KR20150124610A - Method for immobilization of enzyme and immobilized enzyme using the method - Google Patents

Abstract

The present invention relates to a method for immobilization of an enzyme, comprising the following steps: inducing an amine group (-NH_2) on a surface of a carrier by reacting an amino alkyl silane derivative with a carrier; manufacturing an activated carrier by reacting a multi-functional crosslinking agent with the amine group-induced carrier; pretreating an enzyme by stirring a mixed solution including chitonsan, an enzyme, and a buffer solution at 80-120 rpm at 35-39°C, and reacting the same for 20 to 30 minutes; and adding the activated carrier to the pretreated mixed solution to induce adsorption of the carrier and enzyme or a covalent bond.

Description

METHOD FOR IMMOBILIZATION OF ENZYME AND IMMOBILIZED ENZYME USING THE METHOD BACKGROUND OF THE INVENTION 1. Field of the Invention < RTI ID = 0.0 >

The present invention relates to a method for immobilizing an enzyme on a carrier and an immobilized enzyme immobilized on the carrier.

More particularly, the present invention relates to a method for preparing a metal complex, comprising the steps of: reacting a carrier with an aminoalkylsilane derivative to introduce an amine group (-NH ₂ ) to the surface of the carrier; Reacting the amine group-introduced carrier with a polyfunctional crosslinking agent to prepare an activated carrier; Chitosan, enzyme, and buffer solution at 35 to 39 DEG C for 20 to 30 minutes with stirring at a rate of 80 to 120 rpm to pre-treat the enzyme; And adding the activated carrier to the pretreated mixed solution to induce adsorption or covalent bonding between the carrier and the enzyme, and to an immobilized enzyme produced by the immobilization method.

Also, the present invention provides a method for preparing a chitosan-containing microorganism, which comprises the steps of: pre-treating a mixed solution containing chitosan, enzyme and buffer at 35 to 39 DEG C with stirring at a rate of 80 to 120 rpm for 20 to 30 minutes; Introducing an amine group (-NH ₂ ) to the surface of the carrier by reacting the carrier with an aminoalkylsilane derivative; Reacting the amine group-introduced carrier with a polyfunctional crosslinking agent to prepare an activated carrier; And adding the activated carrier to the pretreated mixed solution to induce adsorption or covalent bonding between the carrier and the enzyme, and to an immobilized enzyme produced by the immobilization method.

Enzymes are proteins that catalyze biochemical reactions, generally unstable after extraction and can not be used in organic solvents or at high temperatures. Thus, enzymatic reactions have often been achieved through simple batch processes in which substrates and soluble enzymes are mixed and incubated at room temperature. However, in such a method, it is remarkably complicated to replenish fresh enzymes while keeping the reaction conditions constant, or to recover enzymes after the reaction, and the reaction products are separated and refined remarkably.

In order to solve such a problem, it has been investigated that the enzyme reaction is carried out continuously by immobilizing the enzyme on a water-soluble carrier.

Methods for immobilizing an enzyme on a carrier include an adsorption method, an ion-binding method, a crosslinking method, a capturing method, and a covalent bonding method. Among these immobilization methods, the covalent bonding method is suitable for industrial processes because of its strong binding force between the carrier and the enzyme. The covalent bonding method is a method in which an enzyme-supported carrier is surface-treated with a chemical product to change the state of an electron-affinity functional group to a fixed state, and then a carrier and an amino group in the enzyme are bound to each other using a crosslinking substance such as glutaraldehyde .

However, the covalent bonding method requires a large amount of enzyme in order to exhibit the same efficiency as other immobilization methods because the activity of the enzyme binds with the chemical substance when the enzyme and the carrier bind to each other and the activity decreases, . In particular, since the binding between the enzyme and the carrier occurs randomly, the probability that the active site of the enzyme binds to the carrier increases, and the active site of the enzyme is partially lost. Therefore, in order to obtain the same effect as the enzyme immobilized, a larger amount of enzyme must be immobilized on the carrier, resulting in an increase in the cost of the enzyme system and an increase in the size of the enzyme reactor.

Therefore, there is a demand for the development of immobilized enzymes capable of maintaining long-term activity by immobilizing highly active enzymes, and being able to use them more stably and over a long period of time.

On the other hand, studies on chitin and chitosan and its derivatives as active new functional materials have been actively carried out because various physiological activities such as immunity enhancement, anticancer and antimicrobial activity, cholesterol improving action and hypertension inhibiting action have been reported.

