JPS6148919B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a method for immobilizing enzymes using ampholyte-type crosslinked polymers. Enzymes are substances with extremely high action specificity that are used as catalysts in a wide range of fields such as the textile industry, food processing industry, and pharmaceutical manufacturing industry. However, in reaction systems that use enzymes, 1. The enzyme itself is unstable to heat, strong acids, strong alkalis, organic solvents, etc., and is deactivated relatively quickly even under conditions suitable for enzymatic reactions, resulting in short reaction times. The reaction rate decreases over time.2 Enzyme reactions use a batch method in which the enzyme is dissolved in water and acts on the substrate.After the desired reaction is completed, the enzyme is denatured and removed to form a product. 3. When using a crude enzyme solution for industrial enzymatic reactions, proteins other than enzymes and colored substances may not be present in the catalyst. Since such impurities are present, an operation to remove them is required, which complicates the separation and purification of the reaction product, resulting in a decrease in yield. Recently, in order to eliminate the drawbacks mentioned above, so-called immobilized enzymes have been changed to stable, water-insoluble types that have enzymatic activity and can be treated in the same way as solid catalysts used in general chemical reactions. has been developed, and its immobilization methods include (a) a carrier-binding method in which the enzyme is bound to a water-insoluble carrier; (b) a cross-linking method in which the enzyme is cross-linked by reacting with a bifunctional reagent; and (c) the enzyme is bonded to a gel lattice. Encapsulation methods have been proposed in which the membrane is wrapped in a membrane or covered with a semipermeable polymer. Among the above methods, the carrier binding method involves immobilizing the enzyme on an insoluble solid surface by chemical bonding, ionic bonding, or physical adsorption, but enzyme activity is sensitive to changes in its molecular structure. However, when it is chemically bonded to other molecules, the probability of deactivation increases significantly. On the other hand, in the case of ionic bonding or physical adsorption, although it is easy to operate, enzymes tend to fall off from the carrier due to changes in ionic strength or pH, and the original purpose of enzyme immobilization may not be fully achieved. do not have. In addition, the cross-linking method is a method in which enzyme molecules are cross-linked and immobilized using a bifunctional reagent using the functional groups in the enzyme molecules, and because it involves a chemical reaction, it not only reduces the activity of the enzyme but also creates complex In addition, encapsulant activity is achieved by incorporating the enzyme into a polymer gel lattice or by using a coating of polymers such as nylon or polyurea. The currently proposed method of incorporating enzymes into polyacrylamide gels involves microencapsulation, but the enzyme itself is not chemically bonded to the polymer that makes up the gel, and the gel lattice is too large. However, if the lattice is too small, the speed of the enzyme reaction will be slow, making it difficult to adjust the lattice size. In the present invention, the gel that confines the enzyme is composed of a polymer that has an extremely high affinity for the enzyme, and it does not have the above-mentioned drawbacks, that is, it has stable efficacy over a long period of time without reducing the activity of the enzyme. This has made it possible to produce immobilized enzymes that can exhibit the following effects. That is, the present inventors believe that since enzymatic reactions that utilize the specificity of enzymes are generally carried out using an aqueous system, the carrier for immobilizing the enzyme, that is, the gel that confines the enzyme, is water-insoluble. However, in order to express the activity of the enzyme, it is desirable that the polymer used as a carrier be hydrophilic. It was discovered that the compound exhibits a stable catalytic action over a long period of time, leading to the completion of the present invention. The present invention is based on the general formula (In the formula, R ₁ is hydrogen or -CH ₃ , R ₂ is -
CH ₂ CH ₂ -, R ₃ and R ₄ are -CH ₃ or -C ₂ H ₅
, R ₅ is hydrogen or

[Formula], X represents -Cl, -Br or -OH) and a cationic monomer represented by the general formula (In the formula, R ₆ is hydrogen or -CH ₃ , Y is -
COOH, ―SO ₃ H or

