JPH0320231B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to an enzyme-immobilized film and a method for producing the same. In recent years, in the pharmaceutical industry, food industry, etc., enzymes have been increasingly applied to industrial processes as extremely useful catalysts that take advantage of the substrate specificity of enzyme reactions. In conventional enzyme reactions, the reaction is carried out by dissolving in water, and it is not easy to separate the enzyme and the reaction product after the reaction is completed. Therefore, it is difficult to collect and reuse enzymes each time. Therefore, in conventional industrial processes, a batch method has to be adopted, which results in poor enzyme utilization efficiency and is extremely uneconomical. Therefore, studies have been actively conducted on immobilized enzymes, including methods of binding and immobilizing enzymes to substrates using physical adsorption methods, ionic bonding methods, covalent bonding methods to prevent enzymes from eluting during reactions, and methods of immobilizing enzymes in porous gels. Research is currently underway on entrapment methods, such as physically confining or encapsulating with high molecular weight polymers. However, for example, in the ionic bonding method, it is easily affected by the type of buffer solution or pH, and if the reaction is performed in a state of high ionic strength, the enzyme may be released from the carrier. becomes limited. Among these several immobilization methods, the comprehensive method has been studied the most. This has the advantage that the comprehensive method can be applied to a large number of enzymes since the enzyme protein itself is not chemically bonded to the immobilization carrier. Immobilized enzymes encapsulated in acrylamide gels polymerized using radiation or high-speed electron beams are a good example of industrial use. However, these methods of using radiation to cause polymerization and entrapment have the disadvantage that the radiation passes through the monomer layer, damaging the enzyme and reducing its activity. In addition, the polymerization temperature must be maintained at a very low temperature, and the facility must be equipped with large-scale equipment from the viewpoint of radiation sources and safety protection, and although it requires high energy, it is not efficient and economical. bad. Under these circumstances, an enzyme immobilization method using new technology has been awaited. On the other hand, the stability of enzymes has been found to be such that enzymes can remain active for long periods of time when they are protected by appropriate compounds such as stabilization in sugar or glycerin. The present inventors completed the present invention as a result of intensive study and research in order to eliminate the conventional defects. An object of the present invention is to provide a novel enzyme-immobilized film that maintains enzyme activity for a long period of time. Another object is to provide a method for manufacturing such a film industrially easily and inexpensively. The present invention provides a hydrophilic film in which a hydrophilic unsaturated monomer is graft-polymerized to a hydrophobic polymer forming a hydrophobic film in the presence of ionized gas plasma, and a complex is formed with the graft polymer obtained by the polymerization. This is an enzyme-immobilized film in which an enzyme is entrapping immobilized in a complex using a complementary polymer having the following properties.The method of the present invention involves injecting a hydrophilic polymer into a hydrophobic polymer in the presence of ionized gas plasma to form a hydrophobic film. A hydrophilic film on which a saturated monomer has been graft-polymerized is immersed in an aqueous solution containing an enzyme and a complementary polymer that has the ability to form a complex with the graft polymer obtained by the polymerization, thereby entrapping and immobilizing the enzyme in the complex. Characterized by urging. The hydrophobic film applied to the present invention may be any film made of a hydrophobic polymer, but from the viewpoint of quality and cost, hydrophobic films made of polyethylene, polypropylene, or polyethylene terephthalate are preferred. The hydrophilic unsaturated monomer is not particularly limited as long as it has a vinyl group or an allyl group and is hydrophilic and can be subjected to normal radical polymerization. Generally acrylamide, methacrylamide, N-vinylpyrrolidone; acrylic acid, methacrylic acid, P-styrenesulfonic acid, vinylsulfonic acid, 2-methacryloyloxyethylsulfonic acid, 3-methacryloyloxy-
2-hydroxypropylsulfonic acid, allylsulfonic acid, methacrylsulfonic acid, ammonium salts and alkali metal salts of these acids, N,
N dimethylaminoethyl acrylate, N,N dimethylaminoethyl methacrylate, diethylaminoethyl acrylate and diethylaminoethyl methacrylate, bases of 2-vinylpyridine and 4-vinylpyridine, quaternized products of nitric acid, dimethyl sulfate, diethyl sulfate or ethyl chloride. Preferred hydrophilic unsaturated monomers include acrylic acid, methacrylic acid, 2hydroxyethyl methacrylate,
2-hydroxyethyl acrylate, acrylamide, methacrylamide, methacrylamide, 2-acrylamido-2-methylpropane sulfonic acid,
N,N dimethylaminoethyl methacrylate or N,N dimethylaminoethyl acrylate can be mentioned. In addition, the graft polymerization rate in the graft polymerization in the present invention can be freely varied depending on the purpose and use, but is preferably at least 1% by weight, preferably 10% by weight, based on the hydrophobic polymer forming the film.
