JPH03251168A - Two phase-based bioreactor and method for carrying out enzymatic reaction using the same reactor - Google Patents

Abstract

PURPOSE:To improve the reaction efficiency by introducing two kinds of reaction substrates ad a pulse flow into a mixing vessel in which an immobilized enzyme is housed and then agitating the contents. CONSTITUTION:Stationary tanks 2 and 8 positioned in the upper and lower ends of a reactor and one or more mixing vesseles in the intermediate part are partitioned by a partition plate 5 capable of passing through a liquid and having micro-through-holes incapable of permeating immobilized enzyme. Then the pulse generator 6 is stopped and a discharge tube 9 having high specific gravity is closed and then a substrate (e.g. glycerin) having high specific gravity is fed from a feed tube 3 into the stationary tank 2 and a substrate (e.g. highly oxidized oil as a model) having low specific gravity is fed from a feed tube 7 into the stationary tank 8. These substrates are packed in the reactor and made to react with an immobilized enzyme. Then, a discharge tube 9 is opened and the substrate having high specific gravity is discharged through an emulsion breakage device 10 and, simultaneously, is fed from a feed tube 3. A pulse generator 6 is simultaneously operated and the substrate having low specific gravity is introduced as pulse flow from the feed tube 7 into the mixing vessel 4 and the contents are made to react with the immobilized enzyme while subjecting the contents to pulse agitation. Then the product having low specific gravity is discharged from a discharged tube 1 through the device 10 and the product having high specific gravity is discharged from the discharge tube 9.

Description

Detailed Description of the Invention [Field of Industrial Application] The present invention relates to a bioreactor and an enzyme reaction method for reacting a reactant that forms two phases using an immobilized enzyme, and is applicable to the oil and fat industry, It is expected to have wide applications in the pharmaceutical industry, food industry, etc.

[Conventional technology]

As a two-phase bioreactor using immobilized enzyme,
A method of filling a column with lipase entrappingly immobilized in a photocrosslinkable gel, stirring water and oil in advance, and refluxing the oil-water mixture in an emulsion state to a packed column (Y,
kimura et, al., Eur, J., Appl, M.
icrol, Biotechnol, 17,107 (
1983)), and using the base-immobilized reversegalam previously developed by the present inventors, a water-soluble substrate with a high specific gravity from the middle upper stage and an oily substrate with a low specific gravity from the middle lower stage were continuously applied. A method of continuously collecting oily products with low specific gravity from the upper end and water-soluble products with high specific gravity from the lower end (Kosugi et al.
(Japanese Patent Publication No. 62-51111), when performing a reaction using immobilized lipase in a multistage reaction tank, the immobilized lipase, oily substrate, and water-soluble substrate are separated and mixed repeatedly, and the immobilized lipase is combined with the oily substrate. Method of self-current contact with water-soluble substrate (Kosugi et al., JP-A-63-59896
In order to countercurrently contact a low specific gravity substrate and a high specific gravity substrate with the immobilized enzyme and immobilized enzyme, a static tank is provided at the upper and lower ends, and a stirring and static tank are provided in the middle section in alternating combinations. A continuous reaction method using an immobilized enzyme characterized by supplying a high specific gravity substrate from the middle upper stage, recovering a low specific gravity product from the upper end, and recovering a high specific gravity product from the lower end (Kosugi et al., JP-A-1-9849) / No. 1) etc.

[Problem to be solved by the invention]

In the previously reported method of passing a pre-emulsified mixture through an immobilized enzyme column, the movement speed of emulsion particles diffusing into the immobilized carrier is extremely slow, so the reaction solution must be circulated through the immobilized enzyme column many times. It is difficult to make it continuous.

Furthermore, it is not possible to continuously separate and collect low specific gravity products and high specific gravity products.

