JPH0574351B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a method for purifying polygalactosamine. More specifically, the present invention specifically precipitates PF-101 produced by Pecilomyces bacteria and polygalactosamine produced by Aspergillus parasitigus without using an organic solvent,
This invention relates to an efficient purification method. In general, polygalactosamine produced by microorganisms includes PF-
101 (Special Publication No. 56-12639) and polygalactosamine (JBC) produced by Aspergis parasiticus.
vol.235 No.9 2538-2541 (1960)) are known. Conventionally, to purify these polygalactosamines, a solvent such as alcohol is added to the culture solution to separate it as a precipitate, washed, and then dissolved in hot water, or for further purification, Sepharose 6B This was accomplished by adsorbing the polygalactosamine to a molecule, eluting it with a buffer solution, and collecting the polygalactosamine fraction (Japanese Patent Publication No. 12639/1983). However, the culture solution contains large amounts of neutral polysaccharides and low-molecular sugars, which are accompanied by precipitation from the solvent, requiring repeated purification and extremely complicated operations to obtain a purified product. However, there were also problems such as a very poor recovery rate. Prior to the present invention, since polygalactosamine itself has an isoelectric point, clarified culture fluid was
They attempted isoelectric precipitation by adjusting the pH, but pH adjustment alone did not lead to precipitation of polygalactosamine. The present inventor conducted research based on the idea that purification would be significantly easier if polygalactosamine was caused to undergo isoelectric precipitation. They learned that if left to dry, precipitates would form even in the presence of impurities such as neutral polysaccharides. In addition, in the present invention, it has been found that precipitates due to the presence of salts are well dissolved in acids, so using these methods, significantly purified polygalactosamine can be produced very easily and at low cost. This made it possible. The present invention is characterized in that 2 to 50% of salt is added to a polygalactosamine-containing liquid without using an organic solvent, and if necessary, the pH is adjusted to 7.0 to 9.0, which causes precipitation, to form a precipitate. This is a method for purifying polygalactosamine. In addition, the present invention adds 2 to 50% salt to a polygalactosamine-containing liquid without using an organic solvent, adjusts the pH to 7.0 to 9.0 to cause precipitation as necessary, and controls the resulting precipitate as necessary. This is a method for purifying polygalactosamine, which is characterized by washing the precipitate with water and dissolving the precipitate in acid. In addition, the present invention adds 2 to 50% salt to a polygalactosamine-containing liquid without using an organic solvent,
If necessary, adjust the pH to 7.0 to 9.0, which will cause precipitation.
The obtained precipitate is washed with water as necessary, the precipitate is dissolved in acid, the pH is adjusted to 7.0 to 9.0 at which precipitation occurs, the obtained precipitate is washed as necessary, and the precipitate is dissolved again. Dissolve in acid and precipitate as above if necessary.
This is a polygalactosamine purification method characterized by repeating washing and dissolving operations. The polygalactosamine-containing solution that can be applied to the purification method of the present invention may be any culture solution containing a large amount of contaminants, a culture solution from which protein has been removed, or a culture solution that has been significantly purified by removing low-molecular substances. The salt added to the polygalactosamine-containing solution is one or more salts including the following exemplified salts. Namely, hydrochlorides such as potassium chloride, sodium chloride, calcium chloride, ammonium chloride, nitrates such as potassium nitrate, sodium nitrate, acetates such as sodium acetate, sulfates such as dipotassium sulfate, ammonium sulfate, calcium sulfate, copper sulfate, Examples include phosphates such as dipotassium phosphate, monopotassium phosphate, disodic phosphate, and monosodium phosphate. Any amount of salt may be added as long as it is in a dissolved state, but it is preferably 0.5 to 50%, more preferably 2 to 40%, based on the polygalactosamine-containing liquid.
That's about it. Depending on the type of salt added, the pH will be 7 or more, so in this case, polygalactosamine will precipitate without adjusting the pH, so it is sufficient to separate the precipitate. If precipitation does not occur even after adding salt, the pH may be adjusted to 7 to 9, preferably around 8.5, which is the isoelectric point, using an alkali such as caustic soda. If salt is added to the polygalactosamine-containing solution and the pH is adjusted to 7 to 9 in some cases, polygalactosamine, which was not easily precipitated due to the interference of impurities, will precipitate and be separated from the impurities. and precipitate.
This precipitate can be separated by centrifugation or filtration with a filter cloth. Considering that no precipitation of polygalactosamine occurred even when the culture solution was subjected to isoelectric point treatment at pH 8.5, it is extremely surprising that the precipitation of polygalactosamine completely occurs just by adding salt. That's true. Since the separated precipitate contains a large amount of salt,
This is washed with water or a solvent to desalt it, and then dissolved in an acid. The acid may be any acid such as an organic acid such as acetic acid or an inorganic acid such as hydrochloric acid, and the concentration may be
It is preferably about 0.01 to 3 moles. After dissolving the precipitate in acid, the precipitate can be easily precipitated only by treatment near the isoelectric point of pH 7 to 9, so add an alkali such as caustic soda to the pH.
The pH is adjusted to 7 to 9, preferably 8.5, to obtain a precipitate. For further purification, the precipitate can be obtained by washing the precipitate with water or the like, dissolving it again in acid, and adjusting the pH to 7 to 9. This purification process can be repeated any number of times, making it possible to obtain almost pure polygalactosamine. Next, test examples and examples of the present invention will be shown. Test Example Glucose 3% Polypeptone 0.3% CaCl ₂ 0.5% PH 7.0 Using the above medium, Paecilomyces sp I-1,
FERM-P3928 was cultured and culture filtrate 20 was obtained.
While heating this to 55-60℃, limit molecular weight 16
We remove and circulate low-molecular substances using ten thousand UF membranes.
A liquid volume of 10 was obtained. Twenty-one bottles of 100 ml each of this clear concentrated culture filtrate were prepared, the salts shown in Table 1 were added thereto, the pH was adjusted as shown in Table 1, and the amount of precipitate was measured. The results obtained are shown in Table 1.
is shown. Recovery rate in the table = Dry weight of precipitate/Measure the amount of galactosamine in the culture solution using the indole-hydrochloric acid method and calculate the total amount x 10
Displayed as 0.

