JPH02276565A - Apparatus for continuous enzymic reaction - Google Patents

Abstract

PURPOSE:To safely, stably and readily enable reaction without microbial contamination by providing an apparatus with a specific O2 dissolver and bioreactor of an immobilized enzyme. CONSTITUTION:A substrate solution in a substrate solution reservoir tank 5 is fed to an ultraviolet ray irradiator 8 with a pump 6, sterilized and then continuously passed through one side (A) divided with a membrane 2 consisting essentially of a hollow fibrous silicone rubber having properties of passing gases therethrough without permeating liquids in an O2 dissolver 1. Oxygen gas is subsequently fed from an O2 gas cylinder 7 through a pressure reducing valve 9 to the other side (B) of the O2 dissolver 1 to increase the pressure in the dissolver 1 to 2-5kg/cm<2> and regulate the dissolved O2 concentration in the substrate solution to 50-60ppm. The resultant substrate solution is then continuously fed to a bioreactor 3 and continuously subjected to enzymic reaction with an immobilized enzyme 4 arranged in the reactor 3.

Description

[Detailed Description of the Invention] <Industrial Application Field> The present invention relates to a reaction device using an immobilized enzyme used in fields such as the food industry. This invention relates to a continuous enzyme reaction device that can suppress microbial contamination within a bioreactor without inhibiting enzyme activity.

<Prior Art> Microbial contamination in bioreticctors used in the food industry is a serious problem, but it is no exaggeration to say that there is still no perfect countermeasure against contamination.

The first problem with contamination control is that because the target is food, there are very few safe disinfectants, and even if there are, it is difficult to put them into practical use economically. The second problem is that the immobilized enzymes used in bioreactors are subject to strict environmental conditions such as heat, pH, and flow rate, and under conditions that deviate significantly from these conditions, the immobilized enzymes themselves may be damaged. It's at the point where you can put it away.

Therefore, conventional contamination countermeasures include UV sterilization of the substrate solution passed through the bioreactor, high temperature sterilization, or sterilization by adjusting the pH within a pH range that does not affect the immobilized enzyme. However, even if the substrate solution is completely sterilized, as the bioreactor is operated continuously for a long period of time, bacteria may enter the bioreactor from outside and cause damage to the bioreactor. Bacterial growth occurs. As a countermeasure to this problem, a method is used in which the inside of the bioreactor and the immobilized enzyme are periodically cleaned with a chemical, but this method also has the disadvantage that the concentration of the chemical and the contact time affect the activity of the immobilized enzyme. .

Therefore, various studies have been conducted on methods to prevent microbial contamination, but recently Takahashi et al. Studies on the growth of microorganisms under high dissolved enzyme concentrations have reported that the growth of all microorganisms is significantly suppressed under dissolved oxygen concentrations of about 50 to 90 ppm.

(For example, J, Fer-ent, Technol, +
62 + 71-75 (1984), Agric
, Biol, Che-, , 51, 257-258 (1
987), Journal of the Fermentation Engineering Society, Volume 65, Nos. 501-506
(1987)).

In addition, Takahashi et al. applied the above findings to enzymatic reactions and performed the enzyme reaction under conditions where the dissolved oxygen concentration in the substrate solution was maintained at a high concentration as described above. It has been reported that only the proliferation of can be suppressed (for example, 1985 Japanese Fermentation Engineering Abstracts P2O4).

The high-concentration dissolved oxygen method described above is considered to be an excellent method that has the potential to prevent microbial contamination in a bioreactor using enzymes, but when implementing this method into a practical device, it is necessary to The most important point is how to dissolve a large amount of oxygen.

That is, in order to add the above-mentioned high concentration of dissolved oxygen to the substrate liquid, a normal oxygen gas or an oxygen-enriched gas with an oxygen concentration of, for example, 50% or more is used as the oxygen source, and the gas and the substrate are mixed together. It is necessary to contact the liquid under pressure of 2 to 5-/d. However, oxygen gas or oxygen-enriched gas is expensive (therefore, unless an oxygen dissolving device with high dissolution efficiency is used, running costs will be extremely high).

