JPH07313163A - Method for continuous reaction using biocatalyst - Google Patents

Abstract

PURPOSE:To sufficiently carry out an enzymic reaction while stably maintaining the activities of a biocatalyst for a long period by performing the initial reaction of the enzymic reaction in a completely mixing type reactional vessel containing the biocatalyst therein and completing the enzymic reaction in a packed column or a fluidized bed type reactor filled with the immobilized biocatalyst. CONSTITUTION:This method for continuous reaction using a biocatalyst is to carry out the initial reaction of an enzymic reaction in a completely mixing type reactional vessel containing a biocatalyst therein, properly performing the condition control such as temperature, preventing the temperature from rising and an enzyme in the biocatalyst from being inactivated and subsequently completing the enzymic reaction in a packed column or a fluidized bed reactor filled with the immobilized biocatalyst. Thereby, the reactional time is shortened or the amount of the biocatalyst is reduced while preventing the size of the reactional equipment from increasing.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for efficiently carrying out a reaction in a method for producing various useful substances by reacting a biocatalyst such as microbial cells or a crushed product thereof or an enzyme with a substrate. It is about.

[0002]

2. Description of the Related Art Biocatalysts such as microorganisms and enzymes have high substrate specificity and can be efficiently catalyzed at room temperature and atmospheric pressure, so that they have been used for producing various useful substances in recent years. . In addition, by binding the enzyme or microbial cells having enzyme activity to a carrier insoluble in the solvent of the reaction or comprehensively wrapping with the carrier or partitioning with an ultrafiltration membrane, etc., so that it does not substantially go out of the reaction system. As a result, the biocatalyst is charged into a reactor, the substrate solution is continuously supplied to the reaction to carry out the reaction, and the product is recovered from the reaction solution flowing out by various methods. ing.

However, in a reaction using a biocatalyst, the activity of the reaction is lowered by the reaction for a long period of time, so that the concentration of the residual substrate in the liquid flowing out from the reactor is increased and the purity and production of the product are increased. In consideration of cost and the like, under the present circumstances, it is necessary to replace the biocatalyst after carrying out the reaction for a certain period of time. Such a decrease in activity is due to the fact that the biocatalyst is composed of proteins,
Degradation due to factors such as pH change of the solution, reaction solution concentration, and impurities in the solution is unavoidable because it is unavoidable, and the development of reaction methods and devices in which biocatalyst activity is less likely to occur is required. .

Particularly when the reaction by the biocatalyst is an exothermic reaction, the activity and stability of the biocatalyst are likely to be deteriorated. Therefore, in order to avoid the activity decrease, it is necessary to use a reactor which can easily remove the heat of reaction. It is necessary to devise it. The method of producing a useful substance by using an immobilized biocatalyst has technical characteristics such as easy separation of the catalyst and product, and repeated use of the catalyst, which is advantageous for continuous reaction by the biocatalyst. It is considered to be a reaction mode.

[0005] However, when the immobilized biocatalyst is packed in a cylindrical reactor or a fluidized bed reactor for use in the reaction, it is difficult to cool it efficiently, and if the cooling is insufficient, the reaction of the biocatalyst will occur. There is a problem that the activity or the life of the biocatalyst is shortened. In particular, when the reaction amount becomes large, the amount of heat generated by the reaction also becomes large, which makes it difficult to control the temperature in the reactor and increases the possibility that the activity or stability of the biocatalyst is reduced.

[0006]

Therefore, according to the present invention, by appropriately controlling the conditions such as the temperature in the initial stage of the reaction, the enzyme reaction is sufficiently performed while maintaining the activity of the biocatalyst for a long time. Methods and apparatus are provided for doing so.

[0007]

In order to solve the above-mentioned problems in the present invention, as a result of intensive studies on the development of a continuous reaction method using a biocatalyst, as a result, a complete mixing type reaction vessel containing a biocatalyst was obtained. Reaction product obtained by using two or more reactors consisting of a packed column or a fluidized bed reactor filled with immobilized biocatalyst and using a completely mixed reaction tank containing biocatalyst The size of the reaction tank is reduced by passing the filtrate containing the liquid to a packed column or a fluidized bed reactor filled with immobilized biocatalyst to complete the reaction,
Further, they have found that it is possible to easily control factors such as heat generation in the reaction system, which cause a decrease in the activity or stability of the biocatalyst, and have developed the present invention.

