JPH0646855A - Immobilization of enzyme - Google Patents

Abstract

PURPOSE:To provide a method for immobilizing enzyme free from problems in terms of safety, enabling enzyme activity decline to be relatively delayed even during and after its continuous use. CONSTITUTION:The objective safe immobilized enzyme stable even during and after its continuous use can be obtained by making transglutaminase on enzyme(s) (protease and/or amylase) adsorbed onto as an ion exchange resin a carrier to form a crosslinked enzyme film on the carrier.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for immobilizing a carrier by coating the carrier with an enzyme active substance adsorbed on the carrier. Specifically, the enzyme-active substances adsorbed on the carrier are covalently bonded to each other without using a chemical reagent that may affect the living body, and the carrier is coated economically and safely for edible materials. The present invention relates to a method of immobilizing an enzyme such that

[0002]

2. Description of the Related Art There have been many attempts to continuously and economically immobilize a physiologically active substance, particularly an enzyme, on an insoluble carrier. Conventionally, 1) a method of adsorbing and immobilizing the enzyme on the insoluble carrier. 2) Method of immobilizing by covalent bond to insoluble carrier, and 3) Cross-linking of enzymes adsorbed on carrier with a chemical cross-linking reagent such as glutaraldehyde to form an enzyme film on the carrier for immobilization. The method of doing has been used. However, since 1) has a weak binding force, the enzyme is easily released from the carrier depending on the external environment such as pH and buffer solution. In the case of 2), since it is immobilized under relatively violent conditions, there is a risk that the enzyme will be deactivated, the chemical reagent used for immobilization will remain, and it is difficult to reuse the relatively expensive carrier. In addition, the method of 3), like the method of 2), may be accompanied by inactivation of the enzyme, or the crosslinking reagent may remain and affect the living body. There is a drawback that it is difficult to use for processing materials and the like.

[0003]

SUMMARY OF THE INVENTION An object of the present invention is to provide an enzyme immobilization method which has no problem in terms of safety and in which the activity decrease of the enzyme can be relatively slowed even when continuously used. .

[0004]

Means for Solving the Problems As a result of intensive studies to solve the above problems, the present inventors have made transglutaminase act on an enzyme adsorbed on a carrier,
It has been found that the above problems can be solved by immobilizing the enzyme by forming a crosslinked enzyme film on the carrier, and has completed the present invention. That is, the present invention is an enzyme immobilization method characterized in that a transglutaminase is allowed to act on an enzyme adsorbed on a carrier to form a crosslinked enzyme film on the carrier.

The present invention will be described in detail below. The carrier used in the present invention may be any carrier as long as it can adsorb the enzyme. For example, an ion exchange resin, an affinity adsorbent, silica gel particles or the like can be used. But,
Among them, it is preferable to use an ion exchange resin. Further, the enzyme used in the present invention is not particularly limited as long as it does not inhibit the cross-linking reaction of transglutaminase, and enzymes for foods, medicines and the like may be used.
For example, trypsin, amylase, glucose oxidase, catalase and the like can be mentioned. As described above, any enzyme can be used, but it is particularly preferable to use trypsin or amylase. Further, these enzymes may be used alone, but it is also possible to use two or more kinds in combination.

To adsorb the carrier and the enzyme, first, the enzyme may be adsorbed to the carrier by adding and mixing the carrier and the enzyme to a buffer solution. The amount of the enzyme added is not particularly limited, but it may be usually added at a ratio of about 0.1 to 20 g per 1 g of the carrier.
As the buffer solution, a commonly used phosphate buffer solution, Tris-hydrochloric acid buffer solution or the like may be used. Next, transglutaminase may be added to form a crosslinked enzyme film on the carrier. Thereafter, the target immobilized enzyme can be obtained by washing with a buffer solution, distilled water or the like. Since it is not necessary that crosslinks are formed between the carrier and the enzyme, as long as crosslinks are formed between the enzymes, the pH of the buffer solution and the type of the carrier may be within the range where transglutaminase can act , PH at which the enzyme is easily adsorbed according to the properties of the enzyme used on the carrier
It is preferable to select the carrier in advance.

