JPS6178397A - Reaction process of enzyme and microorganism - Google Patents

Abstract

PURPOSE:To carry out the reaction of an enzyme and microorganism, in high efficiency, by placing a pair of membranes made of porous substance impermeable to the enzyme and the microorganism interposing a gap therebetween, charging the enzyme and microorganism in the gap, and supplying a substrate to the outside of the membrane pair, thereby separating the reaction product from the reaction system. CONSTITUTION:For example, a fatty acid and glycerol are produced as reaction product from the oil and water used as a substrate using lipase. In the above process, a polypropylene or polyethylene film 1 is used as the porous membrane passing only the oil and fatty acid, and a film 2 made of cellulose acetate or acrylonitrile polymer is used as the porous membrane passing only water and glycerol. Both films are arranged with a gap 4, microorganism and enzyme are charged to the gap, an oil is supplied to the outer surface 3 of one of the membrane to separate the fatty acid, and water is supplied to the surface 5 of the other membrane to separate glycerol.

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, in an enzymatic reaction or atomized reaction involving two or more types of substances, the present invention provides an enzyme chamber in an inner space (enzyme chamber) partitioned by two membranes made of a porous material that do not allow enzymes or microorganisms to freely pass through. These enzymes and microorganisms are allowed to exist, and substances involved in the reaction are made to exist in the space on both outsides that contact the two porous material membranes, and these substances interface with the porous material membranes to form the inside. The chemical products pass through the membrane of the porous material to the outside space, allowing efficient separation of the products as well as enzymatic or microbial reactions. This invention relates to a reaction method for enzymes and microorganisms using a bioreactor constructed as described above.

[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. In addition, not only a single reaction product but also two or more types of chemical compounds are often produced. In this way, in enzymatic reactions and microbial reactions, not only one reaction substrate but also one reaction product has 2f41! There are many reactions involving substances of a similar or higher class. 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 inhibitory factor. Considering these factors, it is possible to use inhibitory substances in the enzyme reaction system,
Or control the amount given to the microbial reaction system,
Alternatively, it is believed that enzymatic reactions and microbial reactions can be carried out more efficiently if inhibiting factors can be quickly removed from the system. If an actor is used, it is possible to eliminate the step of separating the product since it becomes possible to separate the product at the same time as the breaking reaction. On the other hand, enzymes are generally very expensive, and reusing them is an essential condition for reducing production costs. When considering bioreactors, it is of course important to be able to easily remove enzymes and microorganisms from the reaction system and reuse them.

To date, a bioreactor that satisfies all of these issues has not yet been developed. For example, with immobilized enzymes and immobilized microorganisms, enzymes and microorganisms can be easily separated from the reaction system, or
Although it may be possible to solve problems such as the reuse of enzymes and microorganisms, it is impossible to solve problems such as simultaneous reaction and separation operations and control of inhibitors. Recently, immobilized enzymes and immobilized microorganism immobilized carriers have a product separation function (adsorption capacity).
There have been attempts 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]

The present inventors have developed an enzyme that has various problems as described above.
As a result of intensive research on i-microbial reaction glue actors, we have developed an epoch-making glue actor that can solve these problems.
developed.

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 space on both sides of 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.

In the reaction method according to the present invention, a set of two membranes made of a porous material that does not allow enzymes or microorganisms to freely pass through is used, the enzyme or microorganism is allowed to exist in a space partitioned by these, and the reactant passes through the porous material. It is characterized by undergoing an enzymatic or microbial reaction in the space of the fifth chamber, and passing the reaction product through a porous material to remove it 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 = 6-. - For example, in the lipase decomposition reaction, A+B→C+D (A and B are substances involved in the reaction, hereinafter referred to as substrates. C2D is the product. In the lipase reaction, A-oil, B=water, C- fatty acid,
To explain the enzyme reaction system represented by D=glycerin using FIG. 1, the enzyme is present in the inner space 4 (enzyme chamber) partitioned by porous materials 1 and 2, and Substrate A is present in space 5 (reactant chamber), and substrate B is present in the other outer space 5 (reactant chamber). The porous material 1.2 is impermeable to the enzyme and is constructed in such a way that the substrate A can pass through the porous material 1 and the substrate B can pass through the porous material 2. Therefore, in the glue actor in Figure 1,
Substrate A passed from the respective reactant chambers 3 and 5 to the enzyme chamber 4
, B undergo an enzymatic reaction by the enzyme present in the enzyme chamber 4 to become products C and D. Here, the porous material 1 is selected to be one that allows substrate A to pass through, but not B, and allows only one of the reaction products ○ and D (temporarily assumed to be C) to pass through. 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 formed. For example, in the fat and oil decomposition reaction of lipase, 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, any pore size that does not allow this emulsion to pass through is sufficient, and a pore size of 0.1 to several μm can prevent passage of the enzyme. 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 space 4 in order to prevent the enzyme from being concentrated in one part. Alternatively, the enzyme solution or microbial solution may be circulated into this space, that is, the enzyme chamber 4, using a pump, as will be described later. Note that the material, form, etc. of the spacer provided in the space 4 are not particularly limited.

