JPS60202737A - Manufacture of affinity adsorbent - Google Patents

Abstract

PURPOSE:To manufacture the titled affinity adsorbent which is convenient and economical and can be widely applied by using chitosan or a chitosan derivative as an insoluble supporting carrier, and immobilizing a substance having affinity for biopolymers to the carrier with a covalent bond. CONSTITUTION:Chitosan or chitosan derivatives which can be easily manufactured from chitin contained in Crustacea and Insecta which is abundantly present in nature and is scarcely utilized is used as an insoluble supporting carrier, and a substance having affinity for biopolymers is immobilized onto said carrier by utilizing the high reactivity of free amino groups in the molecule. The complicated activation reaction of the insoluble supporting body as before can be omitted, and the affinity adsorbent which is convenient and economical an is widely applied can be manufactured.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing a novel affinity adsorbent using chitosan or a chitosan enzyme conductor as an insoluble support carrier.

Affinity chromatography is a fractional purification method that utilizes the inherent properties of biopolymers, such as specific binding or interaction with specific substances, and its application range has expanded rapidly in recent years. The most important things in the production of affinity adsorbents are insoluble support carriers, substances with affinity for biopolymers (hereinafter referred to as ligands), synthetic polymers such as acrylamide and polyvinyl derivatives, agarose, dextran, cellulose, and glass particles. Natural products such as

However, these have weak hydrophilicity, low ligand synthesis efficiency and target substance adsorption ability, low physical and chemical stability, low swelling rate, lack of porosity, and target substance Currently, agarose is the most widely used among these, as it has some drawbacks such as nonspecific adsorption. This agarose is commercially available as Sepharose, but in order to immobilize a ligand in the production of an affinity adsorbent, it is necessary to convert the agarose into an active derivative in advance, making it complicated and expensive to handle.

Chitosan is an aminopolysaccharide that is obtained by treating chitin with a concentrated alkali and N-deacetylating it (hereinafter referred to as a structure). Chitosan contains many free amino groups within its molecule.

Easily forms salts and dissolves in dilute acids. In addition, many chitosan and chitosan derivatives exhibit gelation function.
Affinity adsorbents utilizing these properties have already been reported. However, in all of these methods, chitosan or a chitosan derivative itself was used for affinity adsorption, and the ligand was not selectively immobilized as a support carrier, so the range of application was extremely limited.

The present inventor used chitosan, which is easily produced from chitin contained in crustaceans and insects, which is abundant in nature and has low utilization, as an affinity support carrier. We focused on this and discovered that by immobilizing various ligands to this functional group, it is possible to produce affinity adsorbents that are simple, economical, and have a wide range of applications.

This invention will be explained in detail below.

The chitosan used in the present invention may be prepared by a conventional method, and examples of its preparation are as follows. Chitin obtained from crabgrass is treated in a 45-50% sodium hydroxide solution at 90°C for 5 hours, then washed with water and dried. This product was used in the examples. Also, this chitosan.

In order to improve the physical and chemical stability as an affinity adsorbent, glutaraldehyde, 1,4-butanediol glycidyl ether, epichlorohydrin, etc. are used in advance to cross-link in such a way that a large number of free amino groups remain. It is preferable to use a derivative as a derivative thereof, or to perform N-acylation or crosslinking after the immobilization reaction between chitosan and the ligand is completed. On the other hand, the ligand to be bound to this carrier may be either a low-molecular or a high-molecular one. For example, if the target of the purified substance is an enzyme, substrates, inhibitors, allosteric effectors, coenzymes, antagonists, etc. and other binding proteins depending on the purpose.

complex carbohydrates, phospholipids, hormones, vitamins, antibiotics,
Nucleic acids, antibodies, habuten, etc. can be used. If the ligand does not have the functional group necessary to bind to the amino group of chitosan or a chitosan derivative, or if you want to improve the immobilization rate of the ligand to the support carrier, use a derivative that easily binds to the amino group in advance. It is necessary to

The present invention utilizes the highly reactive amino groups present in chitosan and chitosan derivatives to directly immobilize a ligand, and the reaction with the ligand is carried out using the knowledge of immobilized enzymes and conventional methods for producing affinity adsorbents. This can be carried out by the diazo method, peptide method, Shipp salt formation method, Yugi reaction method, or cyanogen bromide activation method based on . According to the present invention, in the immobilization reaction of a ligand and a supporting carrier.

Conventionally, the support carrier was an insolubilized material, but chitosan has the property of being soluble in dilute acids, so it is possible to carry out immobilization reactions in the weak acid region even in liquid-liquid reactions. . When immobilized using this method, the support carrier can be insolubilized by alkali gelation, N-acylation, crosslinking, etc., and can be prepared as an adsorbent, and even ligands with a low immobilization rate can be quickly prepared. It has the characteristic of being able to produce affinity adsorbents with high immobilization rates.

