JPS5931520B2 - Method for producing silk fibroin peptide aqueous solution - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing an aqueous silk fibroin peptide solution.

Silk fiproin has been used for a long time as a raw material for cosmetics and other purposes.

When used as a cosmetic raw material, for example, silk fiproin can be swollen or dissolved in acid or alkali, and silk fiproin can be neutralized or treated with an organic solvent to form a water-insoluble precipitate without causing any major chemical changes. Alternatively, a method is adopted in which other synthetic polymer substances and basic colloid of silk fiproin are mixed and adhered to an inorganic cosmetic base, or a method is adopted in which other materials such as condensed thread resin are mixed to form a dry powder. However, since the products obtained by these methods are water-insoluble, they are mainly limited to use in make-up cosmetics. In addition, there are products that make silk fiproin into a paste form with acids, alkalis, salts, etc., or cream forms by mixing fatty acids, etc., but these are made by simply turning fibrous silk fiproin into a paste or cream form. Its purpose is to simply add it to creamy cosmetics. Furthermore, there is a method of decomposing silk fiproin with acid until it shows no protein reaction, but this is an amino acid rather than silk fiproin, and furthermore, it is poorly soluble in water, so its uses are naturally limited. On the other hand, the special public
No. 17030 discloses that a solution obtained by treating silk fiproin with highly concentrated phosphoric acid is mixed with a precipitant made of a specific organic solvent such as acetone to form a precipitate, which is dispersed and neutralized in water, and then subjected to radiation therapy. Although it is described that a water-soluble substance is obtained by using a fungal secreted proteolytic enzyme and again using the above-mentioned precipitant to form a precipitate, the molecular weight distribution must be wide because it is essentially a solid-liquid reaction. Furthermore, if the average degree of polymerization is lowered, the amino acid content inevitably increases, which is a quality problem, and the process is complicated, making it unsatisfactory in terms of operation and cost. It is.

The present inventors have completed the present invention as a result of intensive research in order to eliminate the above-mentioned defects and obtain a high-quality silk fiproin peptide useful as a raw material for cosmetics and other uses from silk fiproin. An object of the present invention is to provide a water-soluble silk fiproin peptide that has a low amino acid content compared to the average degree of polymerization, a low salt content, and high solubility in an aqueous ethanol solution, and is useful for cosmetics and other applications. It is in. Another object is to provide an aqueous silk fiproin peptide solution that has a lower amino acid content and a lower salt content than the average degree of polymerization, and can be applied as is to cosmetics and the like. Still another object is to provide a method for industrially producing the silk fiproin peptide aqueous solution easily and at low cost. The method of the present invention uses copper-ethylenediamine aqueous solution, copper hydroxide-ammonia aqueous solution, copper hydroxide-alkaline glycerin aqueous solution, lithium bromide aqueous solution, calcium, magnesium or zinc hydrochloride, nitrate or thiocyanate aqueous solution, sodium thiocyanate aqueous solution. The method is characterized in that a 0.5 to 20% by weight aqueous silk fiproin solution obtained by dissolving a refined silk raw material in at least one solvent selected from the group consisting of the following and dialysis is hydrolyzed with an enzyme, an acid, or an alkali. The solvent of the silk fiproin aqueous solution applied to the present invention is copper-
Ethylenediamine aqueous solution, copper hydroxide-ammonia aqueous solution (Schweisser's reagent), copper hydroxide alkali-glycerin aqueous solution (Rohe reagent), lithium bromide aqueous solution, aqueous solution of calcium, magnesium or zinc hydrochloride, nitrate or thiocyanate, thiocyanide Examples include aqueous sodium chloride solutions, but calcium or magnesium hydrochloride or nitrate are preferred from the viewpoint of composition and use.

Silk as a raw material used in the present invention can be raw cocoon silk, cocoon waste, raw silk waste, screws, fried cotton, silk cloth waste, boulet, etc., and these can be prepared in accordance with a conventional method, and if necessary, Sericin is removed in warm water in the presence of an activator or in room temperature water in the presence of an enzyme and dried.

The obtained refined silk raw material was added to the above solvent and heated to a temperature of 60 to 9.
Uniformly melt at 5°C, preferably from 70 to 85°C in an apparatus such as a secondary oven.

Here, the bath ratio is usually 2 to 50 times, preferably 3 to 30 times, relative to the refined silk raw material. Calcium chloride and calcium nitrate are preferred as the solvent in view of solubility and economy, and the concentration of these metal salts is usually 15 to 80% by weight, preferably 30 to 70% by weight, particularly preferably 40 to 60% by weight.

