JPS62278988A - Process for enzymatic or microbial reaction - Google Patents

Abstract

PURPOSE:To enable the separation of reaction product simultaneous to the reaction while keeping high conversion, by using a reactor wherein a non- aqueous solution phase and a water phase are separated into upper and lower layers and mixing both phases near their interface. CONSTITUTION:In a reactor 1 containing a non-aqueous solution phase and a water phase separated from each other into upper and lower layers, the non- aqueous solution phase and the water phase are mixed together near the interface remaining an unmixed part of the non-aqueous solution phase and the water phase e.g. by using a stirring blade 20 in a draft tube 3. The non-aqueous solution and water as a substrate or a substrate existing in the non-aqueous solution phase and/or the water phase is converted to the objective product with an enzyme or microorganism and the product is separated from the unmixed non-aqueous solution phase and/or the water phase. The process enables the separation of the product simultaneous to the conversion of the substrate to the product and facilitates the control of various reaction conditions and the continuous operation of the reaction.

Description

Detailed Description of the Invention 3. Detailed Description of the Invention [Field of Industrial Application] The present invention relates to a method for converting a substrate into a product using enzymes or microorganisms.

[Conventional technology and its problems]

In recent years, bioreactors that use enzymes or microorganisms as catalysts have been actively developed, but almost all of the compounds used in these reactions are water-soluble, and therefore reactions are primarily conducted in aqueous solutions. However, there are many reactions that are useful for compounds that are sparingly soluble or insoluble in water. In various peptide synthesis reactions such as those seen in the synthesis of the artificial sweetener aspartame,
These glycerides, fatty acids, and peptides are generally poorly soluble in water. Therefore, the reaction is usually a batch operation in which a fine emulsion is formed, the reaction is stopped after a predetermined decomposition rate is reached, and the product is separated into two phases and recovered. Furthermore, as a method for continuously carrying out enzyme or microbial reactions, it is known to fill a column with immobilized enzymes or immobilized microorganisms and continuously supply a substrate solution. It is necessary to mix and supply the phase and the aqueous phase, and after the reaction is completed, it is necessary to separate them into two phases again.

In this way, in a reaction in a two-phase dispersion system that does not dissolve in each other, it is necessary to separate the two phases not only for the separation and recovery of enzymes or microorganisms after the reaction, but also for the recovery of the product. Generally, methods such as static separation, centrifugation, or membrane separation are used, but combining these separation steps after the reaction results in system complexity and high cost, making it difficult to industrialize. Sometimes there are problems.

(Means for Solving the Problems) As a result of intensive studies in order to solve the above problems, the present inventors have found that, even in the above-mentioned two-phase system reaction, it is possible to simultaneously maintain a high reaction rate and We have discovered an innovative reaction method that can also separate products, leading to the present invention.

That is, the present invention provides a reactor in which a non-aqueous phase and an aqueous phase are separated into upper and lower 2N parts, and the non-aqueous phase and the aqueous phase are separated at the interface while leaving a portion where the non-aqueous phase and the aqueous phase are immiscible. By mixing in close proximity, the non-aqueous solution and water as substrates, or the substrate present in the non-aqueous phase and/or the aqueous phase, are converted into products by enzymes or microorganisms, and the immiscible parts are converted into products. The present invention provides an enzymatic or microbial reaction method characterized by removing a product present in a non-aqueous phase and/or an aqueous phase.

In the present invention, the non-aqueous solution refers to a hydrophobic liquid that is poorly soluble or insoluble in water.

The present invention will be explained in more detail based on the drawings showing preferred embodiments of the present invention.

- For example, A+B-C+D (A and B are each a substrate, and C and D are each a product. Now assume that A and C are water-soluble and B and D are water-insoluble.

The enzyme or microbial reaction system represented by will be explained using FIG. 1 is a reaction tank having a draft tube inside. Substrate A (aqueous phase) and substrate B are present in this reactor.
(non-aqueous solution phase) is prepared at a fixed ratio from the substrate (aqueous phase) storage tank 11 and the substrate (non-aqueous solution phase) storage tank 12, respectively, and the lower aqueous phase is rolled up by the stirring blade 20 in the draft tube 3, and an enzyme or emulsion is prepared. It allows efficient contact with microorganisms to carry out enzymatic or microbial reactions. Here, 4 is a substrate (non-aqueous solution phase) supply nozzle,
6 is a product (non-aqueous phase) storage tank, 7 is a weir, 8 is an ultrafiltration membrane, 9 is a storage tank for aqueous phase membrane treatment, and 10 is a product (aqueous phase) storage tank.

