JPH04335881A - Enzyme immobilization type bioreactor - Google Patents

Abstract

PURPOSE:To provide the subject device which comprises circulating lines between a reactor and a substrate storage tank and between the tank and a humidifier, respectively, so as to circulate a reaction solution, and capable of uniformly maintaining moisture in the column of the reactor. CONSTITUTION:In the bioreactor, a circulating line 7 is installed between a reactor 2 filled in with a carrier having an enzyme immobilized thereon and a substrate tank 1 storage, and further a circulating line 10 is installed between the tank 1 and a humidifier 8 for humidifying the substrate. A reaction solution is circulated in the lines so ass to supply moisture for the reaction solution in the tank 1 and further uniformize the moisture in the reactor 2.

Description

[Detailed description of the invention]

0001

[Field of Industrial Application] The present invention relates to an enzyme-immobilized bioreactor that is used to perform hydrolysis reactions, dehydration condensation reactions, transfer reactions, etc. of organic substances such as fats and oils using immobilized enzymes. .

0002

BACKGROUND OF THE INVENTION Enzyme-immobilized bioreactors as described above have been widely used in various fields. For enzyme reactions in these bioreactors, there is an optimum amount of water in the reaction field, so controlling the amount of water plays an important role in the progress of the reaction. However, conventionally, the reaction was usually carried out by passing the substrate through a reactor filled with immobilized enzyme in one pass.
For example, in a reactor for causing a hydrolysis reaction, there is a problem in that there is a shortage of water in the column mainly at the outlet side of the reactor that comes into contact with a new substrate, making it impossible to utilize the entire column effectively. Similarly, when a dehydration condensation reaction is caused, water in the column becomes excessive around the outlet side of the reactor, making it impossible to utilize the entire column effectively.

[0003] Furthermore, a method has been proposed in which a moisture adjustment column is installed in front of the reactor in which the hydrolysis reaction is carried out, and the substrate is supplied with appropriate moisture by this moisture adjustment column before being supplied to the reactor. (For example, JP-A-1-1379
Publication No. 88). However, even in this method, it was not possible to prevent the above-described gradient in the amount of water inside the reactor from occurring.

0004

[Problems to be Solved by the Invention] The present invention solves the above-mentioned conventional problems, and it is an object of the present invention to solve the above-mentioned conventional problems, and to solve the problems of the present invention. This was completed in order to provide an enzyme-immobilized bioreactor that allows reactions to proceed while maintaining water content uniformly throughout the column inside the vessel.

[0005]

[Means for Solving the Problems] A first invention made to solve the above problems is to provide a system between a reactor filled with a carrier having an enzyme immobilized on its surface and a tank storing a substrate. A circulation line is formed between the substrate storage tank and the humidifier that humidifies the substrate, and the reaction liquid is circulated to make the moisture inside the reactor uniform. That is. Moreover, the second invention is
a reactor filled with a carrier on which enzymes are immobilized;
A circulation line is formed between the tank that stores the substrate, and a circulation line is also formed between the tank for storing the substrate and the dehydration device that dehydrates the substrate to circulate the reaction liquid and dehydrate the inside of the reactor. It is characterized by uniform moisture content.

[0006]

[Operation] In the enzyme-immobilized bioreactor of the first invention, the substrate in the substrate storage tank is gradually hydrolyzed by the immobilized enzyme while repeatedly circulating inside the reactor through the circulation line. The reaction solution in the substrate storage tank is also circulated between the humidifier and the moisture content is adjusted. For this reason, as will be clear from the later examples, compared to the conventional one-pass type device, the water content in the column of the reactor can be maintained uniformly throughout.

Furthermore, in the enzyme-immobilized bioreactor of the second invention, the reaction solution in the substrate storage tank is gradually dehydrated and condensed by the immobilized enzyme while repeatedly circulating inside the reactor through the circulation line. At the same time, the reaction solution in the substrate storage tank is also circulated to and from the dehydrator to adjust its moisture content. Therefore, the water content in the column of the reactor is maintained uniformly. Note that, as in the embodiment, a humidifying device and a dehydrating device can be incorporated into the same device and used properly depending on the reaction.

