JPS635075B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION Field of Industrial Application The present invention relates to a method for producing gel particles on which enzymes are immobilized, more specifically, using a photocrosslinkable resin.
The present invention relates to a method for producing enzyme-enclosed gel particles having excellent enzyme activity by entrapping enzymes in a gel state.

Conventional technology Up until now, the method for producing gel particles with immobilized enzymes has been to dissolve the enzyme in an aqueous solution of polyvinyl alcohol or its derivatives, and to crosslink the polyvinyl alcohol or its derivatives by irradiating the enzyme with radiation, at the same time immobilizing the enzyme. A method is known (Japanese Patent Publication No. 56-33, Japanese Patent Publication No. 59-13189).

Also known is a method in which radiation is applied not only to polyvinyl alcohol systems but also to general aqueous solutions of water-soluble monomers or polymers, singly or in mixtures (Japanese Patent Application Laid-open No. 157587-1983).

However, since these methods require the use of radiation, they require special equipment and may denature the enzyme, leading to various problems in their practical application.

On the other hand, several methods are known in which enzymes are immobilized using photosensitive resins (Japanese Patent Application Laid-open Nos. 142594-1982, 15703-1980, etc.).

However, according to these methods, enzymes are immobilized in film, particulate, or fibrous resins, so the enzymes involved in the reaction are limited to those exposed on the surfaces of these resins, and are not immobilized. It is inevitable that the amount of enzyme used will be significantly smaller than the amount of enzyme used. This is affected by the thickness of the film, the diameter of the particles, and the size of the fibers, and the efficiency of the enzyme decreases as each increases in size. This is a method of obtaining particles with a target large particle size (Japanese Patent Application Laid-Open No. 157587-1987).

Problems to be Solved by the Invention The first object of the present invention is to provide a method for immobilizing an enzyme in a state in which it can efficiently perform its functions without impairing its action.
A second object of the present invention is to provide a method for producing enzyme-enclosed gel particles that contain water necessary for enzyme reactions and can therefore react smoothly with substrates.

Means for Solving the Problems As a result of extensive research in order to achieve the above object, the present inventors used polyvinyl alcohol having a stilbazolium group, which is a type of water-soluble photosensitive resin, We discovered that this objective could be achieved by photo-crosslinking an aqueous solution containing an enzyme finely dispersed in a hydrophobic medium to form a granular enzyme-encompassing gel with a relatively small particle size. The present invention was made based on this.

That is, the present invention involves dissolving an enzyme in an aqueous solution of polyvinyl alcohol having a stilbazolium group, then adding this aqueous solution to a hydrophobic medium and finely dispersing it to form an emulsion, and then irradiating it with actinic light to photocrosslink it. The present invention provides a method for producing a granular enzyme-enclosed gel having an average particle size of 20 to 100 μm.

The polyvinyl alcohol having a stilbazolium group used in the present invention is known, for example, polyvinyl alcohol or partially saponified polyvinyl acetate, and the general formula (In the formula, R represents a hydrogen atom, an alkyl group, or a lower hydroxyalkyl group, and X ^- represents an anion of a strong acid.) −
(See Publication No. 5761).

In this case, polyvinyl alcohol or partially saponified polyvinyl acetate with a degree of polymerization in the range of 500 to 3000 is suitable, and when partially saponified polyvinyl acetate is used, one with a saponification rate of 70 to 88% is used. is desirable. If the polymerization degree is less than 500, a suitable gel will not be produced even if photocrosslinked,
Furthermore, if the degree of polymerization is greater than 3000 or the degree of saponification of polyvinyl acetate is less than 70%, the resin becomes insolubilized and no gel can be obtained. Similarly, the stilbazolium group is 0.5 to
It is preferable that it is introduced in an amount of 8 mol%.

Next, since immobilization in the method of the present invention does not require conditions that adversely affect the enzyme, such as irradiation, heating, or the use of a crosslinking agent, the enzyme to be immobilized is not particularly limited. For example, hydrolytic enzymes such as amylase, protease, cellulase, hemicellulase, lipase, pectinase, lysozyme, naringinase, hesperidinase, anthocyanase, aminoacylase, urease, invertase, melibiase, dextranase, peptidase, ribonuclease, lactase, glucose redox enzymes such as oxidase, uricase, catalase, riboxygenase, cytochrome C, peroxidase;
Immobilization of isomerases such as glucose isomerase, transferases such as cyclodextrin glucosyltransferase, transaminase, elimination enzymes such as aspartase, hyalorondase, chondroitinase, pectin eliminase, etc. Can be done.

Furthermore, in the present invention, as the hydrophobic medium used to disperse the aqueous solution, an inert solvent that is immiscible with water is used. Examples of such materials include silicone oil, various hydrocarbon oils, liquid paraffin, and the like.

In order to suitably carry out the method of the present invention, 5 to 20% of polyvinyl alcohol having a stilbazolium group is used.
Dissolve in water at a concentration of , add the specified enzyme,
Mix thoroughly. Next, add this aqueous solution to 5 to 20 times the volume of a hydrophobic medium and stir vigorously in a transparent container to finely disperse it to form a W/O emulsion. Irradiate active light over a period of time. In this case, the active light irradiation is performed using a light source commonly used for photocuring of photosensitive materials, such as a high-pressure mercury lamp, a xenon lamp, a metal halide lamp, an arc lamp, a fluorescent lamp, a tungsten lamp, an argon ion laser, a helium cadmium laser, etc. This can be done using By irradiating this active light, polyvinyl alcohol having a stilbazolium group is photo-crosslinked to form gel particles. This photocrosslinking reaction is
Although it varies slightly depending on the type of resin used and the crosslinking conditions, it is usually completed within several minutes to several tens of minutes.

