JPS6058094A - Production of dipeptide - Google Patents

Abstract

PURPOSE:To produce a dipeptide in high yield, by reacting an amino acid derivative in the presence of an immobilized enzyme obtained by immobilizing a proteinase to water-dispersible polymer particle having specific diameter through a covalent bond. CONSTITUTION:The first amino acid or peptide having protected amino group is reacted in an aqueous medium in the presence of an immobilized enzyme obtained by immobilizing a proteinase to a water-dispersible polymer paticle having an average particle diameter of 0.03-2mum through a covalent bond. The produced dipeptide is dissolved in an organic solvent, and the dipeptide is separated and recovered while separating the immobilized enzyme as the solid phase. The water-dispersible polymer is preferably a coplymer of a vinyl monomer such as styrene, styrene derivative, (meth)acrylic acid ester, etc. with a monomer having reactive functional group such as (meth)acrylic acid, and a crosslinking monomer such as divinylbenzene.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing dipeptides, and more specifically, the present invention relates to a method for producing dipeptides, and more specifically, the present invention relates to a method for producing dipeptides, and specifically, the presence of an immobilized protease in which a protease is immobilized on water-dispersed polymer particles having an average particle size of 0.03 to 2 μm. Amino r below! This invention relates to a method for producing dipeptides by peptide bonding I derivatives.

It has been known for a long time that proteolytic enzymes such as papain, chymotrypsin, thermolysin, etc. have a catalytic effect on the reverse reaction, which is the formation reaction of peptide bonds. Recently, after peptide bonding of amino acid derivatives in the presence of such proteolytic enzymes in an aqueous medium,
Methods for separating and recovering proteolytic enzymes from reaction mixtures have been proposed (Japanese Patent Publication Nos. 13267-13267, 13268, and 13268), but separation operations for dipeptides and proteolytic enzymes using organic solvents have been proposed. In this method, the deactivation of the proteolytic enzyme is significant and it is difficult to completely separate and recover the proteolytic enzyme, so that dipeptide production cannot be carried out stably over a long period of time.

In addition, production costs increase due to activation and attrition during recovery of the proteolytic enzyme.

The present inventors conducted intensive research to solve the above-mentioned problems in the production of dipeptides using Kumbak degrading enzyme, and found that a proteolytic enzyme was added to water-dispersed polymer particles with an average particle size of 0.03 to 2 μm. By reacting an amino acid derivative in the presence of an immobilized enzyme that has been covalently immobilized, the desired dipeptide can be obtained in high yield, and the immobilized proteolytic enzyme is resistant to organic solvents. It is stable even when the dipeptide produced is dissolved in an organic solvent and the immobilized protease is separated and recovered as a solid phase, and the enzyme activity after recovery is substantially the same as that at the beginning. This discovery led to the present invention.

In the method for producing dipeptides according to the present invention, a proteolytic enzyme is covalently immobilized on water-dispersed polymer particles having an average particle diameter of 0.03 to 2 μm. a step of reacting a first amino acid or peptide with a protected group and a second amino acid or peptide with a protected carboxyl group in an aqueous medium and dissolving the resulting dipeptide in an organic solvent; It is characterized by comprising the steps of separating and recovering the dipeptide from the inside and also separating and recovering the above-mentioned immobilized enzyme as a solid phase.

In the present invention, some of the water-dispersed polymer particles used as carriers for immobilized enzymes are, for example,
This is already known from Publication No. 0380 and the like. In the present invention, a first vinyl monomer such as styrene or its derivative, (meth)acrylic acid ester, (meth)acrylonitrile, or a mixture thereof; (meth)acrylic acid, (
It is possible to use water-dispersed polymer particles obtained by emulsion copolymerization with a second vinyl monomer having a reactive functional group such as glycidyl meth)acrylate or (meth)acrylamide, or a mixture thereof. However, preferably, in addition to the above monomers, a water-dispersed polymer obtained by copolymerizing a polyfunctional internal crosslinking monomer such as divinylbenzene, poly(meth)acrylate of a polyhydric alcohol, etc. As described above, polymer particles are preferably used because they do not swell and dissolve in the organic solvent used when separating and recovering the dipeptide after the enzymatic reaction. Note that it is preferable not to use an emulsifier in this emulsion copolymerization. This is because when an emulsifier is present, there is a risk that the emulsifier will be mixed into the resulting water-dispersed polymer particles and deactivate the enzyme during enzyme immobilization. However, if there is no such risk, it goes without saying that emulsion copolymerization may be carried out in the presence of an emulsifier.

