JPH05219952A - Carrier for fixation of enzyme - Google Patents

Abstract

PURPOSE:To obtain the subject carrier for fixation of an enzyme, capable of exhibiting a high activity when an enzyme is fixed thereto by bonding a coupling agent having a functional group affinitive to the enzyme and a hydrophobic functional group. CONSTITUTION:The objective carrier is obtained, e.g. by treating the surface of ferrite, kaolinite, etc., preferably having 50mum average particle diameter with a silane coupling agent composed of a compound (e.g. gamma- aminopropyltriethoxysilane) having an affinity to the enzyme and a compound (e.g. trimethoxysilane or triphenylethoxysilane) having a hydrophobia functional group.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a carrier for immobilizing an enzyme and a method for producing the carrier.

[0002]

BACKGROUND OF THE INVENTION Enzymes are biochemical catalysts that can control the initiation, promotion, etc. of chemical reactions and are used in many applications. If such an enzyme is supported on a granular carrier to be stabilized, it can be repeatedly used, and many applications can be expected. Such an enzyme-supporting carrier is, for example, a ceramic such as alumina or silica, an inorganic carrier such as porous glass, and a reactive organic group such as a carboxyl group, an amino group, a hydroxyl group, or a thiol in advance according to the reactivity of the enzyme. The introduced silane coupling agent is bonded, and then reactive groups such as carboxyl group, amino group, azo group and thiol group on the enzyme side are reacted with the reactive organic group of the silane coupling agent to form an amide bond or sulfonamide. A bond, an azo bond, an ether bond, an ester bond, a disulfide bond or the like is formed (for example, Japanese Patent Publication No. 55-3
2357, Japanese Patent Publication No. 56-15231, etc.).

[0003]

[Problems to be Solved by the Invention] However, the conventional enzyme immobilized on a carrier (hereinafter referred to as immobilized enzyme) reacts with starch and other substances to be reacted, but the reaction of an enzyme not immobilized on the carrier (hereinafter referred to as free enzyme). It is very low compared to sex, and is usually at most 1% or less. To compensate for this low reactivity it is necessary to use large amounts of immobilized enzyme, so that for example a bioreactor of a given throughput becomes very large.

Therefore, an object of the present invention is to provide a carrier for immobilizing an enzyme which exhibits high activity when the enzyme is immobilized.

[0005]

The present invention is directed to an enzyme-immobilizing carrier having a functional group having an affinity for an enzyme and a hydrophobic functional group, particularly a functional group having an affinity for an enzyme and a hydrophobic group. The above-mentioned problems could be solved by an enzyme-immobilized inorganic or organic carrier to which a coupling agent having a functional group is attached.

Here, the coupling agent is a compound A having a functional group having an affinity for an enzyme, a compound B having a hydrophobic functional group, and a functional group having an affinity for an enzyme and a hydrophobic property. The compound C having both the functional groups of 1) to 1) is selected from one of the following combinations (1) to (5). (1) Compounds A and B, (2) Compound C, (3) Compound A
And C, (4) Compounds B and C, (5) Compounds A, B and C.

Preferably, the coupling agent is a silane coupling agent, and among them, the compound A having a functional group having an affinity for an enzyme is γ-aminopropyltriethoxysilane, N- (2-aminoethyl). -3-Aminopropyltrimethoxysilane, N- (2-aminoethyl) -3-aminopropyldimethoxysilane, and p-
[N- (2-aminoethyl) aminomethyl] phenethyl trimethoxysilane, which is one or more kinds and hydrophobic compound B is trimethoxyphenylsilane, methyltriethoxysilane, methyltrimethoxysilane, methyldiethoxysilane. , Methyldimethoxysilane, trimethylethoxylan, trimethylmethoxysilane, methylvinyldiethoxysilane, and methylvinyldimethoxysilane, and 3- [N-allyl-N (2-aminoethyl)] amino. The compound C, which is propyltrimethoxysilane and has both an enzyme-affinitive functional group and a hydrophobic functional group, is 3- [N
It has been found that what is -allyl-N (2-aminoethyl)] aminopropyltrimethoxysilane is suitable.

