KR100509738B1 - Method for enzyme immobilization using silicagel or composite silicagel carrier - Google Patents

Abstract

The present invention relates to a method for immobilizing a diamino acid oxidase (DAOD) or glutaryl-7-aminocephalosporanic acid acylase (Gl-7-ACAacylase) enzyme on a silica gel or a composite silica gel carrier. The immobilized enzyme prepared by is excellent in initial activity and activity half-life.

Description

Method for enzyme immobilization using silicagel or composite silicagel carrier

The present invention relates to a method for immobilizing a diamino acid oxidase (DAOD) or glutaryl-7-aminocephalosporanic acid acylase (Gl-7-ACAacylase) enzyme on a silica gel or a composite silica gel carrier.

Enzymes are proteins that catalyze biochemical reactions and are generally unstable after extraction and cannot be used in organic solvents or at high temperatures. Therefore, enzymatic reactions have often been carried out through a simple batch process of culturing a mixture of substrate and soluble enzyme at room temperature. However, in order to reuse the active enzyme, it is necessary to separate the enzyme from the reaction mixture, which is an extremely difficult technical problem. In general, a method of separating enzymes and contaminated proteins from the reaction mixture by changing pH or heat treatment (advances in the chemical industry, 25 (9) , 596 (1985)) is used. Therefore, it is uneconomical.

In order to solve the above problems, a method of physically constraining or localizing an enzyme at a certain place in a space has been studied so that it can repeatedly exhibit catalytic activity, and a representative example thereof is an immobilized enzyme. For example, Korean Patent Application No. 1987-13473 discloses a method of forming a seed base by a condensation reaction by a multifunctional crosslinking agent between an amino functional group on the surface of a carrier and an amino functional group in an enzyme molecule. On the other hand, Korean Patent Application No. 199-123834 discloses a method of immobilizing Gl-7-ACA acylase in resins Doulite A7 and Doulite A568 having glutaraldehyde amino groups. However, the carrier obtained by the above method has a problem that it is difficult to make a high activity immobilized enzyme due to its small surface area. On the other hand, Korean Patent Application No. 1996-703112 describes a method for immobilizing DAOD and Gl-7-ACA acylase to an organosiloxane polymer having a glutaraldehyde hydro group introduced therein. However, when the immobilized enzyme prepared by the above method is repeatedly used, the organosiloxane polymer as a carrier is broken and cannot serve as a carrier, and there is a problem in using it continuously because of poor filterability.

As described above, when the organic material is used as a carrier, there is an advantage that the microstructure is easy to control and the immobilization characteristics of the microorganism are excellent, but the inherent characteristics of the carrier are deteriorated due to swelling or weakening of the carrier itself when used for a long time. have. On the other hand, when an inorganic material is used as a carrier, it is known to have excellent heat resistance, durability, and chemical resistance, and to have excellent immobilization characteristics of microorganisms because silanol groups, which are easily bonded, are present on the surface of the carrier. Therefore, the present inventors have developed a method for immobilizing DAOD and Gl-7-ACA acylase using silica gel or composite silica gel as an inorganic material as a carrier.

SUMMARY OF THE INVENTION An object of the present invention is to provide a method for preparing an immobilized enzyme capable of repeatedly displaying enzyme activity even when used for a long time by immobilizing an enzyme on a silica gel or a composite silica gel carrier. Another object of the present invention is to provide an enzyme immobilized on a carrier obtained by the above production method.

The present invention relates to a method for immobilizing a diamino acid oxidase (DAOD) or glutaryl-7-aminocephalosporanic acid acylase (Gl-7-ACAacylase) enzyme on a silica gel or a composite silica gel carrier. DAOD is a fermentation product of a mutant strain of Trigonopsis variabilis , and Gl-7-ACAacylase is a fermentation product of E. coli .

As the carrier used in the present invention, silica gel or composite silica gel is preferable. The composite silica gel includes TiO ₂ composite silica gel, Al ₂ O ₃ composite silica gel, and ZrO ₂ composite silica gel. The carrier preferably has a pore size of 100 to 1000 mm 3. If the pore size is more than 1000 mm 3, the surface area of the carrier is small, so the enzyme activity per volume is small. If the pore size is less than 100 mm 3, the pore size is smaller than the size of the enzyme, so that the enzyme is not sufficiently immobilized in the pore. On the other hand, the particle size of the carrier is preferably 40 to 80 mesh.

