JPS6070077A - Method for producing enzyme immobilization composition - Google Patents

Abstract

PURPOSE:To produce an immobilized enzyme composition, capable of surely and efficiently immobilizing an enzyme to an immobilized enzymatic agent having high activity, by treating a polysaccharide-containing natural product with periodic acid or its salt. CONSTITUTION:A polysaccharide-containing natural product such as grains, algae, fruit peels, wood chips, etc. or its derivative, is optionally subjected to the pretreatment such as washing with water, rough crushing, etc., and immersed in an aqueous solution of periodic acid or its salt having a concentration of 0.0005-0.01mol/l.

Description

[Detailed Description of the Invention] The present invention relates to a VC method for reliably and efficiently immobilizing enzymes to produce immobilized enzyme preparations with high activity. For many years, attempts have been made to apply it to pharmaceuticals, food industry processes, and analytical chemistry.

(1) However, on the other hand, #element is generally extracted from microorganisms by a complicated process and is therefore very expensive, and when the enzyme is used in industrial processes, etc.
The use of enzymes is limited in these respects since it has to be a non-coupled system. On the other hand, research on so-called -chemotaxis, in which enzymes are retained in all-water inert carriers, is
s.

It started in 1992, and became industrially practical in the 1990s, and now immobilized enzymes are widely applied.

Various methods for immobilizing enzymes have been developed, and these are broadly classified into entrapment methods, adsorption methods, cross-linking 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. In other words, 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. Synthetic polymers used in this case include polyacrylamide, ethylene-vinyl acetate copolymer, and borivinyl alcohol, while natural polymers include collagen, cellulose, alginic acid, agar, and carrageenan. . In the comprehensive method, the operation is relatively simple;
Although enzymes are difficult to elute, there are some disadvantages that cannot be ignored compared to the covalent bond method described below.

14+J The point to keep in mind in the comprehensive method is that the rate of diffusion of the substrate and product within the polymer lattice often determines the overall reaction rate, and especially when high molecular weight substrates and products are involved, the diffusion rate may vary. is difficult to apply because of its small size. In addition, when polymerizing a mixture of a monopolymerase and an enzyme by an appropriate method, all conditions must be set so as not to deactivate the enzyme.Also, when enclosing an enzyme in a natural polymer, all conditions must be set so as not to inactivate the enzyme. Similarly, care must be taken to avoid reducing enzyme activity, such as operating at Klfj temperature.

The stratification method is a method of adsorbing and immobilizing enzymes on an insoluble carrier. Examples of insoluble carriers used here include organic polymers such as DEAF cellulose, cellulose ester, and methacrylate, and inorganic substances such as glass, activated carbon, alumina, and diatomaceous earth. Moreover, since the +y element reaction can pass on the surface of the carrier, not only is there no problem with the diffusion of substrates and products as in the above-mentioned comprehensive method, but the gradual adsorption and desorption is reversible, so the carrier can be used repeatedly. The recurved BY-IL, which has the advantage of being able to perform the following, has the disadvantage of being easily eluted.

The cross-linking method is a method in which a polyfunctional substance with a specific molecular weight of 1 is added to an enzyme bath solution or a mixed solution of an enzyme and a small soluble carrier to form a cross-linked structure by covalent bonds between the insoluble carrier or the insoluble carrier. Geltaraldehyde is the most common polyfunctional substance used in this method to insolubilize enzymes by forming . Insoluble carriers include alkylaminated porous glass, polyethyleneimine-coated glass, gelatin, collagen, and the like. However, although this method is relatively easy to operate, it requires a large amount of enzyme and is low in efficiency, and deactivation of the enzyme by polyfunctional substances poses a problem.

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 directly or through some chemical substance, and the peptide bonding method , diazo method, alkylation method, Schiff salt formation method, Ugi reaction method and the like. Examples of small soluble carriers include natural organic polymers such as dextran, cellulose and collagen, synthetic organic polymers such as nylon, polystyrene, polyacrylamide and polyvinyl alcohol, and inorganic substances such as silica, titania and zirconia. Enzymes immobilized by books generally exhibit high activity, sometimes even higher than the original enzyme. It also has the advantage that the elution of m-thickness is extremely small. However, on the other hand, there are disadvantages in that the substances used for immobilization often cause a decrease in enzyme activity, and furthermore, the operation is complicated.