For example, if a material such as ion-exchange fiber is used as a spacer, the enzyme can be immobilized thereon, and as in this case, the spacer can be used to adsorb enzymes. It is also possible to add new functions.

The substrate present in the reactant chambers 5 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 for promoting diffusion, such as pressure or temperature, to each chamber. Or again. 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 a group of transferases represented by the phosphorolytic reaction of polysaccharides by phosphorylase, a synthesis reaction of malic acid by fumarase, a series of reactions by a group of hydrolytic enzymes such as phosphatase and nucleothiodase, Alternatively, it can be used in enzyme reaction systems involving substances of the 2aI class or higher, 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 Rium glutamieum and the like, glutamic acid is fermented and produced from the substrate glucose, but biotin is required for the growth of baking soda, 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. A reactant chamber 5 is formed between one porous material 9 and a reactant chamber 6 is formed between the other porous material 10, and the substances involved in the reaction are pumped 3a. ,3
It is preferable to circulate the reactants to the respective reactant chambers 5 and 6 at step b. In FIG. 2, enzymes or microorganisms can also be circulated to the enzyme chamber 7 by means of a pump 3. In Figure 2, 1,
2 is a reactant storage container, 4 is an enzyme or microorganism liquid storage container, and 8 is an outer frame. Similarly, the enzymes and microorganisms may be left standing by being sprayed or cast on the spacer provided in the enzyme chamber 7 as described above.

The shape of the reactor used in the method of the present invention is not limited to the flat membrane type shown in Figures 1 and 2, but can also be of various types, such as a tube type as shown in Figure 3 or a spiral type as shown in Figure 4. Any form is possible, and the form is not particularly limited. In the tube W reactor shown in Fig. 3, 1 and 2 are porous materials, and there is an enzyme chamber 4 between them, and a reactant chamber 3 is located inside the tubular membrane 1 made of porous material. There is a reactant chamber 5 outside the tubular membrane 2, and 6 is an outer frame. Fourth
In the spiral reactor shown in the figure, a membrane 1 of porous material,
A spacer 2 in which an enzyme is placed is sandwiched between the spacers 3 and 3, and this forms an enzyme chamber. The membranes 1 and 3 made of porous material are sandwiched between two outer frames 4, and these are wound into a spiral shape.
, 3, forming a reactant chamber between them.

One feature of the present invention is that, like immobilized enzymes, enzymes and microorganisms can be easily removed from the reaction system, but because the enzymes and microorganisms are immobilized on porous materials, they can be easily removed if the enzymes or microorganisms become inactivated. By replacing porous materials, only enzymes and microorganisms can be replaced. Therefore, loss of expensive porous materials can be prevented.

In addition, as mentioned above, if there are inhibitors to enzymes or microorganisms, the amount of these inhibitors applied to the enzyme reaction system or microorganism reaction system can be reduced by controlling the material and pore size of the porous membrane. If the product is controlled or inhibits the reaction, the product can be quickly removed from the reaction system. Therefore, enzymes suppress the inactivation of microorganisms,
It becomes possible to reuse these.

For example, in a lipase decomposition reaction, water, which is a reaction substrate, becomes a factor in deactivating the lipase, and if the amount of water is too large, the enzyme will be significantly deactivated.

Therefore, by controlling the amount of water in the porous material, only the amount of water necessary for the reaction can be provided 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 the enzymatic reaction or microbial reaction in the bioreactor of the present invention largely depends on the permeation rate through the membrane of the porous material such as the substrate (reactant) and the surface area of the membrane of the 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 of the present invention is a method in which enzymes and microorganisms are immobilized macroscopically 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 reuse, prevention of inactivation, and simultaneous reaction and separation, and its industrial utility. Gender is a huge thing.

〔Example〕

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

Example 1 A total of 10 polyacrylonitrile porous membranes (ultra-filter membrane, molecular weight cut off 20,000) and Teflon porous membranes (pore size 0.1 μm) each having an area of 0.02 m2 were prepared. Oil-fat degrading enzyme (lipase) 2f was suspended in a small amount of soybean oil, cast in approximately 1/10 portion onto the spacer between both membranes, and set so that it was sandwiched between the two membranes. A flat membrane reactor was created. However, although the enzyme solution in FIG. 2 is circulated by a pump, here a method was adopted in which it was cast onto a spacer and left standing.

400F of soybean oil and 400F of water were circulated so that the soybean oil was in contact with the Teflon membrane and the water was in 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%, and the glycerin concentration in Shiki 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, the fatty acid solution and glycerin aqueous solution were extracted after the reaction, and fresh soybean oil 400F and water 400F were added to Example-1.
The solution was pumped in exactly the same manner. The soybean oil decomposition rate after 24 hours was 70%, the same as in Example-1.

Furthermore, the enzyme was lysed in the same manner, and 400 tons each of fresh soybean oil and water were fed again. A soybean oil decomposition rate of 70% was obtained after 24 hours in exactly the same manner for the fifth time.

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 5 The conditions were the same as in Example 1, but a polyvinyl chloride membrane with a pore size of 2.5μ (surface area 0) was used as the porous membrane in contact with oil.
．． 02m2) were used. 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, soybean oil 400F and water 400F. was circulated to carry out an oil and fat decomposition reaction. In addition, with the PVC membrane, water diffusion to the oil side was observed, so approximately
o A pressure difference of 3Kt/an2 was applied to prevent 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%. Further, 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 there were differences in the reaction rate 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. Soybean oil 400? When water was fed at 400 V 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. 1 and 2 are porous materials, 3 and 5 reactant chambers, 4td,
6 is the outer frame of the enzyme chamber. Fig. 2 is an explanatory diagram of a flat membrane reactor used in the present invention, Fig. 3 is a tubular reactor,
FIG. 4 is a schematic diagram of a snow-like reactor. Applicant's agent Kaoru Furuya Figure 3 Figure 4 Thousand Threads 11j ``νF (Spontaneous) 1981 107] 31++ Patent 1i'-E# Official Manabu Shiga 1, Indication of Case Patent Application 1987-198778 No. 2, Name of the invention Reaction method of enzymes and microorganisms 3, Relationship with amendment Nagisa 1) Matter 41y revised Applicant (091) Hana-1 Ishiki Stock Company Arrival 4, Representative
Feature 3'1 Claims and (1) Specification of the invention, 1
) 2 4th line from directly below ``membrane'' to ``(for example, II!IriJ
Amendment 11 2. Claim 1 In an enzyme reaction or microbial reaction involving two or more types of substances, two sheets of porous +1 material through which these enzymes or microorganisms cannot pass are defined as 1M1, and these porous materials are Enzymes or microorganisms, 1-1-transforming enzymes, or standardized microorganisms are present in the inner space between the two porous materials; 1. Enzyme and microorganism reaction method I, characterized in that the substances involved in the above reaction are placed in front of each other, and one material is enclosed in a porous hole to allow these substances to pass through, and the reaction is carried out in the inner space.
of,. 2. Two porous materials, one that allows only part of the substances involved in the reaction and only one part of the products after the reaction to pass through, and the other that allows only one part of the reaction products to pass through. The method V according to claim 1, which has P1 quality that allows only the remaining substances among the substances involved and the remaining reaction products to easily pass through. If multiple sets of 32 pieces of porous material are the same type of porous material;
One person according to claim 1 or 2, wherein the I's are arranged in sequence so that they face each other. 4. The method according to claim 1, 2, or 3, wherein the substances involved in the reaction are wood and oil, and the enzymatic reaction is a hydrolysis reaction of fats and oils using lipase as an enzyme.

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 substances involved in the above reaction are allowed to coexist in the spaces 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 sheet allows only a part of the substances involved in the reaction and only one part of the products after the reaction to pass through, and the other sheet allows the substances involved in the reaction to pass through. 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. Claim 1, in which a plurality of sets of two porous materials are arranged in sequence so that the porous materials of the same type face each other.
or the method described in paragraph 2. 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.