The present inventor, as exemplified below, has a particle size of 50 μm or less and a primary particle or aggregate size of 50 μm.
The surface of the carrier composed of secondary particles of m or less was treated with various silane coupling agents to support the enzyme, and the reactivity of the immobilized enzyme was examined. As a result, for example, with a composition of 1 mol% γ-aminopropyltriethoxysilane which is a silane coupling agent having an amino group at the terminal and 99 mol% trimethoxyphenylsilane which is a silane coupling agent having a phenyl group at the terminal. It was found that the reactivity of the immobilized enzyme, which was surface-treated to fix glucoamylase as the enzyme, was about 10% of that of the free enzyme. This means that it has about 10 times more reactivity than the conventionally known fixed enzyme.

Although the reason for this is not clear, by using a phenyl group as a reactive terminal group of the silane coupling agent, some of the carrier particles become hydrophobic and repel each other, preventing the carrier particles from aggregating. It seems to be to do. As a result, a large number of enzymes are exposed to the surface overwhelmingly than in the past, and it is considered that the amount of enzymes involved in the reaction increases. In this case, the amount of enzyme used may be the same as the conventional amount.

The carrier usable in the present invention includes metal oxides such as magnetic or non-magnetic ferrite, activated carbon, acid clay, kaolinite, bentonite, porous glass, silica gel, alumina, silica-alumina, titanium oxide-silica-. Alumina, hydroxyapartite, inorganic carriers such as calcium phosphate gel, concanavalin A, gluten,
Organic substances such as starch, chitin, gluten, tannin-aminohexyl cellulose, and phenoxyacetyl cellulose are available. The average particle size of the carrier is preferably about 50 μm or less in the case of primary particles which are not aggregated, and about 50 μm or less in the case of aggregated to form secondary particles. And the activity drops.

Enzymes are generally carboxyl groups, amino groups,
Diazo groups such as tyrosine, cystidine, lysine, -N-O
Since it has a reactive functional group such as M (M is an alkali metal such as Na) and a thiol group, the reactive functional group on the coupling agent side reacts with various functional groups of the enzyme to be immobilized. It must have a functional group to attach. These groups include reactive organic groups such as carboxyl group, amino group, hydroxyl group and thiol (see, for example, Japanese Patent Publication No. 56-15231). Particularly suitable coupling agents are silane coupling agents and titanate coupling agents.

The silane coupling agent may consist of a compound having a functional group having an affinity for the enzyme and a compound having a hydrophobic functional group, or may itself have an affinity for the enzyme. It may be composed of a compound having both a certain functional group and a hydrophobic functional group. In short, the coupling agent may be used alone or in combination as long as the carrier has both groups at the same time and does not inhibit the activity of the enzyme. When the effect of the total length of the silane coupling agent (distance from terminal to terminal) on the amount of coupling agent that can be applied was examined, the shorter the length of the coupling agent, the greater the amount of application. Therefore, from the viewpoint of increasing the amount of the coupling agent applied, it is desirable to select from those having a short total length (for example, γ-aminopropyltriethoxysilane).

The mechanism for binding the coupling agent to the surface of the carrier is as shown in FIG. 3, whereby the alkoxyl group is converted into a hydroxyl group by hydrolysis, these groups are hydrogen-bonded to the hydrophilic group on the surface of the carrier, and then dehydrated. It is believed to condense. Although FIG. 1 shows the binding mechanism of γ-aminopropyltriethoxysilane, a similar mechanism holds for a coupling agent having a hydrophobic group. For example, alkyl-substituted or unsubstituted phenyltrimethoxysilane is γ-
Similar to aminopropyltriethoxysilane, since it has three ethoxy groups or methoxy groups, it causes the same binding reaction with a hydrophilic carrier.

[0014]

[Explanation of Examples]

Example 1 1 g of Ni ferrite powder having an average particle diameter of 0.03 mm as a carrier, and 0.0024 g of γ-aminopropyltriethoxysilane having a parent enzymatic amino group at the terminal and a coupling agent having a hydrophobic phenyl group at the terminal. Trimethoxyphenylsilane 0.237g and acetone 24
The solution diluted with 0 g was mixed for 24 hours, solid-liquid separated, and dried at 110 ° C. for 2 hours. The enzyme-immobilized carrier thus surface-treated was examined for the presence of γ-aminopropyltriethoxysilane and trimethoxyphenylsilane using an infrared spectrophotometer. The results shown in FIG. 1 were obtained. Amino group and phenyl group are fixed.

