JPH0699476B2 - How to collect protamine salts - Google Patents

Description

Description: FIELD OF THE INVENTION The present invention relates to a method for collecting protamine salts from fish larvae.

2. Description of the Related Art Protamine is a relatively small basic protein having a molecular weight (about 4000 to about 10000) and exists as a nuclear protein bound to a nucleic acid (DNA) in sperm of fish such as herring, salmon and trout.

Protamine salts, especially its sulfates, are known to have the action of inhibiting blood coagulation of heparin, the action of sustaining the medicinal effect of insulin, the action of stabilizing enzymes, the action of stabilizing antibiotics, and the like. It is used for various medicines, etc. by utilizing its properties.

Conventionally, as a method of obtaining protamine salts from fish algae, the algae are treated with an aqueous solution of a dilute mineral acid such as sulfuric acid, and protamine and other proteins (hereinafter, other proteins are referred to as “contaminant proteins”).
(Hereinafter referred to as “extraction step”, the solution obtained in this step is referred to as “extraction solution”), and then alcohol such as methanol is added to the extraction solution to produce protamine sulfate and Other contaminant proteins are precipitated, then the precipitate is separated and dried, the solid obtained is extracted with warm water, the warm water is cooled to precipitate protamine salts, and the precipitate is separated from the solution. (Hereinafter, the above process is referred to as a “separation process”), and the separated solid is further purified (hereinafter, this process is referred to as a “purification process”).

In the conventional separation step, since the protamine salts (hereinafter, a typical protamine sulfate is explained) which has been precipitated by adding alcohol to the extract must be taken out once, the alcohol is distilled off and dried, Is complicated and the work flow is very bad.

During precipitation, not only protamine sulfate but also contaminating proteins are precipitated together, so that protamine sulfate and contaminating proteins must be further separated. Protamine sulfate is separated from contaminating proteins by extracting the precipitate with warm water, cooling the solution, and precipitating it.At the same time, the contaminating proteins are also extracted to warm water to the same extent to some extent, and then cooled. If so, the protamine sulfate obtained will inevitably have a low purity with contaminated proteins. A further purification step is required to obtain protamine sulfate having a purity that can be practically used. Since protamine sulfate has a certain degree of solubility even at a cooling temperature, it is not possible to recover all of the protamine sulfate extracted with a dilute mineral acid, resulting in a poor yield.

Furthermore, in order to increase the yield in the conventional method, extraction of the precipitate with warm water must be repeated,
There is also a problem that the operation is complicated.

As another method for separating protamine sulfate, for example, JP-B-59-31519, JP-B-59-31518, and JP-A-59-31518
55-2603, JP 55-2612 or JP 55
-2634, etc.

In the separation process disclosed in these, condensed phosphate is added to the extract to precipitate it as a sparingly soluble protamine phosphate,
This is a method in which protamine sulfate is separated by metathesis of this precipitate with a high-concentration ammonium sulfate solution.

However, this method has a drawback that the work flow is poor and the process is complicated because it must be once taken out as a protamic acid salt.

When a protein is precipitated from the extract using condensed phosphate, not only protamine but also a large amount of other contaminating proteins are precipitated. Therefore, the protamine sulfate obtained by metathesis of it contains contaminating proteins and has a very poor purity. Therefore, in order to obtain protamine sulfate having a purity suitable for practical use, a purification step was always required after the separation step. The purification step is a complicated process in which the separated acidic dilute aqueous solution of protamine sulfate is treated with an adsorbent and then separated and precipitated with an organic solvent.

DISCLOSURE OF THE INVENTION Problems to be Solved by the Invention As a result of intensive investigations by the present inventors on a method for obtaining protamine sulfate in a higher purity and a higher yield, protamine sulfate and contaminant proteins have significantly different solubility depending on the concentration of an inorganic salt. I found that By utilizing this new finding, it has become possible to solve all of the above-mentioned problems and to complete a method by which protamine sulfate with high purity can be easily and efficiently obtained.

Means for Solving the Problems That is, according to the present invention, a fish protamine is treated with a dilute mineral acid to obtain a crude protamine salt extract, and 1.6 to 2.6 mol of ammonium sulfate or sodium sulfate is added to 1 liter of the extract.
Precipitates formed by adding inorganic salts to a concentration of 0.8 to 1.4 mol are removed, and the concentration of ammonium sulfate is 3 mol or more or sodium sulfate is 1.5 mol or more per 1 liter of the supernatant. The present invention relates to a method for collecting protamine salts, which comprises adding inorganic salts to and precipitating and collecting protamine salts.

