JPS6012343B2 - Method for isolating cystine or cysteine and tyrosine - Google Patents

Description

[Detailed Description of the Invention] Conventionally, cystine and tyrosine have been produced by hydrolyzing proteins containing large amounts of cystine such as human hair and feathers with mineral acids, and adding an alkali to the decomposition solution to neutralize it and precipitate it. Individual amino acids are isolated after elutriation-soluble amino acid mixtures are separated.

Cystine and tyrosine contained in the elutriophilic amino acid mixture exhibit various behaviors depending on changes in feed. When a chick-soluble amino acid mixture is dissolved in chick acid and adjusted to a pH of 1 to 2 by adding an alkali, only cystine precipitates, and when a chick-soluble amino acid/acid is dissolved in an alkali and an acid is added to adjust the pH to 9 to 10. In this case, only tyrosine precipitates. These properties are also utilized in their industrial manufacturing methods. However, cystine and tyrosine can be extracted by these methods, and only 80 to 85% of cystine and 40 to 50% of tyrosine are extracted based on the content in the elutri-soluble amino acid mixture. It is also influenced by the ratio of cystine to tyrosine in the amine/acid mixture. Especially meat 1~
In the region 2, there is no problem when the ratio of cystine is high, but when the ratio of tyrosine is high, tyrosine is precipitated together with cystine, making separation difficult. According to the present invention, cystine and tyrosine can be separated regardless of their ratio, and in some cases, cysteine, which is a reduced form of cystine, can be obtained.

Cystine and tyrosine both have the lowest solubility among amino acids, with cystine having the lowest solubility of 11.2% in 100% of water at 2% and tyrosine having the lowest solubility of 45% in 100% of water at 25°C. 2 things [it just dissolves].

In order to separate a mixture of the two, it is sufficient to make one of them easily soluble and utilize the solubility difference. Cystine (1) is
As shown in the following formula, cysteine (0) is easily generated during reduction, and cysteine can be easily oxidized and returned to cystine even under weak oxidizing conditions such as air oxidation, or by oxidizing agents such as hydrogen peroxide. .

Also, cysteine is easily soluble in water.

The present inventor took advantage of this property and attempted to separate cystine and tyrosine by the following method.

A mixture of L-cystine and L-tyrosine is dissolved in hydrochloric acid to prepare a catholyte.

A 5% sulfuric acid solution is used as the anolyte, and a platinum electrode is immersed in the catholyte and anolyte using an ion-exchange membrane as a diaphragm, and reduction is carried out through direct current. After confirming that L-cystine was completely converted into L-cystine by thin layer chromatography, the catholyte was prepared by adding aqueous ammonia dropwise.
Adjust H to 5 and collect the precipitated crystals (L-tyrosine) according to the furnace rules.Additionally, hydrogen peroxide solution is added dropwise to the furnace liquid until it no longer emits blue color using a nitrobrucid test solution to oxidize the precipitated crystals. Add hydrochloric acid until dissolved, add aqueous ammonia dropwise to adjust the pH to 2.5, and re-precipitate crystals (L
Cystiso) is separated from the furnace and collected. The results obtained are shown in Table 1. Table 1 L-cystine used in this experiment has specific rotation [Q] color.

-221.70 and content is 99.8% (iodine titration method)
, L-tyrosine with a specific optical rotation [Q] of 0-11.60 and a content of 99.2% (Macrogerdal method) was used, and the results were satisfactory in terms of recovery rate, optical purity, and content. It was confirmed that this is an excellent isolation method. Next, an example in which a protein is actually used will be explained.

Example 1 Six pieces of seaweed-hydrochloric acid are added to 3k9 chicken feathers, and the mixture is heated under reflux for 8 hours for hydrolysis.

Cool the hydrolyzed solution and adjust the pH to 5 with caustic soda solution (20%).
．． 0 and filter the resulting precipitate. 3.5 parts of water and 800 parts of hydrochloric acid are added to the precipitate collected in the oven to dissolve it, and activated carbon is added and filtered for decolorization. While stirring the furnace liquid, add ammonia water dropwise to adjust the pH to 4.5 to 5.5.
Crystals precipitate. The crystals were filtered and dried and the yield was 255 kg.

This crystal mixture contained 63% cystine and 31% tyrosine, and only small amounts of other amino acids. Add 800% of Su-HCl to this crystal mixture and dissolve it to form a catholyte.

In addition, the anolyte is 5% sulfuric acid 900%, and the cation exchange membrane is 0.1 strength as the corneal membrane.A silver plate electrode is immersed in the catholyte, and a carbon plate electrode is immersed in the anolyte. Electrolytic reduction is performed through direct current of IA per ld. After confirming that cystine has been completely reduced to cysteine by thin layer chromatography, the catholyte is taken out. While stirring well, aqueous ammonia is added dropwise to adjust the pH to 5.0, and crystals are precipitated. The crystals were separated in a furnace (the furnace liquid was saved), dissolved in 500 g of aqueous ammonia, activated carbon was added, and the furnace liquid was concentrated under reduced pressure. The precipitated crystals were then dried in a furnace. It was 6th evening.

The crystals are needle-shaped and have a specific rotation [Q] color. -11. Spots o, C
:59.6%, N:7.69%, which corresponded to L-tyrosine.

The recovery rate was 69% based on tyrosine in the crystal mixture. In addition, while cooling the storage solution well, hydrogen peroxide solution was added dropwise until the nitroprusside mysterious solution no longer showed blue color, and then hydrochloric acid 160% was added to the precipitated crystals.
If you add aqueous ammonia and adjust the temperature to 2.5, crystals will precipitate out.

The yield obtained by filtering and drying the precipitated crystals was 157 pieces. This crystal has a specific optical rotation [Q] of 221.9
0, C: 29.7%, N: 11.58%, which corresponded to L cystine. The recovery rate was 97.7% based on cystine in the crystal mixture. Example 2 people hair 3k
9, hydrolysis and pH adjustment were carried out in the same manner as in Example 1 to obtain 346 mixed crystals.

The mixed crystal contains 78% cystine and 1 tyrosine.
It was 7.6%. When this mixed crystal was subjected to electrolytic reduction and crystallization in the same machine as in Example 1, L-tyrosine 43.2
Evening ([Q] effect.

-11-210・C: 59-62%, N: 7-71%)
I got it. The recovery rate is 72% calculated from the crystal mixture. Also, L-cystine is 2629 ([Q] color 0-220.
950, C: 29.73%, N: 11.61%). Recovery rate is 97% based on cystine in the crystal mixture
It is. Example 3 Using human hair 3k9, hydrolysis and pH adjustment were carried out in the same manner as in Example 1.
After adjustment, 330 pieces of mixed crystals were obtained.

The mixed crystal contained 78.6% cystine and 16.9% tyrosine. When this mixed crystal was subjected to electrolytic reduction and crystallization in the same manner as in Example 1 (however, the storage solution was treated separately), L-tyrosine 41.8 hours ([Q] Keiichi 11.3
20, C: 59.52%, N: 7.58%) was obtained.

Claims (1)

[Claims] 1 Dissolve a mixture containing cystine and tyrosine in mineral acid, electrolytically reduce the cystine in the solution to convert it to cysteine, neutralize it by adding an alkali, collect the precipitated tyrosine by filtration, and collect the filtrate. A method for isolating cystine or cysteine and tyrosine, which comprises concentrating to precipitate and separate cysteine, or oxidizing cysteine in the filtrate and recovering it as cystine.