JPS5810077B2 - Method for producing immobilized enzyme - Google Patents

Description

[Detailed description of the invention] The present invention relates to a method for producing an immobilized enzyme.

Enzyme reactions are also carried out industrially in the process of manufacturing pharmaceuticals, foods, etc., but conventionally enzymes are dissolved in an aqueous solution of a substrate and the reaction is carried out in the solution.

However, with this method, it is difficult to maintain constant reaction conditions, replenish fresh enzyme, and recover the enzyme after the reaction. It's not easy either.

In order to solve these problems, it has already been proposed to use an immobilized enzyme on a carrier to react with a substrate. Enzyme immobilization methods are known.

However, the carrier binding method requires a special carrier and
Complex operations are required to immobilize the enzyme on this carrier,
In general, highly active immobilized enzymes cannot be obtained.

In addition, in the physical adsorption method, since the bond between the carrier and the enzyme is not sufficient, the enzyme is likely to be detached from the carrier during use, and the enzymatic reaction cannot be carried out stably.

Compared to these methods, the entrapment method involves adding an enzyme to a water-soluble polymer and then gelling the polymer using a cross-linking agent or radiation treatment to immobilize the enzyme within the gel lattice. This method is particularly useful industrially because it can be produced easily and in a low-cost manner, and has already been put into practical use to some extent. There is a drawback that there is no

Reducing the crosslink density can prevent a decrease in enzyme activity to some extent, but on the other hand, if the spacing of the enzyme immobilization lattice is wide (
As a result, the elution of the enzyme becomes significant.

This is fatal when enzymatic reactions are carried out continuously and stably on an industrial scale.

The present invention was made in order to solve the various problems described above, and it is possible to easily produce a carrier using a water-insoluble polymer, and the enzyme is firmly bonded to the carrier by a covalent bond. Moreover, it is an object of the present invention to provide a method for producing an immobilized enzyme that maintains high enzyme activity.

In the method for producing an immobilized enzyme according to the present invention, an ethylene-vinyl acetate copolymer comprising 20 to 97% by weight of vinyl acetate units, in which 80 mol% or more of the vinyl acetate units have been saponified, is combined with - molecules. The polymer used in the present invention is characterized in that after a polyaldehyde having at least two aldehyde groups is reacted to form a carrier, an enzyme is covalently bonded to the free aldehyde groups of the carrier.The polymer used in the present invention is ethylene-acetic acid. Saponified vinyl copolymer (hereinafter referred to as EVA copolymer).

) is 3 to 80% by weight of ethylene units, preferably 5 to 60
In the ethylene-vinyl acetate copolymer, 80 mol % or more of the vinyl acetate units are saponified.

In the present invention, a terpolymer in which two parts of the ethylene units are substituted with an ethylenically unsaturated acid such as acrylic acid or maleic anhydride can also be used, and the term "EVA copolymer" It contains such a terpolymer.

In such an EVA-based copolymer, when the ethylene unit content is less than 3% by weight, the stability of the copolymer in water becomes poor, and for example, after forming the copolymer into a film, it is difficult to dissolve the copolymer in water or an aqueous organic solvent. There is a risk that the shape of the molded product will be destroyed when reacting with polyaldehyde.
EVA
The number of hydroxyl groups in the system copolymer is not sufficiently large, so the number of free aldehyde groups after reacting with polyaldehyde is 1.
Furthermore, it is difficult to obtain a sufficiently high enzyme oil level per unit weight or unit area of the carrier.
This is because the hydrophilicity of the copolymer is not sufficient and therefore it is generally suitable for enzymatic reactions which are carried out in an aqueous atmosphere.

In the present invention, the EVA copolymer is dissolved in an appropriate solvent such as dimethyl sulfoxide or a mixed solvent of water and n-solopanol, and then reacted with polyaldehyde in the presence of an acid catalyst to form a gel. The enzyme may be covalently bonded to this carrier after it is made into fine particles by an appropriate method, or the EVA copolymer alone is molded into an appropriate shape by any conventionally known method. Thereafter, the molded product may be immersed in a solvent that does not destroy its shape, such as water or methanol, and reacted with polyaldehyde in the presence of an acid catalyst in this solvent, followed by covalent bonding of an enzyme.

