JPS5817202B2 - Shinsuisei Polymer Noseizouhou - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing a substantially water-insoluble hydrophilic polymer used as a carrier for immobilized enzymes, a carrier for protein separation or purification, and the like.

Carriers for immobilized enzymes and carriers for protein separation and purification can be broadly classified as follows.

(1) Natural polymers obtained from starch, cellulose, dextran and their derivatives, etc. (2) Polyacrylamide, polyaminopolystyrene,
Synthetic polymers obtained from amino acid copolymers, maleic anhydride-ethylene copolymers, polymethacrylic acid, etc. (3) Inorganic materials such as porous glass, activated carbon, alumina, and acid clay Easily immobilize enzymes in these Natural products and their derivatives (1), such as Sephadex (product name of Pharmacia, Sweden), which is a cross-linked product of dextran, and Sepharose (Sweden, a trade name of Pharmacia, Sweden), which is a cross-linked product of agarose, are used because of the ease of protein separation and purification. , Pharmacia Co., Ltd. product name) are often used as Affinitei chromatography.

However, carriers obtained from these natural products are generally inferior to synthetic polymer carriers (2) in terms of mechanical strength, and therefore are difficult to fully demonstrate their performance due to destruction or compression when used in large-scale equipment.

The inorganic material (3) has sufficient mechanical strength, but is difficult to handle in an aqueous solution due to its hydrophilicity.

The main purpose of the present invention is to take advantage of the advantages of the natural polymer (1) and supplement the mechanical strength with the synthetic polymer (2).

That is, a polymer is used which has a -CONH2 group as the base polymer, and in which all or some of the nitrogen atoms of the amide bond or carbamoyl group are methylolated, and this polymer has two or more hydroxyl groups in the molecule. The gist is acidic grafting of compounds to obtain hydrophilic polymers that are substantially water-insoluble.

To explain the present invention in detail, examples of the basic synthetic polymer include the following.

(a) Partially N-methylolated polyacrylamide or polymethacrylamide obtained by formalin treatment of polyacrylamide or polymethacrylamide ('b) Polyacrylamide or polymethacrylamide obtained in the presence of a crosslinking agent such as methylenebisacrylamide (c) Polymer of N-methylol acrylamide or N-methylol methacrylamide (d) Polymer of N-methylolated polyacrylamide or polymethacrylamide obtained by formalin treatment of -Polymer of methylol acrylamide or N-methylol methacrylamide (e) N-methylolated 8-nylon, 6-nylon, etc. obtained by formalin treatment of 8-nylon, 6-nylon, etc. Examples of compounds having two or more hydroxyl groups include the following.

(a) Saccharides (1) Monosaccharides Glucose, etc. (1i) Polysaccharides Dextrin, soluble starch, dextran, etc. (b) Sugar alcohols Water is preferable as the reaction solvent, but the base polymer and the saccharides to be grafted, polyhydric alcohols Any solvent that easily dissolves or swells sugar alcohol, etc. is suitable.

In this respect, water is most preferred and other aprotic polar solvents such as dimethyl sulfoxide and dimethyl formamide can also be used.

Since the grafting reaction proceeds under acidic conditions (pH 0.1 to 6.5), any acidic reagent can be used as a catalyst, regardless of the type.
A variety of organic and inorganic acidic reagents can be used.

In addition, since a condensation dehydration reaction between N-methylol groups proceeds under acidic conditions, a crosslinking reaction due to this reaction also proceeds during the grafting reaction.

The reaction is generally carried out as follows.

Saccharides and polyhydric alcohols that are grafted onto the backbone polymer,
Sugar alcohols, etc. (dissolved homogeneously in a solvent such as water in advance) are mixed in a solvent to make it homogeneous.

In this case, when using a polymer that is difficult to dissolve in a solvent, such as a crosslinked backbone polymer, it is uniformly blended with sugars, polyalconopesaccharide alcohols, etc. in a swollen state.