Further, in order to further improve solubility, it is preferable to add alcohols to these aqueous solutions. The timing of adding alcohol is preferably before or during the dissolution of the silk raw material, and the amount added is 20 to 60% by weight, preferably 2% by weight, based on the aqueous solution of metal salt.
It is 5 to 50% by weight. The salts are almost completely removed from the resulting solution using a dialysis membrane such as a cellophane membrane or a dialysis machine using hollow fibers.

In order to obtain silk fiproin peptide that can be used in cosmetics and the like, it is desirable that the residual salt concentration be approximately 0.003 to 0.06%. From this point of view, it is preferable to adjust the dialysis amount and dialysis membrane area when performing dialysis. For example, it is preferable to adjust the dialysis amount and dialysis membrane area. Preferably salted.

The priming capacity here refers to the internal volume of the dialysis tube or between the membranes. Further, this condition can be achieved, for example, by keeping the membrane spacing at 2' or less in the case of a multilayer structure, or by keeping the diameter of the hollow fibers at 4 or less in the case of a hollow fiber bundle structure. Next, the concentration of the obtained fiproin aqueous solution is adjusted to a concentration suitable for hydrolysis, that is, 0.5 to 20% by weight, preferably 1% by weight.
Adjust to ~15% by weight.

If the concentration of silk fiproin is less than 0.5% by weight, the content of metal salts will be high in the produced silk fiproin peptide, which will not only cause quality problems but also be inferior in terms of cost.
If it exceeds 0% by weight, silk fiproin will coagulate and precipitate during the process, which will not only cause problems in operation but also cause defects such as an increase in amino acid content and a decrease in yield. Concentration 0.

The silk fiproin aqueous solution adjusted to 5 to 20% by weight is then hydrolyzed using an enzyme, acid, or alkali.

Enzymes that can be applied to the present invention include common proteolytic enzymes, such as pronase obtained from actinomycetes and prolase obtained from papaya, which are considered to be a mixture of several proteases, or trypsin, chymotrypsin,
Examples include papain and promeline, and these can be used alone or in a mixture of two or more.

The amount of enzyme used varies depending on the type of enzyme, purity, reaction conditions, average degree of polymerization of the desired silk fiproin peptide, etc., but is usually 100 PPm to 1 for silk fiproin.
0% by weight, preferably 0.1-5.0% by weight. The conditions for hydrolysis in this case vary depending on the type and concentration of the enzyme used, but the pH is usually 5 to 9, preferably 6 to 8.5.
1 at a temperature of 20-50°C, preferably 30-45°C.
~72 hours, preferably 4 to 16 hours. Although the reaction proceeds even if it is left standing, it is preferable to stir or shake it for homogenization and acceleration. Although a buffer such as sodium phosphate or Tris-HCl can be used to adjust the pH, since the pH of the silk fiproin aqueous solution of the present invention is around 7, it would be better not to use a buffer such as silk fifurin peptide salt. This is preferable because the amount is reduced. As the enzymes used, papain and chymotrypsin are preferable in terms of the quality and yield of the product obtained and the working soil. Examples of acids applicable to the present invention include inorganic or organic acids such as hydrochloric acid, sulfuric acid, nitric acid, tartaric acid, malonic acid, succinic acid, and maleic acid. is preferred.

In addition, as the alkali applicable to the present invention, sodium hydroxide, potassium hydroxide, lithium hydroxide, polyamines such as polyethyleneimine, etc. can be used, but from the viewpoint of reactivity, economy, and stability, sodium hydroxide, hydroxide Potassium is preferred. The conditions for hydrolysis with acid or alkali vary depending on the type of acid or alkali used, the average degree of polymerization and distribution of the degree of polymerization of the target silk fiproin peptide, but are usually 0.03 to 10N, preferably 0.3N or less. After the reaction is carried out at a temperature of 20 to 110° C., preferably 30 to 100° C., for 0.5 to 50 hours, preferably 1 to 25 hours, an alkali or acid is added to neutralize.

The silk fiproin peptide aqueous solution of the present invention obtained by hydrolysis can be applied as it is or after being concentrated or diluted, for example, in cosmetics, and can also be desalted to make it of higher quality. By doing so, a powdered silk fiproin peptide can also be obtained.

After desalting and dehydration, high quality powdered silk fiproin peptide can be obtained. Desalting is carried out using, for example, an ion exchange resin, but when an acid catalyst is used, neutralization and desalting can be carried out simultaneously by treating with a weakly basic anion exchange resin after the reaction.

