JPH01165558A - Separation of amino acid compound - Google Patents

Abstract

PURPOSE:To efficiently separate an amino acid compound by migration effects caused by difference in pressure and electric field, by adjusting a solution to be separated to a proper pH and feeding the solution to a separating membrane arranged between electrodes under pressure. CONSTITUTION:An amino acid compound-containing solution to be separated is adjusted to a proper pH, either a component to be separated or the other component is provided with electric charge, the other is made an isoelectric point or provided with the opposite charge and an electric field is generated by electrodes 3 and 4. In this state, the solution 1 to be separated is fed to a separating membrane (e. g., precision filter membrane, ultrafilter or reverse osmosis membrane) M, the component of isoelectric point and the component having the same charge as that of the electrode 3 are passed through the separating membrane M by migration effects resulting from difference in pressure and the electric field and the permeated solution 2 is obtained at the opposite side of the separating membrane M to separate the aimed component.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to a method for separating amino acid compounds such as amino acids and proteins by a membrane separation method using electrophoresis.

[Conventional technology]

In recent years, there has been significant interest in the production of useful substances derived from microorganisms, and the usefulness of membrane separation methods is being recognized as a method for quickly and efficiently separating microbial products from bacterial cells or substrates, etc. . However, there are problems, such as the formation of a gel layer due to the concentration polarization phenomenon and the difficulty in separating particles of similar size (molecular weight) due to differences in the size of solutes (Makoto Nakao).・Takashi Kimura: Chemical Engineering,...Snoring, 545 (1986)).

The present inventors would like to investigate the possibility of practical application of electric ultrafiltration as a method for preventing the formation of the gel layer.
Tsuyoshi Nomura: Membrane, Uniσ, 245 (1982)) and charged ultrafiltration method were used to study the separation of amino acids (S, Ki@ura and A, Tamano: “
5eparation of Am1noacids
by Charged Ultra-filtrat
ion Membranas” in “Membranas”
es and Membrane Processes”
ed, by E, Drioli and M, Naka.
gaki P 191-197. Plenum Pr
ess.

New York, 1986L [Problems to be Solved by the Invention] However, the electric ultrafiltration method has been studied as a means to prevent gel layer formation, and it has not been predicted that it will separate amino acid compounds such as amino acids and proteins. do not have.

Charged ultrafiltration is a method that uses a membrane with a predetermined charge to separate amino acids, etc. Amino acids, proteins, and other amino acid compounds can be charged positively or negatively depending on the type, even at the same solution pH. Since it becomes neutral, it becomes possible to separate substances of similar molecular weight depending on the membrane charge, but since there is a limit to the fixed charge of the membrane, in order to perform separation effectively,
It is necessary to keep the solution concentration below a certain level.

On the other hand, electrophoresis has long been known as a separation method that utilizes the charge of solutes. In recent years, new electrophoresis methods have been developed for the purpose of large-scale separation, but there are still problems such as the throughput of solutions is small and the control of a single separation membrane is not perfect.

This invention is intended to solve the above problems.

The purpose of the present invention is to propose a method for separating amino acid compounds that can efficiently separate particles of similar particle size in a high concentration state with a large processing capacity and that allows easy control of separation membranes.

[Means for solving problems]

In this invention, a liquid to be separated containing an amino acid compound is adjusted to pH
Adjust the voltage to give a charge to either the component to be separated or the other component, and set the other to the isoelectric point or give the opposite charge, and between the separation membrane placed between the electrodes and one electrode. This is a method for separating amino acid compounds, which is characterized in that the amino acid compounds are supplied under pressure and separated by the electrophoretic effect caused by the pressure difference and electric field.

In the present invention, an amino acid compound is a compound containing amino acids such as amino acids and proteins as constituent elements.

The principle of separation in the present invention is as follows, considering a two-component system with comparable molecular weights. Figure 1 is a diagram of the principle,
By adjusting the pH of the liquid to be separated 1 containing the amino acid compound, one side is charged and the other side is made to have an isoelectric point, or the other side is made to have an opposite charge, and an electric field is applied by the electrodes 3 and 4. , when supplied to the separation membrane M under pressure, due to the electrophoresis effect due to the pressure difference and the electric field, components at the isoelectric point and components having the same charge as the electrode 3 pass through the separation membrane M, and components with the opposite sign to the electrode 3 pass through the separation membrane M. Charged components are blocked, a permeate 2 is obtained on the opposite side of the separation membrane M, and the target component is separated.

Examples of the separation membrane M include microfiltration membranes, ultrafiltration membranes, reverse osmosis membranes, and the like.

The device used in the separation method of the present invention is preferably a circulation type device using a porous metal plate as an electrode, one example of which is shown in the sectional view of FIG.

In FIG. 2, a holding plate 5 and a membrane supporting plate 6 are integrally connected via an O-ring 7, and porous electrodes 3 and 4 are integrally connected to the holding plate 5 and membrane supporting plate 6, respectively. . For the porous electrode 4, a porous body or a mesh body made of sintered metal is used.

The form of the electrode 3 is not particularly limited. Electrodes 3 and 4 are connected to power sources 8 and 9 through a holding plate 5 and a membrane support plate 6, respectively.
is connected to. The separation membrane M is provided close to the porous electrode 4, preferably in an overlapping manner.

A liquid to be separated 1 containing a pH-adjusted amino acid compound is supplied under pressure to the space 10 between the electrode 3 and the separation membrane M from the supply port 11 and is circulated to the supply port 11 from the discharge port 12. It has become. 13 is a permeate outlet.

In the separation method using the above device, power is supplied to the electrodes 3 and 4 to form an electric field in the space 10, and the pH-adjusted liquid 1 to be separated is supplied to the space 10 under pressure through the supply port 11 provided in the holding plate 5. , the liquid to be separated 1 is circulated through the discharge port 12 and separated, as indicated by the arrow A1. A2. A.

Amino acid compounds that proceed in the direction of arrow B1. It passes through the separation membrane M and the porous electrode 4 in the direction B2°B, is separated, and is taken out as the permeated liquid 2 from the outlet 13. Further, the amino acid compound having the same charge as the electrode 4 is prevented from permeating the separation membrane M and is concentrated in the liquid to be separated 1.

FIG. 3 is a sectional view showing another example of the device used in the present invention, in which another separation membrane M2 is provided close to the electrode 3, and the permeated liquid 2a is taken out from the outlet 14. and
Using separation membranes M and M2, arrow B1. B2. B3
and/or arrow C1,C,.

It allows amino acid compounds to permeate in the direction of C1.

Furthermore, FIG. 4 is a cross-sectional view of another device for use in the present invention, showing a cross-section perpendicular to the longitudinal direction of the tubular module 20. In this embodiment, porous tubular electrode pairs 4 and 3
Separation membranes M and M2 are provided adjacent to the arrows B1 and B1, respectively. B2 and/or arrow C,,C.

The permeate liquids 2 and 2a are taken out from the take-off ports 13 and 14, respectively.

As described above, the device modules that can be used in the present invention are:
In addition to the flat membrane type illustrated in Figures 2 and 3,
Examples include the tube type, hollow type, and spiral type illustrated in the figure.

〔Example〕

Examples of the present invention will be described below.

Example 1 In order to investigate the effects of pressure, applied voltage, solution pH, etc. on the separation of amino acids in the electric ultrafiltration method, a flat membrane test cell (effective membrane area 57.785 d) was used to investigate the effects of pressure, applied voltage, solution pH, etc. on the separation of amino acids. An experiment was conducted using a circulation type apparatus shown in FIG. 2 using a plate as an electrode. That is, for the following three types of amino acids, the concentration is 5mM, the flow rate is 6Q/win, p) + 6
．． 5. The voltage and operating pressure were varied while keeping the temperature constant at 25°C. The membrane is a polysulfone ultrafiltration membrane (G
5 analysis was performed using TOC (Sumitomo Chemical flI) using R-61PP, molecular weight cutoff 20000).

Three types of amino acids; monosodium L-glutamate (Mw
187.13), glycine (Mw 75.07), and L-lysine hydrochloride (Mw 182.65), each with an isoelectric point of pI
= 3.22, 5.97° 9.59. Therefore, pH
In the solution of 6,5, sodium L-glutamate is negatively charged, glycine is neutral, and L-lysine hydrochloride is positively charged.

Among the experimental results by the electric ultrafiltration method, the results for sodium L-glutamate are shown in Figures 5 and 6, the results for glycine are shown in Figures 7 and 8, and the results for L-lysine hydrochloride are shown in Figure 9. and FIG. 10.

In Figures 5 to 10, ΔP is differential pressure (kg/ci), J
ν indicates the amount of liquid permeated through the membrane (cxl/cxt·S). Rob
s is the apparent rejection rate (-), which is determined by the following equation.

Cb: Initial concentration of the liquid to be separated Cp: Concentration of the permeated liquid From the results above, it can be seen that there is no effect of the electric field on glycine, which is a neutral solute. It can be seen that the change in the apparent rejection rate for sodium L-glutamate and L-lysine hydrochloride is due to the electrophoretic effect of the solute due to the electric field. In particular, when an electric field is applied to a charged solute in the direction of permeation through the membrane, a permeate with a higher concentration than the feed solution is obtained.

Example 2 As an example of protein, bovine serum albumin (B, S, A,)
An experiment was conducted in the same manner as in Example 1 using .

