KR850000717B1 - Process for recovering amino acids from protein hydrolystes - Google Patents

Abstract

Phenylalanine and tyrosine were isolated from protein hydrolysates. Thus, salts-HCl, such as leucine-HCl, isoleucineHCl, phenylalanine- HCl and tyrosine-HCl, were extracted with a H2O-insol. alc. (esp. BuOH or iso-BuOH), concentrated and passed through a weakly, then a strongly, basic anion exchanger, eluting phenylalanine.

Description

Recovery of amino acids from proteolytic products

The present invention relates to a method for recovering amino acids from proteolytic products. To recover amino acids from vegetable or animal proteins, the proteins must first be broken down into their composition, ie the respective amino acids. Acid hydrolysis methods are known in which long-term heating of proteins with hydrochloric acid or sulfuric acid is known. Methods of hydrolyzing proteins with alkali, enzymes, hydrolysis methods and the like are known.

In addition, a variety of special precipitation methods using aromatic sulfonic acid, picric acid, mercury, and copper salts have been proposed for recovering amino acids from proteolytic products.However, these methods must recover the precipitation reagents, It is very complicated because you have to use. In addition, in recent years, a method of separating amino acids by distillation of ethyl esters is rarely used. By using an ion exchanger, amino acids can be separated very effectively. However, this method requires a large separation column, dilutes the solution in a large amount, and contaminates the amino acid by the release agent used for elution.

In order to recover amino acids from proteolytic products, the present inventors have found a variety of methods to find new methods that are simpler and can yield higher yields of products at lower cost, that is, on industrial scale. As a result of repeated studies, the present invention has been achieved. The method according to the invention is characterized in that, after neutralizing the hydrolysis product, fractions with different compositions are obtained as differential agents by fractional filtration.

The inventors have discovered that poorly soluble amino acids are crystallized at different rates upon neutralization of the proteolytic product. That is, leucine is abundantly contained in the first fraction which is precipitated immediately after neutralization, and tyrosine and cystine are abundantly contained in the second fraction obtained after the crystallization and at least two days have elapsed. It is a feature of the present invention to separate these poorly soluble amino acids into at least two fractions.

The raw materials used to carry out the process according to the invention include a variety of protein sources rich in tyrosine, leucine, isoleucine and phenylalanine, such as desugared molasses, cereals, corn embryos, ink microorganisms, in particular It can be obtained from yeast or casein. However, if desired, it is desirable to remove the ballast material from these protein sources and then obtain it by acid hydrolysis.

Preferred steps and the like in the separation method according to the present invention will be described in detail below.

Separation of cystine and tyrosine: Since cystine and tyrosine are bipolar in water vapor and extremely poorly soluble in water, it is difficult to separate these amino acids. The inventors have discovered that the solubility of cystine increases more rapidly than the solubility of tyrosine within a certain pH range when changing from dipolar to anionic form. That is, in solution solution having a pH value of 7.0, tyrosine is dissolved 0.04% and cystine is 0.02%, and in the solution having a pH value of 9.2, 0.04% of tyrosine and 1.0% of cystine are dissolved. Thus, using these amino acid tyrosine and cystine solubility changes within a fairly narrow pH range of 9.0 to 10.0, these two amino acids can be separated. That is, one recoverable method of L-tyrosine is to dissolve tyrosine-rich fractions at pH values of 10 to 11, and to separate the tyrosine precipitated at pH 8.5 to 10, preferably 9.5 by filtering the resulting solution. It is then characterized by purification by recrystallization.

Separation of leucine, isoleucine, methionine and tyrosine: The two isomers, leucine and isoleucine amino acids, have very similar physical properties, making it difficult to separate them.

The inventors have discovered that the solubility of isoleucine increases faster than the solubility of leucine within a certain pH range when changing from dipolar to cationic. That is, in saturated sodium chloride solution, leucine is 0.4% and isoleucine is 0.5% dissolved in a solution having a pH value of 6.0, and leucine is dissolved 1.1% and isoleucine is 1.7% in a solution having a pH value of 1.5. Therefore, by adjusting these solutions to a specific isoleucine concentration, leucine can be precipitated within a pH range of 1.0 to 2.0. However, since these amino acids are coprecipitated with leucine to produce a drug effect containing methionine, it is preferable to oxidize the methionine and convert it into soluble methionine sulfoxide before leucine is precipitated.