When using an immobilized enzyme column to supply low-density substrates and high-density substrates countercurrently, in order to prevent the channeling phenomenon in which the paths of low-density substrates and high-density substrates in the column are completely separated, first It is necessary to secure a flow path through which the low specific gravity substrate and the high specific gravity substrate flow while mixing. Moreover, the processing speed cannot be increased above the oil-water separation speed in the immobilized enzyme column, and since the oil-water separation speed is extremely slow, it is necessary to take measures such as using a thick and short column.

For these reasons, there are many restrictions on operating conditions, making it difficult to fully demonstrate the activity of the immobilized enzyme.

The immobilized enzyme is reacted in a multistage reaction tank, and the immobilized enzyme, low specific gravity substrate, and high specific gravity substrate are separated and mixed repeatedly, and the low specific gravity substrate and the high specific gravity substrate are added to the immobilized enzyme in a countercurrent manner. The contacting method is a batch type using multi-stage reaction vessels, and in order to make this continuous and to continuously collect low specific gravity products and high specific gravity products, a complicated pipeline is required.

The control operation also becomes complicated. In addition, when mixed, a fine emulsion is generated, which impairs the two-phase separation. To prevent this, separation and mixing are performed in separate reaction vessels, which requires separation time in addition to reaction time, making it an efficient method. It's hard to say.

A method in which a static tank and a stirring tank are provided alternately in the middle and agitation is performed using a stirring blade is to increase the volume of the stirring tank for the reaction in which the immobilized enzyme is present relative to the volume of the static tank. Because this is not possible, the reaction field becomes smaller even though the scale of the reaction tank becomes larger. Therefore, a large reaction tank is required for mass treatment. In addition, stirring using vinegar stirring blades results in poor oil-water separation, and therefore it is necessary to alternately provide #100 tanks and stirring tanks. Moreover, air bubbles are often attached to the immobilized enzyme, and when the enzyme is operated for a long time, the air bubbles attached to the enzyme are released from the enzyme and are deposited at the top of the stirring tank, that is, under the partition plate. It accumulates.

If this is not removed, the rate of oil/water separation will be significantly slowed down.
Continuous operation will no longer be possible. Furthermore, if the mixture is stirred for a long time with a stirring blade, the immobilized enzyme will wear out and flow out through the partition plate, resulting in leakage of the immobilized enzyme. Additionally, a 4A stirrer with mechanical seals at the upper and lower ends is required.

[Means for solving problems]

The present inventor completed the present invention as a result of extensive research to improve the various drawbacks of conventional methods.

That is, according to the present invention, the liquid-permeable partition has a static tank at the upper end and the lower end, one or more stirring tanks in the middle part, and has minute holes that do not allow the immobilized enzyme to pass through the various types. partitioned with a plate, and the stirring tank accommodates the immobilized enzyme, furthermore, the upper end of the stationary tank is provided with a high specific gravity substrate supply pipe at the lower part and a low specific gravity product discharge pipe at the northern part thereof,
The lower stationary tank is provided with a two-phase bioreactor characterized in that a high specific gravity product discharge pipe is disposed at the lower part thereof and a low specific gravity substrate supply pipe is disposed at the upper part thereof.

Further, according to the present invention, when two types of reaction substrates are reacted in a stirring tank containing an immobilized enzyme, at least one of the reaction substrates is introduced into the stirring tank as a pulsed flow, and the contents of the stirring tank are Provided is an enzyme reaction method characterized in that stirring is performed.

Hereinafter, one embodiment of a preferred bioreactor of the present invention will be described with reference to FIG. In the figure, 1 is a low specific gravity product discharge pipe, 2 is a static tank at the upper end, 3 is a high specific gravity substrate supply pipe, 4 is a stirring tank, and 5 is a partition plate.

6 is a pulse generator, 7 is a low specific gravity substrate supply pipe, 8 is a static tank at the lower end, 9 is a high specific gravity product discharge pipe, 10 is an emulsion breaking device, 11 is a branch pipe connected to the pulse generator, 12
indicates a branch pipe connected to a metering pump.