The fish spores that can be used in the present invention are not particularly limited as long as they are protamine-containing fish spores such as herring, salmon, trout or mackerel.

Shirako is treated with dilute mineral acid. Treatment is generally carried out by grinding milled Shirako or unmilled Shirako with a dilute mineral acid, eg,
It may be carried out by immersing in an aqueous solution of sulfuric acid or the like, and this method itself may employ a known extraction method.

A sulfuric acid aqueous solution is desirable as the dilute mineral acid.

A suitable concentration of dilute mineral acid is 3 to 20%, especially 6 to 14%.
The treatment temperature is 30 ° C. or lower, and preferably 20 to 30 ° C. The treatment time is preferably within 3 hours.

The ratio of dilute mineral acid to Miko is 1 to 5 times.

Since the obtained protamine extract contains a large amount of contaminating proteins, inorganic salts were added thereto so that the concentration of ammonium sulfate was 1.6 to 2.6 mol or sodium sulfate was 0.8 to 1.4 mol per 1 liter of the extract. Contaminant proteins are precipitated (this step is hereinafter referred to as "separation step" 1).

Salts are added to the extract as it is or as a concentrated aqueous solution. It is desirable that the addition be carried out gradually with stirring, and as the addition is carried out, contaminant proteins will precipitate.

The concentration of salts may be appropriately adjusted depending on the type of extract and the concentration of protamine in the extract. In the present invention, the addition of salts precipitates the contaminant proteins, but it is important to make the concentration of protamine salts as low as possible so that the protamine salts do not precipitate.
For example, if the concentration of protamine sulfate in the extract obtained by treating with dilute mineral acid is 1% by weight or less, ammonium sulfate will be 20%.
It is added so as to be contained in an amount of 1.6 to 2.6 mol, preferably 1.6 to 1.9 mol, per liter of the extract having a liquid temperature of -30 ° C.
If it is less than 1.6 mol, the removal of contaminant proteins will be insufficient.
If it exceeds 2.6 mol, protamine sulfate will also precipitate. When sodium sulfate is used, it is similarly added so as to have a concentration of 0.8 to 1.4 mol, preferably 0.8 to 1.0 mol, per liter of the extract. If it is less than 0.8 mol, the removal of contaminant proteins will be insufficient. If it exceeds 1.0 mol, protamine sulfate will also precipitate. When the concentration of protamine sulfate in the extract is 1% by weight or more, ammonium sulfate is added in an amount of 1.6 to 2.6 mol, preferably 1.9 to 2.4 mol, relative to the extract 1 having a liquid temperature of 30 to 50 ° C. When sodium sulfate is used, it is likewise added so that the concentration thereof is 0.8 to 1.4 mol, preferably 1.0 to 1.2 mol, based on Extract 1.

Adjust the temperature of the extract to 10-50 ° C. Below 10 ° C, protamine precipitates as sulfate. If the temperature is higher than 50 ° C, the contaminant proteins cannot be sufficiently removed as a precipitate.

It is not necessary to adjust the pH of the extract containing salts.
When not prepared, the pH of the extract is usually 1. When it is prepared, the pH is adjusted to 8 or less, preferably 2 to 7 before adding salts. If the pH is less than 2, not only the damage to the equipment will be great, but also the safety of work will be poor. pH is 8
If it is larger than this, the stability of protamine is not preferable.

After adding salts to the extract, the next step can be started immediately.

By the separation step 1, almost all the contaminant proteins contained in the extract can be removed.

The precipitated contaminant protein is separated from the extract by centrifugation or the like.

According to the present invention, after the precipitated protein is separated by centrifugation or the like, the extract can be used immediately in the next step, so that the work flow is fluid and is not interrupted, and the workability is very good.

After the separation step 1, a large amount of inorganic salts is added to the extract from which the contaminant proteins have been separated, and the extract is made into a high-concentration solution of inorganic salt (hereinafter, this solution is referred to as “high-concentration solution”) Are salted out, precipitated and separated (hereinafter, the operation at this stage is referred to as "separation step 2"). The inorganic salts used in the present invention are ammonium sulfate or sodium sulfate.

