JPH0739438B2 - Extraction method of protamine mineral acid salt - Google Patents

Description

Description: FIELD OF THE INVENTION The present invention relates to a method for extracting protamine from fish milts.

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 their sulfates, are known to act on heparin to prevent blood coagulation, to maintain insulin's medicinal effect, to stabilize enzymes, to stabilize antibiotics, and the like. Is used for various medicines.

Conventionally, as a method of obtaining protamine salts from fish agar,
Shirako is treated with an aqueous solution of dilute mineral acid such as sulfuric acid to extract protamine and other proteins (hereinafter, other proteins are referred to as "contamination proteins") (hereinafter, the above process is referred to as "extraction process", The solution obtained in 1. is referred to as "extract", and then alcohol such as methanol is added to the extract to precipitate protamine sulfate and other contaminating proteins, and then the precipitate is separated and dried to obtain further. 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 "separation step"), and the separated solid is further purified. A method (hereinafter, this step is referred to as "purification step") is known.

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.

Furthermore, since the mineral acid aqueous solution used in the extraction step is added with alcohol or the like, it cannot be used again as a solution for extraction. Therefore, a lot of resources are wasted,
And it is very expensive.

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. Moreover, since protamine sulfate has a certain degree of solubility even at a cooling temperature, it is not possible to recover all 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.

The method for extracting protamine is described in, for example, Japanese Patent Publication No. 59-3151.
No. 8, JP-B-59-31519, or JP-A-55-26
No. 34 publication.

The technique described in JP-A-55-2634 has a very poor yield of protamine because it is intended to extract deoxyribonucleic acid together with protamine without decomposing deoxyribonucleic acid using only inorganic salts.

The techniques described in Japanese Examined Patent Publication No. 59-31518 and Japanese Examined Patent Publication No. 59-31519 are to extract shirako only with a dilute mineral acid, and then the extract is separated with sodium metaphosphate to separate protamine. Ammonium sulfate or the like is added. The solution from which protamine has been separated cannot be used again as a solution for extracting algae.

As another method for separating protamine sulfate, for example, JP-B-59-31519, JP-B-59-31518, JP-A-55-2603, JP-A-55-2612 or JP-A-55-2612. -2634 gazette etc. are described in series.

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 the drawback that the work flow is poor and the process becomes complicated because it must be once taken out as protamine phosphate.

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.

Problems to be Solved by the Invention As a result of examining the extraction method of obtaining protamine sulfate with higher purity and high yield, the present inventors have found that protamine sulfate and contaminating proteins are different in solutions having different concentrations of inorganic salts. It was discovered that the degree of extraction from Shirako and the solubility were significantly different. By utilizing this new knowledge, not only can all of the above problems be solved and protamine sulfate of high purity can be obtained simply and efficiently, but the extract after separation of protamine sulfate can be reused. I came to complete a method that can be done.

Means for Solving the Problems A first feature of the present invention relates to a method for extracting protamine mineral acid salt in which protamine-containing fish larvae are extracted with a mixed solution of a mineral acid and a mineral acid salt.

A second feature of the present invention is to extract protamine-containing fish spores with a mixed solution of a mineral acid and a mineral acid salt, and cool the extract solution to precipitate and recover the protamine mineral acid salt and to recover the supernatant liquid with an acid. And a method for extracting a protamine mineral salt, which comprises adjusting the salt concentration and using the solution again as an extraction solution.

The present invention will be described with reference to FIGS.

1 to 3 are flowcharts of the extraction method of the present invention. Shirako is ground if necessary and extracted with a mineral acid / salt mixture as shown in Fig. 1, the extract (containing crude protamine mineral acid salt) is collected, and the residue (shirako extract residue and contaminants) is collected. Protein is removed) and the extract is cooled to remove the supernatant (which is predominantly contaminated protein and contains a small amount of protamine mineral acid salt and mineral acid / salt) and the precipitate is collected.
The protamine mineral acid salt is contained in this precipitate in a relatively high purity, but it may be subjected to a purification step as shown in FIG. 3 in order to further increase the purity. FIG. 2 shows that the protamine mineral salt in the supernatant liquid shown in FIG. 1 is collected without being discarded, and the drainage problem caused by discharging the acid and salt is eliminated. 2 shows a method of recycling and reusing as a solvent of. When reusing, the acid / salt concentration in the supernatant liquid is readjusted to a concentration suitable for extraction. Shirako of fish that can be used in the extraction of the present invention may be ground or unground Shirako and contains protamine. It is not particularly limited as long as it is a white spore of a fish, such as herring, salmon, trout or mackerel.

