JPH0583234B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION Field of Industrial Application The present invention relates to a method of immobilizing an enzyme within the resin by using a water-absorbing resin as a holding carrier and subjecting the resin to a crosslinking reaction.

PRIOR ART Enzymes have substrate specificity, positional specificity, and stereospecificity in that they stereoselectively react with a specific position of a substance with a specific structure. For example, compared to general organic chemical reactions, there are fewer side reaction products, and it is possible to improve yields, reduce purification costs, and suppress emissions of environmental pollutants. Furthermore, the enzymatic reaction usually proceeds at room temperature and pressure, which has the advantage of significantly reducing the amount of energy required. For these reasons, in recent years, various applied researches have been conducted on reactions using enzymes, particularly in the fields of manufacturing pharmaceuticals, foodstuffs, etc., and in the field of analytical chemistry. However, enzymes are generally extracted and purified from microorganisms, animals and plants through complicated routes, and are very expensive.Moreover, they are unstable to heat, organic solvents, acidic or alkaline conditions, etc., and easily lose their activity. It is discarded after each reaction, making it extremely uneconomical and unsuitable for use in continuous industrial production processes.

In order to eliminate these disadvantages and take advantage of the advantages of enzymes, research on so-called enzyme immobilization technology, in which enzymes are held in water-insoluble carriers, has recently become active.
There are already many examples of practical use on an industrial scale.

The currently established methods for immobilizing the above enzymes are:
It is broadly classified into inclusion method, adsorption method, crosslinking method and covalent bonding method. Among these, the entrapment method involves enclosing the enzyme in a space within a polymer lattice serving as a holding carrier, and is a method in which the enzyme is encapsulated in microcapsules. In this method, enzyme molecules are physically immobilized within a three-dimensional network structure formed by a polymer or a shell formed by a polymer membrane. Known polymers used here include various synthetic polymers such as polyacrylamide, ethylene-vinyl acetate copolymer, and polyvinyl alcohol, and various natural polymers such as collagen, cellulose, alginic acid, and carrageenan. .
Although the inclusion method has the advantage that the immobilization operation is relatively simple and enzyme elution is less likely to occur compared to the adsorption method, the enzyme elution rate is still lower than that of the covalent feeding method, and This method has the disadvantage that the diffusion rate of the product within the polymer lattice is slow, and the enzyme reaction rate is slow compared to the adsorption method.

The adsorption method is a method in which an enzyme is physically adsorbed and immobilized on an insoluble carrier made of an organic polymer such as a cellulose derivative or polymethacrylate or an inorganic substance such as activated carbon or alumina. This method is easy to operate, and the immobilized enzyme obtained has the advantage of high reaction efficiency because the contact reaction between the substrate and the enzyme occurs on the surface of the carrier, but it has the disadvantage that the enzyme is easily eluted.

The crosslinking method is a method in which a low molecular weight polyfunctional substance is added in the presence of an enzyme and an insoluble carrier to form a crosslinked structure by covalent bonds between the enzymes or between the enzyme and the insoluble carrier, thereby rendering the enzyme insolubilized. Although this method also has the advantage of being relatively simple to operate, it often has the disadvantage that enzyme reaction efficiency is often reduced due to diffusion rate limitation, and enzyme deactivation due to the polyfunctional substance used is often a problem.

In addition, the covalent feeding method is a method in which the active group of the insoluble carrier and the enzyme molecule are chemically bonded directly or through some kind of chemical substance to make the enzyme insolubilized. It is divided into the chemical conversion method, Schiff base formation method, etc. Enzymes immobilized by any of these methods generally have the advantage of having high activity and low elution, but on the other hand, the immobilization procedure is complicated and the types of chemicals used during immobilization are difficult. This has the drawback that enzyme activity may be extremely reduced due to chemical substances, and selection of the chemical substance is difficult.

As mentioned above, each immobilization method has its advantages and disadvantages.Currently, when immobilizing enzymes, the types of enzymes to be immobilized, the purpose of use of the obtained immobilized enzyme, and the reaction conditions between the enzyme and the substrate are Depending on the situation, one of the above methods has been adopted, but it is also important to develop an excellent immobilization method with fewer drawbacks, in particular, the immobilization operation is simple, the resulting immobilized enzyme is less likely to be deactivated, and is stable. Therefore, there is a demand in the industry for the establishment of a new immobilization technology that can be easily applied to industrial processes.

