JPH06245756A - Loop type two phase system immobilized enzyme reactor and reaction using the same reactor - Google Patents

Abstract

PURPOSE:To provide a two phase system immobilized enzyme reactor having a simple structure and useful for an enzymatic reaction such as hydrolysis of fats and oils. CONSTITUTION:There is provided a loop type two phase system immobilized enzyme reactor characteristically equipped with a fixed bed type reaction unit packed with an immobilized enzyme, with a two phase separation tank for separating the reaction products obtained in the fixed bed type reaction unit into two phases of a low specific gravity product and a high specific gravity product, with a pipe for returning a part of the low specific gravity product in the two phase separation tank to the fixed bed type reaction unit, with a pipe for supplying the reaction product obtained in the fixed-bed type reaction unit to the two phase separation tank, with pipes respectively for supplying a low specific gravity substrate and a high specific gravity substrate to the two phase separation tank and with pipes respectively for discharging the low specific gravity product and the high specific gravity product from the two phase separation tank.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a bioreactor and an enzymatic reaction method for carrying out a reaction such as decomposition of fats and oils, which is a reaction for forming two phases of oily water using an immobilized enzyme. It is expected to have wide applications in industry, pharmaceutical industry, food industry and the like.

[0002]

As a two-layer bioreactor using an immobilized enzyme, the present inventors have previously developed stationary tanks at the upper and lower ends, and two or more stirring tanks in the middle part. , Each of these tanks is partitioned by a liquid-permeable partition plate having microscopic holes that do not allow the immobilized enzyme to pass through, and the immobilized enzyme is contained in the stirring tank, and the upper end of the stationary tank at the upper end is further divided. A low specific gravity product discharge pipe, a high specific gravity substrate supply pipe for the stirring tank at the upper end, a low specific gravity substrate supply pipe for the stirring tank at the lower end, and a high specific gravity at the lower end of the stationary tank at the lower end. A two-phase immobilized enzyme reactor is known in which a product discharge pipe is arranged and a pulse generator is connected to the bottom of a stationary tank at the lower end (Kosugi, Shiraki, Japanese Patent Application No. 2). No. 49797).
In addition, as a loop reactor, a method in which a column is packed with lipase immobilized on a photocrosslinkable gel and water and oil are previously stirred in this packed column to reflux an oil-water mixture that has become an emulsion. Things (Y. Kimura
et.al.Eur.J.Appl.Microl.Biotechnol., 17.107 (198
3)) has been reported.

[0003]

In the two-phase system bioreactor in which the above-mentioned pulse generator is connected, the two-phase substrate is supplied countercurrently at room temperature and the reaction is performed while separating the two phases. A complicated reactor equipped with a stirring device is required, and two-phase separation is difficult. In addition, in the loop type reactor of Kimura et al., Stirring is required to make the emulsion state, and the moving speed of emulsion particles into the immobilized carrier is slow, so that low specific gravity products and high specific gravity products can be obtained. It is impossible to continuously separate and discharge, and it is also not possible to continuously concentrate the high specific gravity product concentration.

[0004]

The inventors of the present invention have completed the present invention as a result of intensive studies to improve the drawbacks of the conventional method. That is, according to the present invention, a fixed bed reactor packed with an immobilized enzyme and a two-phase separation for separating a reaction product obtained by the fixed bed reactor into two phases, a low specific gravity product and a high specific gravity product. A tank, a pipe for returning a part of the low-gravity product phase of the two-phase separation tank to the fixed bed reactor, and a pipe for supplying the reaction product obtained in the fixed bed reactor to the two-phase separation tank And a pipe for supplying a low specific gravity substrate and a high specific gravity substrate to the two-phase separation tank, and a pipe for discharging a low specific gravity product and a high specific gravity product from the two-phase separation tank, A loop type two-phase system immobilized enzyme reactor and an enzyme reaction method using the same are proposed. The reactor of the present invention is
Continuously discharging low specific gravity products and high specific gravity products from the upper and lower layers of the two phase separation tank while continuously supplying low specific gravity substrate and high specific gravity substrate to the two phase separation tank It is possible. It is also possible to continuously separate and discharge low-density products and high-density products. Moreover, in the fat and oil decomposition reaction, the high specific gravity product can be obtained by making the supply rate of the low specific gravity substrate and the discharge rate of the low specific gravity product faster than the supply rate of the high specific gravity substrate and the discharge rate of the high specific gravity product, respectively. It is also possible to continuously concentrate the concentration. Also,
The fluidity of the reaction solution can be improved by using a low specific gravity substrate mixed with a hydrophobic organic solvent or by maintaining the temperature at the melting point or higher of the reaction solution.