Examples of the mineral acid that can be used in the extraction of the present invention include sulfuric acid, hydrochloric acid and the like, and any mineral acid that is commonly used in the extraction of protamine can be used. Sulfuric acid is particularly preferred.

The mineral acid is used in an aqueous solution with a concentration of 3 to 20% by weight. When used at a concentration lower than 3% by weight, the extraction efficiency of protamine is reduced. When the concentration is higher than 20% by weight, the advantage of the present invention that the amount of the contaminant proteins extracted is small is lost as compared with the case of using the mineral acid alone.

As the mineral acid salt used together with the mineral acid, it is preferable to use an acid salt of the same kind as the acid used for extraction, and hydrochloride, sulfate or the like, particularly ammonium sulfate or sodium sulfate is desirable.

The concentration of the mineral acid salt is appropriately adjusted depending on the mineral acid used or its concentration, the type of the mineral acid salt, and the like. 6 for mineral acid
When 14% by weight sulfuric acid is used and ammonium sulfate is used as the mineral acid salt, ammonium sulfate is added so as to be contained in 1.8 to 3.2 mol, preferably 2.26 to 2.5 mol per liter of the sulfuric acid aqueous solution. When used in an amount less than 1.8 moles, the advantage of the present invention that the amount of extracted contaminant protein is smaller than that in the case of using the mineral acid alone is lost. If it is used in an amount of more than 3.2 mol, the extraction efficiency of protamine is reduced. When using sodium sulfate as a mineral acid salt,
For the same reason as above, sodium sulfate is added so that it is contained in an amount of 0.8 to 1.8 mol, preferably 1.0 to 1.4 mol, per liter of aqueous sulfuric acid solution.

The mixed solution of the mineral acid and the mineral acid salt is used in an amount of 1 to 5 times, preferably 2 times the weight of extracted white matter. For example, 1kg of Shirako
It is preferable to use 2 liters of the mixed solution when extracting. When the amount is more than 5 times, the concentration of protamine mineral acid salt in the extract is low, and the protamine mineral acid salt cannot be sufficiently recovered from the extract in the subsequent separation step. If it is less than 1-fold, the amount of protamine extracted will decrease.

The mixed solution of the mineral acid and the mineral acid salt is 20 to 40 ° C, preferably 25 to 30 ° C.
Use at a liquid temperature of ℃. When used at a temperature lower than 20 ° C, the extraction efficiency of protamine decreases. If it is used at a temperature higher than 40 ° C, the extracted protamine mineral salt will decompose within a short time.

The extraction time depends on the ground condition of the Shirako or the extraction means, but usually 30 to 60 minutes or less is sufficient.

Next, the extract obtained according to the present invention is cooled to separate into a precipitate and a supernatant liquid (hereinafter, this process is referred to as a separation step: that is, the extraction method according to the second aspect of the present invention includes an extraction step and an extraction step). A separation step of separating protamine mineral salt from the liquid).

The precipitate is a protamine mineral salt having a sufficiently high degree of purity for practical use.

A feature of the present invention is that the protamine mineral acid salt can be extracted and separated by an extremely simple method, and the work can be carried out continuously without interruption, so that the workability is very good.

Another feature of the present invention is that it can be reused as an extraction solution by adjusting the concentration of the mineral acid and the salt of the supernatant as shown in FIG.

The concentration of mineral acid and salt when reusing the supernatant liquid is
The concentration may be adjusted during extraction as described above.

When reusing the supernatant, it is desirable to first neutralize the supernatant with an alkali such as ammonia. At that time the liquid temperature is 20
-30 ° C is preferred. By carrying out this operation, only the contaminating protein existing in the supernatant can be precipitated and removed.

By reusing the supernatant according to the present invention, the protamine mineral salt remaining in the supernatant is not wasted.

Further, when the supernatant liquid is reused according to the present invention, the amount of waste liquid can be reduced, which is also effective in terms of pollution.

According to the present invention, a highly pure protamine mineral salt can be obtained because the amount of contaminant protein extracted with protamine in the extraction step is small, and the contaminant protein hardly precipitates even when the extract is cooled. This is due to the fact that only the mineral acid salt is deposited.

Table 1 shows an example of a mixed solution of sulfuric acid / ammonium sulfate and sulfuric acid / sodium sulfate, which is used to extract white mite and
Ultraviolet absorption based on the contaminated protein of the double diluted solution (28
(0 nm) is shown.

