JPS58188846A - Separation of cystine or cysteine and tyrosine - Google Patents

Abstract

PURPOSE:To separate the titled substances from a mixture containing cystine and tyrosine, by dissolving the mixture in a mineral acid, converting the cystine in the solution to cysteine by reduction, neutralizing the solution with an alkali, separating the precipitated tyrosine by filtration, and concentrating the filtrate. CONSTITUTION:A mixture of L-cystine and L-tyrosine is dissolved in hydrochloric acid and the solution is used as the catholyte. The anolyte is 5% sulfuric acid solution, and a cation exchange membrane is used as the membrane. The catholyte is reduced by dipping platinum electrodes in the catholyte and the anolyte, and passing DC current through the membrane. After ascertaining the complete reduction of L-cystine to L-cysteine by thin-layer chromatography, the catholyte is adjusted to pH 5 by dripping ammonia water, and the precipitated crystal (L-tyrosine) is recovered by filtration. The filtrate is oxidized by adding hydrogen peroxide solution dropwise to the filtrate until the filtrate does not develop blue color by nitroprusside reagent. The oxidized filtrate is mixed with hydrochloric acid to dissolve the precipitated crystal, added ammonia water dropwise to attain 2.5pH, and the reprecipitated crystal (L-cystine) is recovered by filtration.

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 then neutralizing the decomposition solution by adding an alkali. The precipitated poorly soluble amino acid mixture is separated in a furnace and then the individual amino acids are isolated. Cystine and tyrodine contained in the poorly soluble amino acid mixture exhibit various behaviors depending on the change in -. When a poorly soluble amino acid mixture is dissolved in mineral acid and adjusted to -1 to 2 by adding alkali, only cystine precipitates,
In addition, by dissolving poorly soluble amino acids in alkali and adding acid,
When adjusting to 9 to 10, only tyrosine is precipitated. These properties are also utilized in their industrial manufacturing methods.

However, cystine and tyro-zine that can be extracted by these methods are 80 to 859 c for cystine and 40 to 5 for tyrodane, relative to the content in the sparingly soluble amino acid mixture.
Only 0% is extracted.

The ratio of cystine to tyrosine in the poorly soluble amino acid mixture also has an effect. Especially in the area of -1 to 2,
If the ratio of cystine is high, there is no problem, but if the ratio of tyrosine is high, tyrosine will precipitate along with cystine, making separation difficult.

According to the present invention, cystine and tyrosine can be separated regardless of their ratio, and in some cases, cystine, which is a reduced form of cystine, can be obtained.

Cystine and tyrodane both have the lowest solubility among the amino acids, with cystine having the lowest solubility in 1 ooWd of water at 25°C. Tyrosine dissolves in only 45.319 ml of water at 25°C.

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) easily generates cystine (1) when reduced, as shown in the following formula, and cystine is easily oxidized even under weak oxidizing conditions such as air oxidation or by oxidizing agents such as hydrogen peroxide. Can be converted back to cystine.

(1) (1) 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 form a catholyte. A 5% sulfuric acid solution was used as the anolyte, and a platinum electrode was immersed in the catholyte and anolyte using a cation exchange membrane as a diaphragm, and the reduction was carried out through direct current. The catholyte is
By thin layer chromatography, shim-cystine was completely converted to L-
After confirming that it has been reduced to cysteine, aqueous ammonia is added dropwise to adjust the - value to 5, and the precipitated crystals (L-thyrodine) are collected from house to house. Further, the filtrate is oxidized by adding hydrogen peroxide solution dropwise to the nitroprusside test solution until it no longer emits blue color. Hydrochloric acid is added until the precipitated crystals are dissolved, aqueous ammonia is added dropwise to adjust the - to 2.5, and the precipitated crystals (L-cystine) are separated and recovered again. The results obtained are shown in Table 1.

7−″ 7≦−−−−−−□−−−−−−□−−−−−−−−
−−−−−−−−−−1 Table 1 L-cystine used in this experiment has a specific optical rotation [α-2
2t, 7' and th 99.8 N ("1 urination titration method), L-tyrosine/ is the specific optical rotation [α]!0-11.
6° and a content of 99.2% (macrogelmer method) was used, and the recovery rate, optical purity, and content were all satisfactory, confirming that it is an excellent isolation method. Ta.

Next, an example in which protein is actually used will be described.

Example 1 6 tons of 8N hydrochloric acid is added to chicken feathers 3-, and the mixture is heated under reflux for 8 hours for hydrolysis.