Preferably, the upper end standing tank and the lower end standing tank are conical. The reason is that even if a fine emulsion is generated, the emulsion particles will hit the vessel wall and break before reaching the low-density product discharge pipe or the high-density product discharge pipe 9, making it easier to separate the two phases. be. By providing the emulsion breaking device 10, the two-phase separation can be completed and the substrate can be prevented from being mixed into the product. The emulsion breaking device can be of a conventionally well-known structure. For example, it may be a simple styrene-free steel sieve plate, but preferably it is filled with glass beads or the like to completely destroy the emulsion. It is better to do so. At the top of the static tank 2 at the upper end is a low specific gravity product discharge pipe 1. A high specific gravity substrate supply pipe 3 is provided at the bottom. Further, a high specific gravity product discharge pipe 9 is provided at the lower part of the static tank 8 at the lower end, and a low specific gravity substrate supply pipe 7 is provided at the upper part. Setting the temperature of the static tank to 50°C or higher is appropriate for two-phase separation, and since setting the temperature above 50°C also prevents bacterial contamination, it is now possible to set the temperature above 50°C using a jacket tube, etc. It is desirable that

In the present invention, the stirring tank provided in the middle part is a reaction tank in which immobilized enzyme is present. This stirring is normally performed by pulsed flow, but angled paddle blades or the like may be attached to assist pulsed flow stirring. The stirring tank can also be set to a predetermined temperature. In addition, the static tanks 2 and 8 and the stirring tank 4, or the partition plate 5 that partitions the stirring tanks 4 from each other, are liquid-permeable, and are made of stainless steel sieve plates or perforated plates with a mesh structure that makes it difficult for the immobilized element to pass through. It is desirable to use the following.

In order to completely destroy the emulsion particles, a layer of sieve plates or a sieve plate layered with glass beads may be used.

A pulse generator is used to stir the reaction solution used in the present invention. As the pulse generator, a pulsating liquid pump may be used as is, or a pump that repeats reciprocating motion or perpendicular to the flow in the reactor may be attached in addition to the liquid pump. Although it is possible to attach the pulse generator to a high specific gravity substrate supply pipe, it is preferable to attach it to a low specific gravity substrate supply pipe.

Pulse flow is easy to perform because it only requires a short period of reciprocating motion using a pump. The period and amplitude can take any value, but if they are too slow, the movement speed of the reactant on the surface of the immobilized enzyme becomes slow, resulting in external diffusion resistance and poor oil/water separation.

The low-density substrate in the present invention is a substrate for an immobilized enzyme reaction, and is a substance such as oil, wax, phospholipid, various esters, glycerides, fatty acids, etc., which is usually difficult to dissolve in water, and its specific gravity is the same as that of water. lighter. When non-polar solvents such as isooctane, hexane, heptane, etc. are added, they are also treated as low density substrates. Also, high specific gravity substrate is
Water, a water-soluble substance such as glycerin, glycerophosphoric acid, alcohol, or an aqueous solution thereof.

Enzymes used in the present invention include lipases, phospholipases, proteases, and the like that act on ester bonds and have hydrolysis, ester synthesis, or transesterification abilities. As the enzyme immobilization method, any technique known as an enzyme immobilization method such as a carrier binding method or an entrapping immobilization method can be employed. Enzymes immobilized on phenolic resins have not been used in fluidized beds because they are easily abraded, but since the present invention uses pulsed flow for stirring, even enzymes immobilized on such resins can be used continuously for long periods of time. It is possible.

As an example of operation of the two-phase bioreactor of the present invention, oil is supplied from the low specific gravity substrate supply pipe 7, and oil is supplied from the high specific gravity substrate supply pipe 3.
When more glycerin is supplied, fatty acids and glycerides are produced from the low-density product discharge pipe 1.

Glycerin and water are recovered from the high specific gravity product discharge pipe 9. In addition, high acid value oil is supplied from the low specific gravity substrate supply pipe 7,
When glycerin is supplied from the high specific gravity substrate supply pipe 3, a mixture containing triglycerides and monoglycerides is obtained from the low specific gravity product discharge pipe 1 at the upper end, and the high specific gravity substrate discharge pipe 9
More unreacted glycerin is obtained. Incidentally, unreacted glycerin is dehydrated by vacuum distillation, molecular sieve, dry nitrogen, etc., and then supplied again from the high-density substrate supply pipe.