An example of a preferred loop type two-phase immobilized enzyme reactor of the present invention will be described below with reference to FIG. In the figure, 1 is an immobilized enzyme fixed bed reactor, 2 is a two-phase separation tank, 3 is a circulation pump that returns the reaction product (reaction solution) to the immobilized enzyme fixed bed reactor and then sends it again to the two-phase separation tank. 4 is a pump for supplying a low specific gravity substrate, 5 is a pump for discharging a low specific gravity product, 6 is a pump for supplying a high specific gravity substrate, 7 is a pump for discharging a high specific gravity product, 8 is a reaction solution discharge port, 9 is a reaction solution supply port, 10 is a low specific gravity substrate supply port, 1
1 is a low specific gravity product discharge port, 12 is a high specific gravity substrate supply port, 1
Reference numeral 3 denotes a high-density product discharge port.

The immobilized enzyme fixed bed reactor 1 is a column packed with immobilized enzyme. It is desirable that the temperature of the immobilized enzyme can be adjusted by the outer tube. As the immobilized enzyme used, one suitable for the intended reaction is selected.
For example, for hydrolysis of oils and fats, immobilized lipase (Entilon PF, manufactured by Rakuto Kasei) in which Pseudomonas lipase is immobilized on an anion exchange resin can be used even at 50-60 ° C where the reaction product has fluidity. It is a desirable immobilized enzyme because it is stable over time, has good degradability of various fats and oils, and has stable properties in hydrophobic organic solvents. As an appropriate liquid method, either an ascending method or a descending method can be adopted. Further, it is desirable to ensure that the passages of the low specific gravity reactant and the high specific gravity reactant are mixed in the column. Therefore, it is advisable to mix the low specific gravity substrate and the high specific gravity substrate with the immobilized enzyme, and after the immobilized enzyme having an affinity for both substrates is formed, a treatment such as packing in a column is preferably performed.

The low specific gravity substrate serves as a substrate for an immobilized enzyme reaction, and includes rice bran oil, coconut oil, tallow butter fat, sardine fat, liver oil,
Oils and fats such as cutting fat and castor fat, waste oil that is drawing attention as a soap raw material, various esters, acyl glycerol, etc.
It is a substance that is usually insoluble in water, and its specific gravity is lighter than that of water. The high specific gravity substrate is water, a water-soluble substance such as glycerin, or an aqueous solution thereof. The operating temperature of the reactor is maintained above the temperature at which the substrate and the product remain liquid.
The low specific gravity substrate may contain a high melting point compound such as wax, palmitic acid or stearic acid, which may make loop circulation of the reaction solution difficult. In that case, a hydrophobic organic solvent such as hexane, isooctane, or heptane is added in an amount of 20 to 4
The performance of the reactor can be further improved by adding 0 wt% and forming a low specific gravity miscella as a substrate of the reactor.

The two-phase separation tank 2 is a container having a supply port for various substrates and a discharge port for various products as shown in FIG. 1, and is a device capable of maintaining the reaction liquid at a melting point or higher. The reaction liquid (reaction product) is a mixed liquid of a substrate and a product having a low specific gravity and a high specific gravity, and when a hydrophobic organic solvent is added, the mixed liquid also includes the organic solvent. The reaction solution discharge port 8 is provided in the upper layer of the two-phase separation tank 2. Further, when the reaction liquid discharge port 8 is provided at the lowermost part of the upper layer, the discharged reaction liquid is also entangled and discharged with the reaction liquid of the lower layer, so that a fat and oil decomposition reaction or the like is likely to occur. The reaction liquid supply port 9 is provided at a position apart from the product discharge ports 11 and 13 in order to prevent the reaction liquid from being caught in the product. In addition, when the reaction solution supply port 9 is provided in the lower layer during the decomposition of fats and oils, the water-soluble product is recovered in the lower layer and the oily reaction solution is saturated with water while passing through the lower layer. . The low specific gravity substrate supply port 10 is provided with a product discharge port 11 in order to prevent the substrate from being caught in the product.
Provided at a position away from 13. In addition, when decomposing fats and oils, it is desirable to provide the low specific gravity substrate supply port 10 in the lower layer because the substrate is saturated with water while passing through the lower layer. The low specific gravity product discharge port 11 is provided in the upper layer. Since the fatty acids tend to be concentrated in the uppermost part of the upper layer during the decomposition of fats and oils, it is desirable to provide the low specific gravity product discharge port 11 in the uppermost part of the upper layer. The high specific gravity substrate supply port 12 is also provided at a position apart from the product discharge ports 11 and 13 in order to prevent the substrate from being caught in the product. In addition, in the case of decomposing oils and fats, it is desirable to provide the high specific gravity substrate supply port 12 in the upper layer because the oily substrate is saturated with water while passing through the upper layer. The high specific gravity product outlet 13 is provided in the lower layer. Since glycerin tends to be concentrated in the lowermost part of the lower layer during the decomposition of fats and oils, it is desirable to provide the high specific gravity product discharge port 13 in the lowermost part of the lower layer. Figure 1
The reactor shown in 1 is an example of one loop, but a plurality of loops can be connected to achieve a high reaction rate. In this case, the product outlet of the previous loop is connected as the substrate feed of the latter loop.

