JPH0699478B2 - 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 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 separately precipitated with an organic solvent.

DISCLOSURE OF THE INVENTION Problems to be Solved by the Invention The present inventors diligently studied a method of obtaining protamine sulfate more simply and efficiently, and as a result, the solubility of protamine sulfate in an aqueous sulfuric acid solution remarkably depends on the temperature of the aqueous solution. It was found that the solubility of the contaminant protein was independent of temperature. By utilizing this new finding, it has been possible to solve all of the above-mentioned problems and to complete a method by which highly pure protamine sulfate can be easily and efficiently obtained.

Means for Solving the Problems That is, the present invention is to extract protamine mineral salt by extracting and extracting protamine-containing fish spores with mineral acid and cooling the extract at 0 to 10 ° C. The present invention relates to a characteristic method for 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 18%.
The treatment temperature is 30 ° C. or lower, and preferably 20 to 30 ° C. A treatment time of 3 hours or less is sufficient.

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

Since the obtained protamine extract contains a large amount of contaminant proteins, only the protamine mineral salt is precipitated and precipitated by cooling this extract.

The processing temperature of the extract is 0 to 10 ° C. If it exceeds 10 ° C, the precipitation of protamine mineral is insufficient. The processing time is not particularly limited.

The precipitated protamine salts are separated from the extract by centrifugation or the like to obtain a transparent oily substance, and freeze-dried to obtain a white powder.

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. FIGS. 1 and 2 show the solubility (% by weight) curves of protamine sulfate and contaminating proteins with respect to 1 liter of a sulfuric acid aqueous solution with the temperature change of the aqueous solution. 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. In the figure, (1) shows the solubility curve of protamine sulfate and (2) shows the solubility curve of contaminating proteins.

The extraction solution usually contains 0.5 to 7% by weight of protamine sulfate.

As can be seen from FIGS. 1 and 2, in the separation step of the present invention, protamine sulfate is significantly reduced in its solubility and precipitates and precipitates in a 5% or 10% sulfuric acid aqueous solution at 10 ° C. or lower, but contaminant proteins are completely removed. It is that it is dissolved in.

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

1 kg of ground salmon shirako was added to 1000 ml of 10% (1N) sulfuric acid, and the mixture was stirred at room temperature of 20 ° C for 1 hour. After stirring, the residue was removed by centrifugation to obtain 1530 ml of protamine sulfate extract.

The extract was cooled to 2 ° C. and left for 16 hours to precipitate protamine sulfate.

The precipitated supernatant of protamine sulfate was decanted off to obtain a transparent oily protamine sulfate. This was dissolved in water, neutralized with sodium hydroxide (1N), and freeze-dried to obtain 71.5 g of white protamine sulfate (yield 7.15%).

Example 2 Protamine sulfate obtained in Example 1 was used as a sample and its purity was measured.

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

High performance liquid chromatography was used for the chromatography. The purity of the sample was compared with the Wako Pure Chemical Industries Protamine Sulfate (salmon) as the standard and its purity was 1.00.
Shown as LC 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 1.

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 obtained by multiplying the specific activity of antibacterial activity and the yield of the sample is 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 1.

Absorption analysis is due to contaminant proteins. Absorbance at 288 nm was measured. The measurement is carried out at a temperature of 20
Performed at ° C. The results are shown in Table 2. Pure protamine sulfate does not absorb light above 220 nm. As can be seen from Table 2, the protamine sulfate obtained according to the present invention has a very small amount of contaminant proteins.

Visual observation of the solution state was performed with a 2% aqueous solution (20 ° C.) of the sample. The protamine sulfate obtained according to the present invention was completely dissolved, and the presence of insoluble matter was not recognized (see No. 2).

Example 3 Frozen salmon roe was thawed, washed with water, drained and ground.

Add 500 g of ground salmon shirako to 500 ml of 10% sulfuric acid, and add room temperature 2
The mixture was stirred at 0 ° C for 1 hour. After stirring, the residue was removed by centrifugation to obtain 740 ml of protamine sulfate extract.

The extract was divided into 3 samples of 100 ml each, and each at 2 ° C, 5
After cooling to ℃ and 10 ℃, after standing for 16 hours, the precipitation of protamine sulfate respectively.

Example 4 Frozen salmon Shirako was thawed, washed with water, drained and ground.

Add 500 g of ground salmon shirako to 500 ml of 18% sulfuric acid, and add room temperature 2
The mixture was stirred at 6 ° C for 1 hour. After stirring, the residue was removed by centrifugation to obtain 750 ml of protamine sulfate extract.

The extract was divided into 3 samples of 100 ml each, and each at 2 ° C, 5
After cooling to ℃ and 10 ℃, after standing for 16 hours, the precipitation of protamine sulfate respectively.

Example 5 Using the extracts obtained in Examples 3 and 4 and the supernatant of the solution after protamine sulfate precipitation as samples, their properties were examined by absorption spectrometry and chromatography.

Absorbance analysis, absorbance at 280 nm due to contaminant proteins was measured. The measurement is performed by using an aqueous solution containing 0.1% by weight of the sample at a temperature of 20 ° C.
I did it in.

The results are shown in Table 3. Pure Protamine Sulfate is 22
There is no absorption for light above 0 nm. As can be seen from Table 3, the protamine sulfate precipitate obtained according to the present invention contains almost no contaminating proteins.

The concentration of protamine sulfate was quantified by high performance liquid chromatography using protamine sulfate (salmon) manufactured by Wako Pure Chemical Industries, Ltd. as a standard.

As can be seen from Table 3, almost no protamine sulfate remains in the supernatant obtained after the protamine sulfate precipitation according to the present invention.

EFFECTS OF THE INVENTION According to the present invention, protamine salts having a high purity can be obtained because the separation of protamine salts from the extract is extremely easy.

[Brief description of drawings]

1 and 2 are diagrams showing the solubility of protamine sulfate. In the figure, (1) shows the solubility curve of protamine sulfate and (2) shows the solubility curve of contaminating proteins.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (56) References Japanese Patent Publication Sho 49-16880 (JP, B1) Japanese Patent Publication Sho 59-31518 (JP, B2)

Claims (1)

[Claims] 1. A method for collecting a protamine salt, which comprises extracting protamine-containing white spores of a fish with a mineral acid, and cooling the extract to 0 to 10 ° C. to precipitate and collect a protamine mineral salt.