In particular, low molecular weight chitooligosaccharides produced by chitin and chitosanase are of great interest both domestically and externally due to their diverse and high physiological activities. Chitooligosaccharide has been widely used as a functional oligosaccharide in terms of enhancing immune activity and various physiological activities It has the most excellent pharmacological and physiological activity, and oligomers of higher polymerization degree than pentamer are currently used as intermediates in foods and medicines.

Currently, chitooligosaccharides are generally prepared by hydrolysis of chitosan with concentrated hydrochloric acid, which is not only the safety of the product, but also the stability of the product. The chitosan oligosaccharide is produced by a chemical decomposition method using strong acid and a biological method using chitosanase But also environmental pollution and a high water-soluble oligosaccharide storage rate.

In the prior art related to the present invention, Korean Patent Registration No. 1994-0005581 discloses a method of forming a Schiff base by a condensation reaction between a polyfunctional crosslinking agent between an amino functional group in an enzyme molecule and an amino functional group on a surface of a carrier. However, the carrier prepared by the above method has a problem in that it is difficult to produce a highly active immobilized enzyme with a small surface area.

SUMMARY OF THE INVENTION Accordingly, it is an object of the present invention to provide a method for immobilizing an active site of an enzyme in a method for immobilizing an enzyme, To thereby provide an enzyme immobilization method capable of exhibiting enzyme activity.

Another object of the present invention is to provide an immobilized enzyme produced by the immobilization method.

According to an aspect of the present invention, there is provided a method for preparing a metal complex, comprising: reacting a support with an aminoalkylsilane derivative to introduce an amine group (-NH ₂ ) to the surface of the support; Reacting the amine group-introduced carrier with a polyfunctional crosslinking agent to prepare an activated carrier; Chitosan, enzyme, and buffer solution at 35 to 39 DEG C for 20 to 30 minutes with stirring at a rate of 80 to 120 rpm to pre-treat the enzyme; And adding the activated carrier to the pretreated mixed solution to induce adsorption or covalent bonding between the carrier and the enzyme, and a method for immobilizing the enzyme on a carrier.

Also, the present invention provides a method for preparing a chitosan-containing microorganism, which comprises the steps of: pre-treating a mixed solution containing chitosan, enzyme and buffer at 35 to 39 DEG C with stirring at a rate of 80 to 120 rpm for 20 to 30 minutes; Introducing an amine group (-NH ₂ ) to the surface of the carrier by reacting the carrier with an aminoalkylsilane derivative; Reacting the amine group-introduced carrier with a polyfunctional crosslinking agent to prepare an activated carrier; And adding the activated carrier to the pretreated mixed solution to induce adsorption or covalent bonding between the carrier and the enzyme, and a method for immobilizing the enzyme on a carrier.

According to one embodiment of the present invention, the carrier is a silica gel, a porous glass, a zeolite or a bentonite.

According to another embodiment of the present invention, the aminoalkylsilane derivative is at least one compound selected from the group consisting of? -Aminoethyl-? -Aminopropylmethyldimethoxysilane, aminopropylmethyldiethoxysilane,? -Aminopropyltrimethoxysilane and? Lt; / RTI > silane.

According to another embodiment of the present invention, the polyfunctional crosslinking agent is selected from the group consisting of glutaraldehyde, succinic acid aldehyde, formaldehyde and dextrin aldehyde.

According to another embodiment of the present invention, the mixed solution contains chitosan at a concentration of 0.5 to 2% (w / v).

According to another embodiment of the present invention, the pH of the mixed solution is 4 to 6.

According to another embodiment of the present invention, the enzyme is a chitosanase or a chitinase.

Another aspect of the present invention provides an immobilized enzyme produced by the immobilization method.

According to the present invention, it is possible to provide an immobilized enzyme capable of stably immobilizing an enzyme in a state where the carrier is maintained in a high activity, and capable of a continuous enzyme reaction over a long period of time.

Therefore, by using the immobilized enzyme of the present invention, it is possible to improve the purity of the product obtained by the enzyme reaction, reduce the amount of enzyme used, and lighten the enzyme reactor.

1 is a graph showing the activity of an immobilized enzyme according to an embodiment of the present invention.

