JP2635705B2 - Method and apparatus for producing target substance by recycling enzyme - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION (Industrial application field) The present invention is to obtain a target substance by utilizing a reaction between an enzyme and a substrate, and is capable of circulating the enzyme for a long time without deactivating the enzyme. And a device for producing a target substance.

(Prior art) Conventionally, the production method of a target substance using an enzyme is such that the enzyme is reacted with a substrate as it is or adhered to an immobilized carrier, and after the reaction, the enzyme is inactivated and sterilized by heating or the like. The product was obtained after the product was removed or removed by centrifugation of the enzyme.

There are various product forms such as liquid and powder, but the enzyme according to the above-mentioned conventional method is inactivated and remains in the product, or the immobilized product is separated and used again after washing.

As a method for obtaining a recoverable enzyme, a method disclosed in
-105281 and JP-A-59-14791 are known,
According to these methods, it is known that the enzyme can be repeatedly used for a long time without losing the activity. As such a conventional technique relating to a specific apparatus for repeated use, there is a technique provided with a membrane for separating an enzyme and a substrate in a reactor as disclosed in, for example, JP-A-60-27830.

In addition, general literature describes methods for separating enzymes by centrifugation or membrane separation.

(Problems to be Solved by the Invention) As described above, in the conventional technology, there is a problem that the enzyme is used only once, or even if the enzyme is reused, attention must be paid to contamination.

In order to prevent contamination of the enzyme reactant, there is a method of using a large amount of enzyme to shorten the reaction time, but the cost is high, and as another method, the pH of the substrate is changed to the alkali side or the acid side. Although there is a method of reacting, the target substance may not be obtained in some cases because the enzyme to be used is limited.

Furthermore, as a method of permeating target substances using filters and reusing enzymes, "Reasonable design and optimal operation of bioreactors" published by Technical Information Center, Tsuneo Yamane,
Although described on pages 112 to 127, there is a problem that the content of a substance that changes the composition of the substrate increases in the process of recycling.

This is because insolubles are generated in the enzyme reaction product after the reaction, especially in the production of peptides reacted with enzymes that hydrolyze proteins, the tyrosine content increases, and the solubility of tyrosine is low, so that the precipitation substance increases. The problem that the filter is clogged sometimes arises.

Furthermore, since both the substrate and the enzyme are not in a sterile state, preventing the growth of various bacteria has been a problem in producing the target substance.

As a countermeasure against various germs, there is a method in which a substrate or an enzyme is put into a sterilized and sealed reaction tank through a sterilization filter, but the apparatus system is expensive, and an open system apparatus is used in the production of substances using food materials. It is generally produced in.

Accordingly, an object of the present invention is to provide a method and apparatus for producing a target substance which can be recycled for a long time without deactivating the enzyme.

(Means for Solving the Problems) In order to achieve the above object, the present invention proposes the following method and apparatus as a method and apparatus for producing a target substance.

That is, after reacting the enzyme with the substrate solution in the reaction tank, insoluble substances and various bacteria are removed from the enzyme reaction solution by the first filtration membrane, and the enzyme reaction is further performed by the second filtration membrane. This is a method for producing a target substance by circulating an enzyme, wherein an enzyme is returned to a reaction tank after permeation of a product, and a substrate is added and reacted. Then, the immobilized enzyme in which the enzyme is immobilized on the water-soluble polymer is allowed to react in a reaction tank with the substrate, and the molecular weight of the enzyme or the immobilized enzyme is 10,000 to 1,
000,000. In the above method, the substrate is a protein, and a peptide mixture containing a free amino acid as a target substance is produced.

 The following devices are used.

That is, a first-stage filtration membrane which forms a circulation path of a reaction solution between a reaction tank for reacting an enzyme and / or an immobilized enzyme with a substrate solution, and a balance tank communicating with the reaction tank, A second-stage filtration membrane that permeates the enzyme reaction product from the liquid that has passed through the second-stage filtration membrane, and a residual solution containing the enzyme and / or immobilized enzyme that has not been permeated through the second-stage filtration membrane. And a flow path returning to the reaction tank. The first-stage filtration membrane is a filter of 0.1 μm to 0.45 μm, and the second-stage filtration membrane has a cut-off molecular weight of 1,000.
It utilizes ~ 500,000 membranes.

(Action) The enzyme and substrate react in a reaction tank to decompose and produce the target substance. At this time, some of the decomposed substances are composed of insolubles.

After the reaction is completed, the reaction tank is sent to the balance tank via a pump, and the balance tank is further sent to the first filtration membrane via a pump. Is transmitted to the second filtration membrane.

In the first stage filtration membrane, undissolved substances and various germs are returned to the balance tank without being permeated and discarded.

In the second stage filtration membrane, the target substance is permeated and becomes a product, and the remaining liquid containing the remaining enzyme and / or immobilized enzyme is returned to the reaction tank.

The enzyme returned to the reaction tank and / or the immobilized enzyme is added with a substrate and used for the re-reaction.

(Example) Hereinafter, an example illustrated in the drawings will be described.

Generally, the molecular weight of the enzyme is tens of thousands to hundreds of thousands, but this is immobilized on a water-soluble polymer, and the molecular weight is 10,000 to 1,000,000.
It is assumed that the light passes through the first-stage filtration membrane but does not pass through the second-stage filtration membrane.

Those that are not immobilized are high-molecular-weight enzymes with a molecular weight of 10,000
Use from 00 to 1,000,000. The enzyme and the substrate are reacted in the reaction tank (1) to generate a target substance.

At this time, a part of the enzyme reaction solution is composed of insoluble matter.

After the completion of the reaction, the enzyme reaction solution is sent from the reaction tank (1) via the pump (2) to the balance tank (3) via the pipe (7), and further from the balance tank (3) via the pump (5) via the pipe (5). In 8), it is sent to the first-stage filtration membrane (4).

The target substance and the immobilized enzyme in the enzyme reaction solution that have passed through the first-stage filtration membrane (4) are permeated, and are passed from the pipe (10) via the pump (11) to the second-stage filtration membrane (12). ).

In the first-stage filtration membrane (4), the insoluble matter and various bacteria are not permeated, but return to the balance tank (3) through the pipe (6) and are circulated and permeated until the inside of the reaction tank becomes empty. Insoluble matter and various bacteria remaining in the balance tank (3) are discarded through the pipe (9).

For the first stage filtration membrane, a filter of 0.1 μm to 0.45 μm is used, but generally a filter of 0.22 μm to 0.45 μm is used for sterilization, and considering the insoluble matter removal efficiency, Those in the above range are preferred.

In this filter, the target substance and the immobilized enzyme permeate.

In the second stage filtration membrane (12), target substance is piped (13)
The remaining solution containing the immobilized enzyme is sent to the reaction tank (1) through the pipe (14).

In the second-stage filtration membrane, the immobilized enzyme-containing residual liquid returned to the reaction tank (1) to permeate the target substance from which insoluble matter and various bacteria have been removed by the first-stage filtration membrane is sterilized. Has been cleaned up.

The second-stage filtration membrane uses a membrane having a molecular weight cut-off of 1,000 to 500,000, and can be recovered without permeating the enzyme and / or immobilized enzyme solution. In addition, since insoluble matter and various bacteria are removed by the first filtration membrane before the second filtration membrane, the load on the second filtration membrane is reduced and clogging is reduced.

The immobilized enzyme returned to the reaction tank (1) is used for re-reaction by adding a substrate to the immobilized enzyme, and can be recycled for a long time.

The substrate to be charged into the reaction tank (1) is effective to prevent the growth of various bacteria by being charged through a sterilization filter.

The case where a peptide mixture containing free amino acids is produced using the method and apparatus of the present invention as described above will be described below.

A water-soluble polymer-immobilized carrier, for example, an enzyme having protease activity or an enzyme having pectidase activity is attached to aminoethylated hydroxyethyl cellulose or dextran, and the substrate is acid casein or soybean protein.
A decomposition reaction is performed in the reaction tank as a protein composition.

