JPS5844401B2 - Naizousurukousoobunsansitenaru Jiyugoutaimaku Narabini Sonoseizouhouhou - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention & relates to a method of immobilizing enzymes in a filtration membrane and novel embodiments of membranes with built-in enzymes.

From a chemical engineering standpoint, enzymes have a number of properties that make them ideal catalysts.

Enzymes work well under conditions of quite mild pH and temperature, yet often induce fast reaction rates, and individual catalysts are highly specific in the type of reaction they induce.

The chemical reactions necessary to maintain living cells almost invariably rely on enzymatic contact mediation, and therefore a wide variety of reactions are regulated by these catalysts.

However, the potentially huge commercial potential for enzymes has not yet been realized due to their high initial cost, lack of stability, and difficulty in separating them from reaction products.

For example, since it is generally uneconomical to dispose of enzymes after completion of the desired reaction, attempts have been made to instead separate the reaction products from the enzymes and then recycle the enzymes into the feedstock. It's here.

However, it has been found that in systems of the type described hereinafter, the enzymes are generally sensitive to the required pumping speed and that the enzymes tend to denature under such conditions.

Another proposed solution to this problem is to immobilize the enzyme by attaching it to an artificial matrix.

Indeed, enzymes have recently been attached to natural polymers, synthetic polymers, and inorganic materials such as glass, metals, and metal salts in a variety of different forms, including beads, microcapsules, sheets, membranes, filter papers, and microtubules. There is.

Several techniques have been used to immobilize enzymes.

In adsorption methods, enzymes are retained by the attraction of opposite charges.

For example, a positively charged enzyme is bound to a negatively charged matrix.

Gel latex also captures enzymes.

The pores of the gel latex must be sufficiently large so that the substrate and the desired product can circulate inside or outside the latex.

A third method involves the creation of covalent chemical bonds where there is a direct chemical bond between the enzyme and the matrix.

Generally, when enzymes are attached to polymers, some form of covalent bonding is the mechanism of attachment.

This mode of attachment has the disadvantage that the chemical properties of the enzyme are necessarily changed, which often has an adverse effect on the reactivity of the enzyme.

Therefore, there is a real need for a method of immobilizing enzymes without adversely affecting their reactivity.

It is an object of the present invention to provide a new method for immobilizing enzymes in ultrafiltration membranes.

Another object of the present invention is to provide a method for achieving immobilization of enzymes in ultrafiltration membranes by entrainment.

Still another object of the present invention is to provide a novel ultrafiltration membrane containing an enzyme inside.

Other objects and advantages of the invention will become apparent from the description below.

Generally speaking, the present invention provides a polymeric ultrafiltration membrane containing an effective amount of a mechanically incorporated enzyme by precipitating the membrane from a solution of a polymer containing a dispersed enzyme additive. Relating to a method of manufacturing.

The enzyme-containing ultrafiltration membrane produced by this method has the ability to perform molecular separations and primarily for ultrafiltrate.
It has the ability to work selectively with biologically active, built-in enzymes that perform specific chemical reactions.

Since this enzyme is catalytic in nature, it is not ring-broken during the reaction and retains its activity (stability) for a long period of time.

More specifically, on October 26, 1971, Mika x
Polymeric, anisotropically porous membranes of the type described in U.S. Pat. No. 3,615,024 to Michaels A. It is produced by adding an enzyme to a polymer solution.

As described in the above patent specification, a solution of the polymer in an organic solvent is produced which is then cast into a thin film.

One side of the film is preferentially contacted with a diluent that is highly miscible with the organic solvent but not sufficiently miscible with the polymer to rapidly form the polymer film. to perform the precipitation.

The diluent is maintained in contact with the membrane until substantially all of the solvent has been replaced by the diluent.

Membranes produced by the method, which typically have a thickness of greater than about 0.002 inches and sometimes less than about 0.050 inches, have average pore diameters in the millimicron range, e.g. ~1ooo millimeter 1 micron or about the thickness of the cortical layer - ~10
It is characterized by a very thin and relatively compact skin layer that constitutes a barrier layer of about 0.1 to 5.0 microns thick of microporous polymer.