The concentration of the inorganic salts may be any concentration as long as protamine sulfate is almost salted out. To 1 liter of the extract after the separation step 1, ammonium sulfate is added in a concentration of 3 mol or more or sodium sulfate of 1.5 mol or more. If the concentration is lower than this, protamine sulfate cannot be sufficiently salted out.

Adjust the temperature of the concentrated solution to 10-30 ° C. Above 30 ° C, the precipitated protamine sulphate redissolves. Even if the temperature is lower than 10 ° C, there is no corresponding effect and it is not preferable in terms of energy.

The pH of the high-concentration solution does not need to be particularly adjusted, but when it is prepared, it is pH 8 or less, preferably pH 2 or less before adding the sulfate.
It is desirable to adjust to ~ 5. When the pH is less than 2, not only the damage to the device becomes great, but also the stability of the work is not good. If the pH is higher than 8, the stability of protamine is not preferable.

The high-concentration solution may be transferred to the next operation immediately after the addition of the inorganic salt.

The precipitated protamine salt is obtained as a transparent oily substance by separating it from the high-concentration solution by centrifugation or the like, and is obtained as a white powder by drying it.

The protamine salts obtained as described above have such a purity that they can be sufficiently put to practical use without further purification.

The operation of the separation method of the present invention will be described below. FIG. 1 and FIG. 2 show solubility (% by weight) curves of protamine sulfate when ammonium sulfate is contained in a low molar amount with respect to 1 liter of an aqueous sulfuric acid solution. In the figure, the solid line, the broken line, and the dashed line indicate the solubility of protamine sulfate when the temperature of the solution was 30 ° C, 20 ° C, and 5 ° C, respectively. FIG. 1 is a solubility curve when a 5% sulfuric acid aqueous solution is used, and FIG. 2 is a solubility curve when a 10% sulfuric acid aqueous solution is used.

The extraction solution usually contains 0.5 to 7% by weight of protamine sulfate. As shown in FIGS. 1 and 2, in the separation step 1 in the present invention, protamine sulfate is completely dissolved in a 5% or 10% sulfuric acid aqueous solution containing 1.6 to 1.9 mol of ammonium sulfate. This is because the contaminant protein is sufficiently precipitated even in the low concentration range.
On the other hand, the separation step 2 according to the present invention utilizes the fact that protamine sulfate is completely salted out at a high concentration of 3.0 mol or more (not shown).

If a higher-purity protamine sulfate obtained from the present invention is required, the protamine sulfate obtained in the separation step 2 is dissolved in warm water together with a small amount of a sulfate salt such as ammonium sulfate or sodium sulfate, and then the The solution may be cooled to precipitate protamine sulfate, a transparent oily protamine sulfate may be separated from the solution, and the protamine sulfate may be dried (hereinafter, the above operation is referred to as "purification step").

The higher the concentration of protamine sulfate in the purification step, the better.

The warm water used in the purification step may be any temperature at which the protamine sulfate to be dissolved is dissolved.

Sulfate used in the purification process is 0.
Add an amount of 05-0.2 mol. If it is less than 0.05 mol or more than 0.2 mol, protamine sulfate cannot be efficiently precipitated after cooling warm water.

The warm water is cooled to 10 ° C or lower, preferably 5 ° C or lower. 10 ° C
If it is higher, protamine sulfate cannot be efficiently precipitated.

It is not necessary to adjust the pH of hot water. When adjusting,
After adding protamine sulfate and before adding sulfate, the pH should be 2-7.
It is desirable to set the range to.

By carrying out the purification, salts, acids and contaminant proteins attached to the precipitate of protamine sulfate in the separation step 2 can be removed.

In the purification process of the present invention, the solubility of protamine sulfate in solution 1
Against ammonium sulfate or sodium sulfate
It is based on the new finding that it becomes minimum in a solution in an extremely low concentration range of 0.05 to 0.2 mol.

Table 1 shows the relationship between the solubility (% by weight) of protamine sulfate and the salt concentration in an aqueous solution having a water temperature of 5 ° C. and a pH of 2.5. In the table, the concentration represents the number of moles of salt dissolved in Solution 1.