For example, absorption towers using packing materials and absorption towers using diffusers such as diffuser plates, which are well-known devices for dissolving gas into liquids, are not very efficient because their dissolution efficiency is not that high. There is a problem that the device becomes larger. Furthermore, if the pure oxygen gas or oxygen-enriched gas that flows out of the tower without being absorbed in the absorption tower is disposed of as is, running costs will be high.On the other hand, if unabsorbed gas is recycled and used, equipment costs will increase. There is also the problem that as the cost increases, the equipment becomes more complex.

<Problems to be Solved by the Invention> The present invention follows the idea of the above-mentioned high-concentration dissolved oxygen method, which has the potential to solve the drawbacks of the conventional microbial contamination method using disinfectants, etc., and solves the above-described method. The purpose of the present invention is to provide a continuous enzyme reaction device that can suppress the growth of microorganisms over a long period of time by being equipped with an oxygen dissolver that can achieve a dissolution efficiency of almost 100%. .

Means for Solving the Problems In order to achieve the above-mentioned object, the inventors conducted extensive research and found that an oxygen dissolving device has the property of allowing gases such as oxygen gas to pass through but not liquids. By using a membrane unit equipped with a hydrophobic membrane, it is possible to efficiently dissolve oxygen in the substrate liquid at a high concentration.
Furthermore, it was found that when a substrate solution with a high dissolved oxygen concentration was continuously passed through a bioreactor, microbial growth could be stably suppressed over a long period of time.

The present invention was made based on this knowledge, and the substrate liquid is brought into contact with one side of a membrane partitioned by a membrane that allows gases to pass through but not liquids, and the other side is provided with oxygen gas or an oxygen-rich membrane. an oxygen dissolver that moves and dissolves oxygen into the substrate liquid through the membrane by contacting with a chemical gas; and a substrate liquid to which dissolved oxygen has been added by the oxygen dissolver. This is a continuous enzyme reaction device that combines a bioreactor that uses immobilized enzymes.

<Effect> The present invention will be explained in detail below.

The membrane used in the oxygen dissolver in the present invention may be any membrane as long as it allows gas to pass through but not liquid. For example, it may be made of silicone rubber as a main component,
A hydrophobic homogeneous membrane without any physical micropores, or a membrane made of tetrafluoroethylene, polypropylene, polyethylene, etc., has a pore size of, for example, 0.02 μm to 10 μm.
Hydrophobic porous membranes with many microscopic pores on the order of micrometers, or membranes coated with the silicone rubber or tetrafluoroethylene resin on the surface of a porous membrane made of a hydrophilic resin such as polysulfone or cellulose resin, etc. Examples include known membranes.

Note that the above-mentioned homogeneous membranes and porous membranes have different oxygen permeation effects. In the case of a homogeneous membrane that does not have micropores, oxygen molecules first dissolve in the membrane material and then diffuse through the membrane. , and is desorbed from the membrane material and dissolved in the substrate liquid, whereas in the case of a porous membrane, oxygen directly passes through the micropores and moves into the substrate liquid and dissolves in the substrate liquid. By bringing the substrate liquid into contact with one side of the membrane and bringing oxygen or an oxygen-containing gas into contact with the other side, oxygen can be transferred and dissolved into the substrate liquid through the membrane. It is.

The specific configuration of the device of the present invention will be explained below.

FIG. 1 is an explanatory diagram of a flow showing an example of an embodiment of the present invention, and 1 is an oxygen dissolver equipped with a membrane 2 that allows gas to pass through but not liquid. The oxygen dissolver 1 is configured so that a substrate liquid can flow through one side A partitioned by the membrane 2, and oxygen gas can be supplied to the other side B. Note that the other side is not provided with an oxygen gas outlet, and is configured so that the entire amount of the supplied oxygen gas is transferred into the substrate liquid. 3 is a bioreactor connected to the rear stage of the oxygen dissolver 2 and using an immobilized enzyme 4. In FIG. , which shows a packed bed type bioreactor. Further, 5 is a substrate liquid storage tank for storing the substrate liquid, and 6 is a pump for supplying the substrate liquid in the storage tank to one side A partitioned by the membrane 2 of the oxygen dissolver l. Further, 7 is an oxygen cylinder containing oxygen gas to be supplied to the other side B partitioned by the membrane 2. Note that 8 is the pump 6,
This is an ultraviolet irradiation device installed between the oxygen dissolver 1 and the ultraviolet irradiation device 8, which is used to sterilize bacteria existing in the substrate liquid from the beginning. If the amount of UV radiation is small, the ultraviolet irradiation device 8 may be omitted. In addition, 9 is an oxygen cylinder 7 and an oxygen dissolver 1
This is a pressure reducing valve installed in the middle of the piping that communicates with the