The transglutaminase to be added is derived from a microorganism (see Japanese Patent Application Laid-Open No. 1-27471), a liver from guinea pig (see Japanese Patent Publication No. 1-50382), and a genetically engineered product (Japanese Patent Application Laid-Open No. -300889), and any type may be used as long as it has transglutaminase activity such as plant-derived one. The amount of transglutaminase added in the present invention may be 0.001 to 100 units, preferably 0.01 to 10 units per 1 mg of the enzyme to be immobilized in the case of microorganism-derived transglutaminase. The reason for using this range is that the amount added is
If it is less than 0.001 unit, the enzyme will not be sufficiently cross-linked.
This is because the effect does not change so much even if added in units or more. The reaction conditions for immobilizing the enzyme with transglutaminase are not particularly limited, but are usually 0 to 60 ° C., preferably 3
The reaction may be performed at -40 ° C for 1 week-1 minute, preferably 24 hours-30 minutes. The pH of the reaction solution is usually pH 3-11, preferably p
It is sufficient to react with H5-9.

[0008]

The present invention will be described below with reference to examples.
The invention is in no way limited by these examples.

(Example 1) Trypsin (manufactured by Sigma) was dissolved in 50 ml of a wide-range buffer solution (prepared with phosphoric acid and citric acid) having a pH of 2 to 12 so as to be 5% by weight, and a cation exchange resin (SP) was used. -Sephadex C-25, manufactured by Pharmacia), 0.5 g,
Shake well for 5 minutes. 6 units of transglutaminase (prepared by the method of JP-A 1-27471) was added thereto per 1 mg of trypsin, and the mixture was reacted at 4 ° C for 15 hours while shaking. After the reaction, while sucking with a Buchner funnel, 0.00
300 ml 1N HCl, 300 ml 1N NaCl, 0.001N HCl
Was added and washed to remove non-immobilized trypsin. Then, it was freeze-dried to prepare an immobilized enzyme (hereinafter referred to as a sample).

On the other hand, a cation exchange resin (SP Sephadex C-25, manufactured by Pharmacia) was added to 50 ml of a wide range buffer having a pH of 2 to 12.
Was added, and 1 ml of a 5% trypsin (manufactured by Sigma) solution was added, and the mixture was shaken well for 5 minutes. Glutaraldehyde (manufactured by Nacalai Tesque, Inc.) was added to it to a concentration of 2.5%,
The mixture was reacted at 4 ° C for 15 hours while shaking. After the reaction, while sucking with a Buchner funnel, 300 ml of 0.001N HCl, 1N NaCl
Was added and 300 ml of 0.001 N HCl was added to wash unfixed trypsin and other reagents. Then, it was freeze-dried to prepare an immobilized enzyme (glutaraldehyde-immobilized enzyme).

The effect of pH at the time of immobilization on the activity of each immobilized enzyme thus prepared was examined. Activity is a method of measuring activity (KAPA) using a small molecule synthetic substrate (BAPA).
Walsh: Method in enzymology, 19, 62 (1970)). 5m of freeze-dried immobilized enzyme
Weigh each g and add 1 ml of 0.001 N HCl to swell, 17.
1 ml of 5 mg of BAPA dissolved in 50 ml of pH 7.8 triethanolamine buffer was added and reacted at 37 ° C for 1 minute. 2.5%
Stop the reaction by adding 1 ml of TCA and centrifuge (3,000 rp
m, 15 minutes) to remove the immobilized enzyme, and then the absorbance of the supernatant was measured at 405 nm. For the activity, the amount of enzyme that increases the absorbance at 405 nm by 1 was defined as 1 unit. The results are shown in Fig. 1.

When immobilized with glutaraldehyde as shown in FIG. 1, maximum activity was obtained at pH 2, but immobilization was not possible from neutral to alkaline. This is because trypsin can be immobilized because it is particularly stable at low pH, suggesting that glutaraldehyde has a detrimental effect on the enzymatic activity of trypsin from neutral to alkaline. . On the other hand, when immobilized with transglutaminase, it can be seen that trypsin can be stably immobilized in a fairly wide range if the pH is 11 or less. From these facts, it is inconvenient to immobilize an enzyme with glutaraldehyde because the pH at the time of immobilization is limited. However, when immobilizing the enzyme with transglutaminase, it can be immobilized in a fairly wide range of pH, so it is a very convenient and versatile technique compared to glutaraldehyde.