That is, one recoverable method of L-leucine is to dissolve leucine rich fractions in acid, add oxidant to oxidize methionine, and then adjust the total amount of isoleucine in the solution to the desired range of 1.0 to 1.5%. And, crude leucine is precipitated within a pH range of 1.0 to 2.0.

In addition, the exact pH value within the range of pH 1.0 to 2.0 can be selected according to the degree of tyrosine content in the fraction rich in leucine, and by repeating the precipitation method within the same pH range, the leucine can be purified better. have.

Extraction of Isoleucine, Phenylalanine, Tyrosine and Luucine: The mother liquor from which leucine is precipitated contains residues of the amino acids isoleucine, phenylalanine, tyrosine and leucine, and the present inventors do not mix hydrochloric acid addition salts of these amino acids with water. The use of non-alcohols, especially isobutanol or butanol, has been found to be able to extract from sodium chloride-containing solutions in extremely good yields, and to extract amino acids and amino acid hydrochloric acid salts from aqueous solutions using butanol or isobutanol. Methods are known. However, since the partition coefficient of amino acid or amino acid hydrochloric acid addition salt in a butanol-water system is 1.0 or less, the yield obtained by the said extraction method is obtained. These partition coefficients can be increased to 10 or more by saturating the solution with sodium chloride.

That is, one extractable method of leucine, isoleucine, phenyl-alanine and tyrosine extracts hydrochloric acid addition solutions of these amino acids saturated with sodium chloride with alcohols, especially isobutanol or butanol, which are not miscible with water. It features.

The valine and tyrosine content in the alcohol extract can be further reduced by washing the extract with water, and washing with water can be performed directly at the head of the extraction tower or directly in the separation unit. After addition of water, then azeotropic distillation can be carried out to remove alcohol from the alcohol extract, which can then be recovered to the extraction tower. Hydrochloric acid can be removed from the aqueous solution of amino acid hydrochloric acid using a weak basic anion exchanger, and the neutral solution can be concentrated to obtain a mixture of leucine, isoleucine, phenyl-alanine and tyrosine.

Recovery of Phenyl-Alanine: Typically, phenyl-alanine is recovered with tyrosine from acid solutions by absorption on activated carbon. This method has several drawbacks. That is, activated carbon must be activated by dissipation treatment, and phenyl-alanine must be eluted with an organic solvent such as pyridine or aniline because very little phenyl-alanine is absorbed compared to the volume of activated carbon.

The present inventors treated the amino acid solution containing no neutral salt with a strong basic anion exchanger to absorb methyl-alanine by absorbing phenyl-alanine per unit volume five times as compared with the method of absorbing on activated carbon. I found out that you can.

That is, one separable method of phenyl-alanine and tyrosine is characterized by passing a neutral solution containing these amino acid hydrochloric acid addition salts through weakly basic and strongly basic anion exchangers. In addition, the inventors have found that phenyl-alanine and tyrosine can be completely recovered from the ion exchanger by simply eluting the strong basic ion exchanger with a hydrochloric acid solution. In the case of regeneration using 4% sodium hydroxide solution, only a part of fe-nilalanine and tyrosine were recovered.

The anion exchanger eluate contains phenyl-alanine hydrochloric acid addition salts and tyrosine. According to the solubility in 6N HCl, phenyl-alanine hydrochloric acid addition salts are poorly soluble at low temperatures, and when heated, solubility is increased. It is more soluble than hydrochloric acid addition salts. That is, the hydrochloric acid content in the eluate is adjusted to 20 to 25%, the sodium chloride is separated at 70 to 90 ^° C., and then cooled, whereby phenyl-alanine hydrochloric acid addition salt can be crystallized. Next, phenyl-alanine hydrochloric acid addition salt is purified by recrystallization, and hydrochloric acid can be recovered by a weakly basic anion exchanger. Finally, the neutral solution can be concentrated by evaporation to obtain pure L-phenyl-alanine.