In the case of Fig. 1, a filler 2 is packed on the outside of the draft tube in order to increase the contact efficiency between the substrate and enzymes or microorganisms, and to efficiently retain these enzymes or microorganisms in the reactor by adsorption, etc. Even if no filler is used, if these conditions are met, there is no need to use a filler or the like.

In addition, there are obstacles 5 and 17 at the top and bottom of the reactor, respectively.
Providing a non-aqueous solution prevents complete mixing of the liquid. 8 It is preferable because it can form a part in which the liquid phase and the aqueous phase are separated.

By using the method of the present invention, it is possible to separate the product at the same time as the reaction, so it is possible not only to perform a batch operation but also a continuous reaction or a semi-continuous reaction in which the substrate is supplied while continuously extracting the product.

In the case of this reactor, most of the enzymes or microorganisms used in the reaction are retained within the reactor, but in order to more efficiently recover and reuse the enzymes or microorganisms, it is necessary to It is preferable to concentrate and recover. Preferably, an ultrafiltration membrane is used for this purpose. The ultrafiltration membrane to be used is not particularly limited in terms of material and shape, as long as it does not allow enzymes or microorganisms to pass through. For example, it may be made of any material such as cellulose acetate membrane, polyacrylonitrile membrane, polysulfone membrane, polyamide membrane, etc. It can also be used in any shape, such as a flat membrane, a tube, a spiral, or a hollow fiber.

The molecular weight cutoff of the ultrafiltration membrane varies depending on the enzyme or microorganism used in the reaction, and is not particularly limited as long as it has a pore size that can prevent passage of the enzyme or microorganism, but it is generally about 3,000 to 50,000. is preferred. An aqueous phase free of enzymes or microorganisms may be continuously extracted by ultrafiltration, and a concentrated solution of enzymes or microorganisms may be continuously or semi-continuously returned to the reaction system.

Note that if most of the enzymes or microorganisms are retained within the reactor and dissolution into the aqueous phase can be ignored, there is no need to separate the enzymes or microorganisms by ultrafiltration. Furthermore, it is also possible to fill the container with an immobilized enzyme or immobilized microorganism that has been immobilized in advance using various methods, and in this case as well, the enzyme recovery step by ultrafiltration is not necessary.

Alternatively, it is also possible to omit the ultrafiltration step and add fresh enzymes or microorganisms corresponding to the enzymes or microorganisms dissolved in the aqueous phase.

Using the method of the present invention, no special pretreatment is required (
Enzymes or microorganisms can be retained in the reactor and these enzymes or microorganisms can be efficiently recovered and reused. Enzymes or microorganisms can be retained in the packing material by adsorption or the like without special pretreatment, or they can be filled with immobilized enzymes or microorganisms that have been previously immobilized using various methods, or they can be There are methods such as using it in a free state without using a filler, but which method to use may be appropriately selected depending on the characteristics of the f4 element and microorganisms, reaction conditions, etc.

The diameter of the draft tube when using the draft tube 3 and stirring blade 20 as shown in FIG. A diameter of 90% is preferably used.Also, the rotation speed of the stirring blade is such that the lower layer in the reactor is well rolled up and mixing occurs near the interface between the non-aqueous phase and the aqueous phase, and the upper and lower parts of the reactor are It may be set so that a portion where the non-aqueous solution phase and the aqueous phase are immiscible remains.

When a filler is used as shown in Fig. 1, the form of the filler is not particularly limited, and Raschig rings, Lessing rings, Berl saddles, Interlocks saddles, Pall rings, etc., which are commonly used as fillers, are not particularly limited. It may be filled with a filler or a cylindrical net. The material is not particularly limited either, and metal, stone, plastic, etc. can be used. Also, when filling with immobilized enzymes or immobilized microorganisms,
The immobilization method is not particularly limited, and any method such as the commonly used carrier binding method, crosslinking method, entrapping method, or a combination of these methods may be used.
Just choose appropriately.