An amphoteric electrolyte obtained by radical copolymerization of an anionic monomer represented by [Formula] or an alkali metal salt or ammonium salt thereof) in the presence of a crosslinking monomer having two ethylenically unsaturated groups. The enzyme is immobilized by trapping the enzyme in the gel lattice of the type polymer. Therefore, the polymer used as a carrier has water insolubility due to the crosslinking introduced by the crosslinking monomer, and compatibility with the reaction medium due to the cationic and anionic groups derived from the cationic and anionic monomers. This imparts hydrophilicity to the carrier, thereby simultaneously imparting water insolubility and hydrophilicity necessary for a carrier when immobilizing an enzyme. Furthermore, in the present invention, the polymer that serves as an enzyme carrier and confines the enzyme in the gel lattice is an ampholyte type that retains the ampholyte electrolyte derived from the monomer represented by the above general formulas () and (). Because of this, they have a very high affinity for enzymes, which are the same ampholyte compounds, and keep the enzymes very compatible with the surrounding carrier, thus providing them with a stable and favorable environment. Catalytic action by enzymes takes place at the enzyme's active center (binding site), but enzymes have the property of being affected by the surrounding environment, such as electric fields, which tends to reduce their activity. However, in the present invention, since the polymer used as a carrier is an ampholyte, the polymer itself exhibits affinity with the enzyme due to its positive and negative charges, while the monomer composition of general formulas () and () By selecting a suitable amount, it is possible to impart electrolyte properties that do not adversely affect enzyme activity. The present invention is constructed as described above, and the monomers represented by the general formulas () and () transform the polymer used as a carrier into an ampholyte type, thereby imparting affinity for the enzyme. In addition, water insolubility is imparted by the crosslinking structure introduced by the crosslinking monomer present when the monomers represented by the general formulas () and () are radically copolymerized. This is a new enzyme immobilization method that utilizes a polymer that has all the properties necessary for a polymer carrier. Examples of the cationic monomer represented by the general formula () used in the present invention include dimethylaminoethyl acrylate hydrochloride, diethylaminoethyl acrylate hydrochloride, dimethylbenzylacryloxyethylammonium chloride, diethylbenzylacryloxyethylammonium Examples of anionic monomers represented by the above general formula () include chloride, bromide compounds corresponding to these chloride compounds, oxide compounds, and methacrylate compounds corresponding to acrylate compounds. acids, methacrylic acid, sodium acrylate, sodium methacrylate, potassium acrylate, potassium methacrylate, ammonium acrylate, ammonium methacrylate, and vinylsulfonic acid, p-styrenesulfonic acid, and alkali metal salts thereof or a compound such as an ammonium salt, and the same general formulas () and ()
Examples of crosslinking monomers having two ethylenically unsaturated groups that are used with are methylenebisacrylamide, divinylbenzene or (In the formula, R ₇ , R ₈ and R ₉ are hydrogen or -
diethylene glycol diacrylate, diethylene glycol dimethacrylate, expressed as ( _CH3 , k represents 0 or an integer from 1 to 20);
and triethylene glycol diacrylate, hexaethylene glycol diacrylate, decaethylene glycol diacrylate, tetradecaethylene glycol diacrylate, octadecaethylene glycol diacrylate, eicosaethylene glycol diacrylate, nonapropylene glycol diacrylate, tetradecapropylene glycol Compounds such as monoethylene glycol di(meth)acrylate and polyethylene glycol (meth)acrylate, such as diacrylate and methacrylate compounds corresponding to these acrylate compounds, can be used. In the enzyme immobilization method of the present invention, the molar ratio of each monomer and crosslinking monomer represented by the general formula () and general formula () used when performing radical copolymerization in the presence of an enzyme. can be selected experimentally, but is preferably selected within the following ranges: 0.85 to 0.1 mol of the monomer represented by the general formula () 0.85 to 0.1 mol of the monomer represented by the general formula () 0.01 to 0.4 mol of the crosslinking monomer . In particular, the molar ratio between the monomers represented by the general formula () and the monomers represented by the general formula () is selected by balancing the enzyme activity to be immobilized and the charge amount of the produced polymer. One of the features of the present invention is that the amount of polymer charge can be changed in consideration of the balance with enzyme activity, and therefore it is possible to immobilize a wide range of enzymes depending on the purpose of use. In addition to these three monomers, nonionic water-soluble monomers such as acrylamide, acrylonitrile, hydroxyethyl acrylate, and hydroxyethyl methacrylate can also be copolymerized. The present invention copolymerizes the monomers described above in the presence of an enzyme, and any general radical polymerization initiator can be used as the polymerization initiator, such as tertiary butyl hydroxide. peroxide, organic peroxides such as cumene hydroperoxide, aliphatic azo compounds such as α,α′-azobisisobutyronitrile, 2,2′-azobis(2-amidinepropane) hydrochloride, ammonium persulfate. , inorganic peroxides such as potassium persulfate,