Further, in the case of an alkali catalyst, the same treatment can be performed. Dehydration is usually accomplished by distillation under reduced pressure or by adding a non-solvent such as acetone to cause precipitation. The silk fibroin peptide obtained by the method of the present invention usually has an average degree of polymerization of 2 to 20, preferably 4 to 15, and contains only 15% by weight or less, preferably 10% by weight or less, particularly 5% by weight or less of amino acids. Moreover, it is water-soluble, has high solubility in aqueous ethanol solution, and has a low salt content, so it can be widely used in cosmetics and other applications.

Particularly when used as a cosmetic raw material, it has moisturizing properties and has excellent cosmetic effects, and can prevent itching, itching, and bad odors. Also, allergic symptoms are almost non-existent compared to amino acids. In addition, the silk fiproin peptide of the present invention is characterized by a narrow distribution of polymerization degrees, and even when the average degree of polymerization is low, the amino acid content is low, and even when the average degree of polymerization is relatively high, the degree of polymerization is relatively high. In addition, it does not contain substances with low solubility and/or stability in water, so it does not cause precipitation or turbidity when used in transparent lotions, etc., and does not cause nozzle clogging when used in sprays, etc. . The method of the present invention will be explained below with reference to Examples, and the measurements in Example 5 were carried out by the following method.

(Peptide average degree of polymerization measurement method) The total amount of peptide (including contaminating amino acids) is measured, and the molar amount of amino acids when the peptide is completely hydrolyzed is determined, and this is designated as (a).

Measure the amount of terminal groups and define this as (b). It is determined as average degree of polymerization=(a)Ab). To obtain (a), (1
) The ash content is subtracted from the absolute dry solid content and determined from this and the average molecular weight of the fiproin-constituting amino acids. (2) The nitrogen atomic weight determined by geltal nitrogen measurement is the amino acid molar amount.

(This is because the amount of basic amino acids that make up fiproin is very small, so the error is actually very small.) (3) After hydrolysis with sodium hydroxide or hydrochloric acid, the amino acids produced are measured by colorimetry using ninhydrin, etc. Depends on the method. Although there are some differences between each measurement method, there is generally good agreement. For (b), the terminal CO2H group can be measured by formol titration method, but
The amino acids that make up fiproin are essentially neutral amino acids, so the accuracy is very high. (Amino acid content measurement method) Dissolve sample solid content of approximately 10mf7 in 1CC of pure water,
This is developed in a column packed with Ceftudex GlO (manufactured by Seikagaku Kogyo Co., Ltd.) with an inner diameter of 21 mm and a length of 75 cm.

The solution used was 0.05N-NaCl, and the flow rate was 4.8ml.
/Hr fractionation was set to 1.4WL1 per tube. Each fraction is hydrolyzed with 2.5N NaOH at 90°C for 2.5 hours, then neutralized with 30% acetic acid, and the amount of amino acids contained is measured by ninhydrin colorimetry. Glycine, alanine, serine, and tyrosine were flowed in advance to determine the outflow volume, and what entered this fraction was determined as the amino acid content in the peptide sample. A small amount of dimer such as glycyl-glycine may be mixed into this fraction, but since this was also measured as an amino acid, the amino acid content determined in this way is determined as the maximum value. (30
% ethyl alcohol solubility) 2.5% by weight of the peptide sample was added to a 30% ethyl alcohol aqueous solution, stirred and left at 10°C for 1 day, the amount dissolved was measured and the percentage of the added amount was indicated.

Example 1 Refined silk raw material 1407 is heated and dissolved in 60% by weight aqueous solution 11 of calcium chloride.

At this time, to facilitate dissolution, use 400ml of ethyl alcohol.
was added. Next, this was dialyzed for 24 hours to obtain 2.81% of a 5.0% by weight aqueous solution of fiproin. The residual salt concentration determined by conductivity measurement was 0.004%. Concentrated hydrochloric acid was added to this 11 to make a 0.2N-HCl solution, and after heating at 94°C for 3.8 hours with stirring, 5N-NaO
The mixture was neutralized with H solution to a pH of 6.8. This was decolorized with activated carbon and passed through the mouth to obtain a slightly yellow and transparent aqueous solution of fiproin peptide. (Jikkenya 1) For comparison, scouring raw silk 50
V was immersed in 1N-HCIll, and reacted by heating and stirring at 100° C. for 12 hours in a nitrogen stream. Next, after removing the solvent under reduced pressure using a rotary evaporator, 100 cc of water was added, and the solvent was removed under reduced pressure again. After adding pure water 11 to remove insoluble materials, the solution was neutralized to pH 6.7 with concentrated NaOH solution. (Experimental Washing 2) These two samples were analyzed for average degree of polymerization, amino acid content, salt content (relative to the produced peptide mixture), peptide (including amino acid) yield, solubility in 30% ethyl alcohol aqueous solution, aqueous solution (5 wt. %) stability was measured and observed. The results are shown in Table 1. As can be seen from Table 1, for comparison, Experiment Black 2, in which solid silk fiproin was hydrolyzed with hydrochloric acid, had a low yield, but also had a low average degree of polymerization and a high amino acid content.