Next, to explain the operation, first, the pump 6 is driven to supply the substrate liquid in the substrate liquid storage tank 5 into the ultraviolet irradiation device 8, and the substrate liquid is sterilized in advance by ultraviolet irradiation. The subsequent substrate liquid is supplied to one side A of the oxygen dissolver 1 partitioned by the membrane 2 and allowed to flow continuously through the oxygen dissolver 1, while the substrate liquid is supplied to one side A of the oxygen dissolver 1 partitioned by the membrane 2, Oxygen gas is supplied to the other partitioned side B from an oxygen cylinder 7 via a pressure reducing valve 9.

Such a flow allows oxygen to be moved and dissolved into the substrate liquid through the membrane 2. In this case, the solubility of oxygen increases as the pressure within the oxygen dissolver 1 system increases, but in order to suppress the growth of bacteria in the subsequent bioreactor 3, the dissolved oxygen concentration in the substrate solution must be reduced to 50 to 60 pp.
m or more, so usually an oxygen dissolver 1
The pressure in the system is preferably increased to about 2 to 5 kg/d, and the pressure on the oxygen gas side in the oxygen dissolver 1 system is preferably controlled to be slightly higher than the pressure on the substrate liquid side.

Next, the substrate liquid to which a predetermined amount of dissolved oxygen has been added by the oxygen dissolver 1 is continuously supplied to the subsequent bioreactor 3 to perform an enzyme reaction. In addition, in the apparatus of the present invention, the inside of the piping after the oxygen dissolver 1 and the inside of the bioreactor 3 are maintained at almost the same pressure conditions as the inside of the oxygen dissolver 1 system, so that the dissolved oxygen added in the substrate liquid is kept within the piping. It is necessary to prevent the gas from being released as bubbles inside the bioreactor 3 or the like. In the bioreactor 3, the growth of various bacteria within the column can be reliably prevented by high-concentration dissolved oxygenation of the inflowing substrate liquid, and therefore a stable enzymatic reaction can be performed for a long period of time.

The shape of the membrane used in the oxygen dissolver in the present invention includes hollow fiber shape, tube shape, spiral shape, flat membrane shape, etc.
Any type of material may be used, but it is usually best to use a hollow fiber or tube shape. By passing oxygen gas or oxygen-enriched gas to the outside of the membrane, oxygen is moved from the outside to the inside of these membranes, and the substrate liquid with dissolved oxygen added thereto is taken out from the other end of the hollow fiber or tube. Configure.

Next, the bioreactor used in the present invention is not limited to the packed bed type described above, but may be any bioreactor commonly used for enzyme reactions, such as a control, calibration, membrane or film type so-called membrane bioreactor. Any type of bioreactor may be used, but a bioreactor with a structure that can be pressurized is particularly suitable.

In addition, in the above-mentioned embodiment, an example was explained in which oxygen gas was used as the gas supplied to the oxygen dissolver, but in the present invention, oxygen-enriched gas can also be used instead of oxygen gas, and in that case, the The higher the oxygen concentration, the more preferable the oxygen-enriched gas is, but it is usually best to use an oxygen-enriched gas with an oxygen concentration of 50% or more.