Therefore, the present invention provides a method for producing a useful substance by continuously converting a substrate into a useful substance by an enzymatic reaction using a biocatalyst. (1) In a completely mixed reaction tank, the biocatalyst and the substrate By reacting with a substrate reaction solution containing the compound in a completely mixed state to convert a part of the substrate into a useful substance to obtain an intermediate reaction liquid containing a biocatalyst, an unreacted substrate and a useful substance. (2) The intermediate reaction liquid is separated into a biocatalyst and an intermediate substrate reaction liquid containing an unreacted substrate and a useful substance, and the separated biocatalyst is reused in the step (1); And (3) converting the unreacted substrate in the intermediate substrate reaction solution into a useful substance by passing the intermediate substrate reaction solution through a packed column or a fluidized bed reactor filled with an immobilized biocatalyst, which is useful. Including substance It provides a method, comprising the steps; final reaction solution obtain composed of.

According to one embodiment of the present invention, the separation in the step (2) is carried out by filtration at the outlet of the complete mixing type reaction tank to obtain an intermediate reaction substrate liquid containing an unreacted substrate and a target substance. Take out from the complete mixing type reaction tank. According to another aspect of the present invention, the separation in the step (2) is performed after the intermediate reaction solution is taken out from the completely mixed reaction tank, and the separated biocatalyst is returned to the completely mixed reaction tank. The intermediate reaction substrate reused and separated in step (1) is used in step (3). According to one aspect of the present invention, the reaction in step (1) is an exothermic reaction, and the temperature of the reaction liquid in the completely mixed reaction tank is kept constant by cooling. According to one embodiment of the present invention, the biocatalyst in the completely mixed reaction tank in step (1) is an immobilized biocatalyst, and the biocatalyst in the packed column or fluidized bed reactor in step (3) is immobilized. The biocatalyst is of the same type.

The present invention further provides (1) a complete mixing type reaction vessel containing a biocatalyst in a continuous enzyme reaction apparatus, wherein the biocatalyst and a substrate medium containing a substrate are mixed to prepare a substrate For converting a part to useful substances,
(2) means for separating the biocatalyst from the intermediate reaction medium obtained in (1), (3) a packed column or a fluidized bed reactor filled with immobilized biocatalyst, wherein the intermediate reaction medium An apparatus for converting an unreacted substrate therein to a useful substance is provided. According to one aspect of the present apparatus, the biocatalyst in the completely mixed reaction tank in step (1) is the immobilized biocatalyst. Further, according to a preferred aspect of the present invention, the complete mixing type reaction tank is a tank equipped with a stirrer or an air lift stirring tank. According to a preferred aspect of the present invention, the separating means is a filter or a centrifuge.

[0011]

EFFECTS OF THE INVENTION According to the present invention, in the first reactor into which a high-concentration substrate flows, the reaction rate is high because the substrate concentration is high, and therefore the calorific value is large, and the loss of biocatalyst such as a change in the pH of the liquid is lost. Although the factors that lead to activity also become large, the control of factors that lead to a decrease in the activity or stability of various biocatalysts such as temperature control and pH control is better than in packed columns or fluidized bed reactors because it is a completely mixed reaction tank. It also has the characteristic that it is easy to do.

[0012] However, there are problems that the reaction efficiency is poor to complete the reaction using only the complete mixing type reaction tank, the reaction equipment becomes large, and the versatility is poor. Therefore, without completing the reaction in the complete mixing type reaction tank, the reaction liquid in the middle of the reaction in which the reaction product and the raw material are mixed is flowed into the packed column or the fluidized bed type reactor filled with the immobilized biocatalyst to carry out the reaction. It has been found that the above problems can be solved by completing them.

In this case, since the reaction liquid flowing into the packed column or the fluidized bed type reactor packed with the immobilized biocatalyst is a solution having a low substrate concentration, the calorific value is also small, and therefore the temperature control is performed. It is possible to prevent the decrease in the activity or stability of the biocatalyst due to heat generation and the like. In addition, the change in the liquid in the reaction tank, which leads to a decrease in the activity of the biocatalyst such as pH, is also reduced. Further, the reaction time can be shortened because the liquid during the reaction is circulated, or the amount of the biocatalyst to be charged can be reduced.

[0014]

DETAILED DESCRIPTION The embodiments of the present invention will be described below, but the present invention is not limited to such embodiments. The completely mixed reaction tank used in the present invention is, for example, a so-called stirred tank equipped with a stirrer, and is an apparatus for completely mixing and reacting so that all compound concentrations in the liquid become completely uniform. Alternatively, it may be a so-called air-lift type reaction tank in which stirring is performed by blowing gas. The above reaction tank may be a single reaction tank or a plurality of reaction tanks connected in series or in parallel. Further, if necessary, a storage tank can be provided at the connecting portion of the reaction tank.