Next, examples of the present invention will be shown. In FIG. 1, 1 is a tank for storing substrates, and 2 is a reactor filled with immobilized enzyme. A tank 1 for storing substrates is equipped with a stirrer 3, a water concentration meter 4, and a thermometer 5, and the reaction temperature inside the tank 1 is maintained at 30°C. Inside reactor 2, carriers with enzymes immobilized on their surfaces are packed vertically in multiple stages, but such carriers are at risk of being deformed or destroyed when the substrate is circulated at high speed by a pump. It is necessary to use a carrier with a small amount of carbon, and carriers made of ceramics, glass, metal, etc. can be used. Among these, it is desirable to use calcined sepiolite as a carrier containing a large amount of enzyme immobilization material. In the example, immobilized lipase prepared by immobilizing 20 mg of lipase per 1 g of calcined sepiolite and supplied with 400 mg/g of water to a carrier was packed into a reaction column (capacity: 3 liters) with a movable adjuster, and the reaction temperature was increased. is maintained at 30°C. A pump 6 is also installed between the substrate storage tank 1 and the reactor 2.
A circulation line 7 is formed.

[0009] Reference numeral 8 denotes a humidifying device, which consists of a column in which porous ceramic is moistened with water. A circulation line 10 including a pump 9 is formed between the humidification device 8 and the substrate storage tank 1, and the reaction liquid in the substrate storage tank 1 is circulated through the humidification device 8 by this circulation line 10. and hydrated.

Reference numeral 12 denotes a dewatering device, and in the embodiment a flash evaporator is used. This dehydration device 12
A circulation line 14 including a pump 13 is formed between the substrate storage tank 1 and the substrate storage tank 1.
The reaction liquid inside is dehydrated through this circulation line 14 and its moisture content is adjusted.

[Example of hydrolysis reaction: production of oleic acid] Triolein dissolved in hexane at a concentration of 10 g/L was used as a substrate, and 2.
It was supplied to tank 1 for substrate storage with a capacity of 50 liters at a flow rate of 4 L/HR, and after 10 hours, the flow rate was 4 L/Min. A humidifier 8 is supplied by a circulation line 10 containing a pump 9 at a flow rate of
The circulation of substrate was started. Changes in the water concentration within the substrate storage tank 1 after the start of circulation are as shown in FIG. 20 hours after starting the substrate supply, the water-saturated substrate in the tank 1 is pumped at a flow rate of 4 L/Min by the pump 6.
．． pump 16 from inside tank 1 while circulating to reactor 2.
Withdrawal was started at a flow rate of 2.4 L/HR. Figure 3 shows the changes over time in the oleic acid and glycerol concentrations in the drawing liquid. Further, after the continuous operation was completed, the water content of the immobilized lipase packed bed in the reactor 2 was measured by the Karl Fischer method, and the results shown in FIG. 4 were obtained. As shown in FIG. 4, according to the present invention, the water content in the column within the reactor 2 can be maintained uniformly.

[Comparative Example] Next, when the operation of the pump 9 was stopped and the circulation of the reaction liquid to the humidifier 8 was eliminated, the oleic acid and glycerol concentrations in the drawn liquid changed over time as shown in FIG. became. As described above, the oleic acid concentration rapidly decreases due to the lack of water after the start of the reaction. In addition, the water distribution of the immobilized lipase packed bed after the reaction is shown in Figure 6.
As shown in Fig. 4, it can be seen that a significant water shortage has occurred compared to Fig. 4.

[Conventional Example] Next, the flow rate of the pump 6 was set to 2.4.
L/HR was changed, and the reaction liquid coming out of the reactor 2 was directly drawn out in one pass without being circulated into the tank 1. FIG. 7 shows the change over time of the product in the drawn liquid at this time. In this case, the oleic acid concentration tends to gradually decrease. Further, the moisture distribution of the immobilized lipase packed bed after the reaction is as shown in FIG. 8, and when compared with FIG. 4, it can be seen that there is non-uniformity of moisture.

[Example of Dehydration Condensation Reaction: Production of Tristearin] Using the apparatus shown in FIG. 1, tristearin was produced by a dehydration condensation reaction of glycerin and stearic acid. However, the reaction temperature is 40°C. The substrate was glycerin and stearic acid dissolved in hexane to a concentration of 100 mM each, and this was supplied to the substrate storage tank 1 with a capacity of 50 liters by a pump 15 at a flow rate of 1 L/HR, and after 20 hours, 2L/M
in. Circulation of the substrate to the dehydrator 12 was started by the pump 13 at a flow rate of . Changes in the water concentration in the substrate storage tank 1 after the start of circulation are as shown in FIG.