Problems to be Solved by the Invention The present invention solves the drawbacks and difficulties found in the various enzyme immobilization techniques described above, and in particular, the immobilization operation is simple, the resulting immobilized enzyme is less likely to be deactivated, and is stable. The purpose of this study is to establish a new immobilization technology that is easy to apply to industrial processes and has high practical value.

Means for Solving the Problems According to the present invention, after an aqueous enzyme solution is absorbed into a water-absorbing resin having a carboxyl group in the molecule,
A method for producing an immobilized enzyme agent is provided, which comprises bringing the resin into contact with at least one selected from a polyvalent metal salt aqueous solution and a polyamine aqueous solution to cause water release and a crosslinking reaction.

The present inventors have focused on enzyme immobilization techniques, particularly the above-mentioned entrapment method, and in the process of diligently researching the holding carriers used for this, the crosslinking method thereof, the conditions for enclosing enzymes on the carriers, etc. recent years,
When water-absorbing resins, which are increasingly being used as sanitary products and sanitary products, use a buffer solution containing enzymes as the water to be treated, their inherent water-absorbing properties allow them to absorb this into the resin, resulting in It was also discovered that the enzyme was also incorporated into the resin, and that the amount of the enzyme absorbed almost completely matched the enzyme concentration in the water to be treated, and was independent of the type of resin and its absorption capacity. However, the uptake of the enzyme by the above resin is
It is reversible, and once the enzyme is taken up in this state, it is released out of the resin together with the buffer solution and is not immobilized on the resin at all. However, in the case of water-absorbing resins that have carboxyl groups in their molecules, the enzymes When the resin that has incorporated the ions is then put into a polyvalent metal salt aqueous solution or a polyamine solution, the coexistence of the polyvalent metal ions or polyamines reduces the water absorption ability of the resin, and the buffer solution absorbed into the resin is released. However, an ionic crosslinking reaction occurs between the polyvalent metal ion or polyamine and the resin, and as a result, the resin expanded by water absorption shrinks and crosslinks,
Most of the enzyme is enclosed within the network structure of the resin, and some is adsorbed on the resin surface, thus immobilizing the desired enzyme. We obtained a new finding that almost no enzymes entrapped and adsorbed within the resin or on the resin surface are released even if The present invention was completed based on this knowledge. The enzyme immobilization method of the present invention immobilizes the enzyme on a water-absorbing resin by crosslinking and adsorption as described above, and the method itself has the advantages of the conventional entrapment method and adsorption method. That is, in the immobilized enzyme preparation obtained by the method of the present invention, firstly, the enzyme adsorbed on the resin surface can easily contact the substrate on the resin surface, and in this respect, the enzyme reaction efficiency is high, and secondly, the water absorbency used Judging from the crosslinking density of the resin, the network structure formed within the resin is also moderate enough to facilitate contact between the enzyme enclosed within the lattice and the substrate, making it difficult to use conventional enclosing immobilization. The enzyme reaction efficiency is superior compared to enzyme agents, and the enzyme immobilization rate can also be maintained at a high level. As described above, the immobilized enzyme preparation obtained by the present invention has the characteristic that it can simultaneously satisfy the antinomy of enzyme reaction efficiency and enzyme immobilization rate, which were completely unsatisfactory with conventional immobilized enzyme preparations of this kind. ing.

In addition, the water-absorbing resin used in the present invention has excellent mechanical strength, physical strength, chemical stability, etc., and has the advantage that it can be easily handled and molded into any shape. By using the resin, in the method of the present invention, an arbitrary amount of enzyme can be immobilized in the resin depending on the enzyme concentration of the enzyme aqueous liquid as the water to be treated.

The method for producing the immobilized enzyme agent of the present invention will be described in detail below.

In the method of the present invention, the enzyme used is not particularly limited, and various conventionally known enzymes can be used alone or in a mixture of two or more.
Typical examples include oxidizing enzymes such as catalase, glucose oxidase, and peroxidase, transferases such as glycine aminotransferase and leucine aminopeptidase, hydrolytic enzymes such as asparaginase, ilvertase, urease, and lipase, and glucose isomerase. Examples include isomerases such as alanine racemase and glutamate racemase, and ligases such as gratathione, synthetase, and asparagine synthetase.