In order to immobilize the enzyme on the carrier, a process of activating the carrier by first introducing an amino group onto the surface of the carrier is required. Scheme 1 below shows a process of activating a carrier. That is, when the aminoalkylsilane derivative of the following formula (2) is reacted with the carrier of the formula (1), an activating carrier into which the amino group of the following formula (3) is introduced by a silane coupling reaction between the silanol group present on the surface of the carrier and the aminoalkylsilane derivative You can get it.

Wherein R is a methyl group or an ethyl group; m, n and o are each independently an integer from 0 to 3; Carriers represent silica gel or composite silica gel.

The aminoalkyl silane derivative of Formula 2 is β-aminoethyl-γ-aminopropylmethyldimethoxysilane, aminopropylmethyldiethoxy silane, γ-aminopropyltrimeth Reagents, such as oxy-aminopropyltrimethoxy silane and γ-aminopropyltriethoxy silane, can be used.

Referring to the activation process of the carrier in more detail as follows. That is, the silica gel or the composite silica gel of the formula (1) in the organic solvent, the aminoalkyl silane derivative of the formula (2), and then reflux at 100 to 110 ℃ preferably the amino group of the formula (3) by the silane coupling reaction To make the introduced water insoluble carrier. The amount of the aminoalkylsilane derivative is preferably 1 to 20% by weight, preferably 2 to 15% by weight based on the carrier.

On the other hand, the enzyme can be immobilized by reacting the crosslinking agent with the obtained activation carrier. Scheme 2 below shows the immobilization of the enzyme. That is, a crosslinking agent having an aldehyde group represented by the following Formula 4 may be reacted with a carrier having an amino group represented by Formula 3 below to obtain a compound represented by Formula 5, and then reacted with an enzyme having an amino group represented by Formula 6 to immobilize the enzyme on the carrier. have.

Wherein m, n and o are each independently an integer of 0 to 3; Carrier refers to silica gel or composite silica gel; The enzyme represents DAOD or Gl-7-ACAacylase.

As the crosslinking agent of Chemical Formula 4, polyaldehydes such as glutaraldehyde, dialdehyde starch, succinate aldehyde and the like may be used, and among them, glutaraldehyde is preferably used.

The immobilization process of the enzyme is described in more detail as follows. First, the compound of Formula 5 is obtained by reacting the cross-linking agent of Formula 4 with a carrier into which the amino group of Formula 3 is introduced. The amount of the crosslinking agent is preferably 20 to 50% by weight based on the carrier. The reaction is carried out by stirring at a temperature of 0 to 30 ° C. for about 0.5 to 4 hours. Thereafter, the enzyme of Formula 6 may be reacted with the compound of Formula 5 to fix the enzyme on the carrier. It is preferable to use the enzyme in the form of an aqueous solution having a concentration of 30 to 70 U / mL. The reaction is carried out by stirring at a temperature of 10 to 30 ° C. for 0.5 to 15 hours, preferably 1 to 7 hours. PH and temperature of the reaction solution should be made in a range that does not lower the activity of the enzyme.

The amount of immobilized enzyme is preferably 50 to 200 U per gram of carrier. After the crosslinking reaction, it is preferable to sufficiently wash the carrier to which the enzyme is immobilized with a suitable buffer to remove the easily detachable enzyme and excess crosslinking agent having insufficient binding.

The present invention also provides an enzyme immobilized on a carrier, represented by the following formula (7).

Wherein the carrier represents silica gel or composite silica gel; The enzyme represents DAOD or Gl-7-ACAacylase.

Hereinafter, the present invention will be described in more detail with reference to Examples. The following examples illustrate the present invention in detail, and the scope of the present invention is not limited by the following examples.