Further, it is desirable that the reaction vessel be equipped with a jacket or a cooling coil so that the reaction vessel can be cooled. It is also possible to insert a heat exchanger between the reaction tanks. As the biocatalyst used for the reaction in the complete mixing type reaction tank, any biocatalyst can be used as long as it can be used for the reaction with the substrate raw material. In this case, as the biocatalyst, microbial cells can be used as they are, or microbial cell crushed products obtained by physically or biochemically crushing by ultrasonication, grinding, freeze-thawing, enzyme treatment, and the like, and microbial cells. Alternatively, it is also possible to immobilize and use an appropriate natural polymer such as polyacrylamide, alginic acid, κ-carrageenan, or a synthetic polymer as a carrier, which is obtained by disrupting bacterial cells or an enzyme extracted from bacterial cells.

In the reaction in the reaction vessel, the reaction conditions in the reaction vessel may be set in consideration of the activity and stability of the biocatalyst to be used, and the conditions relating to the activity and stability of the biocatalyst are: Examples include reaction temperature, pH of reaction solution, substrate concentration and type, concentration and type of product, stirring speed, reaction time, and the like. The reaction in the complete mixing type reaction tank, for example, continuously introducing the substrate medium into this reaction tank,
It can be carried out by removing the intermediate reaction liquid from the reaction tank at the same rate. However, this operation does not have to be continuous, and for example, the introduction of the substrate medium and the withdrawal of the intermediate reaction medium can be performed simultaneously or intermittently with a time lag.

Means for separating the intermediate reaction liquid into a biocatalyst and an intermediate medium containing an unreacted substrate and the produced useful substance is a filtration means such as a filtration membrane (filter membrane) at the outlet of the complete mixing type reaction tank. ) Is provided and only the liquid is taken out from the complete mixing type reaction tank. Alternatively, the intermediate reaction liquid containing the biocatalyst may be once taken out from the complete mixing type reaction tank and then solid-liquid separated. As the separating means in this case, a commonly used solid-liquid separation device such as a filtration device or a centrifugal separation device can be used. The separated biocatalyst is returned to the completely mixed reaction tank by a conventional means such as a pump.

The filtrate containing the reaction product obtained in the complete mixing type reaction tank is continuously or discontinuously passed through a packed column or a fluidized bed type reactor packed with immobilized biocatalyst to complete the reaction. . It is preferable to use the packed tower or the fluidized-bed reactor equipped with a cooling device such as a jacket or a cooling coil.

A known method can be adopted for the preparation of the immobilized biocatalyst such as the immobilized microbial cells or the immobilized enzyme, and the method of filling the reaction tank can also be the known method. The number of packed towers or fluidized-bed reactors may be one or more. If the number is appropriate depending on the type of reactor, volume, feed rate of influent, reaction temperature, type of immobilized biocatalyst, etc. Good. When using a plurality of reaction tanks, the reaction tanks can be connected in series or in parallel. The flowing method of the inflow liquid may be either a downflow type or an upflow type.

In carrying out the method of the present invention, the reaction progress rate is defined as the composition (raw material, product concentration) of the influent from the completely mixed reaction tank described above, the amount of immobilized biocatalyst, the reaction temperature, and the flow rate. (Especially linear velocity) is affected by others,
For example, in the case of the reaction by the column method, it is easy to find the optimal condition for increasing the reaction progress rate to 100% by appropriately adjusting the flow rate of the inflow solution according to the amount of the immobilized biocatalyst to be packed. is there.

As described above, according to the present invention, in a continuous reaction method using a biocatalyst, when a useful substance is produced by an enzymatic reaction, a complete mixing type reaction tank to which a biocatalyst is added and a packed biocatalyst are filled. A packed column in which two or more reactors consisting of towers or fluidized bed type reactors are used and a reaction is carried out using a complete mixing type reaction tank, and a filtrate containing the obtained reaction product is packed with an immobilized biocatalyst. Alternatively, since it is characterized by conducting the fluidized bed reactor to complete the reaction,
A high-concentration substrate solution can be continuously reacted in a small-scale reaction tank, and the activity of the biocatalyst such as heat generation and pH change during the initial reaction and the factors that reduce stability can be easily controlled, so It has the advantage that the frequency of catalyst replacement can be reduced.