Effect> As explained above, by using an oxygen dissolver equipped with a membrane that allows gas to pass through but not liquid, oxygen can be efficiently dissolved in the substrate liquid at a high concentration. Therefore, the oxygen dissolving device becomes more compact than a conventional absorption tower. Furthermore, by supplying a substrate liquid with increased oxygen concentration to the subsequent bioreactor, it is possible to reliably suppress the growth of germs within the bioreactor.

Furthermore, the device of the present invention allows extremely stable oxygen concentration management with extremely simple operation. ■Almost 100% of the oxygen supplied into the oxygen dissolver transfers to the substrate liquid side, so there is no wastage of oxygen.■Oxygen that has migrated through the membrane is completely dissolved in the substrate liquid and remains as bubbles. Therefore, even if the substrate liquid is introduced into the bioreactor, there is almost no possibility that oxygen gas will accumulate in the space such as the upper part of the bioreactor, and therefore there is no danger of explosion.

■Since no harmful chemicals are used, it is extremely safe in terms of food hygiene. ■It has the characteristics that there is no decrease in enzyme activity even if the dissolved oxygen concentration in the substrate solution is increased. Therefore, the device of the present invention is highly suitable for the food industry, and is also an extremely useful device that can be applied to all other fields.

Examples will be described below to make the effects of the present invention more clear.

<Example> Using the apparatus shown in FIG.
Branched cyclodextrin (hereinafter referred to as branched CD) is produced by reacting the branched dextrin with the action of
A continuous enzymatic reaction was carried out to produce .

That is, a bioreactor as shown in FIG. 1 was prepared in which the column was filled with immobilized enzyme 201 in which CGTase was covalently immobilized on a glycidyl methacrylic acid polymer carrier (primary amine). . On the other hand, 4% branch C
D was dissolved in acetate buffer with pH 6, heated to a temperature of 50 °C, and continuously pumped at a flow rate of 3.21/hr. In addition to sending it to an oxygen dissolver equipped with a hollow fiber-like homogeneous membrane as a component, 5 kg of
/cj, pure oxygen was sent from an oxygen cylinder to the oxygen dissolver and dissolved therein. With such operations,
Approximately 180 ppm of dissolved oxygen could be continuously dissolved in the substrate solution. Next, the substrate solution was sent to the above-mentioned bioreactor and the enzymatic reaction was carried out continuously.
As shown in Figure 2, bacterial growth was suppressed for about 70 days, and as shown in Figure 3, although there was a normal decrease in enzyme activity due to continuous use, the overall effect was very low. Stable generation of branched CDs was observed.

The membrane used in the oxygen dissolver was a commercially available hollow fiber membrane for gas exchange for oxygenators (Merashirock = S, manufactured by Senko Medical Industry Co., Ltd.), with a membrane area of 0.8 mm and a membrane outer diameter of 0.8 mm. It is 400μ and 100μ thick.

[Brief explanation of drawings]

FIG. 1 is an explanatory diagram of a flow showing an example of embodiment a of the present invention, and FIGS. 2 and 3 are graphs showing the results of continuous enzyme reactions in the example, and FIG. (Day)
A graph showing the relationship between the number of viable bacteria in the reaction solution (cells/mA'), and Figure 3 shows the relationship between the number of days in which the solution was passed and the content (wt%) of branched CD formed in the total sugar content in the reaction solution. This is a graph showing 1... Oxygen dissolver 3... Bioreactor 5... Substrate liquid storage tank 7... Oxygen cylinder 9... Pressure reducing valve 2... Membrane 4... Immobilized enzyme 6... Pump 8... Ultraviolet irradiation device

Claims (1)

[Claims] 1. A substrate liquid is brought into contact with one side of the membrane partitioned by a membrane that allows gas to pass through but not a liquid, and oxygen gas or oxygen-enriched gas comes into contact with the other side. An oxygen dissolver configured to move and dissolve oxygen into the substrate liquid through the membrane, and a substrate liquid to which dissolved oxygen has been added by the oxygen dissolver, for reacting.
A continuous enzyme reaction device characterized by combining a bioreactor using an immobilized enzyme. 2. Claim 1, wherein the membrane that allows gas to pass through but not liquid is a membrane whose main component is silicone rubber.
The continuous enzyme reaction apparatus described.