The remainder of the film structure is comprised of an integral support layer of coarsely porous polymer that offers little hydrodynamic resistance to fluids.

According to the teachings of the present invention, the enzyme is introduced into the polymer solution in finely divided form and in an effective amount and then thoroughly dispersed in the polymer solution by mixing.

This enzyme-containing polymer solution is spread to form a film as described above.

When brought into contact with a diluent, usually water, to which 1-3% of a surfactant (e.g. polyethoxyethanol such as Triton x 100) can be added, the polymer precipitates out and becomes different as described above. It produces a tropic membrane that contains enzymes within it, especially along its pore channels.

The following table shows suitable polymer-solvent systems for practicing the present invention.

Although the above polymer-solvent systems are by no means exhaustive, the literature is abundant on other polymer-solvent systems, and this is largely a matter of choice for the person skilled in the art.

Generally, the polymer solids content in the flow solution is in the range of about 5-40% of the polymer-solvent mixture.

Enzymes suitable for inclusion in ultrafiltration membranes include glucoamylase for starch saccharification, glucose imerase for converting glucose pentose to fructose, invertase for converting sucrose to invert sugar, and urea. There is urease to convert it into ammonia.

In addition to filtrate, which is commonly used in all systems in which water acts, there is a use as a diluent in special cases where organic solvents such as methanol, gasoline, fusel oil, etc. are present.

However, if the use of such diluents is indicated, compatibility with the enzyme must first be determined.

Film casting and enzyme-containing membrane deposition are carried out at relatively mild temperatures, usually in the range of about 0°C to about 90°C.

Examples are provided below to further explain the advantages of the present invention.

Example 1 A polymer solution is prepared by mixing 15F of acrylonitrile-vinyl chloride (ratio 4060) copolymer (Dynel) with 85mlI') dimethylformamide (DMF) to give a clear solution.

On the other hand, the urease enzyme is obtained as a powder with a microparticle size of 0.1 micron.

Next, 1.0 of this finely divided enzyme? is added to the solution and the container is agitated on a roller for 5 minutes to obtain a homogeneous dispersion.

The enzyme-containing mixture is applied in a thin film onto a porous paper support and the paper-supported film is then immersed in a water bath for 20 minutes at 25° C. to deposit an anisotropic film.

The membrane thus obtained had a thickness of 0.005 inch;
Visually, it is indistinguishable from anisotropic membranes that do not contain enzymes.

At this point it is calculated that 0.12151 urease is bound per 24 square inches of membrane.

This membrane was then cut into 3 inch diameter disks and Amicon #
401 batch cell through which distilled water is run to elute any remaining solvent and free enzyme.

The activity of this immobilized enzyme is tested by hydrolysis of urea.

Urea (H2NCONH2) is passed through the membrane at a rate of 5-50 GFD (gallons per square foot 17 days).

The catalytic reaction that occurs converts urea into CO2 and NH3.

The organicity of the enzyme is determined by the amount of ammonia in the permeated solution, and the results of this ammonia analysis are compared to the results of the Nessler reagent reaction on the feed material.

This analysis was performed by Bausch &
It is carried out colorimetrically using a spectral type "20" colorimeter from Lomb.

The conversion value obtained at an appropriate flow rate is inversely proportional to the flow, and is, for example, 49% at a flow rate of 5 GFD and 32% at a flow rate of 21 GFD.

At even higher flow rates, the conversion becomes linear, indicating that the limiting factors are the enzyme concentration and contact time in contact with the pore passages.

At low flow rates the conversion is higher than expected.

This is due to a new mechanism called diffusion.

Example 2 An enzyme-immobilized ultrafiltration membrane prepared as described in Example 1 is stored at 40"P for 3 months.

At this point the activity of the enzyme is determined by urea hydrolysis.

No decrease in enzyme activity is observed at this point. There was actually a slight increase in activity.

This is apparently due to polymer stabilization or crystallization, which is known to increase the transport properties of the membrane without affecting the membrane layer or molecular specificity.