[0022]

The present invention will be described in detail below with reference to examples, but the present invention is not limited thereto. The reaction product was analyzed by liquid chromatography. Example 1. Fumaric acid 20 in 2L jar fermenter
g, 1 potassium phosphate 1 g, magnesium sulfate heptahydrate 0.5 g, yeast extract 20 g, corn steep liquor 20 g were dissolved in water and charged with 1 L of a medium whose pH was adjusted to 6.8 with ammonia, followed by sterilization Esc cultivated by adding 50 ml of the same medium to a 500 ml shake flask
herichia coli ATCC 11303 was inoculated and cultured at 37 ° C. with aeration and stirring.

When the fumaric acid in the medium disappeared, acetic acid was added to the cell culture solution to adjust the pH to 5 and left at 45 ° C. for 1 hour, and then the culture solution was centrifuged to separate the cells. did.
The cells were suspended in 50 mM phosphate buffer (pH 7) containing 1 mM magnesium sulfate, and the cells were disrupted using a French press. Using the above buffer, the total protein content is 5
Approximately 1.5 cell disruption solution adjusted to a concentration of g / L
Ion exchange resin Duolite A-7 (US Diamond Shamrock Chemical) 0.5 L was added to L and stirred at 4 ° C. for 24 hours to adsorb the enzyme in the disrupted cell suspension to the ion exchange resin. 14. 1L of ion exchange resin
3 g of enzyme protein containing aspartase activity was adsorbed.

As a reaction solution for the L-aspartic acid formation continuous reaction, an aqueous solution of ammonium fumarate containing 200 g of fumaric acid and 0.2 g of magnesium sulfate heptahydrate in 1 L (pH was adjusted to 8.3 with aqueous ammonia). Was used. Equipped with stirrer, equipped with inlet and outlet of reaction liquid, total volume 50
100 ml of the immobilized enzyme was placed in a 0 ml complete mixing type reaction vessel with an outer tow, and 100 ml of the above reaction solution was added while keeping the reaction temperature at 37 ° C by circulating hot water to the outside under stirring.
The reaction solution was added at a rate of h, and at the same time, an equivalent amount of the reaction solution was withdrawn.
Then, the effluent from the complete mixing type reaction tank of the first tank was kept warm at 37 ° C by circulating warm water in the jacket. 100 on immobilized enzyme column (25φ x 500 mm, filling resin amount 200 ml)
The liquid was passed at a rate of ml / h (flow rate S.V. = 0.5).

The effluent of the first reaction tank and the effluent of the second reaction column were appropriately sampled, and the results of measuring the concentration of fumaric acid remaining in the liquid and the concentration of L-aspartic acid produced are shown below.

[0026]

[Table 1]

Example 2. Acinetobacter tartarogenesu (Acinetobacter tartarog
enes ATCC 31105) per liter, 5 g of disodium cis-epoxysuccinate, 3 g of ammonium sulfate, 1.5 g of potassium phosphate, 1.5 g of disodium phosphate, 0.5 g of magnesium sulfate-7-hydrate, iron sulfate-7-water Inoculate 100 ml of a liquid medium (pH 6.2) containing 10 mg of salt, 10 mg of calcium chloride / dihydrate, 20 mg of manganese sulfate / tetrahydrate, and 0.1 g of yeast extract at 30 ° C, 2
After shaking culture for 4 hours, this was inoculated into a 5 L jar fermenter charged with 3 L of the medium having the same composition as the above, and aeration stirring culture was carried out at 30 ° C. When the concentration of the organic acid in the medium dropped to 0.1% or less, the culture solution was centrifuged to separate the cells. After suspending the cells in 50 mM phosphate buffer (pH 7), the cells were disrupted using a French press.

Approximately 1.5 L of the disrupted bacterial cell solution adjusted to a total protein amount of 5 g / L using the above buffer solution, 0.5 L of ion exchange resin Duolite A-7 (US Diamond Shamrock Chemical) For 24 hours at 4 ° C.
Stirring was carried out to allow the ion exchange resin to adsorb the enzyme in the disrupted cell suspension. 18.8 g of the enzyme protein having a hydrolytic activity was adsorbed per 1 L of the ion exchange resin.