In a first aspect of the present invention, there is provided a method for preparing a metal complex, comprising: reacting a carrier with an aminoalkylsilane derivative to introduce an amine group (-NH ₂ ) to the surface of the carrier; Reacting the amine group-introduced carrier with a polyfunctional crosslinking agent to prepare an activated carrier; Chitosan, enzyme, and buffer solution at 35 to 39 DEG C for 20 to 30 minutes with stirring at a rate of 80 to 120 rpm to pre-treat the enzyme; And adding the activated carrier to the pretreated mixed solution to induce adsorption or covalent bonding between the carrier and the enzyme, and a method for immobilizing the enzyme on a carrier.

In a second aspect of the present invention, there is provided a method for preparing a chitosan, comprising: reacting a mixed solution containing chitosan, an enzyme and a buffer at 35 to 39 DEG C with stirring at a rate of 80 to 120 rpm for 20 to 30 minutes to pretreat the enzyme; Introducing an amine group (-NH ₂ ) to the surface of the carrier by reacting the carrier with an aminoalkylsilane derivative; Reacting the amine group-introduced carrier with a polyfunctional crosslinking agent to prepare an activated carrier; And adding the activated carrier to the pretreated mixed solution to induce adsorption or covalent bonding between the carrier and the enzyme, and a method for immobilizing the enzyme on a carrier.

In order to immobilize the enzyme on the carrier of the present invention, it is necessary to first introduce an amine group (-NH ₂ ) to the surface of the carrier.

That is, when a carrier is reacted with an aminoalkylsilane derivative, a carrier into which an amine group is introduced can be prepared by a silane coupling reaction between a functional group such as a hydroxy group and an aminoalkylsilane derivative present on the surface of the carrier.

According to one embodiment of the present invention, the carrier is placed in an organic solvent to which an aminoalkylsilane derivative is added and reacted at 40 to 60 ° C for 1 to 3 hours to produce an activated carrier having an amine group introduced therein by a silane coupling reaction.

The carrier used in the present invention may be silica gel, porous glass, zeolite, bentonite, etc. The aminoalkylsilane derivative used in the present invention is? -Aminoethyl-? -Aminopropylethyldimethoxysilane -aminopropylmethyldimethoxy silane, aminopropylmethyldiethoxy silane, γ-aminopropyltrimethoxy silane, γ-aminopropyltriethoxy silane and the like can be used. .

Subsequently, a multifunctional crosslinking agent is reacted with a carrier to which an amine group is introduced to prepare an activated carrier.

According to one embodiment of the present invention, an activated carrier can be prepared by adding a carrier and a buffer solution in which an amine group is introduced to a polyfunctional crosslinking agent solution, and then reacting at 10 to 30 ° C for 1 to 3 hours.

The polyfunctional crosslinking agent used in the present invention may be a polyaldehyde such as glutaraldehyde, succinic acid aldehyde, formaldehyde, and dextrin aldehyde, among which glutaraldehyde is preferably used.

The buffer used in the present invention may be a buffer such as phosphate, MOPS (3- (N-morpholino) propanesulfonic acid), MES (2- (Nmorpholino) ethanesulfonic acid), McIlvaine, The range is 7 to 9.

Then, the mixed solution containing chitosan, enzyme, and buffer is reacted at 35 to 39 DEG C with stirring at a rate of 80 to 120 rpm for 20 to 30 minutes to pre-treat the enzyme.

By protecting the active site of the enzyme by the pretreatment process, it is possible to prevent the enzyme from binding to the carrier or the chemical substance in the step of immobilizing the enzyme.

Since the chitosan is competitively bound to the active site of the enzyme, pretreatment of the enzyme with chitosan before the immobilization step reduces the probability that the active site of the enzyme binds to the carrier or chemical substance. That is, the active site of the enzyme binds to the chitosan before binding to the carrier, and the amine group at another site other than the active site binds to the carrier.

The weight average molecular weight of the chitosan usable in the present invention may be 150,000 to 360,000. Chitin is a polysaccharide polymer substance composed of N-acetyl-D-glucosamine units bound to each other in an abundance, and chitosan is obtained by deacetylation of chitin. However, deacetylation is incomplete. In general, chitosan is a polymer mixed with chitin and deacetylated chitin. Therefore, the deacetylation degree of the chitosan which can be used in the present invention is 70 to 90%, preferably 75 to 85%.

The enzyme used in the present invention may be an oxidoreductase, a hydrolase, a transferase, an isomerase, and the like. As an example of the hydrolase, chitosanase or chitinase may be used. But is not limited thereto.

Since the pre-treatment step of the enzyme is affected by the pH, a buffer solution is important, and the buffer solution is preferably acetic acid buffer solution.

