JPS63160586A - Production of immobilized enzyme - Google Patents

Abstract

PURPOSE:To easily obtain an immobilized enzyme having high activity and stability, by impregnating an aqueous solution of an enzyme-containing water-soluble polymer substance in a carrier and forming a thin film of polymer on the surface of the carrier. CONSTITUTION:An aqueous solution of a water-soluble polymer substance such as polysaccharides, proteins, having a concentration of 0.1-5wt% and containing 1-10mg/l of an enzyme such as oxidoreductase, transferase, hydrolase, isomerase, detaching enzyme, synthase, is prepared beforehand. The aqueous solution is impregnated in a preparatorily washed and dried carrier such as activated carbon, cellite, porous glass, natural fiber, and the carrier is made to contact with a organic solvent solution of a film-forming resin having a number-average molecular weight of <=100,000 and soluble in an organic solvent. The solvent is evaporated to form a thin film on the surface of the carrier and immobilize the enzyme.

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to a method for producing an immobilized enzyme used as a catalyst for biological reactions.

[Conventional technology]

In recent years, biological reactions have been widely used in the chemical industry, pharmaceutical industry, food industry, etc.
Its use in various fields is progressing. In particular, biological reactions using enzymes proceed rapidly under mild conditions of room temperature and normal pressure, and are much safer than conventional chemical reactions.
There is no fear of pollution, so it is very advantageous in terms of resources, energy, production equipment, etc. Furthermore, since enzymes have excellent substrate specificity, they also have the advantage of allowing the desired product to be obtained in high yield and drastically reducing the amount of processing required for each reaction product.

By the way, enzymes are water-soluble, and it is difficult to dissolve them in water, perform an enzymatic reaction, and then separate and collect only the enzymes after the reaction for reuse. Therefore, it has been proposed to immobilize enzymes. There is. By immobilizing enzymes, it becomes possible to carry out enzymatic reactions in a continuous manner, and it also makes it easy to recover enzymes, and it also makes it possible to detect specific substances in solutions by utilizing the substrate selectivity of enzymes. It will also be possible to create enzyme sensors.

The conventional manufacturing method for immobilized enzymes involves impregnating a carrier with a water-soluble polymer sol such as polysaccharide or protein in which the enzyme has been dissolved, and then gelling it with a gelling agent such as a calcium salt to coat the enzyme on the carrier. Immobilization method (for example, JP-A-59-109)
No. 173), or a method in which a mixed solution of enzyme and geltaraldehyde is supplied to the surface of the carrier, which has been reaminated as necessary, to gel the enzyme, and further to form a thin polymer film on the surface (for example, JP-A-61-5782). ) have been proposed.

[Problems to be Solved by the Invention] However, in such conventional methods for producing fp5 of immobilized enzymes, relatively sophisticated immobilization reaction reagents such as gelling agents are used, and the reaction time is long. There is a problem in that enzyme denaturation, deactivation, etc. tend to occur during immobilization, resulting in low enzyme immobilization rate, enzyme activity, and activity retention of the resulting immobilized enzyme.

The present invention is intended to solve the above-mentioned problems, and provides a method for producing an immobilized enzyme that has a high enzyme immobilization rate, enzyme activity, and activity retention rate, and is less likely to cause the enzyme to fall off. It is intended to.

[Means for solving problems]

The present invention is a method for producing an immobilized enzyme, which comprises impregnating a carrier with an aqueous solution of a water-soluble polymeric substance containing the enzyme, and then forming a thin polymer film on the surface of the carrier.

In the present invention, a material having a large specific surface area, such as a material having micropores, can be used as the carrier, and examples thereof include activated carbon, celite, porous glass, and natural fibers. These types are not particularly limited.

More than one type can be used. The water-soluble polymeric substance may be any substance that can dissolve in water and impart viscosity, such as alginic acid (including salts such as sodium salt), carrageenan, multi-Ms such as starch, proteins such as collagen, Others are used, and one or more of these can be used.

Enzymes include enzymes used in biological reactions and others, such as hydrolytic enzymes such as invertase, trypsin, amylase, cellulase, hemicellulase, protease, and lipase; oxidoreductases such as alcohol dehydrogenase, glucose oxidase, and amino acid oxidase; methyl These include transferases such as transferase, glycosyltransferase, and 1-lansaminase; elimination enzymes (lyases) such as amino acid decarboxylase and glutamic acid decarboxylase; isomerases (isomerases); and synthesis enzymes (ligases).

One or more types of these can also be used.

The thin polymer film formed on the surface of the carrier may be any material that can form a thin film on the surface of the carrier impregnated with an aqueous solution of a water-soluble polymer substance containing an enzyme, such as polyvinyl acetate, cellulose acetate, polystyrene, polymethacrylic acid, etc. There are resins that have a number average molecular weight of too or less than methyl and are soluble in organic solvents.

One or more types of these can also be used. The organic solvent may be any organic solvent as long as it can dissolve the resin; for example, aromatics, ketones, alcohols, etc. are preferred;
One or more types of these can be used.

In the method for producing an immobilized enzyme, first, a water-soluble polymeric substance and an enzyme are dissolved in water to prepare an aqueous solution of the water-soluble polymeric substance containing the enzyme. At this time, the amount of water-soluble polymer substance added is 0.
．． The amount of enzyme added is about 1 to 1% (lnt!).
, /Q is preferable. On the other hand, a carrier that has been washed and dried in advance is impregnated with an aqueous solution of a water-soluble polymeric substance containing the enzyme. Then, in a solution of the film-forming resin dissolved in an organic solvent,
A carrier impregnated with an aqueous solution of a water-soluble polymeric substance containing the enzyme is brought into contact with the carrier, and then the solvent is evaporated to form a thin film on the carrier surface, thereby immobilizing the enzyme on the carrier.