The water-absorbing resin used in the present invention is not particularly limited, and may be any conventionally known resin, provided that it has water-absorbing ability and can undergo a crosslinking reaction due to the coexistence of polyvalent metal ions or polyamines.

Specifically, the water-absorbing resin used in the present invention has a carboxyl group in its molecule that causes a crosslinking reaction in the presence of a polyvalent metal ion or a polyamine. partially crosslinked products, hydrolysates of starch-acrylonitrile graft copolymers, starch-acrylic acid graft copolymers, poly-acrylic acid or -methacrylic acid (hereinafter referred to as "poly(meth)acrylic acid") Partially crosslinked salts, polyvinyl alcohol-(meth)acrylate copolymers, other polyvinyl alcohol-maleic anhydride-based products, and polyisobutylene-maleic anhydride-based products can be mentioned. Among these, hydrolysates of starch-acrylonitrile graft copolymers, starch-(meth)acrylic acid graft copolymers, partially crosslinked poly(meth)acrylates, and the like are preferred. Each of the above-mentioned water absorbent resins can be manufactured according to a known method. As an example, for example, JP-A-56-
No. 93716, JP-A-56-131608, JP-A-56-
No. 147806, JP-A-56-171559, JP-A-58-
No. 117222, Special Publication No. 1971-30710, Special Publication No. 1977-37994
No. 53-46200, U.S. Pat. No. 4,041,228, etc.

Further, the water absorbing capacity of the water absorbing resin used in the present invention is not particularly limited, but if the water absorbing capacity determined by the following method is usually 10 or more, it can be used without any problem. In general, well-known water absorbent resins, including commercially available products, have a water absorption capacity of approximately 100 to 1000.
within the range of

[Water absorption capacity]

(a) Add 150 g of deionized water and 0.12 g of water-absorbing resin sample to a 200 ml beaker, leave it for 30 minutes, and then
The water was separated with a 200-mesh wire mesh, the weight of the flowing water was measured, and the water absorption capacity was calculated using the following formula.

Water absorption capacity = (weight of water initially added) - (weight of water flowing out) / weight of water absorbent resin sample The gel strength of the above water absorbent resin is determined by the following method, and is usually 1.0 x 10 ³ dynes/ If it is cm ² or more, it can be used without any problem.

[Gel strength]

A gel (hereinafter referred to as 30x gel) was created by mixing 60 g of physiological saline and 2.0 g of a water-absorbing resin sample.
The hardness (surface hardness) of the gel is measured using a Neocard meter manufactured by Iio Electric Co., Ltd. Here, the surface hardness is expressed as a resistance force that prevents the pressure-sensitive shaft from pushing away the gel and entering the sample surface.

In the method of the present invention, first, an aqueous enzyme solution, for example, typically water in which an enzyme is dissolved or dispersed (hereinafter referred to as "enzyme-containing water"), is absorbed into a water-absorbing resin. The enzyme concentration in the enzyme-containing water at this time,
The shape and amount of the water-absorbing resin to be used are appropriately determined depending on the type of enzyme and resin used and the purpose of use of the resulting immobilized enzyme.

The above-mentioned water-absorbing resin may be in a commonly available shape, such as beads, film, flakes, granules,
It can be used in any shape, such as a lump, and its size is not particularly limited, but it is determined as appropriate in consideration of the usage conditions of the immobilized enzyme obtained according to the method of the present invention. The particle size is as follows:
Generally, it is appropriate that it is about 0.1 mm or more, preferably about 5 mm or more. The ratio of the water-absorbing resin to the enzyme-containing water is arbitrarily determined depending on the water-supplying capacity of the water-absorbing resin. Appropriately, the amount of enzyme-containing water is about 100 to 1000 parts by weight per 1 part by weight.