The average particle diameter of water-dispersed polymer particles is 0°03~2
μm, especially 0.07 to 1 μm
It is preferable that The average particle diameter of the polymer particles is 0.0
When it is smaller than 3 μm, it becomes difficult to separate the immobilized enzyme from the substrate and reaction products after dispersing the immobilized enzyme in the reaction system and carrying out the dipeptide production reaction using this carrier as a carrier. , on the other hand, the average particle size is 2μ
When the particle size exceeds m, it becomes difficult to disperse the particles in the reaction system, the surface area per unit volume of the particles is small, the enzyme activity of the immobilized enzyme is relatively reduced, and the reaction efficiency is deteriorated.

The protease used in the method of the present invention is not particularly limited as long as it can be used for peptide synthesis, but examples include acidic proteases such as pepsin, papain,
Thiol proteases such as stem bromelain, ficin, canapsin B1 chymopapain, and streptococcal protease, neutral proteases of actinomycete origin, sptilisin, Aspergillus alkaline protease, elastase, α-lytect protease, chymotrypsin, and serine proteases such as chymotrypsin C. etc. can be mentioned.

Such proteolytic enzymes are required to be covalently immobilized on the water-dispersed polymer particles. Immobilized enzymes in which proteolytic enzymes are immobilized by other methods tend to be easily desorbed during separation operations of generated dipeptides that involve the use of organic solvents, as described above, and the immobilized enzymes lack stability. be.

The first amino acid or peptide with a protected amino group used in the present invention is not particularly limited, but may be a protecting group conventionally used in peptide synthesis, such as a benzyloxycarbonyl group or a tert-butyloxycarbonyl group. Amino acids with protected amino groups such as glycine, alanine, valine, leucine, isoleucine, phenylalanine, methionine, cystine, serine, threonine, aspartic acid, glutamic acid, lysine, arginine, bistidine, proline, oxyproline, tyrosine, tryptophan, etc.; Mention may be made of peptides. Similarly, the second amino acid or peptide protected with a carboxyl group is not particularly limited, but for example, a lower alkoxy group, benzyloxy group, benzhydryloxy group, anilino group, amino group, etc. Examples include the above-mentioned amino acids or peptides with protected .

The concentrations of the first and second amino acids or their peptides in the reaction system are not particularly limited, but usually both are about 0.001 to 7M, and the molar ratio is 1:
The reaction ratio is 5 to 5:1, preferably 1:3 to 3:1.

In the method of the present invention, the substrate can be dissolved in an aqueous medium, water-dispersed polymer particles having immobilized proteolytic enzymes can be dispersed, and the reaction can be carried out under stirring. In the present invention, the aqueous medium includes water or a mixture of water and an organic solvent capable of dissolving the reaction product dipeptide. When water is used as the aqueous medium, dipeptides, which are reaction products, generally have low solubility in water and are precipitated in the medium. However, when the aqueous medium contains an amount of organic solvent sufficient to dissolve the reaction product dipeptide, the resulting dipeptide will dissolve in the organic solvent. In order to easily separate the dipeptide from the immobilized Kumbaku-degrading enzyme after the reaction, it is preferable to use a water-immiscible organic solvent as the organic solvent. It is also possible to use an aqueous solution as the reaction medium using a specific organic solvent.

In the method of the present invention, the amount of immobilized enzyme to be used relative to the substrate is not particularly limited, but in terms of enzyme amount, it is usually in the range of 0.1 to 30% by weight of the substrate. In addition, the pH range in which the reaction is carried out is the pH range in which the enzyme used exhibits activity; for example, in the case of metalloprotease, pH 4 to
It is 9. The reaction temperature is also within a range in which the enzyme retains its activity, usually from 20 to 50°C, and the time required for the reaction is usually about 0.5 to 24 hours under the above conditions.

After the reaction is completed, as mentioned above, if the dipeptide that is the reaction product is not dissolved in the reaction medium, the dipeptide will precipitate in the medium, so the reaction product and unreacted substrate should be added to an appropriate organic solvent with f4M. For example, the immobilized enzyme can be easily separated by means such as membrane separation using a microfiltration membrane or ultrafiltration membrane, or centrifugation, and the reaction product can be separated from the organic phase containing it. Alternatively, if the reaction medium dissolves the reaction product, the immobilized enzyme can be immediately separated from the reaction mixture by the above-described means. According to the method of the present invention, the immobilized enzyme thus separated retains substantially the same enzyme activity as the initial enzyme, and is suitable for use, for example, in continuous reactions.