The grafting reaction is carried out by adjusting the ratio of the raw materials used so that each N-methylol group in the base polymer contains 0.1 to 100 alcoholic hydroxyl groups, preferably 0.2 to 50 alcoholic hydroxyl groups. be exposed.

If this ratio is too small, the grafting reaction will not be carried out sufficiently, and if this ratio is too large, a large amount of unreacted saccharides, sugar alcohols, polyhydric alcohols, etc. will remain, which is economically disadvantageous.

Next, the acidifying reagents used as catalysts include organic acidifying reagents, such as sulfonic acid derivatives such as para-toluenesulfonic acid and trifluoromethanesulfonic acid, and carboxylic acid derivatives such as acetic acid, and examples of inorganic acidifying reagents include, for example, sulfuric acid, There are strong acid and weak acid reagents such as hydrochloric acid, nitric acid, phosphoric acid, boric acid, hydrofluoroboric acid, hydrofluorosilicic acid, and perchloric acid.

Using at least one of these organic and inorganic acidic reagents,
They can also be used in combination.

The amount used is not particularly limited, and the acidic reagent is used so that the pn of the reaction solution is on the acidic side, preferably from 1 to 6.5.

Other acidic salts such as ammonium chloride, zinc chloride, zinc nitrate, and ammonium sulfate may also be used.

If the pH is lower than this value, decomposition of the base polymer and the saccharide, sugar alcohol, etc. to be grafted will occur, while if it is higher than this value, the reactivity of the N-methylol group will decrease and the grafting reaction will not proceed.

Next, water is removed from the aqueous solution containing the base polymer prepared as described above, the saccharide to be grafted, polyhydric alcohol, sugar alcohol, etc., and the acidic reagent.

Generally, the grafting reaction does not proceed in the presence of water.

Any commonly used method for removing water may be used.

For example, azeotropic dehydration methods such as thermal evaporation, reduced pressure dehydration, and addition of a third component that is azeotropic with water, such as benzene or toluene, can be employed.

After sufficient moisture is removed, grafting is performed by heating.

Heating temperature correlates with heating time, but generally 50 to 150
°C, preferably 80 to 120 °C, and heating time is 1 minute to 5 hours, preferably 2 minutes to 2 hours.

If the heating is insufficient (the heating temperature is low and the heating time is short), the grafting reaction will not proceed sufficiently, and if the heating is too high, the grafted polymer may decompose.

When the reaction product heat-treated as described above is sufficiently washed with water used as a solvent, unreacted grafting reagents (saccharides, polyhydric alcohols, sugar alcohols, etc.) can be dissolved and removed.

The desired grafted polymer can be obtained by drying the residue.

By grafting, the grafting agent forms an ether bond with the base polymer and becomes insoluble in water.

That is, a substantially water-insoluble hydrophilic polymer can be obtained.

Note that since the acidic reagent used in the catalyst is removed together with the unreacted grafting reagent by washing with water, the resulting polymer can be easily purified.

In this way, hydrophilic synthetic polymers having polyacrylamide and nylon rubamoyl groups -CONH2 are
A substantially water-insoluble hydrophilic polymer can be produced by chemically bonding a saccharide having an alcoholic hydroxyl group, a sugar alcohol, a polyhydric alcohol, etc. to a methylolated modified polymer in a graft manner.

The substantially water-insoluble hydrophilic polymer of the present invention utilizes the OH group at the end of the graft to further bromine cyanide (BrON).
), it is possible to immobilize various enzymes, and it can also be used for the separation and purification of other proteins.

Further, the substantially water-insoluble hydrophilic polymer obtained in the present invention can be treated with an ion exchange group by treating its terminal OH groups, and thus can be used as a hydrophilic ion exchange resin.

The raw materials for Sephadex and Sepharose, which are currently widely used as carriers of this type, are dextran, agarose, etc., and the former is produced only by microorganisms, and the latter is obtained by extraction from agar, both of which are expensive.

