JPH0530439B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION Field of Industrial Application The present invention relates to a method for continuously producing a dipeptide by reacting N-substituted aspartic acid with a phenylalanine lower alkyl ester, specifically, a method for continuously producing a dipeptide by reacting an N-substituted aspartic acid with a phenylalanine lower alkyl ester. The present invention relates to a method for continuously producing the dipeptide described above using a column prepared by the above method.

Prior Art and its Problems In recent years, attempts have been made to synthesize useful peptides using the reverse reaction of proteolytic enzymes. In reactions using such proteolytic enzymes, the synthesis reaction and the decomposition reaction are in equilibrium, and the equilibrium can be shifted by removing compounds involved in this equilibrium from the system. In many cases (equilibrium systems),
The synthesized condensate is more hydrophobic and has lower solubility in water than the substrate used as a raw material, and this fact can be utilized for peptide synthesis. Recently, various methods have been proposed in which an organic solvent forming two phases with water is added to remove the product from the system by extraction, thereby shifting the equilibrium toward the synthesis side.

By the way, in enzymatic peptide synthesis, it is advantageous in terms of cost to use enzymes repeatedly, and from this and stability standpoints, research has been conducted to immobilize and utilize the enzymes. In the above-mentioned method that utilizes the precipitation of a substance as a precipitate, it is difficult to separate the precipitated product from the immobilized enzyme, which poses a major practical obstacle. On the other hand, it is thought that adding an organic solvent to the system to dissolve or extract the product would enable the use of immobilized enzymes, and based on this idea, Kuhl et al. Dipeptides are synthesized in a two-phase water-dichloromethane system. [P. Kuhl, A. Konnecke, G. Doring,
H. Daumer, H.-D. Jakubke, Tetrahedron
Letters, Vol. 21, p-893-896 (1980)].

Furthermore, in a method for producing a dipeptide from N-substituted aspartic acid and phenylalanine lower alkyl ester, both are reacted in an organic solvent that is immiscible with water in the presence of an immobilized metal protease containing water (such as thermolysin). A method to do this has also been proposed (Japanese Patent Laid-Open No. 135595/1983). In this method, since enzymes have extremely low activity and are unstable in organic solvents, water is impregnated into the pores of the immobilized enzyme and the enzymatic reaction is carried out there.
This appears to be a single-phase reaction using an organic solvent, but if the interior of the immobilized enzyme is considered to be an aqueous phase, it can also be considered to be a two-phase water-organic solvent reaction in which the volume of water is considerably smaller than the volume of the organic solvent.

The present inventors have also conducted intensive studies on peptide synthesis in the above-mentioned water-organic solvent two-phase system, but in general, in such a synthetic reaction, not only the type of enzyme, but also the relationship between the substrates used as raw materials, such as substrates, etc. The yield and reaction rate of the synthesized peptide are greatly influenced by changes in the type of protecting group used, the type of organic solvent used, its concentration or amount used (ratio to water), etc., and the combination of the above factors. The enzymes used are often inactivated or reduced in activity, and the optimal combination of each factor has not yet been elucidated. It was confirmed that the reproducibility was poor, and the enzyme was significantly deactivated when the reaction was carried out continuously, making continuous reaction for industrial implementation practically inappropriate.

As a result of subsequent research, the present inventors determined that the volume ratio of the aqueous phase to the organic phase was approximately 1/1, and by adding and dissolving N-substituted phenylalanine in the organic phase and N-substituted aspartic acid in the aqueous phase. , the inactivation of the enzyme is suppressed (the pH fluctuations in the system due to substrate distribution are maintained within a desirable range during emulsion preparation and the progress of the reaction), and the substrate concentration in the reaction system is improved, and the reaction rate and reaction yield are improved. He discovered a new fact that a desired dipeptide can be obtained very efficiently by repeatedly using an immobilized enzyme, and completed an invention based on this knowledge. See No. 33840].

Furthermore, as an improvement to the above method, the present inventors suspended immobilized metal protease in a raw material solution in which both substrates similar to those described above were dissolved in an organic solvent that is immiscible with water, and added the above raw material solution while stirring. We also established a continuous production method for dipeptides, in which the reaction is carried out while being supplied into the reaction system, and the reaction solution is continuously recovered.
-Refer to Publication No. 1719].

Although the above continuous method developed by the present inventors was suitable for industrial implementation, it did not utilize a column and was still not an ideal continuous synthesis method.
Moreover, when this method was simply applied to a method using a column, the enzyme was significantly inactivated and the yield of the target peptide decreased over time, making it unsuitable for industrialization.