(Example 2) 100 mg of cation exchange resin (SP Sephadex C-25, manufactured by Pharmacia) and trypsin (manufactured by Sigma) were added to a wide range buffer having pH 8.0 (prepared with phosphoric acid and citric acid). 10 mg was added and shaken well for 5 minutes. Add 2.8 units of transglutaminase (obtained by JP-A 1-27471) to 1 mg of trypsin and shake it while shaking.
The reaction was carried out at 4 ° C for 15 hours. It was washed with 10 ml of Tris-HCl buffer (pH 8.0) containing 2 M of NaCl three times, and further with distilled water three times to give trypsin that was not immobilized by cross-linking (adsorbed on the cation exchange resin. Things are naturally included)
Was removed and freeze-dried to prepare an immobilized enzyme (sample).

As a control, trypsin and a cation exchange resin were similarly added to and mixed with a buffer solution to prepare an immobilized enzyme immobilized only by adsorption (control). That is, the control, since the cross-linked membrane of trypsin was not formed because transglutaminase was not added, and therefore trypsin was only adsorbed on the cation exchange resin, it was completely washed away by washing with the buffer solution. Controls are not washed to prevent that.

The continuous activities of both immobilized enzymes thus prepared were compared. The continuous activity test is based on the small molecule synthetic substrate (BA
Activity measurement method using PA (KAWalsh: Method in enzy
mology, 19, 62 (1970)). A column with an internal volume of about 5 ml (φ1.2 x 5 cm) was packed with 0.1 g of immobilized enzyme swollen with a buffer solution (pH 10.5),
The substrate (BAPA) was continuously supplied at a flow rate of 0.4 ml / min by using a peristaltic pump while submerging in a 37 ° C. thermostat. The reaction product eluted from the column was collected with a fraction collector, and the activity was measured in the same manner as in Example 1. The results of the continuous activity test comparing the sample with the control are shown in FIG.

As shown in FIG. 2, the control had an immediate decrease in activity. This indicates that the enzyme is rapidly released from the carrier simply by being immobilized on the carrier by adsorption. On the other hand, the sample immobilized with the addition of transglutaminase showed a slight decrease in activity, but maintained a higher activity than the control.

[0017]

The immobilized enzyme thus obtained is
It retains its activity fairly stably even after continuous use. Further, the immobilized enzyme obtained by the method of the present invention has little effect on the living body and can be easily inactivated by heating even if the washing is insufficient and transglutaminase remains in the immobilized enzyme. it can. In the case of immobilization with glutaraldehyde, for example, the activity of the enzyme to be immobilized is remarkably reduced unless the immobilization is performed under acidic conditions, whereas the activity of the enzyme to be immobilized is reduced by using transglutaminase. There is also an advantage that it can be immobilized in a wide range of pH without being lowered so much. Further, the immobilized enzyme of the present invention has an advantage that the activity decrease of the enzyme can be comparatively delayed even when continuously used. Therefore, the method of the present invention is effective as a method for providing an immobilized enzyme for use in products such as food products and pharmaceutical products.

[Brief description of drawings]

FIG. 1 shows the activities of immobilized trypsin immobilized with glutaraldehyde and immobilized trypsin immobilized with transglutaminase with respect to the pH of a buffer solution at the time of immobilization.

FIG. 2 shows the results of a continuous activity test of immobilized trypsin immobilized with transglutaminase and a control product.

Claims (5)

[Claims] 1. A method for immobilizing an enzyme, which comprises reacting an enzyme adsorbed on a carrier with transglutaminase to coat the enzyme on the carrier. 2. The method for immobilizing an enzyme according to claim 1, wherein the carrier is an ion exchange resin. 3. The method for immobilizing an enzyme according to claim 1, wherein the enzyme is a protease and / or an amylase. 4. The method for immobilizing an enzyme according to claim 1, wherein the transglutaminase is derived from a microorganism. 5. The method for immobilizing an enzyme according to claim 1, wherein 0.001-100 units of transglutaminase act on 1 mg of the enzyme.