[Table] Example 1 Seuculose 2% Yeast extract 0.2% CaCl ₂ 0.5% PH 7.0 Using the above medium, Paecilomyces sp--1,
FERM-P3928 was cultured to obtain 150 ml of culture filtrate. While heating this to 55-60℃, limit molecular weight
160,000 UF membranes remove and concentrate low-molecular substances,
A liquid volume of 50 was obtained. Furthermore, this was centrifuged to remove heat-denatured proteins and bacterial cell residue, to obtain a clear concentrated culture solution. Sodium acetate was added to the obtained clear concentrated culture solution.
15 kg was added, stirred to dissolve, and left overnight. The resulting precipitate was collected on a saran cloth and washed with a large amount of water to remove impurities such as salt and neutral sugar. Furthermore, this precipitate was sterilized in an autoclave, dried, and ground to obtain purified polygalactosamine. Table 2 shows the analytical values of the recovered polygalactosamine. The recovery (%) in the table is the same as in the experimental example, and the galn content (%) is the galactosamine content in the purified product measured by the indole-hydrochloric acid method. Table 2 Recovery (%) galn content (%) Moisture (%) Ash content 97 85 9.0 0.6 galn: Galactosamine Example 2 Glucose 3% Polypeptone 0.3% CaCl ₂ 0.5% PH 7.0 Using the above medium, Paeclomyces sp -1,
FERM-P3928 was cultured to obtain 100 ml of culture filtrate. While heating this to 55-60℃, limit molecular weight
Low molecular weight substances were removed and concentrated using a 160,000 UF membrane, and the liquid volume was reduced to 60,000. Furthermore, this was centrifuged to remove bacterial cell residue and heat-denatured proteins to obtain a clear concentrated culture filtrate. 10.6 kg of ammonium sulfate (30% saturation) was added and dissolved in the obtained clear concentrated culture filtrate. The pH was then brought to 8.5 using 40% caustic soda. The resulting precipitate was collected by centrifugation, dispersed in water, and desalted. Furthermore, after dissolving this in 0.1 mol of hydrochloric acid, the pH was adjusted to 8.5 again with 40% caustic soda.
A precipitate was obtained. This operation was repeated twice to remove impurities. Furthermore, this precipitate was sterilized in an autoclave, dried,
It was pulverized to obtain purified polygalactosamine. The purification results are shown in Table 3. Galn in Table 3 is the galactosamine content in each solution measured by the indole-hydrochloric acid method and the total amount calculated. In addition, the recovery (%) is based on the galn of the culture solution.
It is shown as the ratio of the amount of galn in each purification step when it is set to 100.