Means for Solving the Problems The present invention was developed following the previous invention by the present inventors, and provides an ideal method for continuous production of dipeptides using columns.

That is, in the present invention, when producing a dipeptide by reacting N-substituted aspartic acid and phenylalanine lower alkyl ester, a buffer solution containing calcium ions and the above-mentioned substrates are dissolved in an organic solvent that is immiscible with water. A substrate solution is prepared using the method, and the substrate solution is continuously supplied to a column packed with immobilized metal protease to carry out the reaction at a temperature of 30°C or less. During the reaction, the column is intermittently washed to prevent channeling. The present invention relates to a method for continuous production of dipeptides characterized by preventing.

The N-substituent in the N-substituted aspartic acid used as one substrate in the method of the present invention is an amino group-protecting group commonly used in peptide synthesis reactions, and a typical example thereof is a benzyloxycarbonyl group. . Other representative protecting groups include p-methoxybenzyloxycarbonyl group,
Examples include t-butoxycarbonyl group. The lower alkyl group of the phenylalanine lower alkyl ester used as the other substrate is also a commonly used carboxyl protecting group for amino acids, and a preferred example thereof is an alkyl group having 1 to 4 carbon atoms, particularly a methyl group. These raw material substrates are usually in the L form, but may also be in the DL form, in which case only the L form participates in the reaction.

In the method of the present invention, first, a substrate solution is prepared by dissolving both of the above raw material substrates together with a buffer containing calcium ions in an organic solvent that is immiscible with water. Here, a typical example of the buffer containing calcium ions is an MES [(2-cyanomorpholino)ethanesulfonic acid]-NaOH buffer containing CaCl ₂ . It preferably contains CaCl ₂ in the range of about 20-50 mM, and its pH is not particularly limited, but it is usually suitable to be around 6. The buffer solution is generally dissolved in a water-immiscible organic solvent to a saturation concentration, preferably at a concentration of about 2.5%. A typical example of the water-immiscible organic solvent is ethyl acetate.

The amount of both raw material substrates used in the substrate solution, that is, the concentration of each substrate in the substrate solution is determined as appropriate,
From the viewpoint of reaction rate, it is preferable to use a concentration as high as possible, but usually for phenylalanine lower alkyl ester, the concentration is about 40 to 200 mM, preferably about
The concentration range is preferably around 100mM, and for N-substituted aspartic acid to be reacted with this, it is appropriate to set the concentration to be about 1/3 to 1/2 times the concentration of the above phenylalanine lower alkyl ester. It is.

In the method of the present invention, the substrate solution prepared as described above is then supplied to a column filled with immobilized metal protease, and both substrates in the solution and the immobilized enzyme are brought into contact reaction in the column. . As the immobilized enzyme used here, any of various types can be used, typically a metal protease such as thermolysin immobilized on a suitable support according to a conventional method. The above-mentioned supports include Merckogel [Merckogel SI 1000Å, manufactured by Merck], Amberlite IRC50 [manufactured by Rohm and Haas Co.], and Dowex MWA [manufactured by Dow Chemical Co.].
Dowex MSC [manufactured by Rohm and Haas], Amberlight XAD2 [manufactured by Rohm and Haas], Amberlight XAD7 [manufactured by Rohm and Haas], Amberlight
Examples include porous ion exchange resin carriers such as XAD8 [manufactured by the same company]. Among these, amber light
XAD7 is most preferred. Thermolysin and the like can be immobilized on the support by various methods well known in the art, with glutaraldehyde crosslinking being particularly preferred. The glutaraldehyde concentration in the glutaraldehyde crosslinking method is
Approximately 4% compared to the 2% to 3% commonly adopted
The concentration is preferably ~6 times as high, particularly around 12.5%, and thermolysin and the like are preferably dissolved in a suitable solution such as NaBr, adsorbed onto a support, and then fixed. This method allows the solution concentration to be approximately 50 times higher than when adsorbing the same enzyme on a support as an aqueous solution, increasing the amount of enzyme adsorbed per unit of support, and improving activity and stability compared to conventional methods. Highly immobilized thermolysin can be obtained.

The immobilized thermolysin thus prepared is
Usually, 0.02 to 0.5 g of thermolysin is fixed per 1 g (wet weight) of the support, and the titer (synthetic activity) per g is about 0.15 to 3.0 units/wet g. This synthetic activity is determined by measuring the amount of dipeptide produced by enzymatic reaction using the same procedure as in Example 1 below, using high performance liquid chromatography. It refers to the amount of immobilized enzyme (wet weight) that produces 1 μmol of dipeptide.