When the reaction medium does not dissolve the reaction product, such as water, to separate the immobilized enzyme and the reaction product after the reaction is completed,
Specifically, for example, if a water-miscible organic solvent is added to the reaction mixture and the reaction product is dissolved in the aqueous organic solvent produced by stirring, the immobilized enzyme can be easily separated as a solid phase. .

Examples of the water-miscible organic solvent include methanol,
In addition to lower alcohols such as ethanol and propatool,
Acetone, tetrahydrofuran, dimethylsulfoxide, dimethylformamide, dimethylacetamide, etc. are used. Alternatively, if a water-immiscible organic solvent is added to the reaction mixture and stirred, the reaction product is dissolved in the organic solvent and extracted, and then separated into two layers, an organic layer and an aqueous layer. The reaction product can be obtained from Examples of such water-immiscible organic solvents include chloroform, lower alkyl halides such as ethylene dichloride, ethyl acetate,
Lower aliphatic esters such as isopropyl acetate, benzene,
Aromatic hydrocarbons such as toluene and xylene are used.

Furthermore, as another method, after the completion of the reaction, the reaction product and the immobilized enzyme are separated as a solid phase, and then an organic solvent is added thereto to dissolve the reaction product, so that the immobilized enzyme can be separated by the membrane separation, etc. It is possible to separate and recover the organic layer, and also obtain the reaction product from the organic layer. In order to separate the reaction product from the organic layer, conventional methods known in the art, such as concentration crystallization and extraction, can be used.

As described above, according to the method of the present invention, since water-dispersed polymer particles with an average particle diameter of 0.03 to 2 μm are used as carriers for immobilized enzymes, they move in the reaction system similarly to free enzymes. Therefore, the reaction efficiency is high.

Further, according to the method of the present invention, when recovering the immobilized enzyme and separating the reaction product, the immobilized enzyme can be immediately and completely separated from the reaction mixture as a solid phase, unlike when using a free enzyme. Furthermore, since the immobilized enzyme used in the method of the present invention has excellent resistance to organic solvents, separation operations between the immobilized enzyme and the reaction product including the use of organic solvents as described above are possible. Substantially no inactivation occurs even in the above, and therefore, the recovered immobilized enzyme can be used repeatedly and can also be advantageously used for the synthesis of 9-dipeptide in an aqueous organic solvent.

The present invention will be explained below with reference to Examples, but the present invention is not limited to these Examples in any way.

Example 1 (11 Production of immobilized lipase 26 g of methyl methacrylate 1 12 g of acrylonitrile
, 18 g of glycidyl methacrylate and 4 g of tetraethylene glycol dimethacrylate were added to 300 g of distilled water, and an aqueous polymerization initiator solution prepared by dissolving 0.3 g of potassium persulfate in 40 ml of water was added at a temperature of 70° C. under a nitrogen stream.
Polymerization was carried out for 4 hours by stirring at 200 rpm while maintaining H at 7.0, and then the temperature was raised to 80°C and the reaction was carried out for 1 hour to form a water-dispersed polymer with a solid content of approximately 15% and an average particle size of 0.4 μm. An aqueous dispersion of high molecular weight polymer particles was obtained, and the polymer particles were centrifuged and washed with water. This was redispersed in 340 ml of distilled water to obtain an aqueous dispersion of polymer particles.

Next, 50ml of this aqueous dispersion, 300ml of water, and 50ml of polyethyleneimine aqueous solution (solid content 30%) were mixed and reacted with stirring at room temperature for 3 days and then at 40°C for 1 day to form polyethylene into polymer particles. combined with imine.

After this, the reaction mixture was centrifuged, washed with water, and diluted with 0.1 M
It was washed with Tris buffer (pH 6.5) and redispersed in distilled water to make 5Qml.

Add 6 ml of 25% glutaraldehyde aqueous solution to 30 ml of this aqueous dispersion, react at room temperature for 2 hours, and then wash with water.
An aldehyde group is introduced by forming a Schiff base with the amino group of the polymer particles, and then 150 μl of thermolysin is added to 40 ml of an equal volume mixture of dimethyl sulfoxide and water.
(containing 5 mmol/cc of calcium acetate) was added to the solution and reacted with gentle stirring. After this, in order to block unreacted aldehyde groups, a Tris-HCl buffer (5 mmol) of pH 8, 0, 0, 1M
/cc of calcium acetate. ) was added in the same amount.

Then dimethyl sulfoxide, water, 0. Washing was repeated in the order of I M Tris-HCl buffer to obtain an immobilized enzyme in which 16 mg of sarsolycin was immobilized per 1 g of dry polymer particles.