Furthermore, when the ligand and support carrier are directly bound, there is no spatial distance between the ligand and the matrix, so the support carrier is susceptible to steric hindrance, and the original affinity adsorbent may not be exerted. In such a case, an arm can be introduced between the ligand and the support carrier via the amino group of chitosan or a chitosan derivative, so that a wide range of affinity adsorbents can be produced.

As described above, the present invention utilizes the amino groups of chitosan or chitosan derivatives, omits the conventional complex activation reaction of an insoluble support carrier, and produces an affinity adsorbent that is simple, economical, and has a wide range of applications. It is. The present invention will be explained in detail with reference to Examples below.

Example 1 After dissolving 1 g of chitosan in 80 ml of 0.5% acetic acid solution, 5N
(Hydroxide) Gradually add IJ solution to pH.

6.0, and added distilled water to make it 100 mJ. After dissolving 601n9 carbodiimide reagent in 5 ml of this solution, 1 ml of concanavalin A (161n9/ml protein concentration) extracted from jack bean and partially purified by ammonium sulfate salting out and column chromatography using Sephadex G-75 was added. N, gas-filled, tightly closed chamber source, 2
．． The reaction mixture was stirred gently for 5 hours. After the reaction is completed, 0.2
M ammonia buffer (PH10) pH to 8-9 w4
The mixture was prepared and gelled, and thoroughly washed with 0.1M phosphate buffer (PH7.2) containing 0M sodium chloride to obtain an affinity adsorbent. The gel amylase (Seikagaku G) Imjl' (t/mJ concentration) equilibrated in advance with 0.1M phosphate buffer (PH7.2) containing -1.0M sodium chloride was developed for 30 minutes at room temperature. After holding, the column was washed with 20 ml of the above buffer solution.
- Elution was carried out with 20 ml of the same buffer containing mannoside. The eluate was separated into 1 ml portions, and each fraction was divided into 2
The enzymatic activity of glucoamylase was measured by protein extraction and the Somogyi Ken-Nelson method at an absorbance of 80□. The result is the first
It was as shown in the figure.

Example 2 After dissolving chitosan IIi in 80ml of 0.5% acetic acid solution, a 5N sodium hydroxide bath solution was gradually added to adjust the pH to 6.0.
Distilled water was added to make the volume 100ml. This bath t5m
20 liters of sodium cyanoborohydride and 0.5 ml
Add a bath solution in which 15 Da of maltose was dissolved in 0.1 M phosphate buffer (PH 6.0), and place in a sealed stopper filled with nitrogen gas for 2 hours.
The mixture was shaken and reacted at 5°C for 244 hours. 25% after completion of reaction
0.02 mJ of glutaraldehyde was added and shaken for 1 hour to form a gel.

This gel was collected by centrifugation and washed with 0.1 M 17-phosphate buffer (PH6,0) to obtain an affinity adsorbent. The above gel column 2 was equilibrated in advance with 0.1M IJ acid buffer (PH7.1) containing 1.0M salt.
ml of Example 1. Concanavalin A (16
Ing/ml protein concentration) 1 ml was developed. After being held at room temperature for 60 minutes, the column was washed with 50 ml of the above buffer and eluted with 20 ml of the same buffer containing 0.5 M α-methyl-D-mannoside. Aliquot the eluate in 1 ml portions,
Protein elution was measured for each fraction at an absorbance of 280 nn+.

The results were as shown in Figure 2. The washing part was concentrated by ultrafiltration, and the eluate part was dialyzed with 0.1 M IJ acid buffer (PH7,1) to remove α-methyl-D-mannoside, and then the 2% concentration in the agar gel for glycogen was determined.
Heavy diffusion was performed and it was confirmed that concanavalin A was localized in the elution area.

Further purified concanavalin A was homogeneous in SDS polyacrylamide gel electrophoresis.

Example 3 5 g of chitosan is dissolved in a 5-thiacetic acid solution. 1 in this solution
096 Hydroxide) Gradually add IJum bath solution to PH8
,0 to gel chitosan. Collect this gel by centrifugation, add 100 ml of distilled water, and stir well. Add 10mj of epichlorohydrin to this gel suspension! Add 1
After reacting at 00° C. for 1 hour, 100 ml of IN hydroxide solution was added and the reaction was further continued for 2 hours. After the reaction was completed, the gel was collected by suction filtration and thoroughly washed with distilled water. This gel was suspended in a 5% acetic acid solution, homogenized using a homogenizer, and fine particles were removed by decantation. The gel obtained here was filtered through a glass filter and thoroughly washed with distilled water to obtain a chitosan epichlorohydrin gel. This chitosan epichlorohydrin gel 10m
1, 100 mg of sodium cyanoborohydride and 0.2
M phosphate buffer (PH6,0) 10mJ, β-1,
A solution of 400 my of 3-glucan (soluble Hachiman) was added, and the mixture was shaken and reacted at 40° C. for 488 hours in a sealed stopper filled with N and gas. After the reaction was completed, the gel was collected by suction filtration and washed with 100 mJ of 1.0M saline to obtain an affinity adsorbent. The β-1,3-glucan soluble batiman used here was obtained by limited hydrolysis of defatted sclerotia of Poria COQ) 5 with formic acid, ethanol fractionation, and dialysis, and the average degree of polymerization was 20. there were. Basidiomycetes Q
0.25 ml (1200 units) of β-1,3-glucanase obtained from M 806 by a known method was developed.

After holding at 4°C for 30 minutes, the column was diluted with 25ml of the above buffer.
After washing with At, it was eluted with 20ml of the same buffer containing 0.1 soluble Panaman. Aliquot the eluate in 2 ml portions,
For each fraction, the protein was eluted using absorbance at 280 mm, and the enzymatic activity of β-1,3-glucanase was measured using the Somogyi-Nelson method. The results were as shown in Figure 3. In addition, β-1,3- purified according to the present invention
Glucanases were homogeneous by gel filtration on Biogel P-60 and SDS polyacrylamide gel electrophoresis.

Example 3. A solution of 2 g of the same β-1,3-glucan (soluble batman) dissolved in 70 ml of IM sodium bicarbonate solution and 2 g of cyanogen bromide in 4 ml of acetonitrile were added.
The same chitosan epichlorohydrin gel 20 as in Example 3 was added to this solution and reacted for 2 minutes at room temperature to obtain cyanogen bromide-activated β-1,3-glucan.
ml was quickly added, and the reaction was allowed to proceed under shaking at 4° C. for 18 hours in a tightly stoppered container. After the reaction is complete, collect the gel by suction filtration and add IM hydrogen carbonate (IM) solution (200ml) - 2M sodium acetate solution (200mA)! , and 200 ml of distilled water to obtain an affinity adsorbent. Affinity Take Of
1 mA of β-1,3-glucanase partially purified from Rh1zoctonia 5olani by a known method. (450 units) in the same manner as in Example 3. The results were as shown in FIG. Note that the β-1,3-glucanase purified according to the present invention was homogeneous in gel filtration using Biogel P-60 and SDS polyacrylamide gel electrophoresis.

[Brief explanation of the drawing]

, 1 Figure 1 shows the affinity chromatography of concanavalin A with glucoamylase (Seikagaku!!J) originating from Chitosandal. Figure 2 shows the adsorption of maltose immobilized on Chitosandal taraldehyde gel. Figure 3 shows the affinity chromatography of concanavalin A using an adsorbent in which β-1,3-glucan (soluble batman) was immobilized on a chitosan epichlorohydrin gel. β-1,3-glucanase of Basidiomycetes QM806. Figure 4 shows the affinity chromatography of cyanogen bromide activated β-1,3-glucan (
This figure shows the affinity chromatography of an adsorbent immobilized on a chitosan-epichlorohydrin gel. Patent Applicant: Yaizu Suisan Kagaku Kogyo Co., Ltd. Figure 2: Fukushikon No. (1m cross) Figure 3: Fukushikon No. (2ml) Figure 4: Fukushikon No. (2m cross) Procedural amendment 1, case Indication Patent Application No. 57542 of 1982 2, Name of the invention Method for manufacturing affinity adsorbent 3, Relationship with the case of the person making the amendment Patent applicant Yaizo Suinan Kagaku Fukugi Mari name Title Yaizu Suisan Kagaku Koei Co., Ltd. Representative Matsu Part 4 of this book, Agent 6, Subject of amendment Specification 7, Contents of amendment (1) As 1 adhesion on page 3, line 12 of the specification.'' (
amend the description as "as an adsorbent". (2) The statement “to be fixed” in the first line of page 4 (
Correct the description to "chemically bond and immobilize with a covalent bond." (3) On the last line of the fourth page, the statement ``Any item is fine'' is amended to read ``One item or either item is fine.'' (4) The description “adsorbent” on page 6, line 12 of the same page was changed to “
It is corrected as "adsorption capacity". (5) 1 adjustment to the statement ``Preparation'' on page 7, line 7.''
and correct it. (6) The statement "Seikagaku Kogyo" on page 8, line 1 and page 13, line 5 is amended to read "Seikagaku Kogyo Co., Ltd." (7) "IN sodium hydroxide" on page 10, line 6
The description has been corrected to read "rlN sodium hydroxide." (8) The statement "1280 nmJ" on page 11, line 13 is corrected to r280nmJ. 9) The description "rIMj" on the 3rd and 11th lines of page 12 will be corrected to "IM". 1. The statement "Shin-to-shi" on page 12, line 9 of the 1st section is amended to read "Shaken-shi."

Claims (1)

[Claims] A method for producing an affinity adsorbent, which comprises using chitosan or a chitosan derivative as an insoluble support carrier, and immobilizing a substance with affinity for biopolymers by covalent bonding thereto.