[Action of the invention]

The present invention provides a reaction system in which a two-phase substrate consisting of a low specific gravity substrate and a high specific gravity substrate is reacted with an immobilized enzyme.

Stirring is necessary to increase the rate of two-phase separation of the two-phase substrate, increase the transfer rate of reactants on the surface of the immobilized enzyme, and reduce external diffusion resistance. When stirred, a fine emulsion is generated. It is necessary to reduce the internal diffusion resistance of the immobilized enzyme by using this fine emulsion as a substrate for the continuous phase again. In the present invention, this emulsion is made into a continuous phase using only the partition plate. Therefore, there is no need to alternately arrange stirring tanks and static tanks in the intermediate portion, and the overall structure of the apparatus can be made compact.

Further, in the present invention, pulse stirring can be preferably used to remove air bubbles accumulated at the lower part of the partition plate. Removing air bubbles required a complicated operation such as passing a conduit through the air bubbles, but by using pulsed agitation that gives vertical reciprocating motion to the partition plate, the air bubbles can pass through the minute holes in the partition plate. This makes it possible to overcome the difficulty of removing bubbles.

Furthermore, since this pulse flow agitation provides a reciprocating motion in the vertical direction, it promotes the oil-water decomposition effect, which involves a large density difference, and has the effect of increasing the oil-water separation speed, and is not subject to physical stimulation such as stirring blades. This also has the advantage that the immobilized enzyme is less likely to wear out.

Furthermore, the pulse stirring device does not require complicated equipment such as mechanical seals, and can be easily implemented by adjusting the liquid pump system for low-density substrates, so equipment can be simplified. can.

〔Example〕

Next, the present invention will be explained in more detail with reference to Examples.

Example A reactor as shown in FIG. 1 was prepared. In this case, the partition plate 5 is a stainless steel partition plate with a 22 micron mesh, and is an emulsion breaking device! 10 is a styrene steel partition plate with a 100 micron mesh. Although not shown in the drawing, the reaction tank is surrounded by a jacket tube to keep the temperature constant. Conical standing tanks 2 and 8 are provided at the upper and lower ends. There is a specific gravity substrate supply pipe 7 and a high specific gravity product discharge pipe 9. The five intermediate stirring tanks 5 each have a diameter of 50 mm and a height of 301,111 mm, and the total volume inside the reactor including the upper and lower static tanks is 346 d. 5 g of immobilized enzyme (dry weight) was added to each stirring tank. The immobilized enzyme is Lipozyme produced by Novo. This enzyme is a lipase produced by the filamentous fungus Mucor mehei immobilized on a phenolic porous anion exchange resin.

A joint having branch pipes divided into three sides in the 120°C direction is connected to the low specific gravity substrate supply pipe 7 via one of the branch pipes, and the remaining branch pipe 11 is used for pulse generation. Machine 6
A metering pump is connected to the remaining branch pipe 12. The pulse generator 6 is configured to generate a pulsating current 30 times per minute. The size of one pulsating flow can be appropriately controlled from 0 to 15-.

At first, the pulse generator 6 is not operated, and the high specific gravity product discharge pipe 9 is
The reactor was closed, and dehydrated glycerin was supplied from the high specific gravity substrate supply pipe 3, and model high acid value oil was supplied from the low specific gravity substrate supply pipe 7, and the model high acid value oil was filled into the reactor so that 100% of the model high acid value oil was contained. The moisture content of dehydrated glycerin is 0.35
It was 4. The model high acid value oil was a 1:1 blend of refined rice bran oil and oleic acid, and had an acid value of 98.0 and a saponification value of 197.0. The degree of esterification is 100 (
It is calculated as saponification value - acid value)/saponification value, and is 50.3 for the model high acid value oil.