In order to carry out an enzymatic reaction using the loop type two-phase immobilized enzyme reactor of the present invention, a low specific gravity substrate and a high specific gravity substrate are placed in a two-phase separation tank for initialization and the immobilized enzyme fixed bed reactor 1 is used. The two-phase separation tank 2 is connected to form a loop, and the reaction solution is circulated at a rate that eliminates the external diffusion resistance of the immobilized enzyme fixed bed reactor 1. The reaction rate increases as the circulation rate increases, but the reaction rate does not increase when the circulation rate increases to some extent, which is the rate at which there is no external diffusion resistance. When the immobilized enzyme reaction is completed, the product can be taken out batchwise, but the continuous method is convenient for large-scale processing. In order to make the reactor continuous, the low specific gravity product and the high specific gravity product are continuously discharged from the upper layer and the lower layer of the two-phase separation tank, respectively, while the substrate is continuously supplied to the two-phase separation tank. The feed rate and the discharge rate are set to be almost the same as the feed rate when the specific gravities of the substrate and the product are almost equal. In the fat and oil decomposition reaction, the high specific gravity product can be obtained by making the supply rate of the low specific gravity substrate and the discharge rate of the low specific gravity product faster than the supply rate of the high specific gravity substrate and the discharge rate of the high specific gravity product, respectively. It is also possible to continuously concentrate the concentration. The air in the reactor used in the present invention is replaced with an inert gas such as nitrogen or argon, and then the reaction is carried out under the condition that the reactant does not come into contact with oxygen in the atmosphere, thereby increasing the peroxide value of the reaction product. Can be prevented. Although argon is expensive, it has a high specific gravity and is a convenient gas for the substitution process. Such treatment is necessary in the case of production of highly unsaturated fatty acids and the like.

[0010]

EXAMPLE A reactor as shown in FIG. 1 was prepared. The immobilized enzyme fixed bed reactor 1 was a column with a jacket tube, and was packed with 18 g of immobilized lipase (ENTILON PF, manufactured by Rakuto Kasei). The immobilized lipase was allowed to react at 60 ° C by circulating ethylene glycol adjusted to 60 ° C in the outer tube. An emulsion of rice bran oil and water was then flowed through the immobilized enzyme fixed bed to create an oil water passage in the fixed bed reactor. The two-phase separation tank 2 has a diameter of 45 mm and a height of 13
In a 0 mm cylindrical tube, put 50 g of rice bran oil and 50 g of water, discharge the reaction liquid from the reaction liquid discharge port 8 so that a small amount of water can be discharged together with the rice bran oil, and pass through the pump 3 circulating in a loop. The reaction solution was passed through the immobilized enzyme fixed bed reactor 1 and the loop reaching the reaction solution supply port 9 was rotated at a speed of 5 ml / min. This loop reactor was placed in a thermostatic chamber at 35 ° C. and heated with a ribbon heater to prevent the flow path from being clogged with palmitic acid, which is a hydrolysis product. The decomposition rate of fats and oils in the upper layer and the concentration of glycerin in the lower layer at each reaction time are as shown in Table 1. The decomposition rate of fats and oils was determined from the ratio of saponification value and acid value, and glycerin was determined by colorimetry using chromotropic acid.

[0011]

[Table 1] It can be seen from Table 1 that the fat and oil decomposition can be carried out batchwise and the initialization of the reactor is completed in about 100 hours.

