JPS606703A - Bonding of biopolymer to polymer - Google Patents

Abstract

PURPOSE:To bond a biopolymer to a polymer safely at a high rate of immobilization, by converting a hydroxyl group-containing polymer into a 1-imidazolylsulfonate derivative and reacting the derivative with an amino group-containing biopolymer. CONSTITUTION:A hydroxyl group-containing polymer (e.g., dextran or hydroxyethylcellulose) is reacted with sulfuryl chloride (reaction formula I , wherein R is a polymer residue), and the product is further reacted with imidazole (reaction formula II). The produced 1-imidazolylsulfonate derivative is reacted with a prim. (or sec.) amino group-containing biopolymer (e.g., enzyme) under a nearly neutral condition to form a polymer-biopolymer conjugate (reaction formula III). This process is safe because no toxic reagent such as a cyanogen halide is used and, besides, the process is applicable also to biopolymers which lose their activities under an alkaline condition.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for bonding a biopolymer having an amino group such as halogen or a proteinaceous group to a polymer having a hydroxyl group such as cellulose.

BACKGROUND ART Conventionally, immobilized enzymes, etc., in which enzymes and proteins are immobilized by chemically bonding them to polymeric substances as carriers, have been used as column packing materials for bioreactors and liquid chromatography. For example, for polymers such as polysaccharides having one or more hydroxyl groups, preferably PH8-13
A method of activating a cyanide halide under alkaline conditions, and then reacting and bonding it with a biopolymer having amide 7 groups under weakly alkaline conditions. It is activated by the action of p-trienesulfonyl, and then is reacted with berocykdase and alcohol dehydrogenase by a nuclear substitution reaction at an activity-fixed rate of about 18 and 25, respectively.
It is known that ='=1 is made constant.

However, in the method using halogenated/ammonium, the halogenated/ammonium is extremely poisonous, so care must be taken when handling it, and it takes time and effort to treat wastewater after use. Since the reaction between the polymer and the biopolymer must be carried out under alkaline conditions, there is a drawback that biopolymers that are deactivated under alkaline conditions cannot be used, and in the latter case, the activity immobilization rate is low. It has the disadvantage of being low.

The present invention addresses the above-mentioned current situation, eliminates the need to use harmful reagents such as cyanogen halides, and furthermore provides a polymer carrier that does not deactivate biopolymers that would otherwise be deactivated under alkaline conditions. The purpose of this research is to provide a method for chemically bonding biopolymers to polymers having hydroxyl groups at a high activity immobilization rate, and the gist of this method is as follows. In a method for covalently bonding a biopolymer containing a primary or secondary amine group to a polymer containing a hydroxyl group, the polymer is reacted with sulfuryl chloride and then with imidazole; The present invention resides in a method for bonding a biopolymer to a polymer characterized by reacting the above-mentioned living body and polymer with the above-mentioned biological polymer.

Polymers containing hydroxyl groups used in the present invention include polysaccharides and derivatives thereof, such as polysaccharides such as dextran, cellulose, starch, dextrin, and agarose, and aqueous groups of polysaccharides such as hydroxyethyl cellulose. Containing gg conductors are preferably used.

Based on the present invention, in order to bond a biological material to a polymeric carrier containing a hydroxyl group such as a polysaccharide, the core polymer is first reacted with sulfuryl chloride to convert the hydroxyl group of the polymer to the formula (I
), and then, imidazole is added thereto and reacted to form a 1-imidanolyl-sulfonate derivative as shown in formula (It), thereby activating the hydroxyl group of the polymer. (R is a polymer residue) In the reaction of formula (I), the polymer from which moisture has been sufficiently removed is usually reacted with a suitable organic medium such as N,N-dimethylformamide.
Row 9 is preferred, in which sulfuryl chloride is suspended or dissolved in ↓ and cooled to -40 to -50°C, and sulfuryl chloride is added dropwise.

Further, it is preferable that the reaction (II) is carried out by adding a large excess of imidazole after dropping sulfuryl chloride in the above procedure, and gradually raising the temperature to room temperature.

The 1-imidazolyl-sulfonate derivative thus produced has the formula (+1
The reaction is performed as in 1) to give a polymer-biopolymer conjugate stably bound by covalent bonds.

In addition, in the above formula (III), the primary amino group (-NH2)
Although we have shown a mechanism in which a biopolymer having Combine with molecules.

Therefore, in the present invention, biopolymers such as enzymes and proteins having a primary or secondary amino group to be bound are preferably bonded to a medium having a neutral P I (value of around 1'fJ). The above conjugate can be obtained by adding K dissolved in a buffer solution and the activated product of the hydroxyl group-containing polymer prepared above, that is, the l-imidazolylsulfonate derivative, and stirring.

In addition, in the present invention, after binding the biopolymer as described above, it is preferable to block the active sites still remaining in the polymer such as cellulose, and for this reason, K is
Approximately P containing about 0.05 T of 2-mercaptoethanol
H80 Tris J bar 1' should be submerged in relaxation solution.

``Ali-like'' - a conjugate of a polysaccharide molecule and a biopolymer such as an enzyme or a protein, prepared according to the present invention, as an immobilized enzyme, etc. Bile can be used as a packing material with a reaction force of 2 μm due to immobilized enzymes used in liquid chromatography for analysis. Since it is strongly bonded, it is not easily decomposed and can be used stably even in a wide range of P II.

The method of the present invention is as described above, and in particular, after a polymer containing one or more hydroxyl groups is activated by reacting with sulfuryl chloride and then with imidazole, The method of the present invention involves reacting with a biopolymer containing a group and covalently bonding the polymer and the biopolymer. The reaction can be carried out safely without using any reagents9, and since the binding reaction can be carried out under neutral conditions, biopolymers that are inactivated by alkalinity can be bound to the polymer carrier. Moreover, it has the effect of being able to bind biopolymers such as enzymes to polymeric carriers at a high activity immobilization rate.

The present invention will be explained below based on Examples.0 Example Activation of carrier: Cellulose beads (trade name) After washing with cell water, dehydration [7]N,N-dimethylpolamide (DMF
) Cellulose beads 2.6
Take out y, stir and suspend in 60mel of DMF,
It was cooled to -40°C in a dry ice-ethanol bath. Add 1.6 m of sulfuryl chloride to this while stirring gently.
When e was added dropwise, the reaction solution turned pale yellow. After 30 minutes, imidazole 1.0.89 was added, the temperature was returned to room temperature with stirring, 4 cellulose beads were separated from the furnace, taken out, and DMF
, distilled water, and DΔ1F in this order, and sufficient suction dehydration was performed to prepare activated cellulose beads.

Enzyme immobilization: Next, 3ol-hydroquinosteroid dehydrogenase (3α-H8D) 12.6M OPI
I 7.0 ノ 0.1 M 9 fi! 2 buffer w, 2
- The activated cellulose beads U and 627 prepared above were added to the nitrified mixture and reacted at room temperature with stirring. After 2 hours, the cells were separated and the active sites remaining on the cellulose beads were removed. 0.1 at pH 8.0 containing 0.05% 2-mercaptoethanol to block
M)! Immersed in salt solution.

After standing at 4°C for 2 hours, it was separated from the furnace and treated with tr containing 0.05% 2-mercaptoethanol and 0.1mM EDTA.
PH7,0(D100mM!J) was stored in rr1 buffer.

Calculation of immobilization rate: In order to investigate the immobilization rate of the 3α-H8D-immobilized cellulose beads obtained in this way, the above 3α-
The enzyme activity before and after immobilization of II SD solution was determined using 20 mM pyrroline 1'i of PI (9,5).
The enzyme activity before immobilization was 0.275 when measured in the presence of coenzyme nicotinamide adenine dinucleotide and substrate (cholic acid) in ims solution (25°C).
22) / 0.1d, the activity of 9 elements after immobilization is 0.
0174 units, 10.1-, and therefore the immobilization rate was calculated to be 93.7%.

Measurement of activity of immobilized enzyme: Coenzyme nicotinamide adenine nucleotide 1mM and substrate (Colefl) 1m in 20mM pyrophosphate laxative solution (25°C) at pH 9.5.
In the presence of M, a precisely weighed immobilized enzyme was added, stirred, and reacted.The activity was examined, and a specific activity value of 8.9 units/2 was measured.

patent applicant Sekisui Chemical Co., Ltd. Representative Mototoshi Numa

Claims (1)

[Claims] 1. In a method of covalently bonding a biopolymer containing a primary or secondary amino group to a polymer containing a hydroxyl group, the polymer is bonded with sul7lyl chloride and then with imidazole. A method for bonding a biopolymer to a polymer, which comprises reacting the reaction and then reacting the biopolymer with the above biopolymer.