More preferably it is 100% by weight, particularly preferably 300% by weight. The polymerization in the present invention is so-called plasma initiated polymerization. That is, plasma-initiated polymerization is one in which a non-equilibrium ionized gas plasma is used to initiate polymerization or create active species, and most of the polymerization is completed in the absence of plasma. Generation of an ionized gas plasma can be accomplished by any of the known methods for generating plasma. For example, J. R. Hollahan and A. T. Bell, “Applied Techniques of Plasma Chemistry”, Wiley, New York 1974 and M. Shen.
ed. “Plasma Chemistry of Polymers” Detzker, New York, 1976. That is, the monomer can be placed under vacuum between parallel plate electrodes connected to a high frequency generator, and the plasma can be generated using the parallel plates either outside or inside the vacuum chamber.
Alternatively, an electric field may be created by an external induction coil to generate a plasma of ionized gas, or a plasma may be generated by placing oppositely charged electrodes spaced apart directly into a vacuum chamber. In the present invention, only a film made of a hydrophobic polymer is held in the presence of an ionized gas plasma, and then a hydrophilic unsaturated monomer is prepared in an aqueous solution of a hydrophilic unsaturated monomer in the absence of the plasma. be graft polymerized onto a film made of a hydrophobic polymer, or
Alternatively, polymerization of a film made of a hydrophobic polymer and an aqueous solution of a hydrophilic unsaturated monomer, or a film made of a hydrophobic polymer and a gas of a hydrophilic unsaturated monomer is initiated in the presence of ionized gas plasma. Thereafter, the film is immersed in an aqueous solution of the monomer to perform graft polymerization onto the film in the absence of the plasma. Instead of polymerizing with an aqueous solution of a hydrophilic unsaturated monomer in the absence of an ionized gas plasma, an emulsion or an organic solvent solution in or of the monomer may be used. To explain the method of the present invention in more detail, the degassing is 10 ^-1
A film made of a hydrophobic polymer is irradiated with plasma using the above method at a pressure of ~10 ^-4 Torr. Glow discharge is preferably performed at 20 to 200 watts, more preferably 40 to 100 watts, to generate active species in the hydrophobic polymer. The irradiation time is usually 1 to 3600 seconds, preferably 1 to 1800 seconds. After plasma irradiation, when the film is polymerized in an aqueous solution of a hydrophilic unsaturated monomer in the absence of the plasma, the monomer is consumed by the active species of the film. So-called graft polymerization begins. Post-polymerization temperature and time vary depending on the type of monomer used and are not particularly limited, but usually the temperature is 1~
A temperature of 60°C for 1 to 25 hours is sufficient. Depending on the type of monomer, if the temperature exceeds 60°C, thermal polymerization may occur and low-molecular polymers may also be produced, so care must be taken. The thus obtained graft polymerization compound of the hydrophilic unsaturated monomer onto the hydrophobic film has a straight chain hydrophilic polymer having a high degree of polymerization grafted onto the surface of the film. It can be seen that the properties are improved, for example, when polyethylene is grafted with 24% by weight of acrylic acid by the method of the present invention, the water contact angle is significantly improved to 56° compared to 101° for untreated polyethylene. The complementary polymer of the graft polymer applied to the present invention is a polymer that forms an intermolecular complex with the graft polymer. For example, when the graft polymer is made of a monomer having a carboxylic acid group such as acrylic acid, methacrylic acid, or maleic anhydride, complementary polymers include polyethylene glycol, polyvinylpyrrolidone, polyxylviologen, polyethyleneimine, etc. Examples include polyvinylpyridinium chloride. In addition, when the graft polymer is a polymer consisting of a basic monomer such as acrylamide, methacrylamide, vinylpyridine, N,N-dimethylaminoethyl methacrylate, or N,N-dimethylaminoethyl acrylate, its complementary polymer Examples include polyacrylic acid, polymethacrylic acid, poly2-acrylamido-2methylpropane sulfonic acid, polystyrene sulfonic acid, and polyvinyl sulfonic acid.