The shape of the molded product is not limited to hair, and may be, for example, granules, pellets, sheets, structures, hollow systems, porous bodies, foams, etc.

In either method, an acid catalyst such as hydrochloric acid, sulfuric acid, P-)luenesulfonic acid, etc. is suitably used for the reaction between the EVA copolymer and polyaldehyde.

In the present invention, polyaldehyde refers to a compound (including a polymer compound) having at least two free aldehyde groups in one molecule.

), specifically dialdehyde compounds such as glyoxal, malondialdehyde, maleic dialdehyde, succinic dialdehyde, adipine dialdehyde, glutardialdehyde, pimeline dialdehyde, speridine dialdehyde, terephthal dialdehyde, etc. Polymeric polyaldehydes such as , dialdehyde starch, and polyacrolein can be used.

In the present invention, when a carrier is prepared by reacting an EVA copolymer with a polyaldehyde, it is necessary to leave free aldehyde in the carrier.

Therefore, when dialdehyde compounds are used, some of the aldehyde groups must acetalize the copolymer, while the other aldehyde groups must remain unreacted.

Furthermore, when a polyaldehyde having three or more aldehyde groups in one molecule is used, a part of the polyaldehyde is bonded to the copolymer through at least one aldehyde group, while the copolymer is cross-linked. It may be used or not, but at least one aldehyde group must remain unreacted.

The required amount of polyaldehyde can be easily determined experimentally depending on the reaction method of polyaldehyde and EVA copolymer.

Generally, when a certain amount of EVA copolymer is reacted with polyaldehyde, when the polyaldehyde exceeds a certain amount, the amount of free aldehyde in the crosslinked copolymer becomes approximately constant.

In the present invention, the crosslinked copolymer thus obtained can be suitably used by reacting a polyaldehyde with a copolymer in an amount close to the minimum amount so that the aldehyde groups in the crosslinked copolymer are almost constant. Of course, there is no particular limit to the amount of unreacted free aldehyde groups left in the crosslinked copolymer as long as the enzyme activity can be obtained in the immobilized enzyme.

In order to covalently bond an enzyme to a carrier made of the acetalized EVA copolymer thus obtained, preferably the enzyme is dissolved or dispersed in a buffer solution at a relatively low temperature, and the carrier is immersed in this solution. Let stand or stir.

However, the reaction conditions between the carrier and the enzyme are not particularly limited as long as the enzyme activity is not lost.

Therefore, the reaction temperature is appropriately selected from 6°C or higher, the temperature up to the inactivation temperature of the enzyme, and the pH from 1 to 11, but usually the temperature is from 0 to 40°C and the pH is from 65 to 8. Let's do it.

The reaction time may range from several minutes to several tens of hours, and is appropriately selected.

The enzyme that can be used in the present invention is an enzyme that has a reactive group that can covalently bond to a free aldehyde group of a carrier, and typically has a free amine group.

Specific examples of such enzymes include oxidoreductases such as glucose oxidase and catalase, transferases such as aspartate amine transferase and histamine transferase, lipase, α-amylase, β-amylase, urease, α-chymotrypsin, etc. Examples include hydrolases, isomerases such as glucose isomerase and alanine racemase, and glue gauzes such as asparagine synthase and glutamine synthase.

The immobilized enzyme, in which the enzyme is covalently immobilized on the carrier as described above, can be extracted from a reaction medium such as a buffer solution,
It can be easily separated by means such as centrifugation,
This is washed, freeze-dried, etc., or stored in a buffer solution at low temperature to produce a product.

In the method of the present invention, as described above, a JEVA copolymer is acetalized with polyaldehyde to form a stable carrier, and an enzyme is covalently immobilized on unreacted free aldehyde groups contained in this carrier. Because of this, there is no elution or separation of the enzyme from the carrier, the enzyme is bound to the carrier without losing its activity, and furthermore, the enzyme activity is maintained at a high level.

In addition, since the EVA copolymer is easy to mold and a carrier can be obtained by reacting it with polyaldehyde, the carrier is also easy to manufacture.

Examples of the present invention are listed below, but the present invention is not limited to these Examples at all.