In addition to the draft tube method shown in Figure 1, there are other ways to successfully roll up the lower layer in the reactor, such as from below near the interface between the upper and lower layers in the reactor, as shown in Figure 2. Methods include blowing inert gas such as nitrogen gas, passing inert gas such as nitrogen gas through a draft tube as shown in Figure 1, or simply stirring the area near the interface without using a draft tube. Any method may be used as long as the upper layer and lower layer are mixed near the interface, and a method in which the upper layer and lower layer are mixed together in the vicinity of the interface may be used.

The feature of the present invention is that the enzyme or microbial reaction is carried out by mixing the upper and lower layers in the reactor near their interface, and the reaction is carried out while leaving unmixed parts in the upper and lower layers of the reactor. Since the reaction is carried out, the non-aqueous liquid phase and the aqueous phase can be taken out independently at the same time as the reaction, and the products can be separated. Therefore, the product can be obtained simultaneously while continuously adding the substrate and the like. Furthermore, since the reaction can be carried out continuously, the concentration of each component in the reactor can be maintained constant, which is very advantageous when considering the stability of the enzyme.

The case of hydrolyzing fats and oils using the method of the present invention will be described below. In this case, the substrates are oil and water, the enzyme is lipase, and the reaction products are fatty acids and glycerin.

The present inventors have discovered that when hydrolyzing fats and oils using lipase, the concentration of the product glycerin greatly contributes to the stability of the lipase.

According to research by the present inventors, when the glycerin concentration in the aqueous phase in the reaction system is within the range of 10 to 40% by weight, the enzyme is stabilized and hydrolysis of fats and oils preferably progresses. The method of the present invention makes it easy to keep the concentrations of various components in the reactor constant, and is therefore preferably applied to the hydrolysis of fats and oils by lipase.

When the method of the present invention is applied to the hydrolysis of fats and oils by lipase, the supply ratio of fats and oils and water can be calculated and determined by the following method to maintain the glycerin concentration at an optimal condition. For example, when trying to maintain the glycerin concentration in the aqueous phase in the reaction system at 20 wt%, the amount of oil m = X (kg/h) supplied continuously or semi-continuously is
r) 〃 〃 Water amount = Y (kg/hr
) Oil decomposition rate = 77 (%), molecular weight of oil = 1, molecular weight of water = 18. When the molecular weight of glycerin is 92, the following formula is established, and the ratio of X and Y can be determined from the formula 0. If M = 900 and η = 95%, then X/Y = 1.84 from ■2. In this way, the continuous, continuous or semi-continuous feed ratio of fat/water is determined.

The method of the present invention can be applied to various enzymatic reactions and microbial reactions in which reactions occur in a two-phase system of a non-aqueous solution phase and an aqueous phase. Applicable to peptide synthesis reactions using proteases, such as the transesterification reaction of thermolysin, or the synthesis of the artificial sweetener aspartame (aspartyl phenylalanine methyl ester) from carbobenzyloxy-1-aspartic acid and T-phenylalanine methyl ester using thermolysin. Yes, but not limited to these.

Another feature of the present invention is that the form of the enzyme or microorganism used may be a so-called immobilized enzyme or immobilized microorganism that has been immobilized in advance by various methods, but no special immobilization treatment is required. It is also possible to fill the reactor with a suitable filler, or to retain enzymes or microorganisms to some extent in the reactor without using these fillers. In addition, since enzymes or microorganisms dissolved in the aqueous phase can be easily recovered using an ultrafiltration membrane, they can be efficiently recovered and reused without any particularly complicated immobilization treatment.

The enzyme or microorganism used in the present invention does not necessarily have to be highly purified, and an extract, a partially purified product, or a fermentation solution can also be used.

The bioreactor according to the present invention is shown in FIG. 1 or 2.
As shown in the figure, only one tank may be used, but in order to carry out the reaction more efficiently, a multi-stage reaction may be used.

〔Example〕

Examples of the present invention will be described below, but the present invention is not limited to these Examples.

Example 1 Soybean oil was hydrolyzed by lipase using the reaction system shown in FIG. In the hydrolysis of fats and oils by lipase, in Figures 1 and 2, 4 is the oil supply nozzle, 6
9 is a fatty acid solution storage tank, 9 is a glycerin aqueous solution membrane treatment storage tank, lo is a glycerin water storage tank, 11 is a water storage tank, and 12 is an oil storage tank.

In reaction tank 1, 1 kg of decomposed fatty acids (fatty acid content 85%) obtained by enzymatically decomposing soybean oil and 1 kg of 20-t% glycerin water were placed.
kg and 2 g of lipase (320,000 units/g) produced from Candida cylindriase to make the reaction tank 30
The reaction was carried out while maintaining the temperature at °C.