The enzyme-containing water can be easily absorbed into the water-absorbing resin by mixing and stirring the two. This mixing and stirring operation is carried out in order to absorb the enzyme-containing water into each particle of the water-absorbing resin while the enzyme is uniformly dispersed in the water, and can be carried out by a normal stirring method. This stirring causes the enzyme-containing water to be absorbed into each particle of the water-absorbing resin. Furthermore, if necessary, a buffer can be added to the enzyme-containing water. By doing so, the pH in the reaction system during the enzyme reaction can be kept almost constant, and the enzyme can be used in the optimum pH range. The reaction can proceed smoothly. An immobilized enzyme preparation can be easily obtained by the above method, but at this time, excess unimmobilized enzyme can also be removed by washing using deionized water, an aqueous buffer solution, or the like.

According to the method of the present invention, the water-absorbing resin swollen by absorbing the enzyme aqueous solution is then brought into contact with a polyvalent metal salt aqueous solution and/or a polyamine aqueous solution,
Allow water release and crosslinking reaction to occur. The polyvalent metal salt used here includes polyvalent metal ions that can crosslink the carboxyl groups present in the water-absorbing resin molecules, such as calcium, magnesium,
Divalent or trivalent materials such as copper, iron, aluminum, cobalt, etc.
Any type of material that can provide valent metal ions may be used.
Specific examples include chlorides such as calcium chloride, magnesium chloride, ferrous chloride, ferric chloride, cupric chloride, aluminum chloride, cobalt chloride, calcium nitrate, magnesium nitrate, ferrous sulfate, and ferrous sulfate. Examples include diiron and aluminum sulfate. Among these, calcium chloride, ferrous sulfate, ferric sulfate, aluminum chloride, aluminum sulfate, etc. are particularly suitable because they are ionized near neutrality and are not likely to be toxic to enzymes.
The above polyvalent metal salt solution is advantageously used in the form of an aqueous solution, usually with a concentration of about 0.1 to 10% by weight. The amount used slightly affects the enzyme immobilization ability of the obtained immobilized enzyme and the physical strength during its use.
It is best to take these points into account and select an appropriate value, and usually the polyvalent metal ion per mole of carboxyl group contained in the water-absorbing resin molecule used is
The amount is preferably 0.03 mol or more. Contact between the polyvalent metal salt aqueous solution and the enzyme-absorbing resin can be easily carried out by simply adding the resin into the polyvalent metal salt aqueous solution and stirring, and this contact reduces the amount of water absorbed due to a decrease in the water absorption capacity of the resin itself. Simultaneously with the release of the resin, a crosslinking reaction of the resin occurs, and as a result, the desired immobilized enzyme with the enzyme trapped inside the resin can be obtained. The thus obtained immobilized enzyme agent has the enzyme trapped in the internal voids of the cross-linked water-absorbing resin and also retains a small amount of water, but this retained water can be removed by drying if necessary. You can also do it.

In addition, the polyamine aqueous solution used together with or as an alternative to the above polyvalent metal salt aqueous solution has water solubility, viscosity (molecular weight), cationic content, etc. It is appropriately selected from known aqueous solutions of polyamines in consideration of properties and the like. Specific examples thereof include polymers containing cationic monomers such as polyallylamine, polyvinylamine, polyalkylene polyamine, polyethyleneimine, polyvinylpyridinium halide, dialkylaminoalkyl (meth)acrylate, dialkylalkyl (meth)acrylamide, etc. Copolymers or copolymers containing the monomers as a main component can be mentioned. The molecular weight of such polyamines is usually about
Preferably, the amine number is in the range of 1000 to 1,000,000, and the amine number is preferably in the range of about 130 to 1,300.
Further, the concentration of the aqueous solution of the polyamine is appropriately determined in consideration of the viscosity of the aqueous solution, but it is usually preferably about the same concentration as the aqueous solution of the polyvalent metal salt.

The amount of the above polyamine aqueous solution to be used varies slightly depending on the amine value of the polyamine used, but it is usually about 0.01 to 50% by weight, preferably about 0.01 to 20% by weight (all solid content ratio) based on the water absorbent resin. It is appropriate to

The polyamine aqueous solution is used in the same manner as the polyvalent metal salt aqueous solution, and similar results are obtained. That is, by using the polyamine aqueous solution and bringing it into contact with an enzyme-absorbing resin, a crosslinking reaction of the resin occurs simultaneously with the release of absorbed water from within the resin, making it possible to confine the enzyme within the resin. Therefore, of course, both the polyamine aqueous solution and the polyvalent metal salt aqueous solution can be used in combination.

