JPS5826316B2 - Continuous reaction method using enzymes or microorganisms - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to an enzyme reaction method using hollow fibers.

Generally, enzymatic reactions are carried out by reacting an enzyme dissolved in water with a substrate, but in this case, there is a method of separating the enzyme without deactivating it when removing the product after the reaction is completed. Since there is no active enzyme, the conventional method has been to denature the enzyme that remains active and separate it from the product.

Therefore, this method has disadvantages in that enzymes are used uneconomically and the work is done in batches, which increases labor costs.

For this reason, in recent years, attempts have been made to perform continuous reactions with enzymes made insoluble, and methods for producing insoluble enzymes include the carrier binding method, in which the enzyme is bound to a water-insoluble carrier, and the carrier binding method, in which the enzyme is reacted with a functional reagent. Known methods include a crosslinking method that makes the enzyme insolubilizable, and an entrapment method that envelops the enzyme in a gel lattice or coats it with a polymer film.

However, this comprehensive method can be used for many enzymes because it does not cause any significant changes to the enzyme itself, but it can be used for gelation using conventionally known acrylamide and N,N'-methylbisacrylamide as polymerization catalysts. The method of enclosing the enzyme in this and the method of entrapping the enzyme in microcapsules or hollow fibers such as ethyl cellulose or polystyrene require complicated and expensive preparation, and also require the use of polymerization catalysts and organic solvents during this entrapment treatment. The disadvantage is that the enzyme may be deactivated due to the use of water or high-temperature treatment, or the activity of the insolubilized enzyme may be significantly lowered.

Furthermore, as seen in Japanese Patent Publication No. 51-25486, there is a method of carrying out an enzyme reaction using hollow fibers made of a copolymer of acrylonitrile and an olefinic unsaturated monomer; If a hollow fiber membrane is used, the product cannot be selectively permeated through the membrane, and therefore the product cannot be efficiently separated and recovered.

The present invention eliminates these drawbacks by flowing an enzyme or microbial solution inside or outside a semipermeable polyvinyl alcohol resin (hereinafter referred to as VPA) hollow fiber membrane, and flowing a substrate solution on the opposite side. , or by flowing a mixture of an enzyme or microbial liquid and a substrate liquid on the inside or outside of the hollow fiber membrane and flowing the dialysate on the opposite side, an enzyme reaction is carried out inside or outside the hollow fiber membrane. This is a continuous reaction method using enzymes or microorganisms, which is characterized by permeating the substrate or product through a membrane and selectively separating and removing the product from the substrate.

According to the present invention, enzymes or microorganisms are not immobilized and used, so there is no need for immobilization, and there is no decrease in activity unlike with immobilized enzymes, which shows high activity and has a low P content.
Since it uses a VA membrane, it is stable over a wide pH range, and the product can be more selectively permeated through the PVA membrane, allowing for efficient separation and recovery of the product. .

Furthermore, since the reaction is carried out by flowing the enzyme or microorganism cell solution, continuous enzymatic reaction is possible even though the enzyme or microorganism is not immobilized.

Furthermore, since PVA hollow fibers are used, the permeation area can be made small, and therefore a highly efficient enzymatic reaction can be carried out with a compact device.

In the present invention, in the enzyme reactor equipped with a PVA hollow fiber membrane, as shown in FIGS. Supported reactors of the so-called heat exchange type are preferred.

In one embodiment of the present invention, the enzyme or microbial liquid is introduced from 5 of the reactor 1 and flows outside the hollow fiber 2 6.
The substrate liquid was introduced from 3, and the hollow fiber was removed from 2.
flow inside.

During this time, the substrate inside the hollow fibers is permeated through the fiber membrane to the outside of the fiber by the dialysis action, where an enzymatic reaction takes place, and the product is permeated to the inside of the hollow fiber membrane by the dialysis action and taken out from 4.

When the reaction proceeds rapidly, no substrate is detected from 4, but when the reaction is relatively slow, a mixture of unreacted substrate and reaction product flows out from 4.

In this case, only the product can be finally obtained by introducing the effluent from step 4 into the same or different reactors and carrying out the reaction over several stages.