Once the photocrosslinking reaction is complete, the hydrophobic medium is washed away with an enzyme-inert solvent such as n-hexane. In this way, the average particle size is 20 to 100 μm,
Enzyme-enclosed gel particles with a water content of 60-85% are obtained.

The enzyme-encompassing gel thus obtained can be subjected to various enzyme reactions in the same way as conventional immobilized enzymes, such as by filling a column and allowing a substrate-containing solution to flow down, or by adding it to a substrate-containing solution and stirring. Can be done.

Effects of the Invention According to the method of the present invention, it is possible to easily obtain enzyme-enclosed gel particles that exhibit stable enzyme activity over a long period of time, have a high substrate diffusion rate, are highly reactive, and are easy to handle. Therefore, it can be widely used as an industrial enzyme immobilization method.

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

The photocrosslinkable resin used in each example has the formula Average degree of polymerization with 3.95 mol% of stilbazolium groups introduced based on vinyl alcohol units
500, partially saponified polyvinyl acetate (hereinafter abbreviated as PVA-SbQ) with a saponification rate of 88%.

Example 1 PVA-SbQ is dissolved in water to prepare an aqueous solution with a concentration of 9.9% and a pH of 6.3. Collect 18 ml of this aqueous solution and add Rhizopa lipase (Seikagaku Kogyo Co., Ltd.,
6000V/mg) was dissolved in distilled water, added, and thoroughly stirred to obtain 20 ml of an enzyme-containing PVA-SbQ aqueous solution.

Next, put 200ml of silicone oil (manufactured by Shin-Etsu Chemical Co., Ltd., Shin-Etsu KF96, viscosity 10cs/25℃) into a 300ml glass beaker, and use a 6-blade standard turbine with a disk diameter of 30mm to rotate at a rotation speed of 1600 rpm.
While stirring, the oxygen-containing PVA-SbQ aqueous solution prepared as described above was added and suspended, and then photoirradiation crosslinking was performed under direct sunlight while stirring for 30 minutes. Next, the generated gel particles are filtered using a centrifugal filter, and the attached silicone oil is washed and removed using n-hexane.
Gel particles with entrapping enzyme immobilized were obtained.

The gel particles thus obtained have a particle size of 20
~100μm range, typical particle size is 50μm
It was hot. Moreover, the water content was 82% by weight.

Next, 1.34 g of PVA-SbQ gel particles containing 1.4 mg of lipase obtained as described above and 100 ml of olive oil were placed in a 100 ml Erlenmeyer flask, and the hydrolysis reaction was carried out at 35°C while stirring using a magnetic stirrer. The initial reaction rate was determined from the water consumption rate. As a result, the initial reaction rate was 500μM−
Triglyceride/min・mg enzyme.

Example 2 Invertase (manufactured by Seikagaku Corporation, 124V/
PVA-SbQ gel particles entrapping and immobilizing enzymes were obtained in exactly the same manner as in Example 1, except that 120 mg of PVA-SbQ gel particles were used. The particle size of this product was almost the same as that of Example 1, and its content was 79% by weight.

Next, in a 100 ml Erlenmeyer flask, PVA-
1.00 g of SbQ gel particles and 0.5 M (100 ml) of sucrose were added, the pH was adjusted to 4.5 with hydrochloric acid, a hydrolysis reaction was carried out while stirring using a magnetic stirrer, and the initial reaction rate was determined. As a result, the initial reaction rate is
It was 37.3 μM-sucrose/min·mg enzyme.

Example 3 Instead of using the enzyme Rhizopus lipase in Example 1, β-galactosidase (manufactured by Sigma,
PVA-enzyme was entrappingly immobilized in exactly the same manner as in Example 1 except that 448 mg (0.11V/mg) was used.
SbQ gel particles were obtained. The particle size of this product is Example 1
It is almost the same as that of , and its moisture content is
It was 88% by weight.

Next, 13.0 mg of β-galactosidase obtained in this way was placed in a 100 ml Erlenmeyer flask.
PVA-SbQ gel particles 1.00g and O-nitrophenyl-β-D-galactoperanoside 33mM (100ml)
was added, the pH was adjusted to 7.5 with a phosphate buffer, a hydrolysis reaction was carried out while stirring using a magnetic stirrer, and the initial rate of the reaction was determined. As a result, the initial reaction rate was 6×10 ⁻³ μM-O-nitrophenyl-β-D-galactopyranoside/min·mg enzyme.

Claims (1)

[Claims] 1. It is characterized by dissolving an enzyme in an aqueous solution of polyvinyl alcohol having a stilbazolium group, then adding this aqueous solution to a hydrophobic medium and finely dispersing it to form an emulsion, and then irradiating it with active light to photocrosslink it. , average particle size 20-100μm
A method for producing a granular enzyme-encompassing gel.