JPH0412108B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a method for producing an immobilized enzyme, particularly a membrane-like immobilized enzyme, which exhibits low enzyme elution and high activity. Enzymes are used in pharmaceuticals, food industry, analytical chemistry, etc. due to their selectivity in reacting with specific substances. However, enzymes are generally extracted and purified from microorganisms using complicated means and are therefore expensive, and when using enzymes industrially, they must be discontinuously processed, which limits their use. .
Regarding this, research began around 1950 on so-called immobilization, which involves holding enzymes on water-insoluble carriers.
Industrial application became possible in the 1960s, and immobilized enzymes are now widely used. Various methods for immobilizing enzymes have been developed, and these are broadly classified into entrapment methods, adsorption methods, crosslinking methods, and covalent bonding methods. Among these, the entrapment method is a method in which the enzyme is enclosed in a space within a polymer lattice or in which the enzyme is encapsulated in a microcapsule. That is, the enzyme molecules are physically immobilized within the three-dimensional network structure formed by the polymer or within the shell formed by the polymer membrane. In this case, the polymers used include synthetic polymers such as polyacrylamide, ethylene-vinyl acetate copolymer, polyvinyl alcohol, etc., and natural polymers such as collagen, cellulose, alginic acid and its salts, agar, carrageenan, etc. The inclusion method has the advantage of being relatively easy to operate and the enzyme is difficult to elute, but it has some disadvantages compared to the covalent bond method described below. Furthermore, it should be noted that the comprehensive method is difficult to apply because the diffusion rate of the substrate and enzyme within the polymer lattice often determines the rate of the overall reaction. be. In addition, when polymerizing a mixture of polymerizable monomers and enzymes by an appropriate method, conditions must be set so as not to deactivate the enzymes, and when enclosing enzymes in natural polymers, Similarly, consideration must be given to operating at low temperatures so as not to reduce enzyme activity. The adsorption method is a method in which enzymes are adsorbed and immobilized on an insoluble carrier. Examples of the insoluble carrier include organic polymers such as DEAE cellulose, cellulose ester, and polymethacrylate, and inorganic substances such as glass, activated carbon, alumina, and diatomaceous earth. This method is easy to operate, and since the enzymatic reaction proceeds on the surface of the carrier, there is no problem of substrate or product diffusion, unlike the above-mentioned inclusive method, and since adsorption and desorption are reversible, It has the advantage that it can be used repeatedly, but it has the disadvantage that the enzyme is easily eluted. The crosslinking method involves adding a low molecular weight polyfunctional substance to an enzyme solution or a mixed solution of an enzyme and an insoluble carrier to form a crosslinked structure between enzyme molecules or between an enzyme and an insoluble carrier through covalent bonds. This is a method to insolubilize. Glutaraldehyde is the most common polyfunctional substance. Insoluble carriers include alkylaminated porous glass, polyethyleneimine-coated glass, gelatin, collagen, and the like. Although this method is relatively easy to operate, it requires a large amount of enzyme, has low efficiency, and often suffers from deactivation of the enzyme by polyfunctional substances. The covalent bonding method is to make the enzyme insolubilized by chemically bonding the active group of the insoluble carrier and the active group of the enzyme molecule, either directly or through some kind of chemical substance. method, diazo method, alkylation method, Schiff salt formation method, Ugi reaction method, etc. Insoluble carriers include natural organic polymers such as dextran, cellulose, and collagen, nylon,
Examples include synthetic organic polymers such as polystyrene, polyacrylamide, and polyvinyl alcohol, and inorganic substances such as silica, titania, and zirconia. Enzymes immobilized by this method generally exhibit high activity, sometimes even higher than the original enzyme. It also has the advantage of extremely little enzyme elution. However, the disadvantage is that the substance used for immobilization may reduce enzyme activity, and furthermore, the procedure is complicated. Examples of the form of the immobilized enzyme include granular, fibrous, and film forms. Among these, membrane-immobilized enzymes have the advantage of almost no pressure loss within the enzyme reaction vessel, and are used in sensors that utilize enzyme reactions, such as enzyme electrodes. Membrane-like immobilized enzymes have been produced using synthetic organic polymer membranes as base materials by various methods including adsorption, crosslinking, and covalent bonding. However, the immobilized enzymes produced by these methods have the above-mentioned drawbacks and cannot be said to have sufficient performance yet. The present inventors focused on the characteristic that enzyme activity is high in the comprehensive method, and as a result of repeated research to improve the shortcomings of the comprehensive method, acidic polysaccharides are extremely excellent as a base material for the comprehensive method. We also discovered that when reacted with a quaternary ammonium salt, it becomes insolubilized, and that this reactant has the extremely unique property of blocking the permeation of high-molecular substances, but allowing low-molecular substances to freely permeate. The invention has been completed. The present invention provides an acidic polysaccharide solution containing an enzyme,
A method for producing an immobilized enzyme characterized by treating with one or more quaternary ammonium salts selected from benzalkonium chloride, cetyltrimethylammonium bromide, cetylpyridinium chloride, and methylene blue. Used in the present invention. Any enzyme can be used, such as oxidizing enzymes such as catalase, glucose oxidase, peroxidase, etc., transfer enzymes such as glycine aminotransferase, leucine aminopeptidase, etc., hydrolytic enzymes such as asparaginase, invertase, urease, lipase, etc. Examples include lyases such as aspartase, glutamate decarboxylase, fumarate hydrolase, etc., isomerases such as glucose isomerase, alanine racemase, glutamate racemase, etc., and ligases such as glutathione synthetase, asparagine synthetase, etc. Examples of acidic polysaccharides include water-soluble polysaccharides having acidic groups such as carboxyl groups and sulfate groups, such as sodium alginate, carrageenan, furcerelan, agar, pectin, gum arabic, and mucopolysaccharides such as heparin and chondroitin sulfate. Modified polysaccharides, in which acidic groups have been introduced into polysaccharides, are used. Examples of neutral polysaccharides include cellulose, starch, guar gum, and locust bean gum.
Acidic groups can be introduced by carboxymethylation or sulfation reactions. In carrying out the present invention, acidic polysaccharides are dissolved in water using conventional methods. At this time, heat as necessary. The enzyme to be immobilized may be added at the same time as the acidic polysaccharide, or at any time before or after. However, when heating and dissolving acidic polysaccharides with enzymes added, it is necessary to do so within a temperature range that does not reduce oxygen activity. In addition, in the case of adding oxygen to an acidic polysaccharide solution, the solution needs to have sufficient fluidity in a temperature range where the activity of the added enzyme is sufficiently high. When used, the lower the concentration, the better. Next, the polysaccharide aqueous solution containing the enzyme is insolubilized using a quaternary ammonium salt. The quaternary ammonium salt used in the present invention is a quaternary ammonium salt selected from benzalkonium chloride, cetyltrimethylammonium bromide, cetylpyridinium chloride, and methylene blue, and these may be used alone or as a mixture of two or more. used. The quaternary ammonium salt can be used either in aqueous solution or in solid form. In the present invention, by using the above-mentioned specific quaternary ammonium salt, it is also possible to use acidic polysaccharides such as gum arabic that do not have gelling ability, which was extremely limited in conventional comprehensive methods. It becomes possible to select the base material from a wide range, and it also becomes possible to use a polysaccharide solution with a low concentration, and it is possible to solve problems such as inhibition of substrate movement and decrease in oxygen activity. Any method may be used as a treatment method as long as the two come into contact. In particular, when producing a film-like immobilized enzyme, a polysaccharide solution containing the enzyme may be made into a solution film and then subjected to water solubilization treatment. In order to form a liquid film, a method such as casting on a water-insoluble plate such as a glass plate, a stainless steel plate, or a synthetic resin plate is used. The insolubilization process is completed in a few seconds. By the insolubilization treatment, a double structure consisting of a thin layer of an insoluble reaction product of an acidic polysaccharide and a quaternary ammonium salt and an acidic polysaccharide solution is formed. Among these, the insoluble reaction product in the outer layer has molecular weight fractionation ability, completely suppressing the outflow of the enzyme inside, and allowing the movement of substrates and reactants to occur extremely freely, making this structure ideal. It is in the form of an immobilized enzyme. Although known entrapment methods can maintain the activity of the enzyme, enzyme efflux is a drawback. In contrast, the immobilized enzyme obtained by the method of the present invention can prevent enzyme leakage while taking advantage of the above characteristics. In the known comprehensive method, the polysaccharide used as the base material is required to have gelling ability, so the selection range of the base material is extremely limited, but the method of the present invention uses an acidic polysaccharide that does not have gelling ability. You can also
You can choose from a wide range. Furthermore, since the known comprehensive method requires that the base material gel,
It is necessary that the concentration of the polysaccharide solution is sufficiently high. In general, the migration speed of the substrate is extremely important in the comprehensive method, and increasing the concentration of polysaccharide as a base material not only inhibits the migration of the substrate, but also imposes restrictions on the three-dimensional structure of the enzyme. Activity may decrease. On the other hand, in the method of the present invention, a low concentration polysaccharide solution can be used, so that the drawbacks of the conventional method can be overcome. The immobilized enzyme obtained by the method of the present invention can be used, for example, in a bioreactor or a sensor, and maintains high activity for a long period of time. Example 1 Katupa carrageenan containing potassium as a cation was dissolved in water at 40°C, and partially purified glucose isomerase obtained from Streptomyces faeochromogenes (225 units/mg) was added to 50 g of the resulting 1.0% carrageenan solution. A mixed solution of 5 mg was poured onto a glass plate to a thickness of 2 mm. A 10% benzalkonium chloride solution was gently added to this to insolubilize the surface layer to obtain 42 g of membrane-like immobilized enzyme (immobilized glucose isomenase A). Partially purified glucose isomerase 5 as described above
2.5mg was dispersed in 2ml of water and kept warm at 40℃.
% Katupa carrageenan solution was added and mixed, and the mixture was similarly poured onto a glass plate to a thickness of 2 mm, and then 1% potassium chloride was added to gel it to obtain 55 g of immobilized enzyme (immobilized glucose isomerase). B: Comparative example). Add 10g each of immobilized glucose isomerase A and B to 300ml of 0.1M glucose solution, and adjust the pH
7. Shake at 37°C for 4 hours and replace the reaction solution. This operation was repeated to examine changes in the amount of fructose produced. The results are shown in Tables 1 and 2. It is known that glucose isomerase A of the present invention has higher activity and longer binding time than Comparative Example B. Note that 1 unit in Table 1 means the activity of producing fructose at a rate of 1 μg/min.

【table】

[Table] Example 2 1.2 in place of Katupa carrageenan in Example 1
Immobilized glucose isomerase was obtained in the same manner using 8% cetyltrimethylammonium bromide solution instead of % sodium alginate solution and benzalkonium chloride. This was subjected to a reaction in the same manner as in Example 1, and the residual activity rate in the 25th reaction was 96%.

Claims (1)

[Scope of Claims] 1. An acidic polysaccharide solution containing an enzyme is mixed with one or more of four selected from benzalkonium chloride, cetyltrimethylammonium bromide, cetylpyridinium chloride, and methylene blue.
1. A method for producing an immobilized enzyme, characterized by treatment with an ammonium salt. 2. Claim 1, characterized in that the acidic polysaccharide solution containing the enzyme is treated as a liquid film.
Method for producing the immobilized enzyme described in Section 1.