[Table] Example 3 Clear concentrated culture filtrate obtained in the same manner as Example 2
160 and 40 kg of salt was added and dissolved. From now on, take 10 samples A, B, and C.
In A, the precipitate generated after adjusting the pH to 8.5 was collected on a saran cloth, washed with a large amount of water, excess salt and impurities were washed away, and then sterilized, dried, and crushed to obtain purified polygalactosamine. In B, the precipitate formed after adjusting the pH to 8.5 was washed with a large amount of water, and then the precipitate was dissolved in 0.1 mol of acetic acid.
Then, PH this acetic acid solution with 40% caustic soda.
Adjust to 8.5 to generate precipitate, wash with water in the same way,
Purified polygalactosamine was obtained by sterilization, drying, and pulverization. After adjusting the pH to 7.5, the resulting precipitate was washed with a large amount of water, followed by sterilization, drying, and pulverization to obtain purified polygalactosamine. Table 4 shows recovery (%) analysis values for each sample.
Shown below. The indications of recovery (%) and galn content (%) in Table 4 are the same as in Example 1.

[Table] Example 4 Using the culture values of Example 2, Paeclomyces −
1. FERM-P 3928 was cultured in liquid to obtain culture filtrate 10. This was heated at 80°C for one hour to denature the protein. Furthermore, this was centrifuged to remove insoluble matter. Next, add 3 kg of ammonium chloride (30 kg) to this solution.
% solution) was added and dissolved, and the pH was adjusted to 8.5. The resulting precipitate was collected by centrifugation. This precipitate was washed with an ammonium salt solution of the same concentration to remove soluble substances. Next, this precipitate was dissolved in 0.01 mol of phosphoric acid, and the pH was adjusted to 8.5 with 40% caustic soda for isoelectric precipitation. Next, in the same manner as in Example 1, desalination, phosphoric acid dissolution, PH
8.5 Repeat the adjustment twice, sterilize, dry, crush,
Purified polygalactosamine was obtained. Recovery (%) and galn content (%) in Table 5 are the same as in Example 1. Recovery (%) and analytical values are shown in Table 5. Table 5 Recovery of purified polygalactosamine and analysis value recovery (%) Galn content (%) Moisture (%) Ash content (%) 73 92.5 5.5 1.9

Claims (1)

[Claims] 1. Adding 2 to 50% salt to a polygalactosamine-containing solution without using an organic solvent, adjusting the pH to 7.0 to 9.0, which causes precipitation if necessary, and causing precipitation to occur. Characteristic polygalactosamine purification method. 2 Add 2 to 50% of salt to the polygalactosamine-containing solution without using an organic solvent, adjust the pH to 7.0 to 9.0 to cause precipitation if necessary, wash the obtained precipitate as necessary, A method for purifying polygalactosamine, which comprises dissolving the precipitate in an acid. 3. Add 2 to 50% of salt to the polygalactosamine-containing solution without using an organic solvent, adjust the pH to 7.0 to 9.0 to cause precipitation if necessary, and wash the obtained precipitate with water if necessary, Dissolving the precipitate in acid,
The method is characterized by adjusting the pH to a level that causes precipitation, washing the obtained precipitate as necessary, dissolving the precipitate in acid again, and repeating the above precipitation-washing-dissolution operation if necessary. A method for purifying polygalactosamine.