A second embodiment is the reverse of the above embodiment, in which the enzyme or microbial liquid is flowed inside the hollow fiber and the substrate liquid is flowed outside.

In a third embodiment, the mixture of enzyme or microbial solution and substrate liquid is passed on the outside of the hollow fibers 2, while the dialysate (for example water) is passed on the inside of the hollow fibers.

During this time, an enzymatic reaction occurs on the outside of the hollow fiber, and most of the products permeate through the fiber membrane to the inside of the fiber due to the dialysis action and are taken out together with the dialysate from 4. The product is 6
taken out from

The fourth embodiment is the reverse flow of the third embodiment.

In the embodiment described above, the unreacted substrate solution, enzyme or microorganism solution may be recycled and used again, or two or more reactors may be arranged and the reactor may be sequentially fed to the next reactor.

With this method, separation of substrate and product approaches completeness.

Further, in the embodiment described above, the liquid flowing inside and outside the hollow fibers flows in parallel, but it is also possible to flow in countercurrent.

The outer diameter of the hollow fibers used in the present invention is preferably 5000μ or less, preferably 20 to 2000μ, particularly preferably 40 to 1000μ.

The smaller the outer diameter of the hollow fiber, the better its mechanical strength and pressure resistance.
Therefore, the film thickness can be reduced, and the reactor can also be made more compact.

The thickness of the hollow fiber membrane is preferably from 5 to 100 microns, preferably from 10 to 80 microns, from the viewpoint of dialysis properties.

PVA, which is the material of hollow fibers, refers to PVA that is normally used in vinylon, etc., and monomers such as ethylene, vinyl pyrrolidone, vinyl chloride, methyl methacrylate, acrylonitrile, and itaconic acid that can be copolymerized with vinyl alcohol. copolymers with the body, etc.

The PVA-based membrane used in the present invention has a swelling degree of 1.01 to 1.8.
0 times, more preferably 1.01 to 1.40 times.

PVA-based membranes have hydrophilic properties as a characteristic of PVA, and can be made into membranes with any degree of swelling.However, in order to improve dialysis and permeability, the degree of swelling must be increased from 1.01 to 1.08 times. It is desirable that there be.

Here, the degree of swelling is a value expressed as the ratio of the cross-sectional thickness or outer diameter of a flat membrane or a hollow fiber membrane to that of a wet membrane or a dry membrane.

Dry thickness or outer diameter measurement at room temperature 20℃, RH60
% after leaving it for a day and night, wet measurement is done after leaving the sample at 25%.
Perform this after leaving it in water at ℃ for a day and night.

Almost all enzymes and bacterial cells can be used in the present invention.

For example, amylase, glucoamylase, trypsin,
Chymotrypsin, pepsin, papain, vancreatin, aminoacylase, nuclease, ribonuclease, phytin, catalase, galactosidase, ATP deaminase, L-lyretamate dehydrogenase, phosphatase, tyrosinase, invertase, flavokinase, streptokinase, avirase, ATP - Creatine phosphotransferase, pectinase, carboxypeptidase, L-asparaginase, maltase, lactase, urease, tannase, lipase, glucose isomerase, melibiase, aldolase, cellulase, anthocyanase, naringinase, hesperidinase, glucose oxidase, Encoli (aspartase) ), Pseuput 1da (arginine deaminase), etc.

A plurality of these enzymes and microbial cells may be used simultaneously.

Industrial applications of the present invention include, for example, DL-amino acid splitting glycoamylase by amino acid acylase, α
- production of glucose from starch by amylase, production of glucose from cellulose by cellulase, production of fructose syrup from glucose by glucose isomerase, production of invert sugar by invertase, protein hydrolysis by protease, production of semi-synthetic penicillin by penicillin amylase, Hydrolysis of lactose by β-galactosidase, Encoli (aspartase)
Production of L-aspartic acid by Pseuput 1
Examples include the production of L-citrulline using da (arginine deaminase).

Among these, the present invention is particularly suitable for reactions in which the substrate is a polymer and the product is a low molecule.

The present invention will be further explained below with reference to Examples.

Example 1 The reactor shown in Figures 1 and 2 (polyvinyl alcohol hollow fiber (outer diameter 600μ, film thickness 50μ, length Im1, degree of swelling 1.2)
5) The following experiment was conducted using 1o5 pieces).