The ratio of the diameter of the reaction tank and the diameter of the draft tube 3 is lO:6
It is. Further, the stirring blade was a ribbon-type blade 20 as shown in FIG. 1, and stirring was performed at a circumferential speed of about 0.5 m/sec.

This reaction tank 1 is supplied with 500 ml of
Soybean oil (fatty acid content 0%) was continuously supplied from the bottom of the reaction tank at a flow rate of g/HR, and from the water storage tank 11 to the ribbon pump 1.
Water was continuously supplied from the top of the reaction tank at a flow rate of 25 g/IIR using 4. That is, the average residence time of soybean oil in the reaction tank was 20 hours, and the glycerin concentration in the aqueous phase was approximately 2.
Each was supplied into the reaction tank so that it could be maintained at 0%. In the reaction tank, the water in the lower layer is once rolled up by the draft tube, and while the water droplets pass through the packed layer outside the draft tube, the lipase, oil, and water come into contact and a hydrolysis reaction takes place.

On the other hand, there are baffle plates 5.17 at the top and bottom of the reaction tank, respectively.
By providing the upper and lower sides thereof, sweet wood containing almost no water or fatty acids or almost no oil can be obtained. In this way, fatty acids are continuously extracted by the amount of soybean oil supplied by overflow, and Amagi is continuously extracted from the lower part of the reaction tank by the pump 15, and once stored in the storage tank 9, the aqueous phase is filtered through the ultrafiltration membrane 8. The enzyme dissolved in the water was concentrated and recovered, and the amount of glycerin water extracted was 25g.
/IIR. In this example, a polyacrylonitrile membrane (fraction molecule: 130,000) was used as an ultrafiltration membrane to semi-continuously concentrate the enzyme and return it to the reaction tank.

Using such a reaction apparatus, the reaction was continued while continuously supplying soybean oil and water and continuously withdrawing the fatty acid solution and the aqueous glycerin solution.

After 20 hours (equal to the average residence time in the reaction tank), the fatty acid solution was collected from the fatty acid solution storage tank 6 and the acid value and saponification value were measured.Acid value = 170, saponification value = 19
4 was obtained. The hydrolysis rate was calculated from the formula below: 8
It was 6%.

Saponification value, glycerin water? The glycerin concentration of the glycerin water in the 8-liquid storage tank 10 was 20-t%.

Similarly, 40 hours, 60 hours, and 8 hours after the start of soybean oil supply.
The decomposition rate and glycerin concentration were measured after 0 hours and 100 hours, and the results were as shown in Table 1.

Table 1: Even after 100 hours of continuous reaction, the enzyme did not deactivate at all, the soybean oil decomposition rate remained at 85-86%, and the glycerin concentration in the aqueous phase remained at 18-20%. did it.

On the other hand, the water content in the fatty acid solution obtained in the fatty acid solution storage tank 6 was 0.5% or less. In addition, the glycerin water that passed through the ultrafiltration membrane was of good quality. As described above, it was found that by using this reaction system, it is possible to simultaneously perform the reaction and separation of the produced proboscis, while efficiently recovering and reusing the enzyme and maintaining a high decomposition rate.

Example 2 Using the same equipment as in Example 1, with the initially charged soybean oil decomposition solution, 20 wt% glycerin aqueous solution, and the same amount of enzyme as in Example 1, soybean oil and water were continuously pumped by pumps 13 and 14. The amount added was changed as follows, and the average residence time of soybean oil was 40 hours.

Soybean oil addition amount 25g/ll R1 water addition amount 12.5g/H
Continuous decomposition was performed while adjusting the R fatty acid solution to overflow and the amount of glycerin water extracted to be 12.5 g/l (R).The soybean oil hydrolysis rate and glycerin concentration were measured for each reaction time. The result is as shown in Table 2.

Table 2 As shown in Table 2, it was found that when the residence time was 40 hours, a decomposition rate of about 90% was obtained.

Example 3 In Example 1, the water in the reaction tank was stirred up by the stirring blade in the draft tube to cause a hydrolysis reaction, but in this example, instead of stirring, the water was stirred as shown in Figure 2. Nitrogen was blown from the lower part of the reaction tank from the nitrogen gas blowing nozzle 22 to raise water droplets into the oil phase, and the lipase held in the packed bed was brought into contact with the oil and water to cause a hydrolysis reaction. A funnel-shaped baffle plate is provided at the top of the reaction tank to collect nitrogen, and care is taken to prevent mixing of liquids above the baffle plate.