Example 2 The enzyme-immobilizing carrier of Example 1 was immersed in a 0.1% glucoamylase aqueous solution for 24 hours to immobilize glucoamylase (Seikagaku Corporation). After the immobilization was completed, it was washed several times with pure water. The immobilized glucoamylase was then added to 6.
0.3 liter and 40 of 7 g (g / l) starch aqueous solution
The reaction was carried out at 30 ° C. for 30 minutes. Table 1 shows the results of measuring the amount of glucose produced with a food analysis reagent (Boehringer Mannheim Yamanouchi).

Example 3 As a carrier, 1 g of the same Ni ferrite powder as in Example 1,
And a solution obtained by diluting 0.0024 g of γ-aminopropyltriethoxysilane having a parent enzymatic amino group at the end and 0.237 g of trimethoxymethylsilane as a coupling agent having a hydrophobic phenyl group at the end with 240 g of acetone. Then, the same surface treatment as in Example 1 was performed. Glucoamylase was immobilized on this enzyme-immobilizing carrier in the same manner as in Example 2, reacted with starch, and the glucose production was measured. The results are shown in Table 1.

Example 4 As a carrier, 1 g of Ni ferrite powder having an average particle size of 0.0003 mm was surface-treated in the same manner as in Example 1. Glucoamylase was immobilized on this enzyme-immobilizing carrier in the same manner as in Example 2, reacted with starch and the glucose production was measured. The results are shown in Table 1.

Example 5 As a carrier, 1 g of Ti-Si composite oxide powder having an average particle size of 0.0005 mm was surface-treated in the same manner as in Example 1.
Glucoamylase was immobilized on this enzyme-immobilizing carrier in the same manner as in Example 2, reacted with starch and the glucose production was measured. The results are shown in Table 1.

Comparative Example 1 1 g of Ni ferrite powder having an average particle diameter of 0.03 mm as a carrier and 0.24 g of γ-aminopropyltriethoxysilane having a parent enzymatic amino group at the end were diluted with 240 g of acetone to prepare a solution. After mixing for 24 hours, solid-liquid separation was performed, and dried at 100 ° C. for 2 hours. Using the infrared spectrophotometer, the enzyme-immobilized carrier surface-treated in this way
The presence of aminopropyltriethoxysilane was investigated. The result of FIG. 2 was obtained. The amino group is fixed to the carrier.

Comparative Example 2 Using the enzyme immobilizing carrier of Comparative Example 1, glucoamylase was immobilized by the method of Example 2. The results of measuring the amount of glucose produced are shown in Table 1.

Comparative Example 3 A surface treatment was carried out in the same manner as in Comparative Example 1 using 100 g of commercially available silane beads having an average particle diameter of 0.5 mm (manufactured by Tokyo Rika Kikai). Then, glucoamylase was immobilized in the same manner as in Example 2 and the amount of glucose produced was measured. The results are shown in Table 1.

Comparative Example 4 Example 1 using 100 g of the same silane beads as in Comparative Example 3
The surface treatment was performed in the same manner as in. Then, glucoamylase was immobilized in the same manner as in Example 2 and the amount of glucose produced was measured. The results are shown in Table 1.

Comparative Example 5 A surface treatment was carried out in the same manner as in Comparative Example 1 by using 1 g of Ti-Si composite oxide powder having an average particle diameter of 0.0005 mm as a carrier. Glucoamylase was immobilized by the method of Comparative Example 2 using this enzyme-immobilizing carrier. The results of measuring the amount of glucose produced are shown in Table 1.

[0024]

[Table 1]

From the above results, in fine particles such as Ni ferrite, a carrier in which a functional group of a parent enzyme such as γ-aminopropyltriethoxysilane and a hydrophobic group such as trimethoxyphenylsilane are bonded has excellent activity. You can see that it provides. About 10 times as much glucose production was obtained as compared with the case of not having a hydrophobic group as in Comparative Example 2. Therefore, it is considered that the hydrophobic functional groups repel the carrier particles to each other to improve the dispersibility. Further, as can be seen from Comparative Example 4, the smaller the particles, the higher the activity, which is related to the surface area of the carrier. However, there is no great difference between Comparative Examples 3 and 4 because coarse beads do not have a problem of particle aggregation.

[0026]

The enzyme-immobilized carrier having a coupling agent having a functional group having an affinity for the enzyme and a hydrophobic functional group has a larger size than the conventional carrier when the enzyme is immobilized. It can be seen that it gives excellent activity.