From Table 1, the solubility of protamine sulphate is such that the salt concentration is 0.05 to 0.
It can be seen that it becomes the minimum in the range of 2 moles.

According to the present invention, the extract contains 1.6-2.6 mol of ammonium sulfate or 0.8-1.4 sodium sulfate per liter of extract.
Contaminant proteins can be removed from the extraction solution without loss of protamine by adding salts to a molar concentration.

The present invention will be described below with reference to examples.

Example 1 Extraction Frozen salmon shirako was thawed, washed with water, drained and ground.

100 g of ground salmon Shirako was added to 500 ml of 5% (1N) sulfuric acid, and the mixture was stirred at room temperature (24 ° C) for 1 hour. After stirring, the residue was removed by centrifugation (8000 rpm, 20 minutes) to obtain 550 ml of protamine sulfate extract.

Separation To the extract, 30 ml of concentrated ammonia water was added at room temperature of 24 ° C. with stirring to adjust the pH to 3, and 124 g of ammonium sulfate was added little by little to precipitate contaminating proteins. The contaminant protein precipitate was removed by centrifugation to obtain 630 ml of a supernatant.

130 g of ammonium sulfate at room temperature 24 ° C. with stirring in the supernatant.
Was gradually added to salt out protamine sulfate. The salted-out protamine sulfate was separated by centrifugation to obtain 10.4 (wet) g of white candy-like protamine sulfate.

Purification Further, 10.4 g of white candy-like protamine sulfate obtained as described above was dissolved in 100 ml of warm water (40 to 50 ° C.), and the concentration of ammonium sulfate was 0.09 mol per liter of the solution. Then, the solution was cooled to 5 ° C. and left at that temperature for 2 days to precipitate protamine sulfate.

The precipitated supernatant of protamine sulfate was decanted off to obtain a transparent oily protamine sulfate. It was freeze-dried to obtain 4.76 g of white protamine sulfate (yield 4.76%).

Example 2 Frozen salmon roe was thawed, washed with water, drained and ground.

100 g of ground salmon shirako was added to 500 ml of 5% (1N) sulfuric acid, and the mixture was stirred at room temperature at 26 ° C for 1 hour. After stirring, the residue was removed by centrifugation to obtain 550 ml of protamine sulfate extract.

82.5 g of sodium sulfate was added little by little to the extract under stirring at room temperature of 27 ° C. to precipitate contaminant proteins. The contaminant protein precipitate was removed by centrifugation to obtain 580 ml of a supernatant. While stirring, 55.0 g of sodium sulfate was gradually added to the supernatant liquid at room temperature of 24 ° C. to salt out protamine sulfate. The salted-out protamine sulfate was separated by centrifugation to obtain 9.15 g of white candy-like protamine sulfate.

Further, 9.15 g of white candy-like protamine sulfate obtained as described above was dissolved in 50 ml of warm water (40 to 50 ° C.), and the concentration of sodium sulfate was adjusted to 0.15 mol per liter of the solution. Then, the solution was cooled to 5 ° C. and left at that temperature for 2 days to precipitate protamine sulfate.

The precipitated supernatant of protamine sulfate was decanted off to obtain a transparent oily protamine sulfate. It was dehydrated with acetone, solidified and dried under reduced pressure to obtain 4.70 g of white protamine sulfate (yield 4.70%).

Example 3 The procedure of Example 1 was repeated, except that the purification of Example 1 was not performed.

The obtained white candy-like protamine sulfate was freeze-dried to obtain 6.30 g (yield 6.30%) of white protamine sulfate.

Comparative Example (conventional method) In the same manner as in Example 1, 550 ml of protamine sulfate extract was obtained.

Methanol (2750 ml) was added to the extract and the mixture was allowed to stand at 5 ° C. overnight to precipitate protamine sulfate and contaminant proteins. The precipitate was separated by centrifugation, and methanol was distilled off under reduced pressure to dry it.

Dry the precipitate twice with 100 ml of warm water (30-40 ℃), 50m
It was extracted once with 1 l of warm water. The extracted aqueous solution was centrifuged at room temperature (10000 rpm, 2 hours) to separate from the unextracted solid, and 180 ml of a turbid liquid was obtained.

The suspension was cooled to 5 ° C. and left at that temperature for 2 days to precipitate protamine sulfate.