(2) 0.1 M l-Lis-HCl buffer (p)I 8.0) was added to 5 ml (solid content 4.5%) of thermolysin-immobilized water-dispersed polymer particles obtained in the dipeptide synthesis reaction. Add 20ynl to make the calcium acetate concentration 10 mmol/12, and then add N-
1 mmol of acryloylphenylalanine and 1 mmol of phenylalanine benzyl ester-p-)luenesulfonate were added and reacted. During this time, the pH of the reaction system was adjusted to 8.0 using IN sodium hydroxide.

After reacting overnight, the precipitated product was extracted with ethyl acetate, washed with 7% ammonia water, 0.5% citric acid, and water in this order, and then distilled under reduced pressure to remove the solvent. The dipeptide was obtained with a yield of 58.3%.

On the other hand, the immobilized enzyme in the aqueous layer was filtered through a membrane with a pore size of 0.2 μm, washed with 0.1 M, pH 8 Tris salt diacid buffer (containing 10 mmol of calcium acetate), and then the aqueous dispersion was separated. 5 ml (solid content 4.5%), add 20 ml of 0.1 Tris-HCl buffer to it in the same manner as above to make the calcium acetate concentration 10 mmol/11, further add the same amount of substrate as above, and add pns. , o.

As a result of repeating the reaction using the immobilized enzyme separated and recovered from the aqueous layer after the completion of the reaction, the dipeptide was able to be obtained five times at almost the same yield as the initial one. The results are shown in the drawing.

For comparison, the substrates were reacted in exactly the same manner as in Example 1, except that 3.6 mm of free thermolysin was used instead of the immobilized enzyme in Example 1, and the reaction product was treated with ethyl acetate. When extracted, the yield of dipeptide was 59.
It was 2%. However, when the same amount of substrate as above was added to the aqueous layer after the above extraction and the reaction was repeated under conditions of pH 8.0, the yield of the dipeptide sharply decreased as shown in the figure.

Example 2 The reaction was carried out in exactly the same manner as in Example 1, except that N-carbobenzyloxy-dis-phenylalanine and L-valine methyl ester hydrochloride were used as substrates, and the yield was 72.3. % dipeptide was obtained.

When the substrate was reacted repeatedly using the immobilized enzyme in the aqueous layer in the same manner as in Example 1, the yield of Jibebu was 70.0% in the second reaction and 70.8% in the third reaction. Met.

Example 3 An immobilized enzyme was obtained in exactly the same manner as in Example 1, except that m-xylylenediamine was used in place of polyethyleneimine. When this was used to react the substrate under the same conditions as in Example 1, the dipeptide was reacted with 58.
Obtained with a yield of 8%. In addition, when the substrate was reacted using the immobilized enzyme repeatedly in the same manner as in Example 1, the second
The yields of the dipeptide in the third and third rounds were 58.
They were 8% and 56.1%.

Example 4 A reaction was carried out in exactly the same manner as in Example 1 except that 25 ml of ethyl acetate was present in the reaction system. In this reaction, the reaction product was dissolved in ethyl acetate. The yield of dipeptide was 54.4%. Also, 24
The yields of dipeptide in the ethyl acetate layer were 55% and 56%, respectively, when 1 ml of each substrate was added every hour.
and 49%.

[Brief explanation of drawings]

The figure is a graph showing the change in dipeptide yield when dipeptide synthesis is repeatedly performed using an immobilized enzyme according to the method of the present invention, and the change in yield when using a free enzyme. 723'1S k 4th Liar Procedure Amendment (Spontaneous) 1982 Patent Application No. 166294 2 Title of invention Method for producing dipeptides 3 Relationship with the person making the amendment Case Address of patent applicant Ibaraki City, Osaka Prefecture 1-1-2 Shimohozumi Name Nitto Electric Industry Co., Ltd. 4 Agent address Shinmachi Shichifuku Building 1-8-3 Shinmachi, Nishi-ku, Osaka Correct “bebu” to “dipeptide”.

Claims (1)

[Claims] (11) In the presence of an immobilized enzyme in which a protease is covalently immobilized on water-dispersed polymer particles with an average particle diameter of 0.03 to 2 μm, the first A step of reacting an amino acid or peptide with a second amino acid or peptide with a protected carboxyl group in an aqueous medium and dissolving the resulting dipeptide in an organic solvent, and separating and recovering the dipeptide from the organic solvent, A method for producing dipeptides, comprising a step of separating and recovering the immobilized enzyme as a solid phase.