When the film and the complementary polymer are immersed in an aqueous solution for a predetermined period of time, an interpolymer complex is formed. Further, the amount of complementary polymer is generally controlled by the amount of graft polymer to the film; for example, if the amount of graft polymer is large, a large amount of complementary polymer is also required. Therefore, the amount of complementary polymer is at least equimolar in terms of repeating units to the grafted polymer. That is, since the amount of the complementary polymer that forms a complex with the graft polymer is attached to the film, the complex can be formed in an aqueous solution containing a large excess of the complementary polymer. The amounts of the complementary polymer and enzyme in the aqueous solution are not particularly limited, but are usually about 0.01 to 1 molar concentration and 0.1 to 30 weight concentration, respectively. The temperature of the aqueous solution is preferably room temperature or lower, and the film is usually immersed under stirring for about 15 minutes to 1 hour. Moreover, an inorganic compound such as common salt can be added to the aqueous solution to increase the ionic strength. The pH of the aqueous solution is adjusted to facilitate complex formation depending on the type of complementary polymer. After soaking,
If necessary, it is washed with water and dried, but drying is usually carried out at room temperature and under reduced pressure if necessary. The types of enzymes that can be used in the present invention are not particularly limited, but examples include invertase, urease, glucose isomerase, glucose oxidase, protease, uricalase, acylase,
Examples include α-amylase, β-amylase, collagenase, arginase, acidic protease, alkaline protease, L-glutamic acid decarboxylase, rebarase, D-amino acid oxidase, β-glucosidase, and glucoamylase.
Enzyme content is 0.1 for immobilized enzyme film
~30% by weight is preferred. In the present invention, the enzyme is dispersed in an aqueous solution with a complementary polymer, and the enzyme is immobilized simultaneously with the formation of an intermolecular complex with a hydrophilized (grafted) film.
In other words, the enzyme is not irradiated with radiation, polymerization heat, or even plasma to comprehensively immobilize the enzyme. Therefore, in the present invention, not only is the enzyme not damaged or its activity reduced at all, but also the enzyme is immobilized in the inter-polymer complex. The resulting enzyme is extremely stable and maintains its activity for a long period of time because the polymer complex exhibits excellent protection against enzyme inhibitors, pH, temperature, etc. Furthermore, since it uses a hydrophobic film as a carrier, it has extremely excellent mechanical strength, which is revolutionary and cannot be obtained using conventional methods. The present invention will be explained below with reference to Examples. During the experiment, the grafting rate, water contact angle, and enzyme activity retention rate were measured by the following methods. a Grafting rate Grafting rate is calculated by weight after drying by washing the grafted film with stirring in a good solvent for 2 days to completely remove unreacted monomers and homopolymers Grafting rate (%) = After treatment Film weight - Initial film weight / Initial film weight x 100 b Water contact angle Measured using contact angle type CA-P manufactured by Kyowa Kagaku Co., Ltd. c Activity retention rate (%) = V ₁ /V ₀ ×100 The enzyme-immobilized film was thoroughly washed, and then 1
The enzyme activity at the time of the second measurement of enzyme activity is V ₀
After that, the film was immersed in a buffer solution for 10 days, and the enzyme activity was defined as _V1 . or,
Enzyme activity was expressed as the rate of substrate decomposition. Example 1 A square polyethylene film with a thickness of 50 μm and a side of 15 mm was placed on the wall of a separable flask with an inner diameter of 35 mm at 10 ^-3 Torr, and was placed between two copper electrode plates connected to a 13.56 MHz high frequency generator. insert
Generate and irradiate plasma for 90 seconds with an output of 100W,
Thereafter, a 50% by weight aqueous acrylic acid solution was added in 10 ^-3 Torr, and post-polymerization was carried out at 25° C. for 12 hours. The obtained graft film was washed with stirring in pure water for 2 days, the solvent was removed, and dried to make a sample. When the contact angle of water was measured, it was significantly hydrophilic at 56° compared to 101° for the original polyethylene film. The graft rate was 24%. The film was treated with invertase (activity: sucrose 4g/ml, 20°C x 30 minutes PH4) 10
An immobilized enzyme film was obtained by immersing it in a 10% aqueous solution of polyethylene glycol with a degree of polymerization of 500 containing 1% by weight and 1% by mole of NaCl to entrapping and immobilize invertase in an interpolymer complex between the film and polyethylene glycol. Ta. After thoroughly washing the immobilized enzyme film with water, add 10% of 0.2M acetate buffer (PH4.6) containing 10% by weight of sucrose.
ml and perform a hydrolysis reaction at 25℃ for 90 minutes.