In the present invention, it is particularly essential that the substrate reaction in the column filled with the immobilized metal protease such as the immobilized thermolysin be carried out at a temperature of 30°C or lower, preferably around 25°C, and this temperature condition is adopted. By employing intermittent column washing operations following the column reaction at this temperature, the substrate, reaction product, and immobilized enzyme in the column, as well as the immobilized enzyme
The pH becomes substantially uniform or constant within each column, ensuring that enzyme deactivation is prevented, and the desired dipeptide is rapidly and continuously synthesized in high yield.
It can be earned.

The intermittent washing operation of the column can be carried out as appropriate on the premise that channeling does not occur during the reaction. The washing solution used in this washing operation can be water, a buffer such as MES-NaOH buffer, or an organic solvent that is immiscible with water such as ethyl acetate.
A 0.01M-MES-NaOH buffer containing _-CaCl2 is preferred, and most preferably a combination of an organic solvent such as ethyl acetate saturated with the above buffer and the above buffer saturated with the organic solvent. Further, it is desirable that the above-mentioned washing operation be carried out at an appropriate time before the formation of a cloudy precipitate due to the continuous reaction is observed.
This is usually at least 1 by about 3 days of continuous reaction.
The treatment is preferably carried out at intervals of about once a day, preferably about once a day. The washing operation can be carried out by passing the above washing solution through the column, and the temperature condition at that time is 30°C.
Hereinafter, the temperature is preferably about 25°C, the space velocity (SV) for liquid passage can be about 5 to 20/hour, and the liquid passage time can be about 10 to 60 minutes.

According to a preferred embodiment of the present invention, phenylalanine methyl ester is first added to ethyl acetate.
N-benzyloxycarbonyl-aspartic acid at a concentration of 100mM and 20mM _CaCl2 at a concentration of 40mM.
0.01M MES−NaOH buffer (pH 6.0) containing
Prepare a raw material solution by dissolving each at a concentration of 2.5%,
Next, this raw material solution was preliminarily saturated with ethyl acetate.
A glass column packed with about 5 g of immobilized thermolysin equilibrated with 0.01 M MES-NaOH buffer (containing 20 mM CaCl ₂ , pH 6.0) was filled with a constant flow rate of about 7.5 ml/hour (SV based on the volume of immobilized enzyme: about
1.75/hour) and carried out a continuous reaction at a temperature of about 25°C. During this reaction, 0.01M MES-NaOH buffer (containing _20mMCaCl2 , pH 6.0 ) and the same buffer saturated with ethyl acetate (flow rate: 50 ml/h, SV =
15/hour) to prevent channeling.

By the above method, the desired dipeptide can be obtained stably and efficiently over a period of about 500 hours or more with a high yield of over 95%.

The reaction liquid flowing out from the column outlet due to the above is
The target dipeptide, that is, N-substituted aspartic acid-phenylalanine lower alkyl erther, is contained as an organic solvent solution, and the target dipeptide can be separated from the reaction solution by, for example, fractionating the organic solvent solution and concentrating it. This can be easily carried out by performing operations such as crystallization or extraction. The dipeptide thus obtained can be further purified by conventional isolation and purification means.

The dipeptide thus obtained according to the invention is
It is useful as a reaction reagent for the synthesis of physiologically active peptides, and as a precursor for L-aspartyl-L-phenylalanine methyl ester (aspartame), a synthetic sweetener that is about 200 times sweeter than sugar.

Examples Examples will be given below to explain the present invention in more detail. In the examples, immobilized thermolysin prepared by the following method was used.

<Preparation of immobilized thermolysin> Thermolysin (manufactured by Daiwa Kasei Co., Ltd., titer
9470PU/mg) 7.5g, 5M-NaBr and 16.6mM
-Dissolve in 120 ml of 1/40M Tris-HCl buffer (pH 7.5) containing CaCl ₂ under ice cooling, and add the immobilization carrier Amberlite
Add 30g (wet weight) of
The enzyme was adsorbed onto the carrier while gently shaking at 4°C for 17 hours. When the amount of residual enzyme protein in the supernatant was quantified using the Biuret method, it was found that approximately 70% of the initial amount of enzyme was adsorbed on the carrier.