This model high acid value oil was also dehydrated using a molecular sieve, placed in an oil bath, and maintained at 60°C. The water content of the model high acid value oil was 0.027%.

After the reactor is filled with glycerin and model high acid value oil, the high-density product discharge pipe 9 is opened, and glycerin is discharged from the high-density product discharge pipe 9 at a rate of 1 m12/hour, and the high-density substrate is Dehydrated glycerin was supplied from supply pipe 3 at a rate of 1727 hours. On the other hand, if model high acid value oil is supplied from the low specific gravity substrate supply pipe 7 for 3 to 7 hours while operating the pulse generator, the product with a low acid value will be released from the low specific gravity product discharge pipe 1 at approximately the same rate. Recovered. The reactor was maintained at a constant temperature by flowing liquid paraffin at 60° C. through the jacket tube.

After 107.5 hours after the reaction, the degree of esterification remained almost constant. The continuous reaction progress up to 156 hours after the reaction was as shown in Table 1.

After 156 hours in Table 1, stirring was increased and continuous operation was continued until 433.5 hours. The reaction progress is shown in Table 2. 262.5
The reason why the degree of esterification decreased from around 300 to 3000 hrs. is probably due to the inactivation of the immobilized enzyme. The reason for the increase in the degree of esterification compared to Table 1 is considered to be that the external diffusion resistance was reduced due to stronger stirring.

Table 2 Next, reduce the feed rate of the model high acid value oil to 1-7 hours,
The other conditions were the same as those in Table 2, and the reaction was continued for up to 724.5 hours. The results are shown in Table 3.

The degree of esterification in Table 3 increases again because the substrate feed rate was reduced. As described above, with this reactor, even if a phenolic ion exchange resin with weak physical strength is used, the reaction can be carried out stably for a long time, and in addition to continuous reaction, continuous fractional collection of low specific gravity products and high specific gravity products is possible. It turned out that it is possible.

〔Effect of the invention〕

The present invention provides an efficient reaction system in which an immobilized enzyme is reacted with a two-phase reaction system consisting of a low specific gravity substrate and a high specific gravity substrate. Two-phase reactions are reactions that are widely practiced throughout the oil chemical industry, pharmaceutical industry, and food industry, such as fat and oil decomposition and glyceride synthesis. In addition, it is an enzymatic reaction that can be carried out at room temperature and pressure, causes no denaturation of the product, and causes little damage to the environment, so it will be widely used in the future as a bioindustry production process that poses no problems for people and the environment.

[Brief explanation of drawings]

FIG. 1 is an explanatory cross-sectional view of the two-phase bioreactor of the present invention. 1. Low specific gravity product discharge pipe 2, upper end standing tank 3, high specific gravity substrate supply pipe 4, stirring tank 5, partition plate 6, pulse generator 7, low specific gravity substrate supply pipe 8, lower end standing tank 9 , high specific gravity product discharge pipe 10 , emulsion breaking device 11 . Branch pipe connected to the 12° pulse generator Branch pipe connected to the metering pump

Claims (3)

[Claims] (1) Has a static tank at the upper and lower ends, has one or more stirring tanks in the middle, and partitions each tank with a liquid-permeable partition plate with microscopic holes that do not allow the immobilized enzyme to pass through. , and the stirring tank accommodates the immobilized enzyme, and the stationary tank at the upper end is provided with a high specific gravity substrate supply pipe at the bottom and a low specific gravity product discharge pipe at the top, A two-phase bioreactor characterized in that the tank is provided with a high specific gravity product discharge pipe in the lower part and a low specific gravity substrate supply pipe in the upper part thereof. (2) The two-phase bioreactor according to claim 1, wherein a pulse generator is connected to the low specific gravity substrate supply pipe. (3) When reacting two types of reaction substrates in a stirring tank containing immobilized enzymes, at least one of the reaction substrates is introduced into the stirring tank as a pulse flow to stir the contents of the stirring tank. An enzyme reaction method characterized by: