JPS60237993A - Method of immobilizing enzyme to hydroxyl group-containing fiber - Google Patents

Abstract

PURPOSE:In a method of immobilizing an enzyme, to raise immobilization ratio and to lessen deactivation of enzyme, by deying hydroxyl group-containing fiber with a reactive dye, reacting the remaining reactive group with the enzyme. CONSTITUTION:Hydroxyl group-containing fiber such as cellulose fiber, polyvinyl alcohol fiber, etc. is made cationic with a cation agent such as a quaternary ammonium compound, shown by the formula I, formula II, etc. It is dyed with reactive dye, and the remaining reactive group is reacted with an enzyme such as urease, lactase, trypsin, ribonuclease, etc.

Description

DETAILED DESCRIPTION OF THE INVENTION "Field of Industrial Application" The present invention relates to a method for immobilizing enzymes on hydroxyl group-containing fibers.

``Prior art'' Conventionally, known methods for immobilizing enzymes include embedding enzymes in water-insoluble substances or microcapsules, and binding enzymes to insoluble carriers through ionic bonds or physical force. It is being Among them, the adsorption method, in which the enzyme is immobilized by ionic binding or physical force on the surface of an insoluble carrier, has extremely mild immobilization conditions, and the bond between the enzyme and the carrier is loose, so it is difficult to immobilize the enzyme. Although it has the characteristics of less damage to the enzyme and less deactivation of the enzyme once immobilized, it has low immobilization efficiency and requires a long time for immobilization.

It has drawbacks such as that adsorption is largely dependent on the surface condition 11 of the carrier and that enzyme desorption is likely to occur.

``Object of the Invention'' The purpose of the present invention is to develop an enzyme immobilization method that has a high immobilization yield and that causes less desorption after immobilization.

Another goal is to develop an immobilization method that causes less enzyme deactivation.

Another object of the present invention is to provide an immobilized enzyme carrier that is highly efficient in use as a catalyst, and has excellent strength and ease of operation.

``Structure of the Invention The present invention is a method for immobilizing enzymes on hydroxyl group-containing fibers, which comprises dyeing the hydroxyl group-containing fibers with a reactive dye and then allowing the remaining reactive groups to react with the enzyme.

And after cationizing the hydroxyl group-containing fiber with a cationizing agent, it is dyed with an anionic group-containing reactive dye.

This is a method for immobilizing enzymes on hydroxyl group-containing fibers, which is characterized by reacting the remaining reactive groups with the enzyme.

Examples of the hydroxyl group-containing fibers used in the present invention include natural or regenerated cellulose fibers, polyvinyl alcohol fibers, etc., and cellulose fibers are particularly preferred because they have a large active surface area. Note that it is particularly preferable to cationize the hydroxyl group-containing fiber in advance with a cationizing agent in order to improve the exhaustability and reactivity of the reactive dye. As the cationizing agent, for example, a method of intrafiber polymerization or graft polymerization of a vinyl monomer having a quaternary ammonium base; Examples include a method of reacting a compound, and a method of quaternizing after introducing a tertiary nitrogen-containing compound.

However, a method in which a quaternary ammonium compound having a group capable of covalently reacting with a hydroxyl group is reacted is particularly preferred from the viewpoint of ease of reaction, first reaction efficiency, and the like. Examples of such compounds include compounds represented by the following general formulas CI) and [II).

Specifically, R is epoxypropyltrimethylammonium chloride, epoxypropyltrimethylammonium chloride, N-meflL'-N-(2+3epoxypropyl)morpholinium chloride.

N-mef#-N-(2,3-epoxyprobyl)piperazine and their precursors, the corresponding hemohydrin compounds, but especially 3-mef#-N-(2,3-epoxypropyl)piperazine, the precursor of epoxypropyltrimethylammonium chloride. Particularly preferred is chloro-2-hydroxypropyltrimethylammonium chloride. Modification of cellulose fibers with these compounds is usually carried out in the presence of an alkali catalyst, but when an epoxy group-containing compound is used as a modifier and the process is carried out at a high temperature of 100°C or higher by a bud-steam method or a bud-cure method. can also be carried out in neutral to slightly alkaline conditions.

In particular, when the treatment is carried out by the immersion method or the bud-ripening method, it is preferable to use an alkali species and amount sufficient to make the pH of the treatment solution alkaline at 10 or higher, particularly at 12 or higher. Alkali hydroxide is usually used. The forms of fibers are fibers and threads.

Any fabric such as woven fabric, knitted fabric, non-woven fabric, etc. may be used, and other fibers 5- It may also be a mixed product.

The reactive dye used in the present invention varies depending on whether or not the fiber is cationized. That is, in the case of cationized fibers, since the dye is ionically bonded by the anion group of the 5-reactive dye, the reactive group of the dye can be left to immobilize the enzyme after dyeing in the absence of alkali. Therefore, any dye having one or more reactive groups can be used. However, when reactive dyeing is carried out using covalent bonds in the presence of an alkali, or when a reactive dye is fixed to unmodified fibers in the presence of an alkali, it is necessary to use a dichlorotriazine type reactive dye. , utilizes one reactive group for reaction with the fiber.

The remaining one reactive group is utilized for enzyme immobilization.

Examples of this type of dye include C,1,! j Active Yellow 1.4.7. C, 1, Reactive Red 1
, C, 1, Reactidiorange 18. Examples include C,1, Reactive A/-4131, and the like.

In the case of cationized fibers, dyeing can be carried out without a catalyst; however, the dyeing is usually carried out in a dyeing solution containing a reactive dye and an alkali catalyst, preferably an alkali carbonate, an alkali bicarbonate and, if desired, Glauber's salt.

