JP2897966B2 - Enzyme-immobilized bioreactor - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an enzyme-immobilized bioreactor used for performing a hydrolysis reaction, a dehydration-condensation reaction, a transfer reaction, and the like of an organic substance such as oil and fat using an immobilized enzyme. .

[0002]

2. Description of the Related Art Enzyme-immobilized bioreactors as described above have been widely used in various fields. In the enzymatic reaction in these bioreactors, control of the water content plays an important role in the progress of the reaction because there is an optimum water content in the reaction field. However, in the past, it was common to use a single-pass reaction of the substrate inside a reactor filled with immobilized enzyme.
For example, in a reactor that causes a hydrolysis reaction, there is a problem that water in the column is insufficient around the outlet side of the reactor in contact with a new substrate, and the entire column cannot be used effectively. Similarly, when a dehydration-condensation reaction is caused, the water in the column becomes excessive around the outlet side of the reactor, so that the entire column could not be used effectively.

Further, there has been proposed a method in which a moisture control column is provided in front of a reactor in which a hydrolysis reaction is performed, and the substrate is supplied with appropriate water by the water control column and then supplied to the reactor. (For example, JP-A-1-137988). However, even in this method, it was not possible to prevent the occurrence of the water content gradient inside the reactor as described above.

[0004]

SUMMARY OF THE INVENTION The present invention solves the above-mentioned conventional problems, and is also applicable to a case where a hydrolysis reaction or a dehydration condensation reaction is carried out in a reactor filled with immobilized enzyme. The present invention has been completed in order to provide an enzyme-immobilized bioreactor capable of promoting a reaction while maintaining moisture uniformly throughout the entire column inside the vessel.

[0005]

Means for Solving the Problems A first invention made to solve the above-mentioned problem is that a reactor filled with a carrier having an enzyme immobilized on a surface thereof and a tank for storing a substrate. In addition to forming a circulation line, a circulation line is also formed between the substrate storage tank and the humidifier that humidifies the substrate, and the reaction liquid is circulated to make the water in the reactor uniform. It is assumed that. In the second invention,
A reactor filled with a carrier having an enzyme immobilized on its surface,
A circulation line is formed between the substrate storage tank and the substrate storage tank and a dehydrator for dehydrating the substrate, and a circulation line is formed between the substrate storage tank and the substrate dehydration device. It is characterized by making the water uniform.

[0006]

In the enzyme-immobilized type 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 liquid in the tank for storing the substrate circulates also with the humidifier to regulate the water content. For this reason, as is clear from the following examples, the water content in the column of the reactor can be kept uniform over the whole as compared with the conventional one-pass type apparatus.

Further, 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 tank for storing the substrate circulates also with the dehydration device to regulate the water content. Therefore, the water content in the column of the reactor is kept uniform. As in the embodiment, a humidifying device and a dehydrating device may be incorporated in the same device and used properly according to the reaction.

Next, an embodiment of the present invention will be described. In FIG. 1, 1 is a tank for storing a substrate, and 2 is a reactor filled with immobilized enzyme. The tank 1 for storing the substrate is a stirrer 3,
It has a moisture concentration meter 4 and a thermometer 5, and the inside is maintained at a reaction temperature of 30 ° C. The inside of the reactor 2 is packed vertically in multiple stages with a carrier having an enzyme immobilized on the surface. Such a carrier may be deformed or destroyed when the substrate is circulated at high speed by a pump. It is necessary to use a carrier having a small amount of carrier, and a carrier made of ceramics, glass, metal, or the like can be used. Of these, calcined sepiolite is desirably used as the carrier having a large amount of the enzyme immobilizing agent. In the examples, a fixed lipase prepared by immobilizing 20 mg of lipase per 1 g of calcined sepiolite and supplying 400 mg / g of water to a carrier was packed in a reaction column (3 liter capacity) with a movable adjuster, and the reaction temperature was adjusted. Is maintained at 30 ° C. A circulation line 7 including a pump 6 is formed between the substrate storage tank 1 and the reactor 2.

Reference numeral 8 denotes a humidifier, which comprises a column in which water is moistened in a porous ceramic. A circulation line 10 including a pump 9 is formed between the humidifier 8 and the substrate storage tank 1, and the reaction solution in the substrate storage tank 1 is circulated through the humidifier 8 by the circulation line 10. And be rehydrated.

Reference numeral 12 denotes a dehydrator, and in the embodiment, a flash evaporator is used. A circulation line 14 including a pump 13 is formed between the dehydrator 12 and the substrate storage tank 1, and the reaction solution in the substrate storage tank 1 is dehydrated by the circulation line 14 to remove water. Adjusted.

[Example of hydrolysis reaction: Production of oleic acid] A solution prepared by dissolving triolein in hexane at a concentration of 10 g / L was used as a substrate, and 2.4 L / HR was pumped by a pump 15.
At a flow rate of 50 liters to a substrate storage tank 1 having a capacity of 50 liters, and after 10 hours, 4 L / min. The circulation of the substrate to the humidifier 8 was started by the circulation line 10 including the pump 9 at the flow rate of. Changes in the water concentration in the substrate storage tank 1 after the start of circulation are as shown in FIG. Twenty hours after the supply of the substrate was started, the water-saturated substrate in the tank 1 was pumped by the pump 6 at a flow rate of 4 L / Min. Withdrawing from the tank 1 by the pump 16 at a flow rate of 2.4 L / HR was started while circulating through the reactor 2 with the above. FIG. 3 shows the time-dependent changes in the concentrations of oleic acid and glycerol in the extraction liquid. After the continuous operation, the water content of the immobilized lipase packed layer in the reactor 2 was measured by the Karl Fischer method, and the result as shown in FIG. 4 was obtained. As shown in FIG. 4, according to the present invention, the water in the column in the reactor 2 can be kept uniform.