That is, a buffer solution is used to facilitate the reaction between the active site of the enzyme and chitosan as a reaction substrate, and the pH of the enzyme mixture solution of the present invention is preferably 4 to 6, The activity of the enzyme should not be lowered.

In one embodiment of the present invention, the concentration of chitosan pretreated to prevent the active site of the enzyme from binding to the carrier is 0.5 to 2% (w / v). If the amount of chitosan is less than 0.5% (w / v), the effect of preventing binding of the active site of the enzyme to the carrier is not significant. If the chitosan is present in excess of 2% (w / v) Therefore, there is a problem that the reaction between the enzyme and chitosan is reduced and the protective effect of the active site of the enzyme is lowered.

The enzyme pretreatment step of the present invention maintains a temperature of 40 DEG C or below, preferably 35-39 DEG C, so that the enzyme can be immobilized in an active state.

In one embodiment of the present invention, when the immobilization reaction is carried out at 3 to 5 ° C for 10 to 14 hours, an adsorption or covalent bond is formed between the carrier and the enzyme.

After the immobilization reaction, the carrier immobilized with the enzyme is sufficiently washed with an appropriate buffer to remove the chitosan bound to the active site, whereby the highly active enzyme can be immobilized on the carrier.

The present invention also relates to an immobilized enzyme produced by the aforementioned immobilization method.

Hereinafter, the present invention will be described in detail with reference to Examples.

However, the following examples are intended to illustrate the contents of the present invention, but the scope of the present invention is not limited by the following examples.

Example  1: Activate Carrier  Produce

1 g of the silica gel was added to 20 mL of an acetone solution containing 0.75 M (3-aminopropyl) triethoxysilane, and the mixture was reacted at 50 ° C for 2 hours. After the reaction, the silica gel was washed with distilled water and dried at 60 ° C. for 2 hours. To the 1.6 mL of 2.6 M glutaraldehyde solution was added 18.4 mL of dried silica gel and 100 mM sodium phosphate buffer (pH 8.0) Lt; / RTI > After completion of the reaction, the silica gel was washed with distilled water and dried at 60 ° C. for 2 hours to prepare an activated silica gel carrier.

Example  2: Preparation of pretreated enzyme

Chitosan 1% (w / v) and 3,500 U chitosanase were added to 100 mM sodium acetate buffer (pH 5.0) to prepare 20 mL of the mixed solution. Chitosanase was pre-treated by reacting the reaction solution at 37 ° C and 100 rpm for 30 minutes.

Comparative Example  1: Preparation of non-pretreated enzyme

Chitosan 1% (w / v) and 3,500 U chitosanase were added to 100 mM sodium acetate buffer (pH 5.0) to prepare 20 mL of the mixed solution.

Example  3: Immobilization of pretreated enzyme

The activated silica gel carrier prepared in Example 1 was added to the pretreated enzyme mixture prepared in Example 2, reacted at 4 ° C for 12 hours, filtered, washed with 100 mM sodium acetate buffer (pH 5.0), dried at room temperature To prepare an immobilized enzyme.

Comparative Example  2: immobilization of untreated enzyme

The activated silica gel carrier prepared in Example 1 was added to the untreated enzyme mixture prepared in Comparative Example 1, and the mixture was reacted at 4 ° C for 12 hours, followed by filtration and washing with 100 mM sodium acetate buffer (pH 5.0) And dried to prepare immobilized enzyme.

Experimental Example  1: Measurement of immobilized enzyme activity

1% (w / v) of chitosan and 0.1 g of immobilized chitosanase prepared in Example 3 and Comparative Example 2 were added to 5 mL of 100 mM sodium acetate buffer (pH 5.0), and the mixture was reacted at 37 ° C and 100 rpm for 30 minutes. The reaction was stopped by adding 1 mL of 1N NaOH and centrifuged. 1 mL of the supernatant was reacted with 1 mL of 0.5 M sodium carbonate (anhydrous) (0.5 g potassium ferricyanide solution), boiled for 15 minutes, and then absorbed at 420 nm And the results are shown in Table 1.

1 U of chitosanase activity was defined as the amount of enzyme liberating 1 μmol of reducing sugar per minute, and the free reducing sugar used was a standard reagent of D-glucosamine.