B, S, and A have isoelectric points of pH 4.8. Results first
Shown in Figures 1 to 16. In the figure, ■ indicates voltage (V). B,
S, A, concentration is 1100 pp, Figures 11 and 12
The figure is PH4,8, Figures 13 and 14 are p09. Figures 15 and 16 are p)13. The separation membrane used was a regenerated cellulose membrane manufactured by Fujifilm (pore size 0).
．． It is a microfiltration membrane (FM-22) consisting of 22 μm), has a ΔP 2 kg/am”, a flow rate of 0.IQ/win, and a temperature of 25°C.

From the above results, it can be seen that the same results as in Example 1 can be obtained for proteins as well.

Example 3 The same test as in Example 1 was conducted on the liquid to be separated (amino acid 3 component mixed system) used in Example 1 that simultaneously contained the three types of amino acids. Amino acid concentration was 1mM each, flow ii5
Q/min, no pH adjustment, temperature 25° C., and a polyacrylonitrile ultrafiltration membrane (IRIS 3038, molecular weight cutoff 20,000) manufactured by Loos-Boulin was used as the membrane. The results are shown in FIG.

From the above results, no interaction between the components was observed, and the separation of the components in a mixed system containing two or more types of amino acids or proteins with different charge characteristics was similar to that in a single system like Example 1. It can be seen that results are obtained.

〔Effect of the invention〕

As described above, according to the present invention, the pH of the liquid to be separated is adjusted and it is supplied under pressure to the separation membrane disposed between the electrodes, and the amino acid compounds are separated by the electrophoretic effect caused by the pressure difference and the electric field. , particles of similar particle size can be efficiently separated in a high concentration state with a large throughput, and the separation membrane can be easily controlled.

[Brief explanation of the drawing]

Fig. 1 is a principle diagram showing the principle of the present invention, Figs. 2 to 4 are sectional views showing different devices used in the present invention, and Figs.
FIG. 17 is a graph showing the results of the example. In each figure, the same reference numerals indicate the same or corresponding parts, 1 is the liquid to be separated, 2 and 2a are the permeated liquid, 3 and 4 are the electrodes, 5 is the holding plate, 6 is the membrane support plate, 7 is the O ring, and 11 12 is a supply port, 12 is a discharge port, 13.14 is a take-out port, and M, M are separation membranes. Agent Patent Attorney Sei Yanagihara Figure 1 Figure 2 Figure 3 7゜1/l 60 ε ε 50 Do〉 Q Membrane surface - ・Membrane surface + 10 No place to speak Figure 11 Proceedings amendment belt Commissioner of the Patent Office Kunio Ogawa, 1, Indication of the incident, 62-g2f-'7 Showa 62
Patent application 2, dated December 23, 2013, Name of the invention: Method for separating amino acid compounds Representative: Shogo Takebayashi 4, Agent: 105 Phone: 436-4700 Address:
3-15-8-6 Nishi-Shinbashi, Minato-ku, Tokyo, detailed description of the invention column 1 drawings in the specification subject to amendment, the invention pertaining to the patent application is an invention defined in Article 30, Paragraph 1 of the Patent Act. Document certifying that it is, power of attorney 7, contents of amendment (1) "Membrane" is inserted after "filtration" on page 2, line 13 and page 3, line 7 of the specification. (2) On page 5, line 5, "same as 3" is corrected to "opposite to 4." (3) On page 5, line 6, "opposite to 3" is corrected to "same as 4." (4) Correct page 10, line 7, r2kgJ to rO,1kgJ. (5) On page 10, line 8, ro and IJ are corrected to "2". (6) Among the drawings, Figure 1 will be corrected as shown in the attached sheet. (7) Submit a document certifying that the invention pertaining to the patent application is an invention stipulated in Article 30, Paragraph 1 of the Patent Law. (8) Submit a power of attorney. 8. List of attached documents (1) Document certifying that the invention pertaining to the patent application is an invention stipulated in Article 30, Paragraph 1 of the Patent Law 1 copy (2) Power of attorney 1 copy 1: Yuyu Bunkago Shi Tadasu M, M2: ke1ffi wide

Claims (3)

[Claims] (1) Adjusting the pH of a liquid to be separated containing an amino acid compound to give a charge to either the component to be separated or the other component, and bring the other to the isoelectric point or give it an opposite charge; 1. A method for separating amino acid compounds, which comprises supplying under pressure between a separation membrane disposed between electrodes and one electrode, and separating by the electrophoresis effect due to a pressure difference and an electric field. (2) The method according to claim 1, wherein the electrode is made of porous metal. (3) The method according to claim 1 or 2, wherein the liquid to be separated is supplied in circulation.