On the other hand, the substantially water-insoluble hydrophilic polymer according to the present invention is made from synthetic polymers and readily available natural products, both of which can be produced at low cost.

Next, the present invention will be specifically explained using reference examples and examples, but the present invention is not limited to these examples unless the gist thereof is exceeded.

Reference Example 1 Synthesis of cross-linked polyacrylamide 40% of acrylamide was placed in a separable flask with an internal volume of 11%.
Take g and dissolve it in deionized water 6107111.

Add 5 g of 10% isopropyl alcohol aqueous solution and 20 g of 1% acid disodium phosphate (Ni, HPO,) aqueous solution.
g. Add 0.4 g of methylene bisacrylamide and bubble nitrogen gas into the solution to fully replace the reaction solution and the inside of the reactor with nitrogen. Add 10 ml of 1% potassium persulfate aqueous solution, close the container, and do not stir magnetically. The temperature started to increase from

Polymerization started when the temperature inside the reactor reached 40 to 45°C.

After maintaining the temperature at 50° C. for 2 hours, the resulting polymer was transferred into methanol to precipitate and dried under vacuum at 50° C., whereby a white powdery polymer could be quantitatively obtained.

Reference Example 2 Synthesis of N-methylolated polyacrylamide (method by formalin treatment) The crosslinked polyacrylamide 28.4.9 obtained according to the above method was dispersed in 1100 rulj of deionized water and then 0.5 N hydroxylated. The pH of the dispersion was adjusted to 10.9 by adding an aqueous sodium solution.

Paraformaldehyde 26.2. p was added, stirred, and allowed to stand at room temperature.

After being left at room temperature for 48 hours with stirring, the mixture was transferred to acetone to precipitate the polymer.

After swelling in water again, transfer to acetone and repeat this operation 2 times.
After repeated purification and vacuum drying at 50°C, 35.6 g of white powdery polymer was obtained.

The results of elemental analysis of the polymers obtained in Reference Examples 1 and 2 were as follows.

From this result, considering nitrogen as the standard, carbon, hydrogen, and oxygen are each 0.84.1.67°0.8 due to the reaction.
Only increased by 3.

That is, addition of CH2O was confirmed, and the reaction rate was 84% as judged from the increase in the number of carbon atoms.

Reference example 3 Synthesis of N-methylolated polyacrylamide (when using N-methylol acrylamide) N-methylol acrylamide (Soken Chemical product) CH2=CHCONHC
Take 25.0 g of H20H (methylolation rate 98.02%) into a separable flask with an internal volume of 11, and add 3 ml of deionized water.
53ml of 10% isopropyl alcohol aqueous solution, 5g of 1% acidic disodium phosphate (Na2HP
04) Aqueous solution 209, 'methylenebisacrylamide 0
．． 2! After adding Ill and bubbling nitrogen gas into the liquid to sufficiently replace the reaction liquid and the inside of the reactor with nitrogen, add 6.3 TL of a 1% potassium persulfate aqueous solution, close the container, and start raising the temperature without electromagnetic stirring. did.

Polymerization starts when the temperature inside the reactor reaches around 50℃, and then the temperature reaches 50℃.
℃ for 2 hours, washed once with water, transferred to methanol to precipitate the polymer, and dried under vacuum at 50℃ to obtain a white powdery polymer 24. :l was obtained.

Yield 95.8%.

Reference Example 4 Synthesis of N-methylolated 6-nylon 195.5 g of a 37% formalin aqueous solution and 34 g of calcium chloride were taken into a separable flask with an internal volume of 1 liter and dissolved uniformly, and the pH was adjusted to 0.1 with sulfuric acid. , crushed shida 6-nylon (Mn=1.38X1o') io, og
was added and kept at 90°C for 2 hours while stirring.

The powders adhered to each other over time and floated onto the reaction solution.

After the reaction, the residue was washed with water and neutralized with acidic sodium carbonate.