Forty hours after starting the supply of the substrate, the substrate in the tank 1 is pumped through the pump 6 at a flow rate of 2 L/Min. While circulating to the reactor 2, drawing was started from inside the tank 1 by the pump 16 at a flow rate of 1 L/HR. Figure 10 shows the change over time in the tristearin concentration in the drawn liquid. Further, after the continuous operation was completed, the water content of the reaction solution in the immobilized lipase packed bed in the reactor 2 was measured by the Karl Fischer method, and the results shown in FIG. 11 were obtained. In this manner, according to the present invention, the moisture within the reactor 2 can be maintained uniformly throughout the column.

[Comparative Example] Next, when the operation of the pump 13 was stopped and the circulation of the reaction liquid to the dehydrator 12 was eliminated, the concentration of tristearin in the drawn liquid changed over time as shown in FIG. 12. . As described above, the tristearin concentration in the comparative example is lower than that in the example shown in FIG. Further, the water distribution of the reaction solution in the immobilized lipase packed bed after the reaction is as shown in FIG.

[Conventional Example] Next, the flow rate of the pump 6 is set to 1L/H.
R, and the reaction liquid discharged from the reactor 2 was directly withdrawn in one pass without being circulated into the tank 1. Figure 14 shows the change in the product over time. Further, the water distribution of the reaction solution in the immobilized lipase packed bed after the reaction is as shown in FIG. 15, and when compared with FIG. 11, it can be seen that there is significant non-uniformity of water.

[0018]

Effects of the Invention As is clear from the above description of the embodiments, the enzyme-immobilized bioreactor of the present invention forms a circulation line between the reactor and the tank for storing the substrate or reaction solution, and By forming a circulation line between this substrate storage tank and a device for humidifying or dehydrating the substrate or reaction solution, it is possible to maintain uniform water content in the reactor column. The whole can be used effectively. Therefore, the present invention can greatly contribute to the development of industry as an enzyme-immobilized bioreactor that solves the conventional problems.

[Brief explanation of the drawing]

FIG. 1 is a piping system diagram showing an embodiment of the present invention.

FIG. 2 is a graph showing changes in water concentration in a substrate storage tank after the start of circulation in an example of a hydrolysis reaction.

FIG. 3 is a graph showing changes over time in the oleic acid and glycerol concentrations in the drawing liquid in examples of hydrolysis reactions.

FIG. 4 is a graph showing the water content of the immobilized lipase packed bed in the reactor after completion of the hydrolysis reaction.

FIG. 5 is a graph showing changes over time in the oleic acid and glycerol concentrations in the drawn liquid in a comparative example in which circulation of the substrate to the humidifier was eliminated.

FIG. 6 is a graph showing the water distribution of the reaction solution in the immobilized lipase packed bed after the reaction in Comparative Example.

FIG. 7 is a graph showing the change over time of a product in a conventional example.

FIG. 8 is a graph showing the water distribution of the reaction solution in the immobilized lipase packed bed after the reaction in the conventional example.

FIG. 9 is a graph showing changes in water concentration in a substrate storage tank after the start of circulation in an example of a dehydration condensation reaction.

FIG. 10 is a graph showing the change over time in the tristearin concentration in the drawn liquid in an example of a dehydration condensation reaction.

FIG. 11 is a graph showing the water content of the reaction solution in the immobilized lipase packed bed in the reactor after the completion of the dehydration condensation reaction.

FIG. 12 is a graph showing the change over time in the tristearin concentration in the drawing liquid in a comparative example.

FIG. 13 is a graph showing the water distribution of the reaction solution in the immobilized lipase packed bed after the reaction in Comparative Example.

FIG. 14 is a graph showing the change over time of a product in a conventional example.

FIG. 15 is a graph showing the water distribution of the reaction solution in the immobilized lipase packed bed after the reaction in the conventional example.

[Explanation of symbols]

1 Tank for storing substrate 2 Reactor 7 Circulation line 8 Humidification device 12 Dehydration device 14 Circulation line

Claims (2)

[Claims] Claim 1: A circulation line is formed between a reactor filled with a carrier on which an enzyme is immobilized and a tank for storing a substrate, and a humidifier for humidifying the substrate storage tank and the substrate. A circulation line is also formed between the equipment and the
An enzyme-immobilized bioreactor characterized by circulating the reaction solution to make the moisture content in the reactor uniform. 2. A circulation line is formed between a reactor filled with a carrier having an enzyme immobilized on its surface and a tank for storing the substrate, and a dehydration system for dehydrating the substrate and the tank for storing the substrate. A circulation line is also formed between the equipment and the
An enzyme-immobilized bioreactor characterized by circulating the reaction solution to make the moisture content in the reactor uniform.