What is important here is to leave reactive groups behind after dyeing.

Cationized fibers adsorb reactive dyes quickly and at low temperatures, so there is less decomposition of reactive groups.

Many reactive groups remain as reaction sites with the enzyme, improving the enzyme immobilization efficiency.

The target enzymes of the present invention are enzymes that have a free amino group, phenol group, or sulfhydryl group in the enzyme protein that can bind to the reactive group of a dye, such as urease, lactase, triphen*! J bonuclease, veniciliamylase, catalase, tyro Vnase, lactate dehydrogenase, lysozyme, α-chymotrypsin, pyruvate kinase, phosphodiesterase, phosphomonoesterase, glucoamylase, ATPase.

Examples include avirase and the like.

Enzyme immobilization is usually carried out by contacting reactive dye-dyed fibers with an aqueous enzyme solution.

"Effects of the Invention" According to the present invention, the binding force between the fiber and the enzyme is strong, and the sustainability and activity are also high, so it can be used for continuous reactions. In other words, by filling an appropriate column with enzyme-immobilized fibers and flowing the substrate solution under the optimum temperature and optimum pi (for the enzyme), the reaction product can be taken out continuously. It has the characteristic that it can be easily handled when placed in a solution.It also has the advantage of making it easy to distinguish between types by changing the color depending on the type of enzyme to be immobilized from the dyed area.

Furthermore, it has many features such as high enzyme immobilization efficiency and large surface area, which improves enzyme reaction efficiency.

The present invention will be explained below with reference to Examples. In addition.

Parts in the examples mean parts by weight.

Example 1 3 parts of cotton boblin were put into a treatment solution consisting of 7.5 parts of 3-chloro-2-hydroxypropyltrimethylammonium chloride, 0.45 parts of caustic soda and 68 parts of water, and heated at 60°C while shaking. Heat up to
．． An aqueous solution prepared by dissolving 45 parts in 8 parts of water was added to the treatment liquid, the temperature was raised to 90°C, and the reaction was carried out for 60 minutes. After cooling, it was washed with water and dried to obtain cationized cellulose ma'.

Next, in step A, add the cationized cellulose fibers to 80 parts of water at 20°C, and after 10 minutes, proceed to step C. , Reactive Yellow 4 0.
A dye solution of 0.45 parts dissolved in 20 parts of water was added, and after 10 minutes, 4.5 parts of Glauber's salt/30 parts of water was added, and after another 10 minutes, 1.05 parts of soda ash/10 parts per minute was added.

The temperature was raised to 40°C and dyeing was carried out for 60 minutes. After cooling and washing with water, 9
Washing in a nonionic surfactant 11/J aqueous solution at 0°C,
It was further washed with water and dried.

The obtained dyed cloth was put into an aqueous solution consisting of 1 part of urease (manufactured by Hanbo Chemical Co., Ltd.) and 100 parts of water.

After stirring at room temperature for 8 hours, the mixture was thoroughly washed with water to obtain a urease-immobilized cotton cloth.

When the obtained urease-immobilized cotton cloth was placed in an aqueous urea solution, the pH of the aqueous solution increased and urease enzyme activity was observed. The relationship between reaction time and pH increase at this time is shown in FIG.

Also, apply urease-immobilized cotton cloth twice, three times...
It was observed that the enzyme activity did not decrease even after repeated use.

Example 2゜0.3 part of the dyed cloth obtained in Example 1 was mixed with lysozyme (SIGM).
Company A) 0.2 parts/100 parts of water into a solution.

After stirring at 5°C for 24 hours, the pH was adjusted to 6, after washing thoroughly with water.
It was stored in a cold place in 5 M buffer.

Next, when the enzyme-immobilized cotton cloth was placed in an aqueous suspension of Micrococcus lysodexteix (SIGMA), the enzyme activity of lysozyme was confirmed by increasing the transmittance at 450 μm.9.

The enzyme activity of lysozyme was measured by G, Kay & M.
D, Li1ly; Biochemica et B
iophysica acta, Vol. 198, p. 276 (1970).

Example 3°C, 1, 0.09 parts of reactive yellow and 100 parts of water
3 parts of cotton boblin was added to a dye solution consisting of 3 parts of cotton boblin, 30 parts of 1010-branched 15q6 sodium sulfate aqueous solution was added, and after 10 minutes, 5 parts of cotton boblin was added.
After adding 20 parts of % soda ash aqueous solution and treating at 40°C for 60 minutes, the mixture was submerged in water and dried to produce an enzyme-immobilized carrier. 0.4 part of the carrier thus produced and 1 part of urease were placed in 100 parts of water, stirred at room temperature for 8 hours, and then taken out and thoroughly washed to produce an immobilized enzyme.

The enzyme activity of the obtained immobilized enzyme was tested together with the treated fabric of Example 1, and the results are shown in Table 1 for comparison.

Table 1

[Brief explanation of the drawing]

FIG. 1 is a diagram showing the relationship between the immersion time and the pH change when a urease-immobilized cotton cloth is immersed in a urea aqueous solution. Patent applicant: Toyobo Co., Ltd.

Claims (1)

[Claims] 1. After dyeing a hydroxyl group-containing fiber with a reactive dye. A method for immobilizing enzymes on hydroxyl group-containing fibers, which is characterized by reacting residual reactive groups with enzymes. 2. After cationizing the hydroxyl group-containing fiber with a cationizing agent, it is dyed with an anionic group-containing reactive dye. A method for immobilizing enzymes on hydroxyl group-containing fibers, which comprises reacting residual reactive groups with enzymes.