After removing 75 ml of the above supernatant, add 75 ml of 25% glutaraldehyde solution to the remaining fixed enzyme suspension.
After shaking at 4°C for about 3 hours to carry out the crosslinking reaction,
Cooled 0.1M Tris-HCl buffer (pH 7.5,
Immobilized thermolysin was obtained by washing alternately twice with approximately 1 ml of the same buffer containing 5 mM CaCl ₂ and 1 ml of the same buffer containing 1 M NaCl. The obtained immobilized enzyme was kept at 4°C.
Saved with.

Example 1 20mM-
0.01M-MES containing CaCl2 [ ₂ -cyanomorpholinoethanesulfonic acid, manufactured by Dojindo Laboratories]-
5 ml of NaOH buffer (pH 6.0), 3.58 g (100 mM) of L-phenylalanine methyl ester (L-PheOMe, manufactured by Sigma) and N-benzyloxycarbonyl-L-aspartic acid (Z-Asp).
A substrate solution was prepared by dissolving 2.14g (40mM). On the other hand, the immobilized enzyme (wet amount: 5 g) was added to 0.01M-MES containing 20mM-CaCl ₂ saturated with ethyl acetate.
- Equilibrate with NaOH buffer (pH 6.0) and apply this to a glass column (11 mm diameter x 150 mm, Yamazen Co., Ltd.)
was filled.

The substrate solution was supplied to the glass column filled with the immobilized enzyme at a constant flow rate of about 7.5 ml/hour (SV based on the volume of immobilized enzyme = 1.75/hour), and
Continuous reaction was started at 10°C.

During the above continuous reaction, once every 23.5 hours, 20mM-
The column was washed for 15 minutes with ethyl acetate saturated with 0.01M-MES-NaOH buffer (pH 6.0) containing _CaCl2 , and then for 15 minutes with the same buffer (flow rate: 50 ml/hour each, temperature: 25℃).

During the above continuous reaction, the reaction solution at the column outlet was sampled at appropriate time intervals and subjected to high performance liquid chromatography under the conditions shown below to quantify the amount of product.

<High performance liquid chromatography> Sample: A substrate solution sampled at the column outlet was vacuum dried and then dissolved in an acetonitrile-water mixed solvent (70:30, adjusted to pH 2.5 with phosphoric acid) to prepare.

Equipment: High-performance fluid chromatograph (Shimadzu Corporation LC-3A type) Column: Internal diameter 4.6 _{mm x length 150 mm Packing material: Cosmosil 5C18 -} P (ODS-silica gel) Solvent: Acetonitrile-water (60 mm) :40, pH adjusted to 2.5 with phosphoric acid) Detection: Ultraviolet absorption (254nm) Elution solvent flow rate: 0.8ml/min The results are shown in Figure 1. In Figure 1, the horizontal axis represents continuous reaction time (hours), and the vertical axis represents product yield (%).
shows.

As is clear from FIG. 1, according to the method of the present invention
A yield of over 95% was maintained for over 500 hours.

Comparative Example 1 A continuous reaction was carried out in the same manner as in Example 1 except that the reaction temperature was 40°C.

The reaction solution was sampled at the column outlet and subjected to high performance liquid chromatography under the same conditions as in Example 1 to quantify the amount of product. As a result, the yield of the target dipeptide decreases with the passage of reaction time,
After 150 hours, it decreased to about 80%.

Comparative Example 2 A continuous reaction was carried out in the same manner as in Example 1 except that the column washing operation was not performed.

The results are shown in FIG. 2 in the same manner as in FIG. 1.

From FIG. 2, it can be seen that a yield of about 97% was observed at the beginning of the reaction, but the yield decreased after 80 hours. In addition, after 160 hours, channeling due to the accumulation of white precipitate in the column became significant.
The flow of the liquid was significantly delayed, making continuous reaction difficult.

[Brief explanation of the drawing]

FIG. 1 is a graph showing the relationship between continuous reaction time and yield in the method shown in Example 1, and FIG. 2 is the same graph in the method shown in Comparative Example 2.

Claims (1)

[Claims] 1. When producing a dipeptide by reacting N-substituted aspartic acid and phenylalanine lower alkyl ester, a substrate solution is prepared by dissolving a buffer containing calcium ions and both substrates in an organic solvent that is immiscible with water. The substrate solution is continuously supplied to a column packed with immobilized metal protease to carry out the reaction at a temperature of 30°C or less, and the column is intermittently washed during the reaction to prevent channeling. A method for continuous production of dipeptides characterized by: