JPH0338834B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to a novel reaction method using enzymes and microorganisms. More specifically, the present invention is directed to an inner space (enzyme chamber) partitioned by two porous materials (e.g. membranes) that do not allow enzymes or microorganisms to freely pass through in enzymatic reactions or microbial reactions involving two or more types of substances.
In addition, these enzymes and microorganisms are present, and substances involved in the reaction are present in the spaces on both outer sides that contact the two porous material membranes, and these substances cross the porous material membranes to form the inner space. It permeates into the space, reacts in the central enzyme chamber, and the products also pass through the porous material membrane to the outside space, allowing for efficient separation of the products along with enzyme or microbial reactions. This invention relates to a reaction method for enzymes and microorganisms using a bioreactor.

[Conventional technology and problems]

As is well known, enzymatic reactions and microbial reactions often require two or more types of substrates or a substance other than the substrate as an activator, and there are few reactions in which only a single substance is involved. Furthermore, not only a single reaction product but also two or more types of products are produced very often. As described above, in many enzymatic reactions and microbial reactions, two or more types of substances are involved not only in the reaction substrate but also in the reaction product. However, for example, when there are two or more types of reaction products, it is necessary to separate these reaction products after the reaction is completed in order to obtain the desired product. In addition, in the case of a reaction requiring two or more types of substrates, one of the substrates may sometimes become a factor that inhibits or deactivates the enzyme or microorganism. In addition, the reaction product itself may become an inhibiting factor.

Considering these points, if we could control the amount of inhibitory substances given to the enzyme reaction system or microbial reaction system, or if we could quickly remove the inhibitory factors from the system, we would be able to carry out enzyme reactions and microbial reactions more efficiently. It is thought that this can be done. Furthermore, if a reactor is used that allows product separation at the same time as the reaction, the product separation step may be omitted. On the other hand, enzymes are generally very expensive, and reusing them is an essential condition for reducing production costs. When considering a bioreactor, it is of course important to be able to easily remove enzymes and microorganisms from the reaction system and reuse them.

Until now, a bioreactor that satisfies all of these issues has not yet been developed. For example, with immobilized enzymes and immobilized microorganisms, problems such as easy separation of enzymes and microorganisms from the reaction system or reuse of enzymes and microorganisms may be solved, but simultaneous operation of reaction and separation or inhibition Solving problems such as controlling matter is impossible. Recently, immobilized enzymes and immobilized microorganism immobilized carriers have a product separation function (adsorption capacity).
Some attempts have been made to make it possible, but these have various problems. For example, if a substance that is an inhibitory or inactivating factor is required for the reaction, it is difficult to control the amount of this substance using this method. Furthermore, when the adsorption ability of a carrier is utilized, a very large amount of carrier is required to adsorb the product, which poses a major obstacle in terms of industrialization.

[Means for solving problems]

As a result of intensive studies on enzyme or microbial reaction reactors that have various problems, the present inventors have developed an epoch-making reactor that can solve these problems.

That is, in an enzyme reaction or a microbial reaction involving two or more types of substances, the present invention uses a set of two porous materials through which these enzymes or microorganisms cannot pass, and an inner space sandwiched between these porous materials. Enzymes or microorganisms, or immobilized enzymes or immobilized microorganisms are present in the two porous materials, and substances involved in the above reaction are present in the spaces on both sides of the two porous materials to separate the porous materials. The present invention relates to an enzyme and microorganism reaction method characterized by allowing these substances to permeate and allowing the reaction to occur in the inner space.

The reaction method according to the present invention uses a set of two membranes made of porous material that do not allow enzymes or microorganisms to freely pass through;
Enzymes and microorganisms are present in the space partitioned by these, and the reactant passes through the porous material and undergoes an enzyme or microbial reaction in the central space, and the reaction product is also passed through the porous material. It is characterized in that it is allowed to pass through and removed from the enzymatic reaction system or microbial reaction system.

[Effect]