After completion of the reaction, the first-stage filtration membrane removes and discards insolubles and various bacteria containing tyrosine as a main component, and permeates the peptide mixture containing free amino acids through the second-stage filtration membrane to remove enzymes and / or enzymes. Alternatively, the immobilized enzyme is returned to the reaction tank and recycled.

The reactor itself is a batch production, but depending on the reaction time and the processing capacity of the filter, continuous production can be made possible by a combination of the reactor and the filter.

The resulting peptide mixture containing free amino acids may be used as a flavoring agent, enteral nutritional agent, or a protein agent for preventing milk allergy.

The enzymatic reaction product can be dried and powdered, as in the conventional case.

The molecular weight of the enzyme and the molecular weight cut off of the second-stage filtration membrane are related as shown in the following table, depending on the target substance to be obtained.

In any of these relationships, the first filtration membrane is:
A microfiltration membrane in the range of 0.1 μm to 0.45 μm may be used.

(Effects of the Invention) According to the present invention, the following effects are obtained.

(1) Since enzymes can be circulated aseptically, expensive enzymes can be effectively used.

(2) If the substrate is also introduced through a sterilization filter, the aseptic conditions are uniform, and the growth of various bacteria is suppressed without using a particularly expensive aseptic equipment for the reaction tank, and an operation such as a short-time reaction is not required.

(3) No operation such as pH adjustment is required to suppress the growth of various bacteria, and the enzyme used is not limited.

(4) The filter is divided into two stages, and after removing insolubles and various bacteria, the target substance is permeated, so that the load on the filtration membrane in the second stage is reduced, and the target property can be produced reliably.

Generally, when trying to permeate the enzyme reaction product only through the second-stage filtration membrane, clogging of the membrane occurs due to insolubles such as tyrosine, and long-term operation cannot be performed.

In addition, when the enzyme of claim 3 is used, the first-stage filtration membrane permeates, but the second-stage filtration membrane does not.

When the filter of claim 6 is used, the target substance and the immobilized enzyme pass through the first-stage filtration membrane, and the target substance permeates through the second-stage filtration membrane, and the enzyme and / or the immobilized enzyme are permeated. The residual liquid contained does not permeate and can be recovered.

[Brief description of the drawings]

 The drawing is a flow sheet showing the method and apparatus of the present invention. (1) Reaction tank (3) Balance tank (4) First filtration membrane (12) Second filtration membrane

 ──────────────────────────────────────────────────の Continuing on the front page (72) Inventor Tomoe Yoshida 204, 876-2E Building, Kitaakitsu, Tokorozawa-shi, Saitama

Claims (6)

(57) [Claims] After a reaction between an enzyme and a substrate solution in a reaction tank, insoluble substances and various bacteria are removed from the enzyme reaction solution by a first-stage filtration membrane. A method for producing a target substance by circulating an enzyme, which comprises permeating an enzyme reaction product, returning the enzyme to a reaction tank, and adding a substrate to react. 2. The method according to claim 1, wherein the immobilized enzyme obtained by immobilizing the enzyme on a water-soluble polymer is reacted with a substrate. 3. The enzyme or immobilized enzyme has a molecular weight of 10,000.
3. The method for producing a target substance by recycling enzymes according to claim 1 or 2, wherein the target substance is used. 4. The method according to claim 1, wherein the substrate is a protein and a free amino acid or a peptide or a peptide mixture containing the free amino acid is produced as the target substance. 5. A first stage filtration membrane for forming a reaction solution circulation path between a reaction tank for reacting an enzyme and / or an immobilized enzyme with a substrate solution and a balance tank communicating with the reaction tank. And a second-stage filtration membrane that transmits an enzyme reaction product from a liquid that has passed through the first-stage filtration membrane; and an enzyme and / or immobilized enzyme that has not been permeated through the second-stage filtration membrane. An apparatus for producing a target substance by circulating an enzyme, comprising: a flow path for returning a contained residual liquid to the reaction tank. 6. The first-stage filtration membrane is a 0.1 μm-0.45 μm filter, and the second-stage filtration membrane is a fractionated molecular weight of 1,00.
The apparatus for producing a target substance by circulating an enzyme according to claim 5, wherein a membrane of 0 to 500,000 is used.