Further washing with water gave an elastic white polymer.

In order to avoid condensation reaction between N-methylol groups due to drying, it was stored in a refrigerator in a moist state.

The results of elemental analysis of the obtained white polymer and the raw material 6-nylon were as follows.

Based on these results, when considering nitrogen as the standard, it was confirmed that carbon, hydrogen, and oxygen increased due to the reaction, and addition of CH2O occurred.

The reaction rate was calculated from the increase in carbon to be 56%.

Example 1 A separable flask with an internal volume of 11 contained 100 TL of a deionized water dispersion containing 5.5 I of the N-methylolated polyacrylamide obtained in Reference Example 2 and 9.0.9 of glucose (D-glucose). 100 ml of deionized water and 10 ml of deionized water containing 0.4 g of ammonium chloride were collected and thoroughly stirred, and then the water was removed at 50° C. under reduced pressure using a water jet pump.

After that, vacuum drying at 50°C to remove moisture sufficiently,
The mixture was heated at 100°C for 10 minutes.

After cooling to room temperature, water-soluble substances such as excess glucose and ammonium chloride were removed by thorough washing with deionized water, and the residue was vacuum-dried at 50°C to form a white powder, which was substantially water-insoluble. 7.4 g of hydrophilic polymer was obtained.

For comparison, when the reaction was carried out under exactly the same conditions without using glucose, 5.1 g of a white powdery polymer was obtained.

The results of elemental analysis of the obtained polymer were as follows.

As mentioned above, when glucose is used, an increase in carbon, hydrogen, and oxygen is confirmed compared to when glucose is not used, and it can be seen that something equivalent to glucose residues is added.

In this case, the reaction rate was 31.7%, assuming that the reaction rate when one glucose unit was added was 100%.

Incidentally, we performed the same reaction procedure in the case of glucose, ammonium chloride, and water without using N-methylolated polyacrylamide, but when we washed with water after heating to 100°C, no insoluble matter remained and there was no excess. It was confirmed that glucose and ammonium chloride were sufficiently removed by washing with water.

Examples 2 to 4 Using the N-methylolated polyacrylamide obtained according to Reference Example 2 (reaction rate 59%), various cases were investigated in which the amount of glucose used was varied.

The reaction was carried out as follows.

A reaction solution was prepared from 150 ml of deionized water containing 8.4 g of N-methylolated polyacrylamide, 1801711 deionized water containing a predetermined amount of glucose, and 15 ml of deionized water containing 0.6 g of ammonium chloride.
The reaction was carried out in the same manner.

As a result of the above, it was observed that the reaction rate improved as the amount of glucose used increased.

Example 5 Crosslinked N-methylolated polyacrylamide obtained by polymerizing N-methylol acrylamide in the presence of methylenebisacrylamide according to the method of Reference Example 3 4.05. Glucose 21.4g, ammonium chloride 0. ．． After collecting 28.9 in 10011 Ll of deionized water and mixing thoroughly,
Remove the sewage under reduced pressure using a water jet pump at 0 °C and further
Sufficient dehydration was achieved by vacuum drying at 0° C. for 24 hours.

Thereafter, it was heated to 100° C. for 10 minutes, cooled, and then washed with deionized water to remove soluble materials.

When the residue is vacuum dried at 50°C, it becomes a white powder,
A substantially water-insoluble hydrophilic polymer 4.79 was obtained.

For comparison, when no glucose was used and the other conditions were exactly the same, the white powdery polymer was 3.79%.
I got it.

The elemental analysis values of the obtained polymer were as follows.

As mentioned above, when glucose is used, an increase in carbon, hydrogen, and oxygen is observed compared to when glucose is not used, indicating that something equivalent to glucose residues is added.

The reaction rate was calculated from the increase in carbon number to be 13.7%.