The hydrolyzate was cooled and diluted with caustic soda solution (20%) for -5.
Adjust to 0 and treat the resulting precipitate with F. To the precipitate collected in the oven, 3.5 g of water and ROO- of hydrochloric acid are added to dissolve, and activated carbon is added to decolorize the precipitate by filtration. While stirring the filtrate K, aqueous ammonia is added dropwise to adjust the - to 4.5 to 5.5, and crystals are precipitated.

If the crystals are dried under F treatment, the yield will be 255? Met. Cystinke 63511 contained in this crystal mixture
i' and thyronone were 31eX, and other amino acids were present in very small amounts.

3N hydrochloric acid (800%) is added to this crystal mixture to dissolve it and prepare a catholyte. For the anolyte, 5X sulfuric acid (900%) was used, a cation exchange membrane of 0.1 m' was used as a diaphragm, an iron plate electrode was immersed in the catholyte, and a carbon plate electrode was immersed in the anolyte. Electrolytic reduction is performed through the direct current of this IA. After confirming that cystine has been completely reduced to cysteine by thin layer chromatography, the catholyte is taken out, and aqueous ammonia is added dropwise to the catholyte while stirring well, and crystals are precipitated.

The crystals were distributed door to door (the P solution was saved), and 500% of ammonia water was added.
When the crystals precipitated were filtered and dried, the yield of 7 was 54.61.

The crystal is needle-shaped and has a specific optical rotation [α)-'-xx, 3s°, C
: 59.6%, N-nia, 69%, L-tyrosi'
matched. The recovery rate is 69% based on the tyrodane in the crystal mixture.

In addition, while stirring and cooling the stored solution F well, hydrogen peroxide solution was added dropwise to the nitroprusside test solution until it no longer exhibited a blue color, and then 160-hydrochloric acid was added to the precipitated crystals to dissolve them, and activated carbon was added. Aqueous ammonia is added dropwise to the decolorized filtrate while stirring well to adjust the concentration to 2.5, and crystals are precipitated. The yield C obtained by filtering and drying the precipitated crystals was 157 f. This crystal has a specific optical rotation [α] of 0~
221.9°, C: 29.7%, N: 11.58. %
', which corresponded to L-cystine. The recovery rate was 97.7 N in terms of cystine in the crystal mixture.

Example 2 Human hair 3F4 was hydrolyzed and adjusted in the same manner as in Example 1 to obtain mixed crystals 346v. The mixed crystal contained 78% cystine and 17.6% tyrosine.

When this mixed crystal is subjected to electrolytic reduction and crystallization in the same manner as in Example 1, L-tyrodine 43.2r([α)--1
1,21°, C: 59.62%, Nia, 71 kidneys) were obtained. The recovery rate is 72% calculated from the crystal mixture. In addition, L-cystine is 262 F ((Saki 0-220, 95
°, C: 29.73%, N: 11.61%). The recovery rate is 97% based on cystine in the crystal mixture.

Example 3 Human hair 3 Kf was hydrolyzed and -
After adjustment, 330v of mixed crystals were obtained. Cystine contained in mixed crystals is 78.6%, Chiro-horn? i16.
It was 9%.

This mixed crystal was subjected to electrolytic reduction and crystallization (
However, the stored r solution should be treated separately), then 6. L-chih scene 4! , 8F (cαl, -11,32°1 c:59
, 52%, N near, 58N) was obtained. The recovery rate is 75π in terms of tyrosine in the crystal mixture.

The stored ν liquid is concentrated under reduced pressure in a nitrogen stream to precipitate crystals, cooled by adding methanol, and separated in a furnace.

Dissolve the six crystals in water, add activated carbon, filter,
The liquid is concentrated under reduced pressure in a nitrogen stream to precipitate crystals. After cooling, the crystals were separated in an oven, washed with methanol, and dried under reduced pressure at 50C to obtain 2.2v. This needle-like crystal)
Specific optical rotation [α) 20+s, 9g°, C: 29.82%,
N: 11.68%, the nitroglucide reaction was positive and matched to cysteine.

The content determined by iodometric titration was 99.8X.

The recovery rate is 81.9 in terms of cystine in the crystal mixture.
%.

Claims (1)

[Claims] A mixture containing cystine and tyrodine is dissolved in mineral acid,
After reducing cystine in the solution and converting it to cysteine, neutralize it by adding an alkali, collect the precipitated tyrosine in a separate furnace, concentrate the ν solution and separate the cysteine, or A method for isolating cystine or cystine and tyrodine, which comprises oxidizing cystine and recovering it as cystine.