JPH0634735B2 - Method for producing long-chain amino acid linear saturated alkyl esterified protein - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing an amino acid alkyl esterified protein having a linear saturated alkyl group having a carbon chain length of 14 to 24.

An amino acid alkyl esterified protein having an alkyl group with a carbon chain length of 12 or less has a surface-active effect, and a detergent,
It is used as a dispersant, an emulsifier, etc. in the fields of medicines, foods, cosmetics and the like.

Linear saturated chain having a carbon chain length of 14 to 24, which constitutes an amino acid alkyl esterified protein having a linear saturated alkyl group having a carbon chain length of 14 to 24 (hereinafter referred to as amino acid long chain linear saturated alkyl esterified protein) of the present invention The amino acid alkyl ester having an alkyl group (hereinafter referred to as amino acid long-chain linear saturated alkyl ester) is a condensate of a linear saturated alcohol having a carbon chain length of 14 to 24 and an amino acid. Specific examples of the alcohol, tetradecanol, hexadecanol, octadecanol, eicosanol, docosanol, lignoceryl arrucol, and the like, and specific examples of amino acids, phenylalanine, valine, leucine, Alanine, isoleucine, methionine, serine, lysine, tryptophan, glutamic acid, aspartic acid and the like can be mentioned. In addition, proteins include proteins, peptides, derivatives thereof and salts thereof, which are widely obtained from the natural world, in the range thereof.
As an example, soy protein, wheat protein, glutelin, glucagon, collagen, gelatin, elastin, egg white lysozyme, amylase, fibrinogen,
Myosin, enolase, chymotrypsinogen, histone, fish meat protein, avidin, pepsin, globulin,
Casein, succinylated casein and salts thereof.

In order to produce the above-mentioned amino acid long-chain linear saturated alkyl esterified protein of the present invention, an alcohol and an amino acid are first condensed in the presence of hydrochloric acid or P-toluenesulfonic acid to give an amino acid alkyl ester hydrochloride or P-. Toluenesulfonate is obtained. Next, it is conceivable to dissolve the above salt in a small amount of acetone and introduce it into the protein using papain in a carbonate buffer. However, even if this method is used, the hydrochloride or P-toluenesulfonic acid salt thereof does not dissolve in a small amount of acetone when it becomes an amino acid long-chain linear saturated alkyl ester having a carbon chain length of 14 or more. At a certain temperature around 37 ° C, crystallization occurs in the carbonate buffer and the reaction of introduction into the protein does not proceed.

As a result of various studies on this point, the present inventors have found that the above amino acid long-chain linear saturated alkyl ester hydrochloride or P-toluenesulfonic acid salt is neutralized once with an alkaline aqueous solution to release a free amino acid long-chain linear saturated alkyl ester. It was found that if this ester was subsequently used as the ester in the subsequent protein introduction reaction, the above-mentioned problems would not occur and the desired product could be obtained smoothly. This finding was first discovered by the present inventors.

That is, the present invention provides a method for producing an amino acid long-chain linear saturated alkyl esterified protein, which comprises condensing an amino acid long-chain linear saturated alkyl ester with a protein by papain.

Specifically, the carbon chain length is 14
-24 straight chain saturated alcohols and amino acids are boiled under reflux in an organic solvent such as carbon tetrachloride with hydrochloric acid or P-toluenesulfonic acid as a catalyst to form an ester bond. An aqueous solution of alkali, for example, an aqueous solution of sodium carbonate, sodium hydrogen carbonate, sodium hydroxide, potassium hydroxide, potassium carbonate, sodium borate, disodium hydrogen phosphate, dipotassium hydrogen phosphate is added to the obtained reaction product. Neutralize to give free amino acid long chain straight chain saturated alkyl ester.

Next, the amino acid long-chain linear saturated alkyl ester thus obtained is dissolved in a small amount of acetone, mixed with protein in a carbonate buffer, papain is added, and the mixture is reacted at about 37 ° C. At this time, the protein is hydrolyzed, but at the same time,
The amino acid long-chain linear saturated alkyl ester is amide-bonded to the carboxy terminus of the hydrolyzed protein via the amino group to obtain the target amino acid long-chain linear saturated alkyl esterified protein. The molecular weight of the protein and the like can be arbitrarily changed by changing the reaction time of hydrolysis and amide bond.