Since the immobilized enzyme obtained in this way does not use highly reactive reagents, denaturation and deactivation of the enzyme during immobilization are reduced.

〔Effect of the invention〕

As described above, according to the present invention, after a carrier is impregnated with an aqueous solution of a water-soluble polymeric substance containing an enzyme, a thin film of iii-contacted molecules is formed on the carrier surface.
An immobilized enzyme with high enzyme activity and activity retention and high stability can be easily obtained.

〔Example〕

Next, Examples and Comparative Examples will be described. % in examples
is weight % unless otherwise specified.

Example 1 Activated carbon particles were immersed in 70% HNO at 50°C for 1 hour.
The activated carbon was then thoroughly washed. On the other hand, 5.0B/
10 mQ of a 1% carrageenan aqueous solution containing amylase at a concentration of mQ was prepared. 5K (dry weight) of activated carbon treated as described above was dispersed in this solution, and the activated carbon was impregnated with an enzyme-containing aqueous carrageenan solution. Next, the activated carbon was immersed in a 1% polystyrene benzene solution for 5 seconds and immediately pulled up to volatilize the benzene to obtain an immobilized enzyme.

Comparative Example 1 An enzyme was adsorbed onto activated carbon treated with nitric acid in the same manner as in Example 1, and then treated with glutaraldehyde to obtain an immobilized enzyme.

Table 1 shows the enzyme immobilization rate, enzyme activity, and activity retention rate of the immobilized enzymes obtained in Example 1 and Comparative Example 1.

In the table, the enzyme immobilization rate is a value calculated by the following formula by measuring the enzyme concentration of the aqueous solution before and after immobilization by the Lowry method.

Enzyme activity is a value calculated by performing a hydrolysis reaction using olive oil as a substrate (Yamada et al., Journal of the Society of Agricultural Chemistry, 860 (19(i2))) and titrating the resulting free fatty acids with 1/10N-KOH. The activity retention rate is the ratio of the immobilized enzyme activity to the enzyme activity before immobilization (
%).

Table 1 Table 1 shows that Example 1 has a higher enzyme immobilization rate, enzyme activity, and activity retention rate than Comparative Example 1.

Example 2 Porous glass (20-50 mesh, average pore size 500) was dried under reduced pressure, and the dried porous glass was dispersed in a 1% carrageenan aqueous solution containing lipase at a concentration of 5.0 mg/mQ for 1 hour. The porous glass was impregnated with the enzyme-containing carrageenan aqueous solution by stirring.

After washing the porous glass, it was immersed in a 0.5% polyvinyl acetate methanol solution and immediately pulled up to volatilize the methanol to obtain an immobilized enzyme.

Comparative Example 2 After reacting the same porous glass used in Example 2 with γ-aminopropylethoxylan, the porous glass and lipase were bonded using glutaraldehyde to obtain an immobilized enzyme. .

Table 2 shows the enzyme immobilization rate, enzyme activity, and activity retention rate of the immobilized enzymes obtained in Example 2 and Comparative Example 2.

Table 2 From Table 2, the immobilized enzyme obtained in Example 2 is the same as that of Comparative Example 2.
It can be seen that the enzyme immobilization rate, enzyme activity, and activity retention rate are higher than those obtained in .

Example 3 10 mL of a 1% sodium alginate aqueous solution containing lipase at a concentration of 5 B/m (l) was prepared. Gauze as a carrier was immersed in this solution and then taken out, and the gauze was further immersed in an acetone solution of 1% cellulose acetate. , the immobilized enzyme was obtained by volatilizing the acetone.

Comparative Example 3 Lipase was comprehensively immobilized on a polyacrylamide gel to obtain an immobilized enzyme.

Table 3 shows the enzyme immobilization rate, enzyme activity, and activity retention rate of the immobilized enzymes obtained in Example 3 and Comparative Example 3.

Table 3 From Table 3, the immobilized enzyme obtained in Example 3 is the same as that of Comparative Example 3.
It can be seen that the enzyme immobilization rate, enzyme activity, and activity retention rate are higher than those obtained in .

Next, an enzymatic reaction was carried out using the above-mentioned immobilized enzyme as a catalyst, and the results of measuring the degree of decrease in enzyme activity over time of use were measured in the fourth column.
Shown in the table.

Table 4 From Table 4, the immobilized enzyme obtained in Example 3 is the same as that of Comparative Example 3.
It can be seen that the stability in use is superior to that obtained in .

Claims (5)

[Claims] (1) A method for producing an immobilized enzyme, which comprises impregnating a carrier with an aqueous solution of a water-soluble polymeric substance containing the enzyme, and then forming a thin polymer film on the surface of the carrier. (2) The method for producing an immobilized enzyme according to claim 1, wherein the carrier is one or more selected from activated carbon, celite, porous glass, and natural fiber. (3) The method for producing an immobilized enzyme according to claim 1 or 2, wherein the water-soluble polymeric substance is one or more selected from polysaccharides and proteins. (4) The fixation according to any one of claims 1 to 3, wherein the polymer thin film has a number average molecular weight of 100,000 or less and is one or more selected from resins soluble in organic solvents. Method for producing enzyme. (5) The enzyme is an oxidoreductase, a transferase, a hydrolase,
5. The method for producing an immobilized enzyme according to any one of claims 1 to 4, wherein the immobilized enzyme is one or more selected from isomerase, eliase, and synthase.