Thus, according to the present invention, the target enzyme can be immobilized easily and with a good immobilization rate, and the resulting immobilized enzyme has excellent enzymatic reaction efficiency and has the properties inherent to the immobilized enzyme. activity,
It has mechanical strength, chemical stability, ease of handling, etc., and is extremely useful for application to industrial processes that utilize enzymes.

Examples Hereinafter, examples of producing the immobilized enzyme of the present invention will be described as examples, but the present invention is not limited to these examples.

Example 1 10 mg of aminoacylase (18.6 units/mg) obtained from pig kidney was dissolved in 10 ml of 0.01 M Tris-HCl buffer (PH = 7.0, containing 0.5% cobalt chloride) and added as a water-absorbing resin. 0.45 g of partially crosslinked polyacrylate (manufactured by Arakawa Chemical Co., Ltd., trade name: Arasorb) was added, and water was sufficiently supplied. By washing this with the same buffer solution, 10.7 g (warm weight) of the immobilized aminoacylase agent was obtained.

The concentration of the aminoacylase agent thus obtained is
30 ml of 0.1 M N-acetyl-DL-methionine solution (adjusted to pH 7.0 with potassium hydroxide, containing 0.2% cobalt chloride) was added, and the enzymatic reaction was then carried out at 37°C for 10 minutes with rotational stirring. Thereafter, the L-methionine produced was quantified by ninhydrin colorimetry.

As a result, the activity of the obtained immobilized enzyme agent was
It showed 10.56 units/g, which corresponded to 60.8% of the activity of the enzyme used. In addition, 1 unit of the above-mentioned enzyme activity refers to the enzyme activity that produces 1 μmol of L-methionine per minute.

Example 2 10 mg of aminoacylase (20.1 units/mg) obtained from pig kidney was dissolved in 10 ml of 0.01 M Tris-HCl buffer (PH = 7.1, containing 1% cobalt chloride) and added as a water-absorbing resin. 0.62 g of partially crosslinked polyacrylate (manufactured by Arakawa Chemical Industries, Ltd., trade name: Arasorb) was added, and water was sufficiently supplied. By washing this with the same buffer containing 0.5% cobalt chloride, 10.8 g of immobilized aminoacylase agent was obtained.
(warm weight) was obtained.

The obtained immobilized enzyme agent was subjected to an enzymatic reaction in the same manner as in Example 1, and its activity was measured, and it was found to be 6.29 units/g. This corresponded to 33.8% of the activity of the enzyme used.

Example 3 Plant proteolytic enzyme papain (21.6 units/
mg) 10mg in 0.005M cysteine, 0.01M ethylenediaminetetraacetic acid and 0.02M citric acid-phosphate buffer (PH = 6.2, containing 0.5% calcium chloride)10
mg, and 0.69 g of Arasorb was added thereto, followed by sufficient water absorption. By washing this with the same buffer solution, 9.5 g (warm weight) of immobilized papain agent was obtained.

Add 30 ml of 0.1 M benzoyl arginine ethyl ester (hereinafter referred to as "BAEE") solution dissolved in the same buffer as above to the obtained immobilized papain agent.
was added, and then an enzymatic reaction was carried out at 37°C for 10 minutes with rotational stirring, and the resulting bezoyl arginine was quantified by measuring with an alkali.

As a result, the activity of the immobilized enzyme agent was 4.3 units/g.
showed that. This corresponded to 19% of the activity of the enzyme used. In addition, 1 unit of the above enzyme activity means 1 unit of enzyme activity per minute.
Refers to the enzyme activity that produces 1 μmol of BAEE by hydrolysis.

Example 4 Trypsin (10 units/mg) obtained from bovine pancreas
10mg was added to 0.02M Tris-HCl buffer (PH=7.5,
(containing 0.5% calcium chloride), 0.70 g of Arasorb was added thereto, and water was supplied sufficiently. By washing this with the same buffer solution, 10.3 g (warm weight) of immobilized trypsin agent was obtained.