Example 6 8.1 g of N-methylated polyacrylamide obtained by formalin treatment of crosslinked polyacrylamide according to the method of Reference Example 2, soluble starch: Junsei Kagaku, first grade reagent, chloride 250 TL of deionized water containing 0.4 g of ammonium was treated in the same manner as in Example 1 to obtain a white powdery, substantially water-insoluble hydrophilic polymer 9.
．． 7g was obtained.

The elemental analysis values of the obtained polymer were as follows.

Analytical values when using soluble starch are also listed.

As shown above, when soluble starch is used, an increase in carbon, hydrogen, and oxygen is confirmed compared to when soluble starch is not used, and it can be seen that something equivalent to glucose residues is added.

The reaction rate was calculated from the increase in carbon number to be 71.8%.

Reference Example 5 12.0 g of polyvinyl alcohol (Nippon Gosei NL-05 polymerization degree 520) was used instead of glucose, the pH was adjusted to 3.2 with para-toluenesulfonic acid, and the other conditions were the same as in Example 5. N-methylolated polyacrylamide and polyvinyl alcohol were reacted.

As a result, 3.6 g of a substantially water-insoluble hydrophilic polymer was obtained in the form of a white powder.

The elemental analysis values of the polymer were as follows.

As shown above, when polyvinyl alcohol was used, an increase in carbon, hydrogen, and oxygen was confirmed compared to when polyvinyl alcohol was not used, indicating that something equivalent to polyvinyl alcohol residues was added. Understood.

The reaction rate was calculated from the increase in carbon number to be 11.5%.

Example 7 N-methylolated polyacrylamide and dextrin (genuine chemical system, first grade reagent) were prepared in the same manner as in Example 5 except that 19.2 g of dextrin was used instead of glucose.
reacted.

As a result, 5.9 g of a substantially water-insoluble hydrophilic polymer was obtained in the form of a white powder.

The elemental analysis values of the polymer were as follows.

As mentioned above, when IJ was used, an increase in carbon, hydrogen, and oxygen was confirmed compared to when it was not used, and it was found that something equivalent to a glucose residue was added.

The reaction rate was calculated from the increase in carbon number to be 44.7%.

Example 8 N-methylolated polyacrylamide and dextran (manufactured by Wako Tsumugi Pharmaceutical Co., Ltd., MW = 60000) were prepared in the same manner as in Example 5 except that 6.0 g of dextran was used instead of glucose.
~90,000) were reacted.

As a result, a white powdery, substantially water-insoluble hydrophilic polymer 3.8. ! I got a 9.

The results of elemental analysis of the polymer were as follows.

As mentioned above, when dextran was used, an increase in carbon, hydrogen, and oxygen was confirmed compared to when dextran was not used.

The reaction rate was calculated from the increase in carbon number to be 3.3%.

Example 9 N-methylolated polyacrylamide and glucose were mixed in the same manner as in Example 5 except that para-toluenesulfonic acid was used instead of ammonium chloride, and the pH of the mixture was adjusted to 2.19. It was then treated in the same manner as in Example 1 to obtain 8.2 g of a substantially water-insoluble hydrophilic polymer in the form of a white powder.

For comparison, when no glucose was used and the other conditions were the same, the white powdery polymer was 3.4.
I got g.

The elemental analysis values of the obtained polymer were as follows.

As mentioned above, when glucose is used, an increase in carbon, hydrogen, and oxygen is observed compared to when glucose is not used, indicating that something equivalent to glucose residues is added.

The reaction rate calculated from the increase in carbon was 74.8%.

Examples 10 to 14 N-methylolated polyacrylamide and glucose were mixed in the same manner as in Example 5 except that sulfuric acid was used instead of ammonium chloride, and the pH of the mixture was adjusted to 1 as shown below.
601 to 3.24 and then treated in the same manner as in Example 1 to obtain a white powdery, substantially water-insoluble hydrophilic polymer.

An increase in weight was observed in both cases.