Example 1 Ethylene-vinyl acetate copolymer (ethylene unit content 13
Weight%, 98 of Nippon Gosei Kagaku Kogyo Soarex FH)
Mol% 20 g of saponified product 10 ml of water - 20 ml of acetone
-Dissolved in 70ml of dimethyl sulfoxide mixed solvent, applied to a thickness of 300μ on a glass plate, immersed in water at 50°C for 1 hour to solidify and form a film, peeled off from the glass plate and thoroughly washed with water. 100% IN hydrochloric acid at 30°C.
1 g of terephthalaldehyde was dispersed in m1, and the membrane was immersed in this dispersion for 24 hours, and then thoroughly washed with water and methanol in that order to obtain a membrane carrier.

Phosphate buffer (0.02M) containing urease 100m9
The membranous carrier was immersed in 1.00 ml of urease-free solution at 4° C. for 12 hours, and then thoroughly washed with the same buffer solution as above, which does not contain urease, to obtain an immobilized enzyme.

When the immobilized enzyme thus obtained was immersed in a 3% by weight aqueous urea solution (tris(oxymethyl)aminomethane-hydrochloric acid buffer, 0.1M, pH 6,7) at a temperature of 20°C, 7.
It showed an enzyme activity of 9 U/cm2.

Note that the ammonia generated in the reaction with the urea aqueous solution is 0. The enzyme activity that generated 1 mg of ammonia in 5 minutes was defined as 1 unit (U) by titration with IN hydrochloric acid.

Reference Example The film-like molded product obtained in Example 1 was soaked in an IN hydrochloric acid aqueous solution containing various concentrations of glutardialdehyde and 10% sulfur salt for 40 minutes.
After being immersed for 3 hours at a temperature of 0.degree. C. to acetalize, the sample was thoroughly washed with an aqueous sodium carbonate solution, distilled water, and methanol in this order, and then dried.

This carrier was prepared by the bisulfite method, that is, 50 ml of IN sulfuric acid was added to 250 ml of 1M sodium bisulfite aqueous solution.
The above-mentioned carrier was immersed in an aqueous solution prepared by gradually adding and reacted, and the amount of free aldehyde groups in the carrier was measured using a method of titration with IN sodium hydroxide.

The results are shown in the drawing. Example 2 20 g of the same EVA copolymer as in Example 1 was dissolved in 100 g of dimethyl sulfoxide, 30 g of a 50% by weight aqueous glutardialdehyde solution was added, and then 5 g of p-)luenesulfonic acid was added to the solution. The mixture was stirred.

This aqueous solution was applied to a thickness of 100μ on a glass plate, and
A film-like material was obtained by standing overnight at a temperature of .degree. C., which was washed with water and then thoroughly washed with a phosphate buffer (0.05M, pH 7.5) to obtain a carrier.

Lysozyme (manufactured by Shikuma, 40800U/m9) 0.1
2 was dissolved in phosphate buffer (0.05M, pH 7.5), and 10cnf of the above membrane carrier was immersed overnight at a temperature of 70°C. )
The immobilized enzyme was obtained by thorough washing with water.

The absorbance at 660 nm is 0.0 for a cell with an optical path length of 1 cm.
Micrococcus Iy adjusted to be 7
sodeikticus suspension phosphate buffer (0,05
M.

Add 14 cm2 of the above immobilized enzyme to 5 ml of pH 7.0).
The absorbance at 660 nm was measured at 0 per minute.
．． When the enzyme activity when reducing 001 is IU,
22. It showed an activity of IU/cm.

This immobilized enzyme was added to the same freshly prepared buffer as above for 1 hour.
After soaking for 0 minutes, enzyme activity was measured.

The same reaction was repeated five times in this way, and changes in enzyme activity were examined.

The results are shown in the table below.

[Brief explanation of drawings]

The figure is a graph showing the relationship between the concentration of glutardialdehyde reacted with an EVA-based copolymer and the amount of free aldehyde groups in the acetalized copolymer.

Claims (1)

[Claims] 1 80 mol% of vinyl acetate units in an ethylene-vinyl acetate copolymer consisting of 20 to 97% by weight of vinyl acetate units
The above-saponified polymer is reacted with polyaldehyde, which has a small number of aldehyde groups in the molecule (both have two aldehyde groups), and is used as a carrier, and an enzyme is covalently bonded to the free aldehyde groups of this phase. Characteristic method for producing immobilized enzymes.