pH 7.5.
A mixed solution was prepared by uniformly mixing 20 parts of ferric chloride and 0.1 part of ferric chloride.

Inner frame 5CT with spacers 0.3 mvt thick on a horizontally arranged polypropylene sheet 0.1 mrn thick
A square of t x 5CrfL was made, and the above mixed solution was poured into the inner frame of the Delrin net, and a thickness of 0.
A 1 mm polypropylene sheet was placed in close contact with the sheet and irradiated for 3 minutes with a 1 kW low-pressure mercury lamp from a distance of 5CrfL from the upper surface of the sheet to obtain a membrane-like immobilized enzyme reinforced with Delrin net.

This membrane-like immobilized enzyme did not cause negative tears in the membrane even after shrinking (0.8 times as much as when immobilized) and swelling (1.87 times as much as when being immobilized) 10 times.

Claims (1)

[Claims] 1. A photopolymerization initiator and a hydrophilic photopolymerizable resin having a number average molecular weight of 300 to 30,000 are uniformly mixed into an aqueous suspension of enzymes or microbial cells, and the mixture is irradiated with active light. 1. A method for immobilizing enzymes or microbial cells, the method comprising reinforcing with a highly elastic reinforcing base material. 2. Reinforcing substrates with high elasticity should shrink to 0.9 or less when dry and reach the maximum water absorption state, assuming that one side of the immobilized material immediately after immobilization of enzymes or microorganisms is 1. 2. The method for immobilizing enzymes or microorganisms according to claim 1, wherein the enzyme or microbial cell expands to 1.3 or more when the enzyme is expanded. 3 Reinforcement base materials with high elasticity can be in the form of fibers, nets, woven fabrics,
3. The method for immobilizing enzymes or microorganisms according to claim 1 or 2, characterized in that the immobilization method is in the form of a nonwoven fabric, a woven fabric, or a sheet. 4 Photopolymerization initiators are α-carbonyl alcohols, acyloin ethers, polycyclic aromatic compounds, α-monosubstituted acyloins, azoamide compounds, metal salts, mercaptans, disulfides, halokenides, and dyes. A method for immobilizing enzymes or microbial cells according to claim 1. 5 Hydrophilic photopolymerizable resins are unsaturated polyesters, unsaturated acrylic-urethane resins, unsaturated acrylic resins, unsaturated epoxy resins, unsaturated polyester-urethane resins, and unsaturated urethane resins that have photopolymerizable double bonds in the resin. 1000
．． The method for immobilizing enzymes or microorganisms according to claim 1, wherein the resin has 0.5 to 3 hydrophilic groups per gram and has ionic or nonionic hydrophilic groups introduced into the resin.