Example 1 Activation of Silica Gel

100 g of silica gel was added to 1000 mL of toluene and stirred for 30 minutes. 10 g of β-aminoethyl-γ-aminopropylmethyl-dimethoxysilane was added thereto, refluxed at 110 ° C. for 3 hours, and filtered to obtain an activated silica gel.

Example 2 Immobilization of DAOD

50 g of silica gel, 40 to 80 mesh, activated by the method of Example 1 was added to 200 mL of 1 M phosphate buffer (pH 7.5), and 20 g of glutaraldehyde was slowly injected. The reaction was stirred at 20 ° C. for 2 hours and then filtered. 150 mL of an aqueous DAOD-containing aqueous solution having a concentration of 50 U / mL was added thereto, stirred at 10 ° C. for 3 hours, and then filtered to obtain a fixed DAOD.

Example 3 Immobilization of Gl-7-ACA Acylase

30 g of silica gel, 40-80 mesh activated by the method of Example 1, was added to 500 mL of 1 M phosphate buffer (pH 8.0), and 10 g of glutaraldehyde was slowly injected. The reaction was stirred at 20 ° C. for 2 hours and then filtered. 100 mL of an aqueous solution containing Gl-7-ACA acylase at a concentration of 40 U / mL was added thereto, followed by stirring at 10 ° C. for 3 hours, followed by filtration to obtain immobilized Gl-7-ACA acylase.

Experimental Example 1: Measurement of the activity and half-life of DAOD according to pore size

DAOD titer measurement

In a 100 mL Elenmeyer flask, 100 mL of a substrate [20 mM cephalosporin C solution dissolved in 100 mM KPO ₄ buffer (pH8.0)] was added, saturated with oxygen, and DAOD 300 mg was added thereto and stirred. At this time, the amount of oxygen consumption by DAOD was measured to obtain the activity of DAOD, and the amount of oxygen consumption was measured by using a DO meter at 25 ° C. The activity unit (U) of DAOD represents the amount of enzyme that produces 1 μmole of Gl-7-ACA per minute.

Half-life measurement

DAOD 1.2 kU was added and stirred to 1,000 mL of cephalosporin C solution (20 g of purity 78% cephalosporin C dissolved in 0.1 M phosphate buffer at pH 7.2). The temperature was maintained at 25 ° C. and the pH was maintained at 7.5 by injecting 5% NH ₄ OH. At this time, oxygen was injected at 0.1 vvm (volume / solution volume / min), and the internal pressure was 1 kgf / cm ² . When cephalosporin C was converted to Gl-7-ACA by 95% or more by HPLC, the reaction was terminated and the half-life was determined by measuring the titer of DAOD.

Table 1 shows the results of measuring the initial activity and half-life of DAOD according to the pore size of silica gel.

Pore size (Å) Initial activity (U / g) Active half-life (reactions) 10-100 50 82 100-300 70 99 300-500 66 105 500-700 65 98 700-1000 64 99 1000-1500 42 71 1500-2000 21 42

As shown in Table 1, the initial activity and half-life of the immobilized DAOD enzyme was greatly affected by the pore size of the silica gel carrier, it was found that the suitable pore size is 100 to 1000Å.

Experimental Example 2 Measurement of Activity and Half-Life of Gl-7-ACAacylase According to Pore Size

-Gl-7-ACA acylase titer

50 mL of substrate [52 mM Gl-7-ACA solution dissolved in 5 mM KPO ₄ buffer (pH 8.0)] and 150 mg of Gl-7-ACA acylase were added to a 100 mL reactor and stirred, and the pH was adjusted to 8.0-10 with 0.1 M NaOH. Hold for 15 minutes. At this time, the amount of NaOH consumed was measured to determine the activity of Gl-7-ACA acylase, the temperature was 37 ℃. The activity unit (U) of Gl-7-ACA acylase represents the amount of enzyme that produces 1 μmole of 7-ACA per minute.

Half-life measurement

4.8 kU of Gl-7-ACAacylase was added to 1,000 mL of Gl-7-ACA solution (25 g of purity 95% Gl-7-ACA dissolved in 0.1 M phosphate buffer at pH 8.0) and stirred. The temperature was maintained at 25 ° C. under atmospheric pressure, and the pH was maintained at 8.0 by injecting 5% NH ₄ OH. When Gl-7-ACA was converted to 7-ACA by 95% or more by HPLC, the reaction was terminated, and half-life was determined by measuring the titer of Gl-7-ACAacylase.