If the reaction is carried out for a longer period of time, the yield will improve, but the average degree of polymerization will remain the same or even decrease. However, in Experimental Black 1, in which a fiproin solution dialysate was hydrolyzed, a satisfactory average degree of polymerization and amino acid content were obtained, and the yield was also good. Example 2 Dissolution solution 11 of 100y of refined silk listed in Table 2? After dissolving impurities in the solution and separating the impurities, it is dialyzed to obtain an approximately 5% by weight aqueous solution of fiproin.

Concentrated hydrochloric acid was added to each to form a 0.2N-HCl solution, and the mixture was heated and stirred at 90°C for about 3.8 hours, with an average polymerization degree of 5.0.
~ 5.3. Table 2 shows the results of measuring the amino acid content, yield, and solubility in a 30% aqueous ethyl alcohol solution for each product after neutralization. As can be seen from Table 2, by using these solutions, a uniform suspended fiproin aqueous solution can be obtained, and by hydrolyzing this, a peptide mixture with a low amino acid content can be obtained in good yield. I was able to do it.

Example 3 In the same manner as in Example 1, a refined silk raw material is dissolved in a 60% by weight aqueous solution 11 of calcium nitrate, passed through the mouth, and then dialyzed for 24 hours to obtain a fiproin aqueous solution.

At this time, the scouring silk raw material is 6y) 30y) 100>v, 2
00y, and the fiproin concentration after dialysis is 0.
3, 1.5, and 5.10% by weight were obtained. Furthermore, this 10% fiproin solution was carefully concentrated under a nitrogen stream,
15% and 30% were obtained. For each of these, 0.
An IN-HCI solution was used and hydrolysis was carried out at 94°C for 4.6 hours. After neutralizing to pH 6.8 with 5N-NaOH,
Table 3 shows the results of measuring the yield, amino acid content, salt content relative to the peptide (including amino acids), and average degree of polymerization after separating the insoluble matter.

As is clear from Table 3, as the concentration of fiproin increases, the average degree of polymerization tends to be higher.

When the concentration of fiproin exceeds 20% by weight, the solution becomes unstable and fiproin tends to coagulate and precipitate. Even in the case of 30% by weight as shown in the table above, coagulated precipitates are formed as soon as hydrochloric acid is added, and when the reaction is completed. Since it remained undissolved until then, the yield obtained was low. The amino acid content is also relatively high, which is not preferable from an operational point of view. Furthermore, if the fiproin concentration is low, the salt content will also be relatively high, which is also disadvantageous in terms of economy and post-processing operations. Example 4 A fiproin aqueous solution is obtained in the same manner as in Example 1.

This concentration was performed in a rotary evaporator at 90° C. under a nitrogen stream to give a concentration of 5% by weight. This was hydrolyzed under various alkaline conditions shown in Table 4, neutralized, filtered, and optionally decolorized with activated carbon to obtain a fiproin peptide aqueous solution. Regarding these, amino acid content, salt content for peptide (including amino acids), yield,
30% ethanol solubility (2.5% by weight added), aqueous solution stability (5% by weight added) and average degree of polymerization were measured. The results are shown in Table 4. To obtain a peptide mixture powder, the solvent may be removed from the aqueous solution under reduced pressure, and when this is added to water, it is quickly redissolved. As shown in Table 4, the average degree of polymerization can be varied by selecting an appropriate alkali concentration, temperature, and time. Good results were obtained regarding the stability of the aqueous solution. Furthermore, as can be seen from the above table, the amino acid content is low and the salt content is also low. The yield is also good. Also, in the above experiment, neutralization was performed with 6N-HCl,
Alternatively, the alkali can be removed through a weakly acidic cation exchange resin column, resulting in a salt content of about 0 to 3% by weight.

In this case, the yield is about 80-85%. Example 5 Crude papain manufactured by Sigma (Type, 1 to 2
BAEEunits martyrs) 2.57 water extract is added,
The reaction was carried out in a constant temperature bath at 45°C for 4 hours.