The present invention will be explained in more detail based on the drawings showing preferred embodiments of the present invention. As an example, like the fat and oil decomposition reaction by lipase, A+B→C+D
(A and B are substances involved in the reaction, hereinafter referred to as substrates. C and D are products. In the lipase reaction, A =
To explain the enzyme reaction system represented by oil, B=water, C=fatty acid, and D=glycerin using FIG. 1, there is an inner space 4 (enzyme chamber) partitioned by porous materials 1 and 2. Enzyme is present in the chamber, substrate A is present in one outer space 3 (reactant chamber), and substrate B is present in the other outer space 5 (reactant chamber). The porous materials 1 and 2 are constructed in such a way that they do not allow the enzyme to pass through, and allow substrate A to pass through the porous material 1 and substrate B to pass through the porous material 2. Therefore, in the reactor shown in FIG. 1, substrates A and B, which have passed from the respective reactant chambers 3 and 5 to the enzyme chamber 4, undergo an enzymatic reaction by the enzyme present in the enzyme chamber 4 to become products C and D. . Here, porous material 1 is substrate A
, but not B, and only one of the reaction products C and D (temporarily designated as C) is selected. On the other hand, the porous material 2 is selected to allow only substrate B to pass through, but not A, and to allow only D of the reaction products to pass through. In order to impart these properties to a porous material, the material and pore diameter of the porous material may be appropriately selected. For example, in a lipase reaction, a hydrophobic material such as polypropylene or polyethylene may be selected as a porous material that allows only the substrate oil and the reaction product fatty acid to pass, and acetic acid may be selected as a material that only allows water and glycerin to pass through. Hydrophilic materials such as cellulose materials such as cellulose, acrylonitrile polymers, etc. may be selected. In general, the pore diameter of porous materials needs to be large enough to prevent enzymes and microorganisms from passing through.The size of enzymes is said to be several tens to hundreds of angstroms, so pores of this size are necessary. However, depending on the reaction system, such fine pores may not be necessary. For example, in the lipase decomposition reaction, looking at the reaction state in the space 4 partitioned by a porous material, an oil/water emulsion is formed, and the enzyme is present on the surface of this emulsion. . Therefore, the pore diameter should be 0.1 so that this emulsion does not pass through.
Enzyme passage can be blocked with a pore size of ~ several μm. Furthermore, if an immobilized enzyme or an immobilized microorganism is used, fine pores are naturally not required.

Therefore, in the present invention, the material of the porous material and the pore diameter are not particularly limited, and are appropriately selected according to the above-mentioned criteria.

The enzyme or microorganism present in the space 4 partitioned by a porous material may be present in powder form, or may be dissolved or suspended in a solvent that does not inactivate the enzyme or microorganism. . In carrying out the present invention, one preferred method is to provide a spacer in the space 4 and spray or cast the enzyme or microorganism onto the spacer in order to prevent the enzyme from being concentrated in one part. Alternatively, as described below, enzyme solution or microbial solution can be pumped into this space.
That is, it may be circulated to the enzyme chamber 4. In addition, space 4
There are no particular limitations on the material or form of the spacer provided.For example, if a material that has properties that allow enzymes to be adsorbed, such as ion-exchange fibers, is used as the spacer, enzymes can be placed on it. It can be fixed, or, as in this case, some new function can be added to this spacer.

The substrate present in the reactant chambers 3 and 5 in FIG. 1 diffuses into the enzyme chamber 4 by concentration diffusion, and the reaction product also diffuses from the enzyme chamber 4 into the reactant chamber 3 or 5 by concentration diffusion. However, if the diffusion rate is slow and a sufficient reaction rate cannot be obtained by concentration diffusion alone, diffusion can be promoted by applying a force that promotes diffusion, such as pressure or temperature, to each chamber.
Alternatively, it does not inhibit enzyme reactions or microbial reactions,
It is also possible to add a third substance that does not pass through the porous material to each chamber, and use the difference in osmotic pressure of this substance as a diffusion promoting force.

The bioreactor used in the present invention can be applied to various enzymatic reactions and microbial reactions, and in addition to the oil decomposition reaction using lipase described above, it can also be used for triglyceride synthesis using lipase, transesterification reaction of triglyceride, and phospholipid decomposition reaction using phospholipase. , a series of reactions by the transferase group represented by the phosphorolytic reaction of polysaccharides by phosphorylase, a synthesis reaction of malic acid by fumarase, a series of reactions by the hydrolytic enzyme group such as phosphatase and nucleothiodase. It can be used in enzyme reaction systems involving two or more types of substances, such as reactions of oxidoreductase groups such as alcohol dehydrogenase and amino acid oxidoreductase. Furthermore, in microbial reaction systems, in addition to reaction substrates, many substances are required for the growth of microorganisms, so this reactor can be applied to most microbial reactions. For example, Corynebacterium
In glutamic acid fermentation by glutamicum, etc., glutamic acid is fermented and produced from the substrate glucose, but biotin is required for the growth of this bacterium, and the amount of glutamic acid produced increases or decreases depending on the amount of biotin. As described above, microbial reactions often require vitamins such as biotin, inorganic salts, amino acids, etc. in addition to the reaction substrate, and this reactor can be applied to most of these microbial reactions.