Common radical polymerization initiators such as nitrates such as ammonium nitrate and potassium nitrate, as well as redox initiators such as ammonium persulfate-sodium sulfite, ammonium persulfate-sodium sulfite-hydroquinone, and ammonium persulfate-iron sulfate-ascorbic acid. You can also use When using an oxidizing radical polymerization initiator, if the amount of the initiator increases, the enzyme may be deactivated during polymerization, so the amount used must be appropriately controlled. For example, in the case of APS-catalase, it seems to be inactivated when the amount of initiator exceeds 3 to 4% based on the monomer. In addition, when using redox initiators,
When hydroquinone is added, the polymerization yield tends to decrease. Further, it is preferable to use a low temperature redox initiator, ammonium persulfate-iron sulfate-ascorbic acid system, and conduct the polymerization at a low temperature, since this will reduce the influence of temperature on enzyme activity. Polymerization time varies depending on the type of initiator used, but if the time is too long, the enzyme will be oxidized in the case of an oxidizing initiator, reducing enzyme activity, so long polymerization should be avoided. . The monomer concentration in the water solvent in the polymerization reaction system is
It can be carried out within a range of 10 to 90%, and the yield is generally 90% or more. Almost all kinds of enzymes can be immobilized by the method of the present invention, and among them, catalase, glucose oxidase, D-amino acid oxidase, L-amino acid oxidase, α-amylase, β-amylase, arginase, cholinesterase, It has a particularly remarkable effect on β-glycosidase, β-glucuronidase, invertase, lipase, pepsin, peroxidase, ribonuclease, trypsin, urease, uricase, etc. Next, the enzyme immobilization method of the present invention and the activity of the immobilized enzyme will be explained based on Examples. Example 1 Dissolve 3 g of dimethylaminoethyl methacrylate, 2 g of methacrylic acid, and 0.2 g of methylene bisacrylamide in 360 ml of water, adjust the pH to 7 with sodium hydroxide, and then add 250 mg of catalase and 26 mg of ammonium persulfate as a polymerization initiator. , the polymerization reaction was carried out in a constant temperature bath at 20 °C. The reaction solution lost its fluidity and turned into a gel after 20 minutes, but the polymerization reaction was continued for 5 hours, and the gel-like substance formed was taken out, crushed in a mortar, and then added to a phosphate buffer solution with a pH of 6.8. After thorough washing and subsequent filtration, the immobilized catalase was collected. To check the activity of the obtained immobilized catalase, add 10 ml of 30% hydrogen peroxide and phosphate buffer of pH 6.8.
Add 1 g of the gel-like substance obtained by the above-mentioned over-operation to a mixed reaction solution of 50 ml and 190 ml of water, and stir for 30 min.
The reaction was carried out at ℃, and the decomposition rate of hydrogen peroxide was measured after 10 minutes. As a result, a decomposition rate of 95% was obtained. Next, after the reaction was completed, the gel used in the reaction was collected using a glass filter, thoroughly washed with a pH 6.8 buffer, filtered, and the activity was measured again in the same manner as above. Table 1 shows the decomposition rate of hydrogen peroxide in the 1st, 3rd, 5th, and 10th reactions of immobilized catalase measured by this method.

[Table] Example 2 Dissolve predetermined amounts of dimethylaminoethyl methacrylate, acrylic acid, and methylene bisacrylamide as shown in Table 2 in 36 ml of water, and adjust the pH to 7 using sodium hydroxide or hydrochloric acid. 250mg of catalase and polymerization initiator
26 mg was added and reacted for 5 hours in a constant temperature bath at 20°C to obtain a gel-like substance. In order to examine the activity of the immobilized catalase in the obtained gel-like substance, it was purified under the same conditions as in Example 1, and then the decomposition rate of hydrogen peroxide was measured under the same conditions as in Example 1. Table 2 shows the polymerization reaction conditions of this example.

[Table] Example 3 Dissolve 3 g of dimethylaminoethyl methacrylate, 2 g of acrylic acid and 1 g of polyethylene glycol dimethacrylate in 36 ml of water, adjust the pH to 7 with sodium hydroxide, and then add catalase.