No buffer was used, but the pH at the start of the reaction was 6.8.
The pH at the end of the reaction was almost constant at 6.4. In order to terminate the reaction, the mixture was heated in an autoclave at 110°C for 15 minutes, and then a small amount of white precipitate was centrifuged.
The dry weight was 3.97. The supernatant liquid after removing the white precipitate is a transparent pale yellow liquid, and according to bone dry weight measurement, it has a solid content of 4.85%, and the yield is about 97%.
It was hot. Further, the amount of terminal carboxyl groups of this supernatant liquid was determined to be 70 μM/ml by formol titration. After 1 ee of this supernatant liquid was sealed in nitrogen with 1 cc of 36% concentrated hydrochloric acid and hydrolyzed at 110°C for 16 hours, titration of formol was performed, and the amount of terminal carboxyl groups was 55
It was 4 μM/ml. From these results, the papain digest of silk fiproin obtained in this example was a peptide mixture with an average degree of polymerization of 7.9. For comparison, 50V of scouring raw silk was suspended in water 11 and crude papain manufactured by Sigma (Type 1 ~ 2BAEEunits/~) 2
．． A 5V aqueous extract was added and the mixture was reacted for 4 hours at 45°C in a constant temperature bath, but the silk remained in an insoluble state with almost no enzymatic reaction.

BAEEunit here refers to a-N-benzoyl-L-arginine ethyl ester with a pH of 6.2.
, the enzyme activity unit is defined as 1 unit when 1.0 ItM is hydrolyzed for 1 minute at 25°C.

Example 6 5% by weight dialysate of silk fiproin obtained in the same manner as in Example 1 11? Crude trypsin (manufactured by Sigma)
1000-1500BAEEunits/Chymotrypsin activity at W9 500-1000ATEEunits/
) are 0.2 and 1.0, respectively, relative to the weight of silk fiproin.
, 2.0) was added in an amount of 9.0% by weight, and the mixture was reacted at 45° C. for 5 hours with shaking in a constant temperature bath.

The pH of the solution at the start of the reaction was 6.96°, and as the reaction progressed, it gradually decreased to 6.10 after 5 hours. The increase in end groups of each sample was economically measured by formol titration, and the results were as shown in Table 5. For comparison, instead of the 5% dialysate of silk fiproin, finely chopped 50V scouring silk thread was suspended in water in Step 11 and 10.0% was added.
When the above-mentioned enzyme was applied, even after 5 hours, the reaction hardly proceeded and the silk thread remained in an insoluble state.

After terminating the reaction by autoclave heat treatment, each sample (Haruka 1 to 4) was collected by ICC, and the ICC was 36%.
Add concentrated hydrochloric acid ICC at a time and add 11'Cl6 in an ampoule.
After time hydrolysis, colorimetric determination using ninhydrin revealed that the amount of NH2 terminals in each sample was 532.
μM/ml, 550 μM/ml) 553 μM/ml,
It was 586 μM/ml.

From this result, samples 1 to 4 have approximately 14-mer and 12-mer, respectively.
It was estimated to be a decamer, a decamer, and a heptamer. When the above peptide mixtures 1 to 4 were subjected to thin layer chromatography (using silica gel as a carrier in an ammonia gas phase and a phenol:water = 5:1 mixed solution as a developing solvent), most of the amino acids were It was not detected.

Claims (1)

[Claims] 1 Copper-ethylenediamine aqueous solution, copper hydroxide-ammonia aqueous solution, copper hydroxide-alkali-glycerin aqueous solution, lithium bromide aqueous solution, calcium, magnesium, or zinc hydrochloride, nitrate, or thiocyanate aqueous solution, A 0.5 to 20% by weight aqueous silk fibroin solution obtained by dissolving a refined silk raw material in at least one solvent selected from the group consisting of an aqueous sodium thiocyanate solution and dialysis is hydrolyzed with an enzyme, an acid, or an alkali. A method for producing a characteristic silk fibroin peptide aqueous solution. 2. The manufacturing method according to claim 1, wherein the solvent is an aqueous solution of calcium chloride, calcium nitrate, magnesium nitrate, or magnesium chloride. 3. The method of claim 2, wherein the salt concentration in the solvent is at least 15% by weight. 4. The manufacturing method according to claim 1, wherein the concentration of silk fibroin in the silk fibroin aqueous solution is 1 to 15% by weight. 5. The production method according to claim 1, wherein the enzyme is papain, trypsin or chymotrypsin. 6. The manufacturing method according to claim 1, wherein the concentration of acid or alkali is 0.3N or less. 7. The production method according to claim 1, wherein the silk fibroin peptide has an average degree of polymerization of 2 to 20 and an amino acid content of 15% by weight or less. 8. The production method according to claim 1, wherein the silk fibroin peptide has an average degree of polymerization of 4 to 15 and an amino acid content of 10% by weight or less. 9. The production method according to claim 8, wherein the amino acid content is 5% by weight or less.