Table 2 shows the solubility (% by weight) of protamine sulfate at 5 ° C. in an aqueous solution of sulfuric acid, a mixed solution of sulfuric acid / ammonium sulfate and sulfuric acid / sodium sulfate.

In Tables 1 and 2, the concentrations of ammonium sulfate and sodium sulfate refer to the number of moles added to 1 liter of an aqueous sulfuric acid solution.

It can be seen from Table 1 that protamine can be selectively extracted according to the present invention. It can be seen that when the conventional method is used to extract a solution containing only sulfuric acid, the amount of contaminating proteins can be reduced to about half or less as compared with the solution.

From Table 2, for example, the solubility of protamine sulfate is 1.5% by weight in the case of the extract containing only 7% sulfuric acid aqueous solution, but about 0.13% by weight in the case of the extract containing the mixed solution of 7% sulfuric acid and 1.9 mol ammonium sulfate. %, And it can be seen that protamine sulfate is sufficiently precipitated by cooling.

In Table 3, the presence of contaminant proteins in the solution of the extract obtained according to the present invention before cooling and in the solution from which precipitates were removed after cooling and cooling, and the ultraviolet absorption at a wavelength of 280 nm based on the contaminant proteins were measured. The results of the comparison are shown.

It can be seen from Table 3 that even if the extract obtained according to the present invention is cooled, no contaminant protein is deposited.

According to the present invention, there are few contaminating proteins in the extract, and when it is cooled, the protamine mineral salt is sufficiently precipitated but the contaminating proteins are not precipitated, so that the purity is sufficient for practical use. It is possible to obtain a protamine mineral salt having

To further purify the protamine mineral acid salt obtained in the present invention, as shown in FIG. 3, the obtained protamine mineral acid salt is dissolved in warm water together with a small amount of an inorganic salt, preferably a sulfate salt such as ammonium sulfate or sodium sulfate. Let
After that, the solution is cooled to precipitate protamine mineral salt, separated from the solution, and dried (hereinafter, the above operation is referred to as “purification step”). The concentration of the protamine mineral salt in the purification step is The richer the better.

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

Mineral salt used in the purification process is 0 for 1 liter of warm water.
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, the protamine mineral salt cannot be efficiently precipitated.

It is not necessary to adjust the pH of hot water. When adjusting,
After adding protamine minerals and before adding sulfate
A range of 7 is desirable.

By carrying out the purification, it is possible to remove salts, acids and contaminating proteins attached to the precipitate of protamine mineral acid during the separation step.

The purification step of the present invention is based on a new finding that the solubility of protamine mineral salt is minimum in a solution in an extremely low concentration range of 0.05 to 0.2 mol of ammonium sulfate or sodium sulfate concentration in solution 1. It is a thing.

In Table 4, taking protamine sulfate as an example, the relationship between the solubility (% by weight) and the sulfate concentration in an aqueous solution having a water temperature of 5 ° C. and a pH of 2.5 is shown. In the table, the concentration represents the number of moles of sulfate dissolved in Solution 1.

From Table 4, the solubility of protamine sulfate is 0.05 when the concentration of sulfate is 0.05.
It can be seen that there is a minimum in the range of ~ 0.2 mol.

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

1 kg of ground salmon Shirako and 2.35 mol of ammonium sulfate were added to 2 liters of 10% (2N) sulfuric acid, and the mixture was stirred at 30 ° C for 1 hour. After stirring, the residue was removed by centrifugation to obtain 2.5 liters of protamine sulfate extract.

53.3 g (2.13% by weight) of protamine sulfate in the extract
It was included (dry conversion).

Separation step 2.5 liters of the extract was cooled to 5 ° C. and allowed to stand at that temperature for 16 hours to separate a protamine sulfate precipitate and a supernatant. The supernatant was decanted and collected.

The protamine sulfate precipitate was white candy-like, and the yield was 102 g.

The recovered supernatant was 2.43 liters. Component is sulfuric acid
The content was 7.26%, ammonium sulfate 1.72 mol / liter, contaminating protein 1.6% by weight, and protamine sulfate 0.4% by weight (9.72 g).

Purification Step 102 g of protamine sulfate obtained in the separation step was dissolved in 800 ml of warm water (40 to 50 ° C.), and the concentration of ammonium sulfate was about 0.14 mol per liter of the solution. Then the solution is 5 ℃
After cooling to room temperature, the mixture was allowed to stand at that temperature overnight to separate a protamine sulfate precipitate and a supernatant.

