JP2793158B2 - How to separate tyrosine from fibroin - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a method for separating tyrosine from fibroin which is a main component of silk.

[0002]

2. Description of the Related Art Cocoons produced by silkworms contain fibroin, which is a natural protein. Fibroin contains dozens of amino acids and is used in foods, medical supplies, cosmetics and the like. About 12% by weight of tyrosine is present in silk fibroin, which is closely related to the activity regulating function of various enzymes. Specifically, tyrosine is converted into dopa by the action of hydroxylase, and is used to cure patients with Parkinson's disease. The tyrosine residue is phosphorylated at the hydroxyl group by a specific kinase. Phosphorylated tyrosine is deeply involved in cell proliferation and canceration, and is therefore used in pharmaceuticals and biochemical reagents.

[0003]

SUMMARY OF THE INVENTION Fibroin contains glycine and alanine more than tyrosine. Therefore, when tyrosine is used in the medical field, it is more effective to use only tyrosine separated from fibroin as a medicament than to use fibroin itself as a medicament. Further, tyrosine is preferably reduced in molecular weight so as to be easily absorbed in the body.

In order to separate tyrosine from fibroin, a method of hydrolyzing fibroin with a protease and removing substances other than tyrosine from the obtained low-molecular-weight fibroin can be considered. However, this low molecular weight fibroin is not rich in tyrosine. Therefore, tyrosine cannot be efficiently recovered from this low molecular weight fibroin.

[0005] Tyrosine is a required amino acid that promotes the metabolism of the human body and produces adrenaline and melanin.
Since deficiency of tyrosine may cause phenylketonuria, tyrosinosis, and leukoplakia, it is desired that tyrosine be separated from fibroin as much as possible and used effectively as a pharmaceutical.

The present invention has been made to solve the above-mentioned problems, and has as its object to provide a method for separating as much tyrosine as possible from fibroin.

[0007]

Means for Solving the Problems As a result of intensive studies, the present inventors have hydrolyzed fibroin in a fibroin solution, and when the tyrosine is liberated most in the solution, the insoluble matter is removed from the solution. The filtrate (tyrosine solution)
It has been found that by collecting, a large amount of tyrosine can be separated from fibroin, and the present invention has been completed.

That is, the method of the present invention for separating tyrosine from fibroin is characterized in that a fibroin solution obtained by dissolving fibroin in a neutral salt aqueous solution is added to the fibroin solution .
And add 2.0-3.0% by weight of actinase,
Hydrolyze fibroin for 180-300 minutes at ~ 50 ° C
After, it was heated and recovering the tyrosine solution was filtered off insolubles.

[0009] Proteolytic enzymes include actinase. The concentration of actinase is preferably 2.0 to 3.0% by weight, particularly preferably 2.5% by weight, based on fibroin.
If it is less than 2.0% by weight, much tyrosine cannot be recovered from fibroin. If it exceeds 3.0% by weight, tyrosine cannot be obtained in the amount of actinase added even if it is added in a large amount.

[0010] Neutral salts include calcium chloride, lithium bromide, calcium nitrate and magnesium nitrate.
These may be used alone or as a mixture of two or more. Ethanol may be added to the aqueous solution containing a neutral salt.

The pH of the fibroin solution is preferably maintained between 6 and 9, especially 7. When the pH deviates from 6 to 9, the activity of actinase decreases. In particular, when it is less than 4, the activity of actinase is remarkably reduced.

The fibroin solution is at 30 to 50 ° C., especially at
Preferably, it is kept at 7 ° C. When the liquid temperature is lower than 30 ° C., the activity of actinase is too low and fibroin is hardly decomposed. When the temperature exceeds 50 ° C., the activity of actinase decreases rapidly.

[0013] The temperature of the fibroin solution is preferably kept constant for 180 to 300 minutes, especially 240 minutes after the actinase is added. If it is less than 180 minutes, the release of tyrosine has not been completed yet. If the time exceeds 300 minutes, the release of tyrosine has been completely terminated, so that the tyrosine concentration in the fibroin solution does not increase.

[0014]

EXAMPLES Through the following steps, tyrosine can be efficiently separated from fibroin. First, a waste cocoon is immersed in a 0.5% solution of sodium carbonate to obtain a fibroin sample. This fibroin is dissolved in an aqueous solution containing a neutral salt of calcium chloride. After the temperature of the solution reaches room temperature, the solution is dialyzed, and the pH is adjusted to 6 to 9 to obtain a fibroin solution. A proteolytic enzyme such as actinase is added to the fibroin solution. Actinase was added in an amount of 2.0 to 3.0% by weight based on fibroin.
It is. After maintaining the temperature of the fibroin solution at about 30 to 50 ° C. and stirring the solution for 180 to 300 minutes to hydrolyze the fibroin, the solution is boiled to inactivate the enzyme,
Filter immediately to remove insolubles. The filtrate (tyrosine solution) from which insolubles have been removed is concentrated. When crystals precipitate on the liquid surface, the crystals are cooled and the crystals and the tyrosine solution are separated by filtration. Thereafter, the crystals are dried under vacuum to obtain crude tyrosine, and the crude tyrosine is recrystallized to obtain purified tyrosine. The yield of purified tyrosine is determined by the following formula. ｛(Weight of purified tyrosine) / (Weight of fibroin)｝ × 100 = Yield (%) Below, select the optimal conditions (actinase concentration, temperature and pH of fibroin solution, hydrolysis time) to separate tyrosine from fibroin. An example of an experiment actually performed for the purpose will be described.