Example 1

A mixture of amino acids rich in leucine content determined immediately after the acid protein hydrolyzate was neutralized was filtered off, and more than 3 days after the crystallization, a second fraction of poorly soluble amino acid was separated. This amino acid mixture was dissolved in dilute sodium hydroxide at pH 10, and water was added to form a cystine concentration of 1.0 to 1.5%. The solution was filtered to precipitate and crystallize tyrosine at pH9.5, and after 24 hours, the crude tyrosine was filtered off, washed with water, dissolved in acid and heated to precipitate at pH 1.5 to 1.8. As a result, pure tyrosine crystals were obtained under the aforementioned precipitation conditions.

The acid was then added to the crude tyrosine filtrate to adjust the pH to 7.0 to 2.0, preferably 5.0, and after 24 hours the crude crude cystine was separated. This crude cystine was dissolved in an acid, activated carbon was added to absorb xantyrosine and filtered. Cystine was precipitated at pH 2.0 and separated.

Example 2

After neutralizing the protein hydrolysis products, the precipitated poorly soluble amino acids were separated into first fractions rich in leucine. This amino acid mixture was then dissolved at pH 0.5 and water was added to adjust the concentration of isoleucine to about 1.5%. Next, the methionine content was measured, and equimolar hydrogen peroxide was added thereto at 90 ^° C. for oxidation. Next, activated carbon was added to the solution, followed by filtration. The crude leucine was separated after cooling by adjusting the pH to 1.0 to 1.5 with diluted sodium hydroxide. Next, this crude leucine was dissolved again at pH 0.5 and precipitated at pH 1.0 to 2.0 to further purify. This operation was repeated until the purity of L-leucine was reached at the destination. The pH value in the above operation was selected according to the tyrosine content in the solution. That is, when the tyrosine content in the solution was 0.65, 0.8, 1.0, 1.2, 1.4, 1.6 and 1.8%, the pH value was 1.0, 1.65, 1.52, 1.42, 1.33, 1.26 and 1.16 were selected by Ga.

Example 3

Hydrochloric acid was added to the filtrate from which leucine was precipitated to adjust the pH to 0.5, and, if necessary, saturated with sodium chloride. Next, extraction was carried out by carrying out countercurrent contact between the amino acid solution and isobutanol in the extraction column such that the ratio of isobutanol to amino acid solution was 1: 1 or 1: 2. In this operation, about 90% of the amino acids leucine isoleucine and phenyl-alanine were extracted. This isobutanol extract was then washed with water to further reduce valine and tyrosine content. This washing operation was carried out directly at the top of the extraction tower. Next, water was added thereto, followed by azeotropic distillation to distill the isobutanol from the isobutanol extract and recover the extract column. Subsequently, hydrochloric acid was removed from the aqueous solution of amino acid hydrochloric acid addition salt using a weakly basic anion exchanger, and the neutral solution was concentrated to give a mixture of leucine, isoleucine and phenyl-alanine.

Example 4

The hydrochloric acid was separated by passing an amino acid hydrochloric acid addition salt mixture of leucine, isoleucine, and phenyl-alanine obtained by the extraction operation with isobutanol through a weakly basic anion exchanger. This neutral amino acid mixture was then passed through a hydroxide type strong base anion exchanger to obtain a pure solution of liquid leucine and isoleucine released from the tower. Next, pure isoleucine was obtained from the solution by the usual method of extracting the copper complex compound with methyl alcohol.

Example 5

One layer of the anion exchanger column packed with phenyl-alanine and tyrosine was regenerated with 10% hydrochloric acid, and then with 4% aqueous sodium hydroxide solution. The eluate with hydrochloric acid was collected and concentrated, and then concentrated hydrochloric acid was added thereto. The acid concentration was adjusted to 20% or more by the addition of sodium chloride, and the sodium chloride present was separated at a temperature of 70-90 ^° C. After cooling the solution, the phenyl-alanine hydrochloric acid addition salt was crystallized in 6N HCl to purify. Then, in order to recover the free phenyl-alanine, the phenyl-alanine hydrochloric acid addition salt was dissolved in water, the hydrochloric acid was removed by a weakly basic anion exchanger, and the neutral solution was concentrated to give pure L-phenyl-alanine. It became.

Claims (1)

A hydrochloric acid addition salt solution of amino acids such as leucine, isoleucine, phenylalanine and tyrosine saturated with sodium chloride is extracted with an alcohol that is not miscible with water; A method for the recovery of phenyl-alanine and tyrosine, characterized by passing through weakly basic and infectious anion exchange groups.