250mg, 26mg of ammonium persulfate as a polymerization initiator
was added, and the polymerization reaction was carried out in a constant temperature bath at 30°C. The reaction solution lost fluidity and became a gel after 15 minutes, but the reaction was allowed to continue for 5 hours, and then a gel-like substance was collected and purified in the same manner as in Example 1. When used as a hydrogen peroxide decomposition catalyst according to method 1, the decomposition rate of hydrogen peroxide was 96%. Example 4 A polymerization reaction was performed using glucose oxidase instead of catalase in Example 1, and the resulting gel-like substance was subjected to the same purification treatment as the gel-like substance in Example 1 to collect immobilized glucose oxidase. did. In order to check the activity of the obtained immobilized glucose oxidase, 0.9 g of β-D-(+) glucose was dissolved in 100 ml of phosphate buffer solution of pH 6.86, oxygen was blown into this, and then the above immobilized glucose was dissolved. When 2 g of a gel-like substance, which is oxidase, was added and reacted at 30°C, the glucose oxidation rate was measured after 100 minutes, and an oxidation rate of 65% was obtained. Example 5 Dissolve 3 g of dimethylaminoethyl methacrylate, 2 g of methacrylic acid, and 0.2 g of methylene bisacrylamide in 36 ml of water, and PH it with hydrochloric acid.
After adjusting the temperature to 4.8, add 250 mg of invertase and 26 mg of ammonium persulfate as a polymerization initiator, and heat at 20℃.
The polymerization reaction was carried out in a constant temperature bath. The reaction solution lost its fluidity and became a gel after 40 minutes, but the polymerization reaction was continued for 7 hours, and the gel-like substance produced was taken out, crushed in a mortar, and then added to an acetate buffer solution with a pH of 4.8. After thorough washing and subsequent over-operation, a purified gel-like substance of immobilized invertase was collected. Next, in order to examine the activity of this immobilized invertase, 1.7 g of sucrose was added to 100 ml of acetate buffer at pH 4.8.
When 1 g of the gel-like substance of the immobilized invertase was added to a solution containing dissolved invertase and the reaction was carried out with stirring at 30°C, the conversion rate of sucrose after 30 minutes was 52
It was %. Example 6 3 g of dimethylbenzylacryloxyethylammonium chloride and 2 acrylic acid in 36 ml of water
g, polyethylene glycol dimethacrylate 1
After adjusting the pH to 7 with sodium hydroxide, 250 mg of catalase and 26 mg of 2,2'-azobis(2-amidinopropane) hydrochloride as a polymerization initiator were added, and the mixture was heated in a constant temperature bath at 20°C. A polymerization reaction was carried out. The reaction solution lost its fluidity and became a gel after 30 minutes, but the polymerization reaction was allowed to continue for 5 hours and a gel-like substance was collected. When this was purified under the same conditions as in Example 1 and then used as a hydrogen peroxide decomposition catalyst under the same conditions as in Example 1, the decomposition rate of hydrogen peroxide was 89%. Example 7 Dimethylbenzylacryloxyethylammonium chloride in 15 g of phosphate buffer at pH 6.86
200 mg of catalase, 36 mg of ammonium persulfate as a polymerization initiator, 0.4 mg of ferrous sulfate, and 30 mg of ascorbic acid were added to a solution containing 2.5 g of sodium styrene sulfonate, 2.5 g of sodium styrene sulfonate, and 0.2 g of methylenebisacrylamide, and the mixture was heated at 15°C. The reaction was carried out in a constant temperature bath. The reaction solution lost its fluidity and became a gel after 50 minutes, but the polymerization reaction was allowed to continue for 20 hours and then the gel-like substance was collected. This was purified under the same conditions as in Example 1, and then an activity test as a hydrogen peroxide decomposition catalyst was conducted under the same conditions as in Example 1, and the decomposition rate of hydrogen peroxide was 85%.

Claims (1)

[Claims] 1. In the presence of an enzyme, the general formula (In the formula, R ₁ is hydrogen or -CH ₃ , R ₂ is -
CH ₂ CH ₂ -, R ₃ and R ₄ are -CH ₃ or -C ₂ H ₅
, R ₅ is hydrogen or [formula], X is -Cl, -Br or -OH) and the general formula (In the formula, R ₆ is hydrogen or -CH ₃ , Y is -
Anionic monomers represented by COOH, --SO ₃ H or [Formula] or their alkali metal salts or ammonium salts) are radically co-coated in the presence of a crosslinking monomer having two ethylenically unsaturated groups. A method for immobilizing an enzyme using an ampholyte type polymer characterized by polymerization.