(Amino acid composition of actinase) A 200-fold amount of 6N hydrochloric acid was added to actinase (manufactured by Kaken Pharmaceutical Co., Ltd.) and mixed. The mixture was sealed under reduced pressure, and then hydrolyzed at 110 ° C. for 48 hours. did. After completion of the hydrolysis, the hydrochloric acid was removed with a rotary evaporator, and the mixture was diluted with a 1000-fold amount of sodium citrate buffer (pH 2.2). The diluted solution was applied to an automatic amino acid analyzer (LC-5A, manufactured by Shimadzu Corporation) to analyze the amino acids of actinase. The weight ratio of the amino acid composition is shown in FIG. Aspartic acid content is the highest at 15%, Thr, Ser, Glu, Ala, Val, Leu, Tyr, Ph
e and the like were present at 5 to 10% by weight, and no remarkable difference was observed.

(Measurement of actinase activity) The activity of actinase greatly changes depending on pH and reaction temperature. Actinase was dissolved in buffers of various pH and left at room temperature for 5 hours, after which the change in activity was measured. The result is shown in FIG.
At pH 6 to 9, the actinase activity is high, but when it is lower than pH 4, the activity is extremely reduced. 30 actinase
FIG. 3 shows the activity when dissolved in distilled water at a temperature of from 100 ° C. to 100 ° C. and left for 10 minutes. Actinase activity is 30-5
It was confirmed that the temperature increased at 0 ° C., but rapidly decreased when the temperature exceeded 50 ° C. 2 and 3, it was found that the activity of actinase was highest when the solution temperature of the fibroin solution was 37 ° C. and the pH was 7.

(Autolysis of Actinase) 1 g of actinase was placed in 100 ml of distilled water in a test tube, and the test tube was placed in a thermostat to keep the temperature of the distilled water at 37 ° C. By measuring the free amino acid concentration, the relationship between actinase autolysis and time was investigated. Decomposition time is 60 minutes, 12
Performed at 0, 240, 300 and 360 minutes.

Free amino acids were not found in the autolysate of actinase. It was found that the amino acids contained in actinase were not decomposed within 360 minutes.

(Preparation of fibroin solution) Ohno Paper Co., Ltd.
Was treated twice with a boiled 0.5% sodium carbonate solution twice for 30 minutes, and then thoroughly washed with running water to obtain a fibroin sample. This was boiled 3
After dissolving in a 0-fold volume of a 50% calcium chloride solution, the mixture was brought to room temperature, and then placed in a dialysis membrane, and dialyzed against running water for 4 days. The dialysate is filtered to remove insolubles to give a fibroin solution,
Its concentration was adjusted to 2% by weight.

(Amount of actinase added and release of tyrosine) Different concentrations (0.5% by weight, 1.0% by weight, 1.5% by weight relative to fibroin) were added to a 2% by weight fibroin solution in a test tube.
Wt%, 2.0 wt%, 2.5 wt%, 3.0 wt%, 3.5 wt%, 4.0 wt%, 4.5 wt%, 5.0 wt%) of actinase. 240 minutes after the test tube was placed in a thermostat and the temperature of the fibroin solution was maintained at 37 ° C., a 20-fold amount of sodium citrate buffer (pH 2.2) was added. Thereafter, insolubles were removed by centrifugation, and amino acid analysis was performed by an automatic amino acid analyzer to measure the tyrosine concentration of each solution. FIG. 4 shows the results.

As shown in the figure, when the actinase concentration is 2.0 to 3.0% by weight, the tyrosine concentration shows a high value. The highest tyrosine concentration occurs when the actinase concentration is 2.5% by weight. It can be seen that when the actinase concentration exceeds 2.5% by weight, the concentration of tyrosine decreases. From this, it was confirmed that the actinase concentration was optimally 2.5% by weight.

(Hydrolysis of Fibroin) 2.5% by weight of actinase based on fibroin was added to a 2% by weight solution of fibroin in a test tube. The test tube was placed in a thermostat to maintain the temperature of the fibroin solution at 37 ° C., and then hydrolysis was performed. Hydrolysis time is 15 minutes, 30 minutes, 45 minutes, 60
Minutes, 120 minutes, 180 minutes, 240 minutes, 300 minutes, and 360 minutes. A 20-fold volume of sodium citrate buffer (pH 2.2) was added to the fibroin solution, and then the insoluble matter was removed by centrifugation. Was measured.
The result is shown in FIG.