Example 15 A white polymer obtained by treating 6-nylon with formalin in a separable flask with an internal volume of 11. A reaction dispersion is prepared by taking Og (weighed in a wet state), 30.0 g of glucose, and 0.6.9 g of ammonium chloride and adding 55 ml of deionized water.

After removing water at 50° C. under reduced pressure, the product was further vacuum dried to completely remove water.

Thereafter, the mixture was heated at 100°C for 10 minutes, cooled, thoroughly washed with water, and the residue was vacuum-dried at 50°C to obtain 4.21 of a substantially water-insoluble hydrophilic white polymer.

For comparison, 3.8 g of a white polymer was obtained when the same conditions were used without using glucose.

The elemental analysis values of these polymers were as follows.

As shown above, when glucose was used, an increase in carbon, hydrogen, and oxygen was confirmed compared to when glucose was not used.

The reaction rate was calculated from the increase in carbon number to be 4.8%.

Reference Example 6 N-methylol polyacrylamide and glycerin were reacted in the same manner as in Example 5 except that 10.9 g of glycerin was used instead of glucose and the pH was adjusted to 2.26 with 1N sulfuric acid.

As a result, 5.7 g of a substantially water-insoluble hydrophilic polymer was obtained in the form of a white powder.

The elemental analysis values of the polymer were as follows.

As shown above, when glycerin was used, an increase in carbon, hydrogen, and oxygen was confirmed compared to when glycerin was not used.
Further, the reaction rate was calculated from the increase in carbon number to be 81.0%.

Example 16 Using 21.6 g of d-Tsurupit instead of glucose,
N-methylol polyacrylamide and d-sorbitol were reacted in the same manner as in Example 5 except that the pH was adjusted to 2.21 with IN sulfuric acid.

As a result, 8.2 g of a substantially water-insoluble hydrophilic polymer was obtained in the form of a white powder.

The elemental analysis values of the polymer were as follows.

As shown above, when d-sorbitol was used, an increase in carbon, hydrogen, and oxygen was confirmed compared to when it was not used, and the reaction rate was calculated from the increase in carbon number to 81.2.
%Met.

Test Example The water retention rate (g/g) and degree of swelling (rttl/ml) of the polymers obtained in Examples 1 and 6 were measured according to the following method. The results are shown below.

Moisture retention rate Swelling degree When using glucose 1.54 1.76 When using soluble starch 1.53 1°63 When not using 0.45 0.79 Polymers to which glucose and soluble starch have been added as described above are It can be seen that the hydrophilicity is significantly increased compared to the case without using.

Next, the results of similar measurements performed on the polymers obtained in Examples 9, 12, and 16 and Reference Example 6 are shown below.

Moisture retention rate Swelling degree Example 9 2.84 4.16 Blank test 0.39 0.50 Example 12
2.94 3.68 Blank test 0.6
1 0.78 Water retention rate Swelling degree reference example 6 2.33 2.77 Example 16
2.66 3.58 Blank test
0.78 0.61 In this case also glucose or d
-It can be seen that the hydrophilicity is significantly improved by the Nruvit treatment.

(1) Measurement method for degree of swelling A pulverized sample 0.5.9 was placed in a 10ml measuring cylinder and the dry volume was measured.After adding deionized water and shaking thoroughly, it was left to stand for 2 hours at room temperature. put.

The volume at that time was measured and the degree of swelling was calculated using the following formula.

(2) Method for measuring moisture retention The sample whose degree of swelling was measured was thoroughly drained on Qualitative F paper/i62 using a Buchner funnel under reduced pressure with a water pump, and then quickly weighed. do.

This was dried in a vacuum at 50° C. for 24 hours, and then the dry weight was determined.

The moisture retention rate was calculated using the following formula.

Claims (1)

[Claims] Polymers having a moyl group -CONH2 and in which all or some of the nitrogen atoms of the amide bond or carbamoyl group are methylolated (excluding pearl-like polymers), glucose, dextrin, soluble starch, dextran or sorbitol under acidic conditions.