The enzyme or microorganism used in the present invention does not necessarily have to be highly purified, and an extract, a partially purified product, or a fermentation liquid can also be used.

In order to carry out the reaction efficiently in the bioreactor according to the present invention, multiple sets of porous materials 9 and 10 are placed across the enzyme chamber 7, as shown in FIG. The reactant chamber 5 is located between the porous materials 9 on one side and the porous material 10 on the other side.
It is set so that a reactant chamber 6 is formed between the
It is preferable that the substances involved in the reaction are circulated to the respective reactant chambers 5 and 6 by pumps 3a and 3b, respectively. In FIG. 2, enzymes or microorganisms can also be circulated to the enzyme chamber 7 by means of a pump 3. In FIG. 2, 1 and 2 are reactant storage containers, 4 is an enzyme or microorganism liquid storage container, and 8 is an outer frame. still,
Enzymes and microorganisms may be left standing by being dispersed or cast onto a spacer provided in the enzyme chamber 7 as described above.

The form of the reactor used in the method of the present invention is as follows:
It is not limited to the flat membrane type as shown in Figures 1 and 2, but various forms are possible, such as a tube type as shown in Figure 3 and a spiral type as shown in Figure 4. It's not something you do. In the tubular reactor shown in FIG. 3, 1 and 2 are porous materials with an enzyme chamber 4 between them, a reactant chamber 3 inside a tubular membrane 1 made of porous material, and a tubular membrane 1 made of porous material. membrane 2
There is a reactant chamber 5 on the outside of the chamber, and 6 is an outer frame.
In the spiral type reactor shown in FIG. 4, a spacer 2 in which an enzyme is placed is sandwiched in a space between membranes 1 and 3 of porous material, and this forms an enzyme chamber. The membranes 1 and 3 made of porous material are sandwiched between two outer frames 4, which are wound in a spiral, and between the outer frame 4 and each of the membranes 1 and 3 made of porous material. forming a reactant chamber.

One feature of the present invention is that, like immobilized enzymes, enzymes and microorganisms can be easily removed from the reaction system;
Since the enzymes and microorganisms are not immobilized in the porous material, if the enzymes and microorganisms become inactivated, only the enzymes and microorganisms can be replaced without replacing the porous material. Therefore, loss of expensive porous material can be prevented. In addition, as mentioned above, if there are any inhibitors to enzymes or microorganisms, the amount of these inhibitors given to the enzyme reaction system or microorganism reaction system can be controlled by controlling the material and pore size of the porous membrane. or, if a product causes inhibition, it becomes possible to quickly remove this product from the reaction system. Therefore, it becomes possible to suppress deactivation of enzymes and microorganisms and reuse them. For example, in the fat and oil decomposition reaction by lipase,
Water, which is a reaction substrate, is a factor in deactivating lipase, and if the amount of water becomes too large, the enzyme will be significantly deactivated. Therefore, by controlling the amount of water that permeates through the porous material, only the amount of water necessary for the reaction can be supplied to the reaction system. Therefore, it is possible to suppress the deactivation of lipase.

Another major feature of the method of the present invention is that the reaction products can be separated simultaneously with the reaction. For example, in a fat and oil decomposition reaction using lipase, separation of fatty acids and glycerin, which are reaction products, can be performed simultaneously with the reaction.

Furthermore, the rate of enzymatic reaction or microbial reaction in the bioreactor according to the present invention is determined by the rate of permeation through a membrane of a porous material such as a substrate (reactant),
It is highly dependent on the surface area of the membrane of porous material. Therefore, in order to increase the reaction rate, it is important to select a porous material that has a high permeation rate, such as a substrate. Furthermore, since the surface area of the membrane of porous material has a large influence on the reaction rate, it is also possible to significantly shorten the reaction time by increasing the surface area.

As described above, the bioreactor according to the present invention is a method of macroscopically immobilizing enzymes and microorganisms between porous materials, and while enzymes and microorganisms can be easily removed from the reaction system, enzymes and microorganisms are This is a completely new reaction method that has advantages not seen in conventional methods using immobilized enzymes, such as utilization, prevention of deactivation, and simultaneous reaction and separation, and its industrial usefulness. is very large.

〔Example〕

Examples of the present invention will be described below, but the present invention is not limited to these Examples.

Example 1 Ten polyacrylonitrile porous membranes (ultrafiltration membrane, molecular weight cutoff 20,000) and Teflon porous membranes (pore size 0.1 μ) each having an area of 0.02 m ² were prepared. Suspend 2g of fat-and-fat decomposition enzyme (lipase) in a small amount of soybean oil, cast approximately 1/10 of it onto the spacer between the two membranes, and set it so that it is sandwiched between the two membranes, as shown in Figure 2. A flat membrane reactor was constructed. However, although the enzyme solution in FIG. 2 is circulated by a pump, here a method was used in which it was cast onto a spacer and left standing.