Incorporating enzymes into ultrafiltration membranes has certain advantages over prior art immobilization of enzymes.

That is, when enzymes are conventionally encapsulated in capsules, diffusion through the capsule becomes a dynamic limiting factor in enzymatic conversion.

In the present invention, the enzyme is located along the pore channels through which the permeate flows easily, making it possible to obtain very high reaction rates.

Furthermore, in the present invention, the enzyme is immobilized in the liquid-solid separator, so that it reacts only with the permeate.

The membranes of the invention have large pore volumes, are anisotropic, and the nature of the transport mechanism allows very small volumes of permeate to contact high concentrations of enzyme for short periods of time.

In conjunction with the continuity of the turbulent flow path in which the reacted products are replaced by the reactants in a constant proportion, the dynamics of this system are much more optimal than is possible with encapsulated enzyme systems in the prior art. It has become.

Although the present invention has been described in terms of preferred embodiments, it should be understood that various modifications can be made without departing from the spirit and scope of the invention, as will be readily apparent to those skilled in the art.

Such modifications and changes are included within the scope of the invention as defined in the claims.

The present invention can be summarized as follows.

(1) A membrane of an anisotropic porous polymer comprising at least one type of incorporated enzyme dispersed in the anisotropic porous polymer membrane.

(2) The membrane according to item (1) above, which is composed of an acrylonitrile-vinyl chloride copolymer.

(3) forming a solution of the polymer dissolved in an organic solvent and mixing with this solution an effective amount of finely divided enzyme material to form a homogeneous dispersion of said enzyme in said solution; to form a film, one side of which is preferentially contacted with a diluent that is highly miscible with the organic solvent but not sufficiently compatible with the polymer. The diluent is then added to the film until substantially all of the solvent is replaced by the diluent and a polymer film with enzymes incorporated within the film is deposited. A method for producing an anisotropic porous polymeric membrane having at least one incorporated enzyme dispersed therein, the membrane being maintained in contact.

(4) The method of paragraph (3) above, wherein said diluent is water containing about 1-3% surfactant.

(5) The method according to item 4), wherein the membrane is composed of an acrylonitrile-vinyl chloride copolymer, and the solvent is dimethylformamide.

(6) forming a solution of the polymer dissolved in an organic solvent, spreading the polymer solution to form a thin film, and then applying the polymer, which is miscible with the solvent but not anisotropically porous polymers as a matrix for enzymes, where coalescence involves preferential contact with relatively incompatible diluents to rapidly precipitate said polymers into membrane structures; In producing the membrane, the enzyme is dispersed in the polymer solution prepared in the precipitation step,
An improved method comprising mechanically incorporating the enzyme into the membrane simultaneously with the precipitation.

(7) In the method of item (6) above, wherein the polymer is an acrylonitrile-vinyl chloride copolymer, the enzyme is added to the polymer solution by adding the enzyme in a finely divided solid state and then mixing. How to disperse.

(8) In the method of item (6) above, wherein the polymer is an acrylonitrile-vinyl chloride copolymer, the solvent is dimethylformamide, and the diluent is water, the enzyme is finely divided. A method in which the solid form is mixed with a polymer solution to produce a homogeneous dispersion.

Claims (1)

[Scope of Claims] 1. An anisotropic, microporous polymer membrane comprising at least one type of incorporated enzyme dispersed in the anisotropic, microporous polymer membrane. 2. Producing a solution of the polymer dissolved in an organic solvent, mixing with this solution an effective amount of finely divided enzyme material to form a homogeneous dispersion of said enzyme in said solution, and flowing this solution. spreading to form a film, one side of which is preferentially contacted with a diluent that is highly miscible with the organic solvent but not sufficiently compatible with the polymer. Rapid precipitation of the polymer is effected and the diluent is then kept in contact with the film until substantially all of the solvent is replaced by the diluent and a polymer film with enzymes incorporated within the film is deposited. A method of manufacturing an anisotropic porous polymer membrane with a small amount of internally dispersed enzymes.