The hydrolysis rate was determined by quantifying the specific light application degree. In addition, in order to investigate the stability of the enzyme-immobilized film, we calculated the ratio of enzyme activity after immersing the enzyme-immobilized film in the above buffer solution for 10 days to this value as the activity retention rate, which was 0.04Mol/min. , g, and the activity retention rate was 90%. Example 2 Using the same film, apparatus, and experimental conditions as in Example 1, invertase was immobilized by complex formation between the graft polymer and its complementary polymer as shown below. Konatsuta. When the immobilized invertase activity of the obtained immobilized enzyme film was measured under the same conditions as in Example 1, an enzyme immobilized film having the enzyme activity as shown in the table below was obtained.

[Table] Example 3 Using the same method and equipment as in Example 1, a polyethylene film (15 mm x 15 mm) was coated with 50% by weight containing equimolar amounts of acrylic acid and 2-acrylamido-2-methylpropanesulfonic acid (AMPS). aqueous solution
When grafting was performed using 10ml, the grafting rate was
A 240% polyethylene film was obtained. This polyethylene film was grafted with poly(vinyltrimethylammonium chloride) 10
25ml of wt% aqueous solution and 10ml of amylase 5wt% aqueous solution
ml for 30 minutes at room temperature to immobilize amylase at the same time as interpolymer complex formation. This immobilized enzyme film contains 1.0% starch by weight.
When the film was immersed in an aqueous solution containing the enzyme and its enzyme activity was determined from the change in light intensity, it was found that this film had an 8x
It was found to have the ability to hydrolyze starch at a rate of 10 ^-4 mol/min, g.

Claims (1)

[Scope of Claims] 1. After irradiating a hydrophobic polymer forming a hydrophobic film with ionized gas plasma, a hydrophilic film is obtained by graft-polymerizing a hydrophilic unsaturated monomer in the absence of the plasma, An enzyme-immobilized film in which an enzyme is comprehensively immobilized in a complex by the graft polymer obtained by the polymerization and a complementary polymer having a complex-forming ability. 2. The film according to claim 1, wherein the hydrophobic polymer is polyethylene, polypropylene, or polyethylene terephthalate. 3 The hydrophilic unsaturated monomer is acrylic acid, methacrylic acid, 2hydroxyethyl methacrylate, 2hydroxyethyl acrylate, acrylamide,
2. The film according to claim 1, which is methacrylamide, 2-acrylamido-2-methylpropanesulfonic acid, N,N dimethylaminoethyl methacrylate, or N,N dimethylaminoethyl acrylate. 4. The film according to claim 1, wherein the graft ratio of the graft polymerization is at least 1% by weight based on the hydrophobic polymer forming the film. 5. The film according to claim 1, wherein the complementary polymer is polyethylene glycol, polyacrylic acid, polyxylylviologen, or polyethyleneimine. 6. The film according to claim 1, wherein the amount of the complementary polymer is at least equimolar in repeating units to the grafted polymer. 7. The film according to claim 1, wherein the enzyme content is 0.1 to 30% by weight. 8 After irradiating a hydrophobic polymer forming a hydrophobic film with ionized gas plasma, a hydrophilic film is obtained by graft polymerizing a hydrophilic unsaturated monomer in the absence of the plasma, using an enzyme and the polymerization. 1. A method for producing an enzyme-immobilized film, which comprises immersing the film in an aqueous solution containing a complementary polymer having the ability to form a complex with the graft polymer obtained by the above procedure, thereby entrapping and immobilizing the enzyme in the complex. 9. The method according to claim 8, wherein the hydrophobic polymer is polyethylene, polypropylene or polyethylene terephthalate. 10 The hydrophilic unsaturated monomer is acrylic acid, methacrylic acid, 2-hydroxyethyl methacrylate, 2
Hydroxyethyl acrylate, acrylamide, methacrylamide, 2-acrylamide-2-
Claim 8 which is methylpropanesulfonic acid, N,N dimethylaminoethyl methacrylate or N,N dimethylaminoethyl acrylate
The method described in section. 11. The method according to claim 8, wherein the graft ratio in the graft polymerization is at least 1% by weight based on the hydrophobic polymer forming the film. 12 Complementary polymer is polyethylene glycol,
9. The method according to claim 8, which is polyacrylic acid, polyxylylviologen or polyethyleneimine. 13. The method according to claim 8, wherein the amount of the complementary polymer is at least equimolar in repeating units to the grafted polymer. 14. The method according to claim 8, wherein the enzyme content is 0.1 to 30% by weight.