400 g of soybean oil and 400 g of water were circulated so that the soybean oil came into contact with the Teflon membrane and the water came into contact with the polyacrylonitrile membrane. The temperature was maintained at 30°C in a constant temperature bath to carry out the decomposition reaction of soybean oil with lipase.

After 24 hours, the decomposition rate of soybean oil was 70%, while the glycerin concentration in water was 7%. Furthermore, the glycerin in the oil and the fatty acid in the water were each 0.1% or less.

From this result, soybean oil and water permeate through the porous membrane and react in the central enzyme chamber, and after the reaction, fatty acids go to the oil side, glycerin goes to the water side, and then permeates through the porous membrane and is diffused and separated. I can see that. In this way, simultaneous reaction and separation operations were possible.

Example 2 The enzyme used in Example 1 was left as is, and the fatty acid solution and glycerin aqueous solution after the reaction were extracted, and then 400 g of fresh soybean oil and 400 g of water were fed in exactly the same manner as in Example 1. The soybean oil decomposition rate after 24 hours was 70%, the same as in Example 1.

Furthermore, 400 g each of fresh soybean oil and water were fed again in the same manner, leaving the enzyme as it was. In the third test, a soybean oil decomposition rate of 70% was obtained in exactly the same manner after 24 hours.

In this way, there was no enzyme deactivation in this bioreactor.

On the other hand, the reaction could be carried out efficiently by simply extracting the liquid and feeding fresh oil and water while leaving the enzyme as is.

It can be seen that there is no need for a step to separate the enzyme from the system.

Example 3 The conditions were the same as in Example 1, but 10 polyvinyl chloride membranes (surface area 0.02 m ² ) with a pore size of 2.5 μm were used as the porous membranes in contact with oil. The same polyacrylonitrile membrane as in Example 1 was used as the porous membrane in contact with water. Under the same conditions as Example 1, 400 g of soybean oil,
400 g of water was circulated to carry out an oil and fat decomposition reaction.
In addition, with the PVC membrane, water diffusion toward the oil side was observed, so a differential pressure of approximately 0.03 Kg/cm ² was applied to the oil side.
Prevents water from entering the oil side.

After 24 hours, the soybean oil decomposition rate was 80%, and the glycerin concentration in water was 8.1%. Furthermore, after 48 hours, a soybean oil decomposition rate of 90% and a glycerin concentration in water of 9% were obtained.

As described above, when compared with Example 1, it was found that the reaction rate differed depending on the membrane material and pore size.

Example 4 The same porous membranes as in Example 1 were used, and the number of membranes was five each. When 400 g of soybean oil and 400 g of water were sent in the same manner, a decomposition rate of 44% was obtained after 24 hours.

It can thus be seen that the reaction rate is largely dependent on the membrane area.

[Brief explanation of the drawing]

FIG. 1 is a diagram showing the configuration of the apparatus used in the present invention, in which 1 and 2 are porous materials, 3 and 5 are reactant chambers, 4 is an enzyme chamber, and 6 is an outer frame. FIG. 2 is an explanatory diagram of a flat membrane type reactor used in the present invention, FIG. 3 is a schematic diagram of a tube type reactor, and FIG. 4 is a schematic diagram of a spiral type reactor.

Claims (1)

[Claims] 1. In an enzymatic reaction or microbial reaction involving two or more types of substances, a set of two porous materials through which these enzymes or microorganisms cannot pass, and an inner surface sandwiched between these porous materials. An enzyme or a microorganism, or an immobilized enzyme or an immobilized microorganism is present in the space of the porous material, and a substance involved in the above reaction is present in the space on both sides of the two porous materials. An enzyme and microorganism reaction method characterized in that the reaction is carried out in the inner space by allowing these substances to pass through the inner space. 2. Two porous materials, one of which allows only part of the substances involved in the reaction and only part of the products after the reaction to pass through, and the other part of which participates in the reaction. The method according to claim 1, which has a property of allowing only the remaining substances and the remaining reaction products to easily pass through. 3. The method according to claim 1 or 2, wherein a plurality of sets of two porous materials are arranged in sequence so that the porous materials of the same type face each other. 4. The method according to claim 1, 2, or 3, wherein the substances involved in the reaction are water and oil, and the enzymatic reaction is a hydrolysis reaction of fats and oils using lipase as an enzyme.