In view of the current situation, the present inventors have conducted extensive research on a method for easily producing a complete composition for adsorption chemotaxis. He discovered that the purpose could be achieved by a very specific method of treatment with an acid or its salt, and completed the Misexplosion 111.

In other words, by treating a natural product with periodic acid or its salts, aldehyde groups can be easily generated on the surface of the natural product and in the vicinity thereof, and the aldehyde group is The ability to form a so-called Schiff salt with a group? This makes it a covalent carrier.

Seeds and other grains, seaweed stones such as Amanita agaricus, Amanita chinensis, Amanita chinensis, Phasellaria, etc., peels of oranges, zapon, grapefruit, summer mandarin oranges, unshiu mandarin oranges, bananas, apples, etc., all wood chips before washing with water and crushing as necessary. The processed food is served.

(6) Fibers processed into fibers such as hemp and cotton, as well as those chemically processed into fibers such as viscose rayon, cellulose diacetate, and cellulose triacetate fibers, may also be served in pieces. This is possible.

Considering the pressure loss when the immobilized base agent produced from the composition according to the present invention is packed into a column or the like and subjected to an enzyme reaction, it is found that it is a granular or fibrous material with a uniform particle size like grains. □The feature of the present invention is that such a shape can be obtained extremely easily, that is, without relying on conventional methods.
In contrast to the case where a certain shaping/DO process or sieving is necessary to obtain a uniform particle size, the method of the present invention does not require such a process at all or can be extremely simplified.

The natural product provided in carrying out the present invention is washed with water, crushed or cut as necessary, and then treated with periodic acid or a salt thereof. Various methods can be used as the treatment method, but 1 mole/liter of periodic acid or its #A class solution at 1 mol/liter of o, ooos to θ0 at a temperature of 1 to 1 IoC can be used.
It is preferable to soak for a minute to three hours.

The gist of the present invention is to partially oxidize the isolated polysaccharides on the surface of the provided natural product and in its immediate vicinity, but the shape and strength originally possessed by the natural product are substantially oxidized. In order to achieve this, migration is carried out as appropriate under the above processing conditions, but it is important to optimize the process according to the type of natural product being provided. Of course.

After the partial oxidation treatment, it is washed with water, and if necessary,
After the reaction is completed in a 5% ethylene glycol water bath, a composition for enzyme immobilization having an aldehyde group on the surface is obtained by washing with water. As the enzyme to be immobilized, it is clear from the above that any enzyme having an amide 7 group can be used.

Example Each material in the table washed with water go! 30C in sodium periodate solution with 72θOOS mol/l once
After soaking for one hour, it was washed with running water. Next, partially purified glucose isomerase obtained from Streptomyces phaeochromogenes (22! f units/m) 3 mgiPH? The various materials after the above treatment were dispersed in a solution obtained from physiological saline s o o + nl and left at λjC for 30 minutes to obtain an immobilized glucose isomerase agent S 0 of which /39 A column with a diameter of lSm was packed, and a θ/M glucose solution f10rnl/hour was passed as a substrate to generate fructose, and the following table was obtained.

(9) In the table, the unit of activity represents the activity of raw INJT at the ratio of fructose k/t+9/min.

It can be seen from the table that the immobilized enzyme preparation prepared by the method of the present invention is suitable for practical use.

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Claims (1)

[Scope of Claims] l. A method for producing an enzyme immobilization composition, which comprises treating a polysaccharide-containing natural product or a derivative thereof with periodic acid or a salt thereof. Treatment of the natural product or its six conductors with periodic acid or its salts from o, o o o s to θ01 mol/l
2. A method for producing an enzyme immobilization composition according to claim 1, which comprises immersing the enzyme in a water bath solution.