The initial charged soybean oil decomposition solution, 20v L% glycerin aqueous solution, the amount of enzyme charged, and the average residence time of soybean oil were the same as in Example-1, and the nitrogen blowing amount was 100 m7/win.
Continuous hydrolysis of soybean oil was carried out.

The hydrolysis rate and glycerin concentration of soybean oil for each reaction time were measured and the results are shown in Table 3.

Table 3 As shown above, the method of blowing nitrogen from the bottom of the reaction can also achieve the same stirring effect as the stirring method shown in Figure 1.
It was found that a decomposition rate of 85 to 86% could be maintained, and that the fatty acid solution phase and the glycerin aqueous phase could be efficiently separated as in Example-1.

Example 4 In Examples 1, 2, and 3, continuous hydrolysis was performed using only one reactor tank, but in this example, in order to efficiently obtain a high decomposition rate, the reactor shown in Figure 1 was used. Two stages of continuous hydrolysis were carried out using two tanks.

In this case, soybean oil and water were supplied in countercurrent flow. That is, soybean oil is first supplied from the first stage reactor, the obtained fatty acid solution is again supplied to the second stage reactor, and the water is supplied so that the glycerin concentration in the reactor is approximately 20%. Supplied with due consideration.

Soybean oil was supplied at a rate of 50 g/IIR, and 3 g of glycerin water, which was made by mixing fresh water and glycerin water recovered from the second reactor to approximately 15%, was added to the first reactor.
/HR, and to the second stage, the glycerin water recovered from the first stage reactor and fresh water were mixed and supplied at a flow rate of 20 g/IIR.

The hydrolysis rate and glycerin concentration of soybean oil for each reaction time were measured and the results are shown in Table 4.

Table 4 It was found that oils and fats could be efficiently hydrolyzed by performing the multi-stage reaction as described above.

〔Effect of the invention〕

The enzyme or microbial reaction method of the present invention is a method that can convert a substrate into a product and simultaneously take out the product, and can control various reaction conditions,
It is extremely easy to carry out the reaction continuously, which is advantageous for industrialization.

[Brief explanation of the drawing]

FIGS. 1 and 2 are schematic diagrams of examples of the reaction system of the present invention, respectively. 1... Reaction tank 2... Filler 3... Draft tube 4... Substrate (non-aqueous phase) supply nozzle 5... Upper baffle plate 6... Product (non-aqueous phase) storage tank 7 ...Weir 8...Ultrafiltration membrane 9...Aqueous phase membrane treatment storage tank 10...Product (aqueous phase) storage tank 11...Substrate (aqueous phase) storage tank 12...Substrate (non-substrate) Aqueous solution phase) Storage tanks 13 to 16... Pump 17... Lower baffle plates 18 to 19... Pulp 20... Stirring blades 21... Stirring motor

Claims (1)

[Claims] 1. In a reactor in which the non-aqueous phase and the aqueous phase exist separately into two layers, upper and lower, the non-aqueous phase and the aqueous phase are separated while leaving a portion where the non-aqueous phase and the aqueous phase are immiscible. By mixing near the interface, the non-aqueous solution and water as substrates, or the substrate present in the non-aqueous solution phase and/or the aqueous phase, are converted into products by enzymes or microorganisms, and the immiscible parts are converted into products. An enzymatic or microbial reaction method characterized in that a product present in a non-aqueous phase and/or an aqueous phase is removed. 2 Enzymes or microorganisms are adsorbed or retained on the filler without special pretreatment, or are immobilized on immobilization carriers by various immobilization methods and separated between the non-aqueous phase and the aqueous phase in the reactor. The method according to claim 1, wherein the method is installed near an interface of. 3. The method according to claim 1, wherein mixing near the interface is performed using a stirring blade in a draft tube. 4. The method according to claim 1, wherein the mixing near the interface is performed by blowing an inert gas from below near the interface and rolling up the lower layer. 5. The method according to claim 1, wherein the substrate is water and fat, and the enzyme is lipase. 6. The method according to claim 5, wherein the reaction is carried out while maintaining the glycerin concentration in the aqueous phase in the reaction system within a range of 10 to 40% by weight.