JPS5932117B2 - How to use immobilized enzymes or microbial cells - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for using enzymes or microbial cells that have been immobilized by a comprehensive method, and more specifically to a method for using immobilized enzymes or microbial cells while keeping them constantly moist. It is characterized by:

Conventionally, immobilized enzymes or microbial cells generally have weak mechanical strength, and furthermore, immobilized enzymes or microbial cells have a large volumetric deformation in wet and dry states, so compared to that of reinforcing base materials. Due to the extreme difference in temperature, the immobilized material (hereinafter referred to as immobilized enzymes or microbial cells) will eventually separate from the reinforcing substrate when it becomes dry. Ta.

Under such conditions, the immobilized enzyme or microbial cells could not be considered as industrially useful immobilized products.

Therefore, the present inventors completed the present invention by researching a method for solving the above-mentioned defects of immobilized products (and using enzymes or microbial cells continuously and effectively).

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 microbial cells.
In a mixed solution of uniformly mixed hydrophilic photopolymerizable resin,
An immobilized enzyme or microbial cell obtained by irradiating a reinforcing substrate with actinic rays while immersing it or after immersing it and using it while keeping it in a constantly moist state. The invention relates to the use of enzymes or microbial cells.

In the present invention, when only a polymer is used as a raw material, problems such as poor adhesion to the reinforcing base material as described above occur.

Therefore, the present invention has been made to solve these deficiencies.

That is, the present invention uses a hydrophilic photocurable resin with a number average molecular weight of 300 to 30,000 to eliminate the toxicity of the monomer to the human body, and to reduce the particularly high water content and dry state of the hydrophilic photocurable resin. This aims to simultaneously solve the problem of poor adhesion with the reinforcing base material caused by volumetric deformation in and.

Examples of particularly suitable enzymes that can be used in the present 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., and Lactobacillus as a microbial cell. , bulgaricus, Aerobacter aerogenes, Bacillus subtilis, Azotobacter vinellandii, Proteus, vulgaris, Chloerachella, and other yeasts.

A known photopolymerization initiator is added to a mixed solution of an aqueous suspension of a photocurable resin and an enzyme or a microbial cell used in the present invention in order to carry out a photopolymerization reaction.

Examples of such known photopolymerization initiators include α-carbonyl alcohols such as benzoin and acetoin, benzoin methyl ether, benzine ethyl ether, penzoin globyl ether, anituin ethyl ether, hivaloin ethyl ether, etc. acyloin ethers, polycyclic aromatic compounds such as naphthol and hydroxyanthracene, α-monosubstituted acyloin compounds such as methylbenzoin and α-methoxybenzoin, 2-cyano-2
- Azoamide compounds such as butylazoformamides,
Examples include metal salts such as launyl nitrate and ferric chloride, mercaptans, disulfides, halogen compounds, and dyes.

These photopolymerization initiators usually have a concentration of 0.01 to 10 pHR (p
er hundred Re5in).

In addition, the number average molecular weight that can be used in the present invention is 300 to 300.
00 hydrophilic photocurable resin is 0 per 1000P of the resin.
．． It is necessary to have 1 to 6 moles of photopolymerizable double bonds.

If the amount of photopolymerizable double bonds is less than 0.1 mol, the crosslinking density of the immobilized product becomes too high (too much, and the immobilized material becomes too brittle); The disadvantage is that the density becomes too low and the mechanical strength of the immobilized product becomes too weak.

Such photopolymerization initiators include conventionally known photopolymerizable initiators, such as unsaturated polyester, unsaturated acrylic resin, unsaturated epoxy resin, unsaturated acrylic-urethane resin, unsaturated polyester-urethane resin, and unsaturated urethane resin. You can give resin.

Such a photopolymerizable resin must be hydrophilic, and the hydrophilic group may be ionic or nonionic.

These hydrophilic groups may be present in the resin from the beginning or may be introduced later.

These hydrophilic groups are, for example, carboxyl groups, hydroxyl groups, alkoxy groups, amine groups, ether bonds, etc., and are bonded in the resin via ester bonds, ether bonds, urethane bonds, etc.

This hydrophilic photopolymerizable resin can be mixed with an aqueous suspension of enzymes or microorganisms at any ratio, but usually 20 to 1
000% by weight is desirable.

The reinforcing base material used in the present invention is made of natural or synthetic polymeric substances or metals, and in particular, it is preferable to use polymeric substances in consideration of the shape to be used. It is woven.

Particularly suitable as such reinforcing substrates are (non-)woven or knitted (net) fabrics made of natural or synthetic polymers commonly used in daily life.

As detailed above, the reinforcing base material is immersed in an aqueous suspension of enzymes or microorganisms and a homogeneous mixture of a photopolymerization-initiating and hydrophilic photopolymerizable resin and irradiated with active light, or the reinforcing base material is irradiated with active light. Either the homogeneous mixture is cast (spread) on the material and actinic rays are irradiated, or the reinforcing base material is immersed in the homogeneous mixture and then actinic rays are irradiated while or after the base material is pulled up. By irradiating, the immobilized enzyme or microbial cells used in the present invention can be obtained.

In the present invention, the active light source used to immobilize enzymes or microbial cells has a wavelength of 220 to 700.
Any device that generates light of 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, chemical lamps, sunlight, etc. .

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

Furthermore, performing this irradiation in an inert gas is effective in shortening the irradiation time.

When irradiating with actinic rays, the temperature of the irradiated object rises to some extent, so
In order to prevent the rate of decline in the activity of the immobilized enzyme or microbial cells, the object to be irradiated is preferably cooled to below room temperature or kept in a cooled state.