5 in Figure 1, the starch ice solution that had been liquefied in advance with amylase was adjusted to 55-57°C and pH 5.0 (starch concentration 32% W/W1, glucoamylase 3.0-6.0
X 10-2%15 UN I T7g starch) 0
．． Water was flowed outside the hollow fibers of reactor 1 at a rate of 5'' hr, while water from 3 was flowed into the hollow fibers at a rate of 0.5 TLl/hr.

As a result, 0.5”/hr (glucose concentration 13
%) and 5 ml/hr (starch concentration 16%, g/l/I amylase concentration 3.0~
6. An effluent was obtained at a ratio of 10-2% OX and 2% glucose concentration.

1 In this case l unit = (Bl of 10ml of 1% starch solution
ueValuc+40°C, the force that decreases by 1% in 1 minute is defined as 1 unit.

) Example 2 Using a reactor similar to that used in Example 1,
The following experiment was conducted.

After heating the cellulose at 200°C for 25 minutes in air,
It was ground to ~150μ fine particles and suspended in water.

Add cellulase and acid to this and create a mixed aqueous solution of cellulose and cellulase (cellulose concentration 10%, cellulase*).
FP=1°8, pH 4-5) was poured out of the hollow fiber at a rate of 0.08 ml/min from 5, while 0.1 ml of water was poured from 3.
/min into the hollow fiber.

In addition, in order to maintain the temperature inside the reactor system at 40°C, the reactor was
The test was carried out in a water bath at 5°C.

As a result 4, 0.1 m 11 minutes (glucose concentration 5, 1
The effluent was obtained at a rate of ~s, 6 m9/ml), and the effluent was
．． 08ml/min (cellulose concentration 10%, cellulase p
p=t, s, Kurus: l-s (D concentration 0.1-0.5"
%) of the effluent was obtained.

*Method for disintegrating pieces of astringent paper Add paper IXICrrt and measure the time required for complete disintegration.

The required time was 0 minutes, and the enzyme titer was expressed as follows: dry enzyme Y in 5 ml of enzyme solution.

Enzyme titer -150/(X,Y)XIo,000 l~ Example 3 Using a reactor similar to that used in Example 1,
The following experiment was conducted.

5 in Figure 1, a urea-denatured hemoglobin aqueous solution (urea-denatured hemoglobin concentration 0.6%, pH 3.0, M milk 01 acid buffer) and the protease "Bunapsin" (trade name of Nagashio Sangyo) from the filamentous fungus Aspergi Ilus 12
．． 5 ■ at a ratio of 5:1, and this mixed solution is 0.65
Water was allowed to flow outside the hollow fiber at a rate of 0.657711/min from 3 on the outside of the hollow fiber at a rate of 0.657711/min.

Further, the reaction was carried out while maintaining the temperature inside the reactor system at 40°C.

From the result 4, 0.65 m1/min (amino acid concentration 0.2
5%, partial hydrolyzate concentration 0.10%), and 0.65 rnl 1 minute from 6 (hemoglobin concentration 0.2%).
0%, bunapsin 12.5■, amino acid concentration 0.05%
, partial hydrolyzate concentration 0.10%).

[Brief explanation of the drawing]

FIG. 1 is a longitudinal cross-sectional view showing one example of a reactor used in the method of the present invention, and FIG. 2 is a cross-sectional view of the reactor.

Claims (1)

[Scope of Claims] 1 Enzyme or microbial liquid is poured on the inside or outside of a polyvinyl alcohol resin hollow fiber membrane, and a substrate liquid is poured on the opposite side, or enzyme or microbial liquid is poured on the inside or outside of the hollow fiber membrane. By flowing a mixed solution of the substrate solution and flowing the dialysate on the opposite side, the enzyme reaction is carried out inside or outside the hollow fibers, and the substrate or product is permeated through the hollow fiber membrane. A continuous reaction method using enzymes or microorganisms, which is characterized by selectively separating and extracting a substance from a substrate. 2. The method according to claim 1, wherein the degree of swelling of the polyvinyl alcohol resin hollow fiber membrane is 1.01 to 1.80 times.