[Brief description of drawings]

1 shows an infrared spectroscopic analysis of the carrier of Example 1. FIG.

FIG. 2 shows an infrared spectroscopic analysis of the carrier of Example 2.

FIG. 3 illustrates a reaction mechanism that occurs when a silane coupling agent is bound to a carrier.

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[Procedure amendment]

[Submission date] September 24, 1992

[Procedure Amendment 1]

[Document name to be amended] Statement

[Correction target item name] 0015

[Correction method] Change

[Correction content]

Example 2 0.2 g of the enzyme-immobilizing carrier of Example 1 was immersed in a 0.1% glucoamylase aqueous solution for 24 hours to immobilize glucoamylase (Seikagaku Corporation). After the immobilization was completed, it was washed several times with pure water. Then, the immobilized glucoamylase was reacted with 0.3 liter of an aqueous starch solution of 6.7 g (g / l) at 40 ° C. for 30 minutes. Table 1 shows the results of measuring the amount of glucose produced with a food analysis reagent (Boehringer Mannheim Yamanouchi). U / g in the table is 1 g of carrier
Shows the ability to produce 10 mg of glucose in 30 minutes
You

[Procedure Amendment 2]

[Document name to be amended] Statement

[Correction target item name] 0021

[Correction method] Change

[Correction content]

Comparative Example 3 A surface treatment was carried out in the same manner as in Comparative Example 1 using 100 g of commercially available silane beads having an average particle diameter of 0.5 mm (manufactured by Tokyo Rika Kikai). Then, 40 g of glucoamylase was immobilized in the same manner as in Example 2 and the amount of glucose produced was measured. The results are shown in Table 1.

[Procedure 3]

[Document name to be amended] Statement

[Name of item to be corrected] 0023

[Correction method] Change

[Correction content]

Comparative Example 5 A surface treatment was carried out in the same manner as in Comparative Example 1 by using 1 g of Ti-Si composite oxide powder having an average particle diameter of 0.0005 mm as a carrier. Comparative Example 2 using 0.2 g of this enzyme-immobilizing carrier
Glucoamylase was immobilized by the method described in 1. The results of measuring the amount of glucose produced are shown in Table 1.

[Procedure amendment 4]

[Document name to be amended] Statement

[Correction target item name] 0024

[Correction method] Change

[Correction content]

[0024]

[Table 1]

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Misako Ishiyama 1-13-1 Nihonbashi, Chuo-ku, Tokyo TDK Corporation

Claims (6)

[Claims] 1. A carrier for immobilizing an enzyme, to which a coupling agent having a functional group having an affinity for an enzyme and a hydrophobic functional group is bound. 2. The coupling agent comprises a compound A having a functional group having an affinity for an enzyme, a compound B having a hydrophobic functional group, and a compound having a functional group having an affinity for an enzyme and a hydrophobic group. The carrier for enzyme immobilization according to claim 1, which is selected from one of the following combinations (1) to (5) of the compound C having both functional groups. (1) Compounds A and B (2) Compound C (3) Compounds A and C (4) Compounds B and C (5) Compounds A, B and C. 3. The enzyme-binding carrier according to claim 1, wherein the coupling agent is a silane coupling agent. 4. A compound A having a functional group having an affinity for an enzyme is γ-aminopropyltriethoxysilane,
N- (2-aminoethyl) -3-aminopropyltrimethoxysilane, N- (2-aminoethyl) -3-aminopropylmethyldimethoxysilane, and p- [N- (2
The carrier for enzyme binding according to claim 2, which is one or more selected from -aminoethyl) aminomethyl] phenethyltrimethoxysilane. 5. The hydrophobic compound B is trimethoxyphenylsilane, triphenylethoxysilane, methyltriethoxysilane, methyltrimethoxysilane, methyldiethoxysilane, methyldimethoxysilane, trimethylethoxysilane, trimethylmethoxysilane, methylvinyl. The inorganic carrier for enzyme binding according to claim 1, 2 or 3, which is one or more selected from diethoxysilane and methylvinyldimethoxysilane. 6. The compound C having both an enzyme-affinitive functional group and a hydrophobic functional group is 3- [N-allyl-N (2-aminoethyl)] aminopropyltrimethoxysilane. The carrier for enzyme binding according to claim 1, 2 or 3.