The immobilized enzyme or microbial cells thus obtained must be used while being kept in a moist state at all times.

This is because there is a large difference in volumetric deformation between the hydrophilic photocurable resin that immobilizes enzymes or microbial cells and the reinforcing base material between the hydrated state and the dry state. This is because, if transferred, the hydrophilic photocurable resin used for immobilization will cause "cracking" and eventually peel off from the reinforcing base material.

Therefore, do not leave immobilized enzymes or microbial cells in a dry state (when not in use, immobilized enzymes or microbial cells should be immersed in water or stored in an atmosphere with 100% humidity). It is necessary to keep it moist at all times.

In this way, there will be no problem of separation between the reinforcing base material and the hydrophilic photocurable resin used for immobilization.
Immobilized enzymes or microbial cells can be used industrially.

By using such a hydrophilic photocurable resin for immobilizing enzymes or microbial cells, the immobilized product can be formed into a film.

This film-like material can be made into various forms for use.

In this way, since enzymes or microbial cells are immobilized using a hydrophilic photocurable resin in the presence of a reinforcing substrate, the mechanical strength of the immobilized product can be significantly increased, and the immobilized product can be constantly kept moist. Since the condition was maintained, defects such as peeling, which had been a problem in the past, could be prevented while maintaining the adhesion between the hydrophilic photocurable resin and the reinforcing base material.

Furthermore, since a hydrophilic photocurable resin is used and active light is used to obtain the immobilized product, it has the advantage of being able to immobilize enzymes or microorganisms without reducing their activity (which is not possible in the past). ing.

The enzyme or microbial cell immobilized in the present invention can be widely used in industrial fields such as pharmaceuticals, fermentation, catalysts, foods, clinical practice, analysis, and purification.

Next, in order to explain the present invention in further detail, examples will be given.

In the examples, parts or percentages are by weight unless otherwise specified.

Example I 9 parts of NK ester 23G (dimethacrylate of polyethylene glycol with a molecular weight of 1000, manufactured by Shin-Nakamura Chemical Industries), 0 dissolved in a 0.1 molar acetate buffer with a pH of 5,6,
100 parts of a 5% glucose oxidase (including catalase) aqueous solution and 1 part of benzoin ethyl ether were uniformly mixed.

Horizontally arranged thickness 0. Inner frame 5c7 with spacer of thickness 0, 511TL on ITtr/L polyester sheet
A square of rl x 5CrIl was made, Zirx Clean (for silk screen printing) was put into the square, and the above mixed solution was poured into the square.

A polyester sheet with a thickness of 0.1 mm was adhered thereon, and the top surface of the sheet was irradiated with a low-pressure mercury lamp for 3 minutes to obtain a transparent membrane-like immobilized enzyme with a silk screen encapsulated therein.

This immobilized enzyme membrane showed a specific activity of 65%.

When this immobilized enzyme membrane was left at room temperature for 30 hours, many cracks appeared in the membrane, but a solution of 0.1 molar acetate buffer (PH
5, 6) maintained a perfect film state without any damage such as cracks even after 720 hours.

Example 2 A silk screen (for silk screen printing) was immersed in the same mixed solution containing glucose oxidase as in Example 1, and while maintaining an anaerobic atmosphere by blowing nitrogen gas, the silk screen was pulled up and immediately removed. ,
A membrane-like immobilized enzyme similar to that in Example 1 was obtained by irradiating with a high-pressure mercury lamp for 3 minutes.

If this immobilized enzyme membrane was left at room temperature for 30 hours, many cracks would occur in the membrane, but if it was stored in an atmosphere with 100% humidity, no damage to the membrane was observed even after 720 hours.

Example 3 100 parts of polyethylene glycol (molecular weight 2000) dimethacrylate, 150 parts of a 1% glucose isomerase aqueous solution dissolved in 001M phosphate buffer with pH 7.5,
One part of benzoin methyl ether was mixed uniformly.

The gauze is immersed in this mixture, and while maintaining an anaerobic atmosphere by aerating nitrogen gas, the base material is pulled up and immediately irradiated from both sides with a xenon lamp for 5 minutes to immobilize the gauze in a membrane. I got the enzyme.

This immobilized enzyme membrane showed a specific activity of 78%.

When this immobilized enzyme membrane was left at room temperature for 20 hours, cracks appeared in the membrane, and areas where the gauze and resin peeled off.When left to stand further, the resin completely peeled off, leaving areas with only gauze. If stored in a 100% atmosphere, 72
No damage to the membrane was observed even after 0 hours had elapsed.

Example 4 Example 1 Instead of ONK ester, 1.5 mol of adipic acid was reacted with 1 mol of Epicoat 1001 resin (manufactured by Shell Chemical Co., Ltd., trade name), and then esterified with 4.5 mol of succinic anhydride, followed by glycidyl methacrylate. A similar membrane-like immobilized enzyme was obtained using Giga 75 photocurable resin (number average molecular weight about 4100) produced by reacting 75 mol of -1-2, and using invertase instead of glucose oxidase.

By immersing the membrane in 0.1 molar acetate buffer (pH 5, 6), the membrane maintained its perfect state for more than 720 hours.

Claims (1)

[Claims] 1 The reinforcing base material is immersed or immersed in a mixed solution in which 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. A method for using immobilized enzymes or microbial cells, which is characterized in that the immobilized enzymes or microbial cells obtained by subsequent lifting and irradiation with actinic rays are used while being kept in a constantly moist state. .