The precipitated supernatant of protamine sulfate was decanted off to obtain a transparent oily protamine sulfate. It was freeze-dried to obtain 4.30 g of white protamine sulfate (yield 4.30%).

Example 4 Protamine sulfate obtained in Examples 1 to 3 and Comparative Example was used as a sample and its purity was measured.

The purity was examined by chromatography, absorption spectrometry, the solubility of the solution when protamine sulfate was dissolved in water, and the antibacterial activity.

High performance liquid chromatography was used for the chromatography. The purity of the sample was determined by using Wako Pure Chemical Industries, Ltd. Protamine Sulfate (salmon) as the standard, and comparing it with the purity of 1.00.
Shown as purity. In addition, the product obtained by multiplying the HPLC purity by the sample yield is shown as the standard product yield. Here, the standardized yield refers to the yield when the amount of protamine sulfate obtained from 100 g of white roe is converted to the amount of protamine sulfate having a purity of about the standard, and is considered as a scale for comparison of separation efficiency of protamine sulfate. Good. The results are shown in Table 2.

Comparing the HPLC purity, for example, the purity of protamine sulfate obtained according to the present invention in Example 1 is about 1.1 times better than that of the commercially available product and about 1.2 times higher than that obtained by the conventional method.
You can see that it is twice as good. In addition, comparing the sample conversion purities, it can be seen that the separation can be performed with a high yield of about 1.4 times that of the conventional method.

For antibacterial activity, protamine sulfate (salmon) manufactured by Wako Pure Chemical Industries, Ltd. was used as a standard, and B. subtilis ( B.su
The antibacterial activity against btilis IAM 1069 strain) was compared as 1.00 and shown as the antibacterial activity specific activity. In addition, the product of the antibacterial activity and the yield of the sample was shown as the standardized active yield.
The standardized active yield refers to the yield when converted to the amount of the standard required to show the same antibacterial activity as the amount of protamine sulfate obtained from 100 g of Shiroko.Comparison of separation efficiency of protamine sulfate You can think of it as a measure of. The results are shown in Table 2.
Shown inside. Comparing the antibacterial activity, it can be seen that, for example, protamine sulfate obtained according to the present invention in Example 1 is about 1.1 times better than the commercially available product and about 1.2 times better than that obtained by the conventional method. . In addition, comparing the standardized active yields, it can be seen that the separation can be performed approximately 1.3 times more efficiently than the conventional method.

The absorption spectrometry measured the absorbance at 260 nm attributed to nucleic acid and 280 nm attributed to contaminant protein. The measurement was carried out at a temperature of 20 ° C. in an aqueous solution containing 0.1% by weight of the sample. The results are shown in Table 3. Pure protamine sulfate does not absorb light above 220 nm. As can be seen from Table 2, the amount of impurities of protamine sulfate obtained according to the present invention is much smaller than that of protamine sulfate obtained by the conventional method.

The visual observation of the solution state was performed with a 2% aqueous solution (20 ° C.) of each sample. The protamine sulfate obtained according to the present invention was completely dissolved, whereas the protamine sulfate obtained by the conventional method was not completely dissolved and the presence of an insoluble substance was observed.

EFFECTS OF THE INVENTION According to the present invention, highly pure protamine sulfate can be efficiently and continuously separated from fish larvae by a simple process.

[Brief description of drawings]

1 and 2 are diagrams showing the solubility of protamine sulfate.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (56) References Japanese Patent Publication Sho 49-24209 (JP, B1) Japanese Patent Publication Sho 59-31518 (JP, B2) Mar. (Sho 53-6-30) P. 14 ~ P. 19

Claims (1)

[Claims] Claims: 1. Fish sardine is treated with a dilute mineral acid to obtain a crude protamine salt extract solution, in which 1 to 1 liter of the extract solution contains 1.6 to 2.6 mol of ammonium sulfate or 0.8 to sodium sulfate.
Precipitates formed by adding inorganic salts to a concentration of 1.4 mol are removed, and the inorganic liquid is adjusted to a concentration of 3 mol or more of ammonium sulfate or 1.5 mol or more of sodium sulfate per 1 liter of the supernatant. A method for collecting protamine salts, which comprises adding salts to deposit and collect protamine salts.