The papain to be used may be one that is commonly used in industry, and a well-known one that has been conventionally used can be used.

When reacting the above protein with the amino acid long-chain alkyl ester, the reaction is further facilitated by adding a compound having a thiol group such as 2-mercaptoethanol, cysteine, dithiothreitol, dithioerythritol, glutathione, or dimercaprol as an activator. Is.

Next, the present invention will be described in more detail with reference to Examples. Prior to that, the test methods used in each Example are shown below.

(1) Qualitative determination of higher alcohols in amino acid long-chain linear saturated alkyl esterified proteins To 100 mg of sample, 10 ml of 1N sodium hydroxide solution was added, extracted with ether, and alcohol was extracted by gas chromatography (Diasolid ZT, 1 m column). Qualitative quantification was performed.

(2) Qualitative quantification of all amino acids in amino acid long-chain linear saturated alkyl esterified protein 6N hydrochloric acid was added to 10 mg of the sample, and amino acids were analyzed after hydrolysis.

(3) Qualitative quantification of C-terminal amino acid in long-chain linear saturated alkyl esterified protein of amino acid 0.1 μmol of sample was dissolved in 20 ml of 0.2 MN ethylmorpholine-acetic acid buffer (pH 8.5) and 1 nmol of carboxypeptidase was added. Incubate at 37 ℃ for 24 hours. Transfer the reaction solution corresponding to 0.03 μmol protein to a small centrifuge tube and add hydrochloric acid to adjust the pH to 2 to stop the reaction. After centrifuging the precipitate and drying the supernatant
Amino acid analysis was carried out by dissolving the sample in a buffer solution for dissolving an amino acid sample of pH 2.2.

Example 1 10 g of succinylated casein, 15 ml of pH 9 carbonate buffer containing 20 mM 2-mercaptoethanol, and 14 g of methionine tetradecyl ester dissolved in 5 ml of acetone are mixed. This is 100 of papain 1.2 x 10 ^-2 BAPA units
Add mg, react for 15 minutes with stirring, adjust to pH 1 or less with hydrochloric acid to inactivate the enzyme and stop the reaction. Running water dialysis was performed for 3 days, dehydration was performed, and unreacted methionine tetradecyl ester was removed by treatment with acetone, followed by drying to obtain 6 g of methionine tetradecyl esterified succinylated casein.

The results of testing the obtained methionine tetradecyl esterified succinylated casein by the above test method are shown below. As a control, succinylated casein was used.

(1) Tetradecanol amount A peak of tetradecyl alcohol was confirmed at a retention time of 3.32 under gas chromatography conditions with an initial temperature of 120 ° C and a program rate of 5 ° C / min.

Example 1 …… 0.8 mg (sample 100 mg) Control …… 0 (sample 100 mg) (2) Amount of methionine in all amino acids Example 1 …… 7.2 (mol%) Control …… 3.3 (mol%) (3) Analysis of C-terminal amino acids α ₅₁ -casein has a molecular weight of 250 consisting of 199 amino acids.
It is a protein of 00. It is known that when an amino acid alkyl ester is introduced into succinylated α _S1 -casein that has been succinylated using papain, the amino acid alkyl ester is introduced between the 145th phenylalanine and the 146th tyrosine. There is.

As a result of reacting carboxypeptidase with Example 1 by the above-mentioned test method, methionine and phenylalanine were released. The molecular weight of Example 1 was 20,000 (confirmed by SDS electrophoresis).

From the above, it can be seen that Example 1 is a succinylated casein having a methionine tetradecyl ester introduced at the C terminus.

Example 2 10 g of collagen degradation product, valine tetracosyl ester 23
g dissolved in 4 ml of acetone, papain 1.2 × 10 ^-2
0.2 g of BAPA unit was dissolved in 1 mol carbonate buffer and the reaction was carried out for 15 minutes. Then, the same treatment as in Example 1 was carried out to obtain 3.8 g of a valine tetracosyl esterified collagen decomposition product.

The results of testing the obtained valine tetracosyl esterified collagen hydrolyzate by the above test method are shown below.
As a control, a collagen degradation product was used.