Next, as shown in FIG. 1, a pump 4 for supplying a low specific gravity substrate, a pump 5 for discharging a low specific gravity product, a pump 6 for supplying a high specific gravity substrate, a pump 7 for discharging a high specific gravity product, and a reaction liquid. Discharge port 8, reaction liquid supply port 9, low specific gravity substrate supply port 10, low specific gravity product discharge port 11, high specific gravity substrate supply port 1
2. A high specific gravity product discharge port 13 is provided. Next, the pump for supplying the low specific gravity substrate and the pump 5 for discharging the low specific gravity product were operated at a rate of 0.5 g / hour to continuously supply rice bran oil and continuously discharge an oily low specific gravity product. At the same time, the pump 6 for supplying the high specific gravity substrate and the pump 7 for discharging the high specific gravity product were operated at a speed of 0.05 g / hour to continuously supply water and continuously discharge water. Table 2 shows the oil and fat decomposition rate of the low specific gravity product and the glycerin concentration in the high specific gravity product at each reaction time.

[0013]

[Table 2]

From Table 2, it is understood that the reactor can continuously decompose fats and oils, continuously separate and discharge low specific gravity products and high specific gravity products, and continuously concentrate glycerin concentration which is a high specific gravity product. . 288 in the latter half of Table 2
Although the decomposition rate deteriorated after the elapse of time, it was considered that this was because the pump 3 circulating in a loop was clogged and the circulation speed decreased. Therefore, it was decided to use the rice bran oil isooctane miscella obtained by adding 3 g of isooctane to 7 g of rice bran oil as a low specific gravity substrate. In this experiment,
The pump 4 for supplying the low specific gravity substrate isooctane miscella and the pump 5 for discharging the low specific gravity product are 0.714.
Continuous treatment was performed under the same conditions as in Table 2 except that the operation was performed at a speed of g / hour. The results are shown in Table 3.

[0015]

[Table 3]

From Table 3, according to isooctane miscella, the oil and fat decomposition rate was stable, and glycerin was also continuously concentrated to 4.6 mM / ml, that is, 42.36% by weight.

[0017]

EFFECT OF THE INVENTION The reactor of the present invention can be easily assembled if an enzyme column, a pump, and a container used for a two-phase separation tank are provided. It is applied to reactions such as oil and fat decomposition, which is a reaction that forms two phases of oil and water using an immobilized enzyme.
At present, the decomposition of fats and oils requires high-temperature and high-pressure treatment at 250 to 260 ° C. and 50 to 55 atm, and is not suitable for small-to-medium-scale decomposition. Is expected to be applied.

[Brief description of drawings]

FIG. 1 is an explanatory cross-sectional view of a loop type two-phase immobilized enzyme reactor.

[Explanation of symbols]

 1 immobilized enzyme fixed bed 2 two-phase separation tank 3 pump that circulates the reaction solution in a loop 4 pump that supplies low specific gravity substrate 5 pump that discharges low specific gravity product 6 pump that supplies high specific gravity substrate 7 high specific gravity substrate Pump for discharging 8 Reaction liquid outlet 9 Reaction liquid inlet 10 Low specific gravity substrate inlet 11 Low specific gravity product outlet 12 High specific gravity substrate inlet 13 High specific gravity product outlet 14 Low specific gravity reaction liquid 15 High specific gravity reaction liquid

Claims (3)

[Claims] 1. A fixed bed reactor filled with an immobilized enzyme, and a two-phase separation tank for separating a reaction product obtained by the fixed bed reactor into two phases, a low specific gravity product and a high specific gravity product. A pipe for returning a part of the low-gravity product phase of the two-phase separation tank to the fixed-bed reactor, and a pipe for supplying the reaction product obtained in the fixed-bed reactor to the two-phase separation tank, A loop comprising: each pipe for supplying a low specific gravity substrate and a high specific gravity substrate to the two-phase separation tank; and each pipe for discharging a low specific gravity product and a high specific gravity product from the two phase separation tank. Type two-phase immobilized enzyme reactor. 2. A method for enzymatically reacting a low specific gravity substrate and a high specific gravity substrate using the two-phase immobilized enzyme reactor according to claim 1, wherein the high specific gravity substrate is supplied to the two-phase separation tank and the low specific gravity product is obtained. The method for enzymatic reaction is characterized in that the discharge rate from the two-phase separation tank of (1) is made faster than the supply rate of the high-density substrate to the two-phase separation tank and the discharge rate of the high-density product from the two-phase separation tank, respectively. 3. A mixture of a low specific gravity substrate and a hydrophobic organic solvent is used as the low specific gravity substrate.
the method of.