According to Table 1, the activity of Comparative Example 2 in which chitosanase was immobilized without pretreatment was 10.6 U / g matrix, whereas the activity of chitosanase in Example 3, which was pretreated with chitosan and immobilized, was 16.7 U / g matrix , And it was confirmed that the activity was increased by 57.5% by the enzyme pretreatment step of the present invention.

division Activity
(U / g matrix) Comparative Example 2 10.6 Example 3 16.7

Experimental Example  2: Measurement of protein amount of immobilized enzyme

The amount of protein of the chitosanase immobilized on the silica gel was measured by the difference in the amount of protein in the enzyme solution before immobilization (Example 2 and Comparative Example 1) and in the enzyme solution after immobilization (Example 3 and Comparative Example 2) Respectively.

The chitosanase solution was added to 3 mL of Bradford solution, stirred for 1 minute, and allowed to stand at room temperature for 10 minutes to stabilize the color reaction, and the absorbance was measured at 595 nm.

division Amount of protein immobilized on carrier
(mg / g matrix) Comparative Example 2 0.33 Example 3 0.31

Experimental Example  3: Reuse of immobilized enzyme

The immobilized enzyme was reused 10 times and the activity was measured. The results are shown in FIG. 1 and Table 3.

1% (w / v) of chitosan and 0.1 g of the immobilized chitosanase prepared in Example 3 and Comparative Example 2 were added to 5 ml of 100 mM sodium acetate buffer (pH 5.0) as in Experimental Example 1, and the mixture was incubated at 37 ° C and 100 rpm for 30 minutes After the single reaction was completed, the immobilized chitosanase was collected by centrifugation. The collected immobilized chitosanase was washed several times with 100 mM sodium acetate buffer (pH 5.0) and then reused for the next reaction.

As shown in Table 3, when the immobilized enzyme was reused 10 times, the initial activity of Comparative Example 2 in which chitosanase was immobilized without pretreatment was 10.3 U / g matrix, and the activity was 8.1 U / g matrix when reused 10 times . On the other hand, the initial activity of immobilized chitosan after pretreatment with chitosan was 16.2 U / g matrix and the activity after 10 times re - use was 13.3 U / g matrix.

Therefore, according to the enzyme pretreatment step of the present invention, it was confirmed that not only the initial activity of the immobilized enzyme was improved but also the decrease in the activity by continuous reuse was remarkably slowed down.

division Initial activity
(U / g matrix) Activity after 10 reuses
(U / g matrix) Comparative Example 2 10.3 8.1 Example 3 16.2 13.3

Claims (9)

Introducing an amine group (-NH ₂ ) to the surface of the carrier by reacting the carrier with an aminoalkylsilane derivative;
Reacting the amine group-introduced carrier with a polyfunctional crosslinking agent to prepare an activated carrier;
Chitosan, enzyme, and buffer solution at 35 to 39 DEG C for 20 to 30 minutes with stirring at a rate of 80 to 120 rpm to pre-treat the enzyme; And
Adding the activated carrier to the pretreated mixed solution to induce adsorption or covalent bonding between the carrier and the enzyme; and immobilizing the enzyme on the carrier. Chitosan, enzyme, and buffer solution at 35 to 39 DEG C for 20 to 30 minutes with stirring at a rate of 80 to 120 rpm to pre-treat the enzyme;
Introducing an amine group (-NH ₂ ) to the surface of the carrier by reacting the carrier with an aminoalkylsilane derivative;
Reacting the amine group-introduced carrier with a polyfunctional crosslinking agent to prepare an activated carrier; And
Adding the activated carrier to the pretreated mixed solution to induce adsorption or covalent bonding between the carrier and the enzyme; and immobilizing the enzyme on the carrier. The method of claim 1 or 2, wherein the carrier is silica gel, porous glass, zeolite or bentonite. The method according to claim 1 or 2, wherein the aminoalkylsilane derivative is at least one selected from the group consisting of? -Aminoethyl-? -Aminopropylmethyldimethoxysilane, aminopropylmethyldiethoxysilane,? -Aminopropyltrimethoxysilane and? -Aminopropyl ≪ / RTI > triethoxysilane, and triethoxysilane. The method of claim 1 or 2, wherein the polyfunctional crosslinking agent is selected from the group consisting of glutaraldehyde, succinic acid aldehyde, formaldehyde, and dextrin aldehyde. The method according to claim 1 or 2, wherein the mixed solution contains chitosan at a concentration of 0.5 to 2% (w / v). The method for immobilizing an enzyme in a carrier according to any one of claims 1 to 3, wherein the pH of the mixed solution is 4 to 6. [ 3. The method according to claim 1 or 2, wherein the enzyme is chitosanase or chitinase. An immobilized enzyme produced by the method of claim 1 or 2.

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