The supernatant liquid was decanted and removed to obtain a transparent oily protamine sulfate. Lyophilize it and white Protamine Sulfate 43.2
g was obtained (yield 4.32%).

Reuse 240 ml of concentrated aqueous ammonia was added to the supernatant recovered in the separation step to precipitate contaminating proteins. The precipitate was removed by centrifugation and 2.48 liters of solution was recovered. The components of the recovered solution are ammonium sulfate 2.3 mol / l, contaminating proteins
It was 0.14% by weight.

227 g of concentrated sulfuric acid was added to 1.92 liters of the recovered solution, and the solution was reused as an extraction solvent. Solution component is sulfuric acid 10.7
%, And ammonium sulfate was 2.3 mol / liter.

Example 2 The procedure of Example 1 was repeated except that the purification step of Example 1 was not performed. The obtained white candy-like protamine sulfate was freeze-dried to obtain 53.2 g (yield 5.32%) of white protamine sulfate.

Comparative Example Frozen salmon Shiroko 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 of 26 ° C for 1 hour. After stirring, the residue was removed by centrifugation to obtain 550 ml of protamine sulfate extract.

Methanol (2750 ml) was added to the extract and left at 5 ° C. overnight to reprecipitate protamine sulfate and contaminating proteins. The precipitate was separated by centrifugation (8000 rpm, 20 minutes), 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 3 Protamine sulfate obtained in Example 1, Example 2 and Comparative Example was used as a sample and its purity was examined.

Purity was measured by chromatography, absorption spectrometry, solubility of protamine sulfate in water and 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 5.

Comparing the HPLC purities, it can be seen, for example, that the protamine sulphate obtained according to the invention in Example 1 has a purity which is about 1.2 times better than that obtained by the conventional method. In addition, comparing the standardized yields, it can be seen that the present invention can be separated approximately 1.1 times more efficiently than the conventional method.

For antibacterial activity, protamine sulfate (salmon) manufactured by Wako Pure Chemical Industries, Ltd. was used as a standard, and the Bacillus subtilis (B.su
The antibacterial activity against btilis IAM1069 strain) was compared as 1.00 and shown as the antibacterial activity specific activity. Also, the product obtained by multiplying the antibacterial activity and the yield of the sample is shown as the standard product 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 Miko. You can think of it as a measure of. The results are shown in Table 5.

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 that obtained by the conventional method. Further, comparing the standardized active yields, it can be seen that the separation can be performed with about 1.1 times higher efficiency than the conventional method of the present invention.

Absorbance analysis measured the absorbance at 260 nm due to the acid and 280 nm due to contaminant proteins. Measurement is the concentration of the sample
An aqueous solution of 0.1% by weight was carried out at a temperature of 20 ° C. The results are shown in Table 6.

Pure protamine sulfate does not absorb light above 220 nm. As can be seen from Table 6, 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 dissolution state was performed with a 2% aqueous solution (20 ° C.) of each sample. The protamine sulfate obtained according to the present invention (Examples 1 and 2) was completely dissolved, whereas the protamine sulfate obtained by the conventional method (Comparative Example) was not completely dissolved and the presence of insoluble matter was observed. .

EFFECTS OF THE INVENTION According to the present invention, highly pure protamine sulfate can be continuously extracted and separated from fish larvae very simply, efficiently, with no pollution, at an inexpensive and simple process.

[Brief description of drawings]

1 to 3 are charts showing an embodiment of the method for extracting protamine mineral acid salt according to the present invention.

Continuation of the front page (56) References JP-A-55-2634 (JP, A) JP-B-59-31518 (JP, B2) Chemical Society of Japan "New Experimental Chemistry Course 20 Biological Chemistry" Maruzen (SHO 53- 6-20) P. 14-19

Claims (3)

[Claims] 1. A method for extracting protamine mineral acid salt, which comprises extracting protamine-containing fish larvae with a mixed solution of a mineral acid and a mineral acid salt. 2. Protamine-containing fish spores are extracted with a mixed solution of a mineral acid and a mineral acid salt, and the protamine mineral acid salt is extruded and recovered by cooling the extracted solution, and the supernatant liquid is adjusted for acid and salt concentrations. A method for extracting a protamine mineral salt, which is prepared and used again as an extraction solution. 3. As a mixed solution of a mineral acid and a mineral acid salt, ammonium sulphate 1.8 per liter of a 3 to 20% by weight mineral acid aqueous solution.
Extraction method according to claim 1, characterized in that a mixed solution having a concentration of ˜3.2 mol or 0.8-1.8 mol of sodium sulfate is used.