(1) Amount of valine in all amino acids Example 2 …… 5.3 (mol%) Control …… 2.2 (mol%) Example 3 100 g of gelatin, 45 g of leucine hexadecyl ester dissolved in 40 ml of acetone, cysteine 20 g, papain 1.2 × 10 ^-2 BAPA unit 1g 1M carbonate buffer 160
After dissolving in ml and reacting for 10 minutes, the same treatment as in Example 1 was carried out to obtain 66 g of leucine hexadecylated gelatin.

The results of testing the obtained leucine hexadecyl esterified gelatin by the above test method are shown below. In addition,
Gelatin was used as a control.

(1) Hexadecanol amount A peak of retention time 4.22 hexadecanol was confirmed under gas chromatography conditions with an initial temperature of 120 ° C and a program rate of 10 ° C / min.

Example 3 …… 0.7 mg (sample 100 mg) Control …… 0 (sample 100 mg) (2) Leucine content in all amino acids Example 3 …… 4.5 (mol%) Control …… 2.5 (mol%) Example 4 Soybean Protein 10g, Lysine palmitoyl ester 20g dissolved in acetone 4ml, dithiothreitol 20mg
Was reacted and treated in the same manner as in Example 1 to obtain 5.6 g of lysine palmitoylated soybean protein.

The results of testing the obtained lysine palmitoyl esterified soybean protein by the above test method are shown below.

As a control, soybean protein was used.

(1) Amount of lysine in all amino acids Example 4 6.8 (mol%) Control ...... 5.3 (mol%) Example 6 L-valine L-valine L-valine 4 g, leucine cis-
5-eicosenyl ester 28g, dimethylcaprol 18
0 mg was reacted and treated in the same manner as in Example 1 to give leucine cis-5-eicosenyl esterified L-valine L-valine L.
-3.0 g of valine was obtained.

Comparative Example 1 10 g succinylated casein and 2-mercaptoethanol
15 ml of pH 9 carbonate buffer containing 20 mM and 14 g of methionine tetradecyl ester hydrochloride dissolved in 5 ml of acetone were mixed.

After mixing, crystals of methionine tetradecyl ester hydrochloride precipitated and the dissolution did not progress thereafter. After removing the crystals, add 100 mg of papain 1.2 × 10 ^-2 BAPA units, react for 15 minutes while stirring, and adjust to pH 1 or less with hydrochloric acid. Then the enzyme was inactivated to stop the reaction. A mixture containing methionine tetradecyl ester succinylated casein was dried by running water dialysis for 3 days, removing unreacted methionine tetradecyl ester hydrochloride by treating with acetone, and drying.
g was obtained.

The results of testing the obtained methionine tetradecyl esterified succinylated casein by the above test method are shown below.

(1) Tetradecanol amount A peak of tetradecyl alcohol was confirmed at a retention time of 3.32 under gas chromatography conditions with an initial temperature of 120 ° C and a program rate of 5 ° C / min.

Comparative Example 1 ... 0.05 mg (100 mg of sample) (2) Amount of methionine in all amino acids Comparative Example 1 ... 0.6 (mol%) (3) Analysis of C-terminal amino acid In Comparative Example 1, carboxypeptidase was described above. As a result of the reaction by the test method of 1., methionine and phenylalanine were released. The molecular weight of Comparative Example 1 was 21,000 (confirmed by SDS electrophoresis).

From Comparative Example 1, when the hydrochloride of an amino acid alkyl ester having a linear saturated alkyl group having a carbon chain length of 14 to 24 is subjected to an enzymatic reaction as it is without removing the salt, the amino acid alkyl ester is not introduced into the protein. It was found that the compound could not be obtained efficiently.

 ─────────────────────────────────────────────────── ─── Continuation of front page (72) Kenichi Tomita 1050 Shinba-cho, Kohoku-ku, Yokohama City, Kanagawa Prefecture Hiroshi Ueno, Examiner, Shiseido Research Institute Co., Ltd. (56) Reference JP-A-54-163894 (JP, A)

Claims (1)

[Claims] 1. A carbon chain length 14-24 produced by neutralizing an amino acid alkyl ester hydrochloride having a straight chain saturated alkyl group having a carbon chain length 14-24 and / or P-toluenesulfonate with an alkaline aqueous solution. A method for producing an amino acid long-chain linear saturated alkyl esterified protein, comprising condensing an amino acid alkyl ester having a linear saturated alkyl group with a protein with papain.