As shown in the figure, the hydrolysis time was 18 hours.
At 0 to 300 minutes, the tyrosine concentration is high. The release of tyrosine reached the end point in 240 minutes. Among the constituent amino acids of fibroin, glycine occupying the first and second positions,
Alanine is found to release more slowly than tyrosine.

(Solubility of tyrosine) 100 ° C, 75 ° C, 50 ° C, 2
5 ° C., 5 g of tyrosine is added to 100 g of each distilled water at 0 ° C.
Twenty-four hours later, the amount of tyrosine dissolved in each solution was examined. Table 1 shows the solubility of tyrosine.

[0025]

[Table 1]

The solubility of tyrosine is 0.04 at 25 ° C.
It was 5, but it was 0.565 at 100 ° C.
Therefore, tyrosine can be separated from the fibroin solution by utilizing the difference in tyrosine solubility depending on the temperature.

When the 2% by weight fibroin is subjected to enzymatic hydrolysis, the release of tyrosine is completed after 240 minutes. The tyrosine concentration at that time is about 0.24 g / 100 ml from FIG.
This value corresponds to the solubility at 75 ° C. in Table 1. This suggests that 75 ° C. or higher is preferable for efficient recovery of tyrosine from fibroin. Actually, below 75 ° C., the yield of tyrosine decreased.

From these experiments, the fibroin solution was adjusted to a liquid temperature of 37 ° C. and pH 7, and after 240 minutes from the addition of 2.5% by weight of actinase to the fibroin solution, the fibroin solution was heated, insolubles were filtered off, and tyrosine was removed. When the solution was recovered, it was found that tyrosine could be most efficiently separated from fibroin. Hereinafter, an actual experimental example in which tyrosine is separated from fibroin using actinase will be described.

(Separation of tyrosine from fibroin) A 2% by weight fibroin solution (pH 7) and 2.5% by weight actinase were placed in a test tube, and the test tube was placed in a thermostat at 37 ° C. The test tube was stirred in a thermostat for 240 minutes and then boiled to inactivate the actinase, and immediately thereafter, insoluble matter was removed by filtration. The pH of the filtrate (tyrosine solution) was adjusted to 5.7, the isoelectric point of tyrosine, and the filtrate was concentrated on a rotary evaporator. When crystals were deposited on the liquid surface, the supernatant was quickly cooled, and after 12 hours, the crystals were separated from the supernatant by filtration. The obtained crystal was vacuum-dried to obtain a crude tyrosine product. The crude tyrosine was dissolved in a 1% hydrochloric acid solution, heated to 50 ° C. or higher, filtered quickly, and recrystallized to obtain purified tyrosine. Purified tyrosine yield is 9.5
% By weight.

(Amino Acid Analysis of Tyrosine) Amino acid analysis of crude tyrosine and purified tyrosine was performed in the same manner as in the case of actinase. Table 2 shows the weight ratio of the amino acid composition.

[0031]

[Table 2]

As shown in Table 2, the tyrosine content in the crude tyrosine is about 95% by weight. Crude tyrosine also contains small amounts of alanine, glycine, serine, and phenylalanine. It was confirmed that the purified tyrosine did not contain any amino acids other than tyrosine.

[0033]

As described above, according to the method for separating tyrosine from fibroin of the present invention, the yield of tyrosine was 9.5% by weight. The amino acids constituting fibroin contain only about 12% by weight of tyrosine. Therefore, the method of the present invention which can recover 9.5% by weight of tyrosine is effective as a method for separating tyrosine from fibroin.

[Brief description of the drawings]

FIG. 1 is a diagram showing the weight ratio of the amino acid composition of actinase.

FIG. 2 is a graph showing the relationship between actinase activity and pH.

FIG. 3 is a diagram showing the relationship between actinase activity and temperature.

FIG. 4 is a graph showing the relationship between tyrosine concentration and actinase concentration.

FIG. 5 is a graph showing the relationship between free amino acid concentration and hydrolysis time.

Claims (2)

(57) [Claims] A fibroin is dissolved in an aqueous solution of a neutral salt.
Fibroin solution2.0 for the fibroin
~ 3.0% by weight actinaseAdd, liquid temperature 30-50 ℃
Keep in180-300 minutes,FibroinHydrolyzed
After heating, the insolubles are filtered off to remove the tyrosine solution.
Tyrosine from fibroin characterized by recovery
How to separate. Wherein said neutral salt is calcium chloride, lithium bromide, calcium nitrate, tyrosine from fibroin to claim 1 serial <br/> mounting, characterized in that at least one selected from magnesium nitrate How to separate.