The thus obtained immobilized enzyme agent was subjected to an enzymatic reaction using BAEE as a substrate in the same manner as in Example 3, and its activity was measured and found to be 3.26 units/g. This corresponded to 33.6% of the activity of the enzyme used.

Example 5 Fumarase obtained from pig heart (20.5 units/
mg) 10 mg in 0.02M Tris-HCl buffer (PH
= 7.2, containing 0.5% cobalt chloride), 0.79 g of Arasorb was added thereto, and water was sufficiently absorbed. By washing this with the same buffer,
11.7 g (warm weight) of immobilized fumarase agent was obtained.

Add 30 ml of L-malic acid solution with a concentration of 0.2 M (containing 0.1% calcium chloride, adjusted to PH = 7.8 with sodium hydroxide) to the obtained immobilized enzyme agent, and then stir with rotation at 37°C for 10 minutes. After carrying out the enzymatic reaction, the fumaric acid produced is
Quantified by increase in _OD240 .

The resulting immobilized enzyme activity was 0.22
Units/g are shown. This is based on the activity of the enzyme used.
This corresponded to 12.7%. Furthermore, 1 unit of the enzyme means 1
It refers to the activity of producing 1 μmol of fumaric acid per minute.

Example 6 Invertase obtained from yeast (17.4 units/g)
10mg was added to 0.04M citrate-phosphate buffer (PH
= 4.6, containing 0.5% calcium chloride), 0.77 g of Arasorb was added thereto, and water was sufficiently supplied. By washing this with the same buffer,
10.6 g (warm weight) of immobilized invertase agent was obtained.

30 ml of 0.3M sucrose solution dissolved in the above buffer was added to the obtained immobilized enzyme preparation, and then incubated at 37°C for 10
After carrying out the enzymatic reaction with rotational stirring for one minute, the amount of reducing sugar produced was measured by the Wilschüttester-Schüdel method.

As a result, the enzyme activity of the immobilized enzyme agent was 7.36 units/g. This activity corresponded to 44.8% of the activity of the enzyme used. In addition, 1 unit of the above-mentioned enzyme activity refers to the enzyme activity that hydrolyzes 1 μmol of sucrose per minute.

Example 7 10 mg of aminoacylase (9.0 units/mg) obtained from porcine pancreas was mixed with polyallylamine (molecular weight approx.
) was dissolved in 10 ml of 0.01 M Tris-HCl buffer (PH = 7.0) containing 1% solid content, 0.56 g of Arasorb was added thereto, and water was thoroughly absorbed.
By washing this with the same buffer solution, 12.56 g (warm weight) of the immobilized aminoacylase agent was obtained.

The obtained immobilized enzyme agent was subjected to an enzymatic reaction in the same manner as in the example, and its activity was measured.
It showed 3.0 units/g. This corresponded to 42.0% of the activity of the enzyme used.

Example 8 10 mg of aminoacylase (18.3 units/mg) obtained from porcine pancreas was added to polyvinylamine (molecular weight approximately 10
Contains 0.02% solid content (~120,000)
It was dissolved in 10 mg of 0.01M Tris-HCl buffer (PH=7.0), 0.38 g of Arasorb was added thereto, and water was sufficiently absorbed. By washing this with the same buffer, 13.8 g (warm weight) of the immobilized aminoacylase agent was obtained.
I got it.

The obtained immobilized enzyme agent was subjected to an enzymatic reaction in the same manner as in Example 1, and its activity was measured, and it was found to be 7.38 units/g. This corresponded to 55.6% of the activity of the enzyme used.

Claims (1)

[Scope of Claims] 1. After absorbing an aqueous enzyme solution into a water-absorbing resin having a carboxyl group in the molecule, the resin is brought into contact with at least one selected from a polyvalent metal salt aqueous solution and a polyamine aqueous solution to absorb water. 1. A method for producing an immobilized enzyme agent, which comprises performing release and crosslinking reactions. 2 The water-absorbing resin is a hydrolyzate of starch-acrylonitrile graft copolymer, starch-acrylic acid graft copolymer, starch-methacrylic acid graft copolymer, partially crosslinked polyacrylate, and partially crosslinked polymethacrylate The method according to claim 1, wherein the at least one kind selected from the group consisting of: