JPS60137433A - Adsorbent for urea decomposition - Google Patents

Abstract

PURPOSE:To quicken adsorption velocity by bringing substance contg. urea to which urease is immobilized into contact with polysaccharide contg. an aldehyde group which is obtained by oxidizing polysaccharide having at least two adjacent hydroxy groups in a structural unit. CONSTITUTION:Polysaccharide contg. an aldehyde group which is obtained by oxidizing the hydroxy group of polysaccharide such as starch, cellulose, dextrin, mannan and carboxymethyl derivatives of these compounds to aldehyde group is dispersed in water and is caused to react with enzyme while stirring the mixture at 0-5 deg.C for 10-50hr to immobilize it. As enzyme, urease which has urea as base substance and is decomposed to ammonia is used. After the immobilization, it is dried by freezing and stored in a closed state at 0-5 deg.C.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a novel urea decomposition adsorbent in which urease is immobilized on a polysaccharide oxide.

Conventionally, in order to improve the adsorption performance of an adsorbent, the properties and shape of the adsorbent have been improved. but,
These methods are improvement methods from the adsorbent side, and as long as the substance to be adsorbed is the same, there are naturally limits and it is difficult to significantly improve the adsorption performance. Therefore, a dramatic adsorption effect can be expected by an improved method that changes the viewpoint from the side of the adsorbed substance, that is, a method that converts the adsorbed precursor into a low-molecular substance that is more easily adsorbed, and then immediately removes it with an adsorbent. At this time, an enzymatic reaction may be used to effectively convert the adsorbed precursor.

For example, an adsorbent for removing urea has been desired as an adsorbent for artificial detoxification organs from various fields, but no adsorbent effective for urea has been found at present.

Therefore, the enzyme urease is used to convert urea into ammonia, which has a smaller molecular weight, and this is immediately adsorbed using an ammonia adsorbent. In general, conventional adsorption systems using enzymes involve binding the enzyme to a water-insoluble carrier or immobilizing the enzyme by encapsulating it in microcapsules. There is a method in which these are reacted with a solution containing a precursor to be adsorbed, and the reaction product is removed using an adsorbent such as activated carbon or ion exchange resin. However, in this case, a secondary reaction may occur before the reactant is adsorbed by the adsorbent, and a sufficient effect may not be achieved, and more than one device is required, resulting in a large size. There was a drawback.

The inventors of the present invention have conducted extensive research to solve these problems, and as a result have completed a urea decomposition adsorbent that has an extremely high adsorption effect. That is, it is a urea decomposition adsorbent in which urease is immobilized on an aldehyde group-containing polysaccharide prepared by oxidizing a polysaccharide having at least two adjacent hydroxyl groups in the structural unit as an adsorbent base material.

When a urea-containing substance is brought into contact with this adsorbent, the urea is converted into ammonia by the enzymatic reaction of the grass, which is immediately adsorbed onto the adsorbent substrate. Since the enzymatic reaction product is immediately adsorbed by the adsorbent, the occurrence of secondary reactions, which is a disadvantage of the conventional method, is almost completely prevented, and the enzymatic reaction is also promoted. Furthermore, since the urease is immobilized on the adsorbent substrate, the device according to the invention is extremely compact.

As the adsorbent base material used in the present invention, an aldehyde group-containing polysaccharide, which is an oxide prepared by oxidation of a polysaccharide having at least two adjacent hydroxyl groups in its structural unit, may be used. The monomer is a polysaccharide in which the hydroxyl group is oxidized to an aldehyde group, but it may contain a small portion of carboxyl group. Examples of polysaccharides include starch, cellulose, dextrin, mannan and their carboxymethyl derivatives, aminoethyl derivatives,
/ ethylamino derivatives, etc. The enzyme used is urease, which decomposes urea into ammonia using urea as a substrate. The ammonia adsorption rate and adsorption capacity (on a molar basis) of the above-mentioned oxides of these polysaccharides are greater than those of urea, so if urea is efficiently converted to ammonia using urease, the urea concentration will be dramatically reduced. death,
In addition, ammonia produced by the enzymatic reaction is adsorbed at a faster rate than urea having a higher concentration of nitrogen.

According to the present invention, when an enzyme is immobilized using a part of the active aldehyde groups of an aldehyde group-containing polysaccharide, the aldehyde group-containing polysaccharide is dispersed in water,
Immobilization is carried out by reacting with the enzyme under stirring for 50 hours, preferably 20 to 30 hours. In addition, when immobilizing enzymes using other functional groups in aldehyde group-containing polysaccharides, for example, in the case of carboxymethyl groups, it may be immobilized using a condensation reagent such as carbodiimide or a Landward reagent. Alternatively, there are methods such as making an acid azide derivative of a carboxylic acid and then immobilizing the enzyme. In the case of an aminoethyl group, it can also be immobilized using a crosslinking agent such as glutaraltehyde. The aldehyde group-containing polysaccharide on which the enzyme has been immobilized by the method described above is recovered by freeze-drying, and preferably stored tightly at 0 to 5°C until used as an adsorbent.

The urea decomposition adsorbent thus obtained can be used as a medical or pharmaceutical industrial material, an industrial raw material, or as a material for chemical experiments. For example, a urea decomposition adsorbent in which the enzyme urease is immobilized on dialdehyde starch can be used to remove uremic toxins from the blood and body fluids of uremic and hypermonosemic patients and to reduce blood urea nitrogen concentration. It is used as a therapeutic adsorbent for dialysis patients, or as an orally administered drug to reduce the number of dialysis treatments in dialysis patients, or to delay the initiation of dialysis treatment in uremic patients.

Next, the present invention will be explained in more detail with reference to Examples and Reference Examples.

Example 1゜Aldehyde starch 107 obtained by periodic acid oxidation of potato starch was suspended in 100 mJ of water containing urease (2300 international units/1154) and
Stirred at 5°C for 24 hours. Then filter the precipitate,
The powdered urea decomposition adsorbent (U-
I) 8.11- was obtained. The urease content of U-I determined from the nitrogen analysis value was 6.0, and the urease activity per U-I 17 was 18.6 international units.

The results of examining the urea removal ability of the obtained urea decomposition adsorbent are shown in the following table in comparison with the adsorption base material itself for non-immobilized urease. In the urea removal experiment, the urea concentration was 200 ml/
A certain amount of adsorbent was added to a dialysate (p" 7.2) adjusted to di and after incubation for 24 hours, the urea removal rate was estimated from the change in urea concentration.

Example 2 8 of potato starch was dispersed in 500 ml of water and mixed with 2.3 g of epichlorohydrin at 20°C.
50 rnl of N-sulfurium hydroxide water bath solution was added and stirred for 2 hours to obtain 70% crosslinked potato starch. The cross-linked dialdehyde starch 305' obtained by periodic acid oxidation of this cross-linked potato starch was used in place of the dialdehyde starch in Example 1, but the procedure was exactly the same as in Example 1, and the urea was treated with a urease solution. 25 g of decomposed adsorbent (UT) was obtained. The urease content of U-N determined from the nitrogen analysis value was 4.9, and the urease activity per U-IIlg- was 15.5 international units.

The urea removal rate of the obtained urea decomposition adsorbent was investigated according to the method of Example 1. The results are shown in the table below.

Example 3 The dialdehyde mannan 11- obtained by oxidizing mannan with periodic acid was used in place of the dialdehyde starch in Example 1, except that it was treated with a urease solution in the same manner as in Example 1. Urea decomposition adsorbent (U-I
N) 8.3P was obtained. The urease content of U-[ determined from the nitrogen analysis value was 5.2%, and the urease activity per U-1[]J was 156 international units.

The urea removal rate of the obtained urea decomposition adsorbent was investigated according to the method of Example 1. The results are shown in the table below.

Example 4 1 g of dialdehyde carboxymethyl dextran obtained by periodic acid oxidation of carboxymethyl dextran
- was suspended in 10 ml of water, water-soluble carbodiimide 15' was added thereto, and the mixture was stirred for 2 minutes. Next, urease (
Add 2300 international units/y-)6oomg, 0.2N
- No. 11 was adjusted to 8.5 with a sodium hydroxide/rum aqueous solution and slowly stirred at room temperature for 20 minutes and then at 4°C for 5 hours. The generated precipitate was filtered and resuspended in a pH 3.5, O, 1M aqueous sodium carbonate solution containing 0.05M 2-amine ethanol. After standing at 4°C for 10 hours, the precipitate was reduced to 0.15. M-Sodium chloride aqueous solution,
After thoroughly washing with 0.05M calcium chloride aqueous solution and filtering the precipitate, urea decomposition adsorbent (U
-W) o, s6y were obtained. The urease content of U-W was determined from the nitrogen analysis value to be 4.9%, and the urease activity per U-■ IP was 15.2 international units.

The urea removal rate of the obtained urea decomposition adsorbent was investigated according to the method of Example 1. The results are shown in the table below.

Example 5 Potato starch was oxidized with periodic acid to obtain aldehyde starch partially oxidized to carboxylic acid.

Urea decomposition adsorbent (U-v) 3.9P was obtained in exactly the same manner as in Example 4 except that this aldehyde starch 5y- containing a carboxyl group was used in place of dialdehyde carboxymethyl dextran. Ta.

The urease content of U-V determined from the nitrogen analysis value is 48%, and the urease activity per U-v17 is 149%.
It was in units of N.

The urea removal rate of the obtained urea decomposition adsorbent was investigated according to the method of Example 1. The results are shown in the table below.

Example 6 Dialdehyde amine ethyl cellulose 151- obtained by oxidizing aminoethyl cellulose with periodic acid was pl-
17. OIJ acid buffer 15. yil, 4 ml of 50% glutaraldehyde aqueous solution was added thereto, and the mixture was stirred at room temperature for 2 hours. The reaction product is centrifuged and the pH
Washed 6 times with 7.Q phosphate buffer. The resulting powder was then resuspended in 15 ml of pl-17,0'J acid buffer, to which was added 1 ml of an aqueous solution containing 100 #19 urease (2300 international units/P), and the mixture was kept at room temperature. So 2
Stir for hours. The final reaction product was centrifuged and ρl-17
, Q three times with phosphate buffer. Filter the precipitate,
Urea decomposition adsorbent (U4I) by freeze-drying
0.72 P was obtained. The urease content of U-Vl determined from the nitrogen analysis value was 5.4%, and the urease activity per U-Vllg- was 173 international units.

The urea removal rate of the obtained urea decomposition adsorbent was investigated according to the method of Example 1. The results are shown in the table below.

In addition, non-immobilization 1 to 6 in Comparative Examples are Examples 1 to 6.
This is the adsorption substrate itself on which urease is not immobilized. Furthermore, in all of the urea removal experiments using U-4-U-■, almost no residual ammonia was detected in the urea solution after 24 hours. This indicates that all of the converted ammonia is removed by the adsorption substrate.

Furthermore, as is clear from this table, the compounds of the present invention exhibit a dramatic urea removal effect compared to compounds on which urease is not immobilized.

Patent applicant Nippon Carlit Co., Ltd. (2 others)

Claims (2)

[Claims] (1) A urea decomposition adsorbent in which urease is immobilized on an aldehyde group-containing polysaccharide prepared by oxidizing a polysaccharide having at least two adjacent hydroxyl groups in its structural unit. (2) The aldehyde group-containing polysaccharide is selected from the group consisting of starch, cellulose, dextrin, mannan, and carboxymethyl derivatives, aminoethyl derivatives, and diethylamino derivatives thereof containing aldehyde groups. The urea decomposition adsorbent described in .