As a reaction solution for the L-tartaric acid production continuous reaction, an aqueous disodium cis-epoxysuccinate solution (pH 8) containing 200 g of cis-epoxysuccinic acid in 1 L was used. 100 ml of the above-mentioned immobilized enzyme was placed in a total mixing type reaction vessel equipped with a stirrer, equipped with an inlet and an outlet for the reaction solution and having a total volume of 500 ml, and the hot water at 30 ° C. was circulated in the cooling coil under stirring. While maintaining the reaction temperature at 30 ° C., the above reaction solution was added at a rate of 100 ml / h, and at the same time, an equivalent amount of the reaction solution was withdrawn. Then, the effluent from the complete mixing type reaction tank of the first tank was circulated with warm water through the jacket.
The temperature was kept at 0 ° C. Immobilized enzyme column (30φ x 500m
m, and the amount of the filled resin was 300 ml) at a rate of 100 ml / h (flow rate SV = 0.33).

The effluent of the first reaction tank and the effluent of the second reaction column were sampled, and the concentration of cis-epoxysuccinic acid remaining in the liquid and the concentration of L-tartaric acid produced were measured. As a result, the generated L- in the effluent of the first tank
The tartaric acid concentration and the residual cis-epoxysuccinic acid concentration were 78.2% and 20.9%, respectively, and the produced L-tartaric acid concentration and the residual cis-epoxysuccinic acid concentration in the effluent of the second tank were 99.6% and 0, respectively. It was 0.35%.

Continuation of front page (51) Int.Cl. ⁶ Identification code Office reference number FI technical display area C12R 1:01) (C12P 13/20 C12R 1:19)

Claims (12)

[Claims] 1. A method for producing a useful substance by continuously converting a substrate into a useful substance by an enzymatic reaction using a biocatalyst, comprising: (1) including a biocatalyst and a substrate in a completely mixed reaction tank. (2) by reacting the substrate reaction liquid consisting of the above in a completely mixed state to convert a part of the substrate into a useful substance to obtain an intermediate reaction liquid containing a biocatalyst, an unreacted substrate and a useful substance; The intermediate reaction liquid is separated into a biocatalyst and an intermediate substrate reaction liquid containing an unreacted substrate and a useful substance, and the separated biocatalyst is reused in the step (1); and (3) ) By passing the intermediate substrate reaction solution through a packed column or a fluidized bed reactor filled with an immobilized biocatalyst, the unreacted substrate in the intermediate substrate reaction solution is converted into a useful substance to contain the useful substance. Final reaction consisting of Obtaining a liquid; a method comprising the steps. 2. The method according to claim 1, wherein the biocatalyst used in the steps (1) and (2) is immobilized and used. 3. The separation of the step (2) is carried out by filtration at the outlet of the completely mixed reaction tank, and an intermediate reaction substrate liquid containing unreacted substrate and a target substance is taken out from the completely mixed reaction tank. The method according to claim 1 or 2, characterized in that: 4. The separation of the step (2) is performed after the intermediate reaction solution is taken out of the complete mixing reaction tank, and the separated biocatalyst is returned to the complete mixing reaction tank to obtain the biocatalyst in the step (1). Reused and separated intermediate reaction substrate is step (3)
The method according to claim 1, wherein the method is used in. 5. The reaction in step (1) is an exothermic reaction, and the temperature of the reaction solution in the complete mixing type reaction tank is kept constant by cooling. The method described in the section. 6. The immobilized biocatalyst in the completely mixed reaction tank in step (1) and the immobilized biocatalyst in the packed column or fluidized bed reactor in step (3) are of the same type. The method according to any one of claims 1 to 5. 7. The method according to claim 1, wherein the reaction is a production reaction of L-aspartic acid from fumaric acid using aspartase or a substance containing aspartase activity. 8. The reaction for producing L-tartaric acid from cis-epoxysuccinic acid using a tartaric acid epoxidase or a tartaric acid epoxidase activity-containing substance, as claimed in claim 1.
The method according to 6. 9. A continuous enzyme reaction apparatus, comprising: (1) a completely mixed reaction tank containing a biocatalyst,
For mixing the biocatalyst with a substrate medium containing a substrate to convert a part of the substrate into a useful substance, (2) the biocatalyst, and the intermediate reaction medium obtained in (1) above. A device for separating, (3) a packed column or a fluidized bed reactor packed with an immobilized biocatalyst for converting an unreacted substrate in the intermediate reaction medium into a useful substance. 10. The device according to claim 9, wherein the biocatalyst of the device (1) (2) is immobilized and used. 11. The apparatus according to claim 9, wherein the complete mixing type reaction tank is a tank equipped with a stirrer or an air lift stirring tank. 12. The device according to claim 9, wherein the separating means is a filter or a centrifuge.