Table 2 shows the results of measuring the initial activity and half-life of Gl-7-ACAacylase according to the pore size of silica gel.

Pore size (Å) Initial activity (U / g) Active half-life (reactions) 10-100 85 119 100-300 118 153 300-500 113 145 500-700 110 144 700-1000 108 139 1000-1500 75 89 1500-2000 50 67

As shown in Table 2, the initial activity and half-life of the immobilized Gl-7-ACAacylase enzyme were greatly influenced by the pore size of the silica gel carrier, and the suitable pore size was found to be 100 to 1000 mm 3.

Experimental Example 3 Measurement of Activity and Half-Life of Gl-7-ACAacylase According to Silica Gel Type

Gl-7-ACA was immobilized to the composite silica gel having a pore size of 100 to 300 mm 3 in the same manner as in Example 3, and the initial activity and half-life of Gl-7-ACA acylase immobilized in the same manner as in Experiment 2 Measured. The results are shown in Table 3.

Initial activity (U / g) Active half-life (reactions) Silica gel 118 153 TiO ₂ Composite Silica Gel 115 154 Al ₂ O ₃ Composite Silica Gel 117 156 ZrO ₂ Composite Silica Gel 120 150

As shown in Table 3, it was found that even in the case of using the composite silica gel, the initial activity and the activity half-life are excellent as in the case of using the silica gel.

The activity of the enzyme immobilized on the carrier according to the present invention is 64 to 70 U / g for DAOD and 108 to 118 U / g for Gl-7-ACA acylase (see Table 1 on page 24 of Korean Patent Application No. 199-23834). 1, Table 2), the half-life was also very stable at least 98 times for DAOD, 139 times for Gl-7-ACA acylase.

Claims (14)

In a silica gel or a composite silica gel carrier having a pore size of 100 to 1000 mm 3 and a particle size of the carrier of 40 to 80 mesh, After reacting the aminoalkylsilane derivative of Formula 2 to prepare an activating carrier of Formula 3, A crosslinking agent of the following formula (4) is reacted with a carrier of the following formula (3) to obtain a compound of the following formula (5), followed by reaction with an enzyme of the following formula (6) to diamino acid oxidase or glutaryl-7-aminocephalosporanic acid acylase enzyme To freeze: Wherein R is a methyl group or an ethyl group; m, n and o are each independently an integer from 0 to 3; Carriers represent silica gel or composite silica gel, and enzymes represent DAOD or Gl-7-ACA acylase. delete delete delete The aminoalkylsilane derivative according to claim 1, wherein the aminoalkylsilane derivative is β-aminoethyl-γ-aminopropylmethyldimethoxysilane, aminopropylmethyldiethoxysilane, γ-aminopropyltrimethoxysilane and γ-aminopropyltriethoxysilane Method characterized in that the at least one component selected from. The method of claim 1, wherein the amount of the aminoalkylsilane derivative is 1 to 20% by weight based on the carrier. delete The method according to claim 1, wherein the crosslinking agent is polyaldehydes such as glutaraldehyde, dialdehyde starch, succinate aldehyde and the like. The method of claim 1, wherein the amount of the crosslinking agent is 20 to 50% by weight based on the carrier. The method according to claim 1, wherein the enzyme is in an aqueous solution at a concentration of 30 to 70 U / mL. The method according to claim 1, wherein the reaction between the activating carrier and the crosslinking agent is carried out by stirring at a temperature of 0 to 30 ° C for about 2 to 4 hours. The method according to claim 1, wherein the reaction with the enzyme is carried out by stirring at a temperature of 10 to 30 ° C for about 0.5 to 15 hours. The method of claim 1 wherein the amount of immobilized enzyme is between 50 and 200 U per gram of carrier. An enzyme immobilized on a carrier represented by the following Chemical Formula 7: Wherein the carrier represents silica gel or composite silica gel; The enzyme represents DAOD or Gl-7-ACAacylase.