Comparative Example Next, when the operation of the pump 9 was stopped and the circulation of the reaction solution to the humidifier 8 was stopped, the time-dependent changes in the oleic acid and glycerol concentrations in the withdrawal solution were as shown in FIG. became. As described above, the oleic acid concentration is rapidly decreased due to a shortage of water after the start of the reaction. In addition, the water distribution of the immobilized lipase-packed layer after the reaction is as shown in FIG. 6, and it can be seen that there is a significant water shortage as compared with FIG.

[Conventional Example] Next, the flow rate of the pump 6 is set to 2.4 L / H.
R, and the reaction solution discharged from the reactor 2 was directly extracted in one pass without being circulated into the tank 1. FIG. 7 shows the change over time of the product in the withdrawal liquid at this time. In this case, the oleic acid concentration tends to decrease gradually.
Further, the water distribution of the immobilized lipase-packed layer after the reaction is as shown in FIG. 8, and it can be seen from the comparison with FIG. 4 that there is unevenness in water.

Example of Dehydration-Condensation Reaction Production of Tristearin Tristearin was produced by a dehydration condensation reaction of glycerin and stearic acid using the apparatus shown in FIG. However, the reaction temperature is 40 ° C. The substrate was prepared by dissolving glycerin and stearic acid in hexane so that the concentrations of glycerin and stearic acid became 100 mM, respectively. 2L / Min. Pump at a flow rate of
By 13 the circulation of the substrate to the dehydrator 12 was started. The change in the water concentration in the substrate storage tank 1 after the start of circulation is as shown in FIG.

Forty hours after starting the supply of the substrate, the substrate in the tank 1 was pumped by the pump 6 at a flow rate of 2 L / Min. 1L from inside tank 1 by pump 16 while circulating to reactor 2.
Withdrawal was started at a flow rate of / HR. FIG. 10 shows the change over time in the concentration of tristearin in the extraction liquid. After the continuous operation, when the water content of the reaction solution in the immobilized lipase packed layer in the reactor 2 was measured by the Karl Fischer method, the results as shown in FIG. 11 were obtained. As described above, according to the present invention, the water in the reactor 2 can be maintained uniformly over the entire column.

[Comparative Example] Next, when the operation of the pump 13 was stopped to circulate the reaction solution to the dehydrator 12, the time-dependent change in the concentration of tristearin in the withdrawn solution was as shown in FIG. . Thus, in the comparative example, the tristearin concentration is lower than in the example of FIG. Further, the water distribution of the reaction solution in the immobilized lipase packed layer after the reaction is as shown in FIG.

[Conventional Example] Next, the flow rate of the pump 6 is set to 1 L / HR.
And the reaction solution discharged from the reactor 2 was directly drawn out in one pass without being circulated into the tank 1. FIG. 14 shows changes over time of the product at this time. Further, the water distribution of the reaction solution in the immobilized lipase-packed layer after the reaction is as shown in FIG. 15, and it can be seen from the comparison with FIG. 11 that there is significant non-uniformity of water.

[0018]

As is clear from the description of the above embodiments, the enzyme-immobilized bioreactor of the present invention forms a circulation line between a reactor and a tank for storing a substrate or a reaction solution, By forming a circulation line between the tank for storing the substrate and a device for humidifying or dehydrating the substrate or the reaction solution, the water in the column of the reactor can be uniformly maintained, and the column of the reactor can be maintained. The whole can be used effectively. Therefore, the present invention greatly contributes to industrial development as an enzyme-immobilized bioreactor which has solved the conventional problems.

[Brief description of the drawings]

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

FIG. 2 is a graph showing a change 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 the change over time in the concentrations of oleic acid and glycerol in the withdrawn liquid in an example of the hydrolysis reaction.

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

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

FIG. 6 is a graph showing a water distribution of a reaction solution in an immobilized lipase-packed layer after a reaction in a comparative example.

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

FIG. 8 is a graph showing a water distribution of a reaction solution in an immobilized lipase-packed layer after reaction in a conventional example.

FIG. 9 is a graph showing a change in the 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 concentration of tristearin in the withdrawal liquid in an example of the 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 concentration of tristearin in the withdrawal solution in Comparative Example.

FIG. 13 is a graph showing the water distribution of the reaction solution in the immobilized lipase-packed layer after the reaction of the comparative example.

FIG. 14 is a graph showing a 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 layer after the reaction of the conventional example.

[Explanation of symbols]

 1 tank for storing substrate 2 reactor 7 circulation line 8 humidifier 12 dehydrator 14 circulation line

──────────────────────────────────────────────────続 き Continuation of the front page (56) References JP-A-4-39399 (JP, A) JP-A-2-142485 (JP, A) JP-A-1-137988 (JP, A) JP-A-59-1984 210893 (JP, A) JP-A-2-286072 (JP, A) JP-A-2-502875 (JP, A) (58) Fields investigated (Int. Cl. ⁶ , DB name) C12M 1/40 C12N 11 /14

Claims (2)

(57) [Claims] 1. A circulating line is formed between a reactor filled with a carrier having an enzyme immobilized on its surface and a tank for storing a substrate, and a humidifier for humidifying the substrate storage tank and the substrate. An enzyme-immobilized bioreactor, wherein a circulation line is also formed between the reactor and the reaction solution, and the reaction solution is circulated to make the water 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 a substrate, and a tank for storing the substrate and a dehydration for dehydrating the substrate. An enzyme-immobilized bioreactor, wherein a circulation line is also formed between the reactor and the reaction solution, and the reaction solution is circulated to make the water in the reactor uniform.