JPS585193A - Preparation of immobilized enzyme entrapping with complex between high polymers - Google Patents

Abstract

PURPOSE:To control the entrapping ratio of an enzyme into a complex in entrapping and immobilizing the enzyme with the complex formed by the secondary intramolecular bonding strength, e.g. ionic hydrogen bonding or charge transfer interacting force, by changing the pH, solvent, temperature, etc. CONSTITUTION:A complex between high polymers formed by the so-called intramolecular bonding strength, e.g. ionic bonding, hydrogen boinding or charge transfer interacting force, can be obtained by mixing two high polymeric solutions capable of interacting on each other. In preparing the complex, an enzyme is added simultaneously, and the pH, ionic strength, solvent, temperature, etc. are changed to entrap the enzyme in the high polymeric complex. Thus, the entrapping ratio thereof and the reaction activity of the formed immobolized enzyme are controlled. The resultant immobilized enzyme is protected from inhibiting factors of enzymic activity, e.g. pH, temperature, ionic strength, metal, organic or inorganic reagents, by the high polymeric complex.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention utilizes a polymer complex to encapsulate an enzyme, thereby producing an immobilized enzyme.

Conventionally, enzyme immobilization methods include carrier binding method, crosslinking method,
Many comprehensive methods are known, but among these methods, the envelope method has the advantage that it can be applied to a large number of enzymes because the enzyme protein itself is not chemically bonded to the immobilization carrier. poly7
Immobilized enzymes encapsulated in Krylua ξ-dogel are a good example of industrial use, but examples of enzyme immobilization that utilize complex formation between polymers as in the present invention are still unknown. It has not been done.

Polymer axes (or high molecular weight co/plexes) can be obtained by mixing two types of polymer solutions so that they interact with each other. (See chapter 8 of "Functional Polymers" (Kyoritsu Publishing), Vol. 7, "Functional Polymers" (Kyoritsu Publishing)). For example, when an aqueous solution of 41 vinylpyridium salt, which has a positive charge, is mixed with an aqueous solution of polystyrene sulfonic acid having an equal amount of a negative charge, a precipitate is instantly formed, and a so-called polyion complex is obtained. This polyion complex is an intermolecular complex formed by electrostatic bonding force, and is thought to have a network structure or ladder structure crosslinked by high molecular weight salt bonds. Such inter-polymer resistance is not limited to electrostatic bonding force, but is also caused by polymethacrylic acid (PMAA) and polyacrylic acid (F
AA) and polyvinylpyrrolidone (PVPdn) or polyethylene glycol (PEG), etc., formed by inter-polymer complexes formed by linked hydrogen bonds, and formed by charge transfer interactions between polyaminostyrene and polymaleic anhydride, etc. There are various types of inter-polymer complexes, other stereo complexes, etc., and the present invention does not limit the type or adjustment method. In addition, as described in the above-mentioned literature, one component of the polymer complex is made to coexist with the enzyme as a polymer, and the other component is made to coexist as a monomer, using the so-called "matrix polymerization" method. It is also possible to incorporate enzymes simultaneously with polymerization. What is important is a method that utilizes the network or ladder structure formed by the polymer complex to encapsulate the enzyme, thereby producing an immobilized enzyme. The network density formed by the interpolymer complexes is generally very high and stable enough to trap and entrap the enzyme within the network. Moreover, the network obtained by the inter-polymer complex of the present invention differs from the network structure formed by chemical cross-linking methods, such as acrylamide gel, in that it depends on the various conditions of complex formation, such as -pH and ionic strength. It has the characteristic that the crosslink density, its distribution, bond strength, etc. can be easily controlled by the type of solvent, temperature, etc. Moreover, this network can be obtained simply by mixing polymer solutions under appropriately selected conditions, and the process is not only extremely simple, easy, and quick, but also economical and efficient, and can be achieved by methods such as chemical crosslinking. Damage and inactivation of enzymes due to radiation, electron beams, light, etc., heating or cooling for polymerization,
No operations such as depressurization are required. This is the second feature of the present invention.

The third feature that can be realized by the present invention is that it has the function of protecting enzymes from 11 various heavy metals, organic and inorganic compounds, potassium, temperature, salt, and other enzyme inhibitors. Since the inter-polymer crosslinked structure produced by the present invention is not based on chemical bonds, it can be easily constructed by changing the reaction conditions. The crosslinking density, distribution, bonding strength, etc. vary depending on the complex site, resulting in diversity. Some of these complex-forming sites are chemically highly potent, together with non-bonding sites (groups that can form intermolecular complexes but do not actually participate in intermolecular bonding). It has the ability to preferentially bind to other ions and reagents when they coexist. This is the reason why it exhibits the ability to protect enzymes when compounds that have inhibitory effects on enzymes, such as heavy metal ions and klH1 basic organic substances, coexist.

For the same reason, the optimal p, H
It is also possible to change and control the so-called optimal conditions for enzymes, such as temperature, ionic strength, and temperature.

There are no restrictions on the enzymes applicable to the present invention. However, when it comes to polymer complexes, it is important to choose a combination in which the component polymer and the complex itself do not lose or reduce their activity toward enzymes. Also, depending on the binding strength and type of the polymer complex, it may dissociate under conditions other than the complex formation conditions such as the specified IiH range, temperature, and ionic strength, or the bonds may become loose, causing enzyme deviation. It is also necessary to take this into account. On the other hand, there are also cases where the conditions for forming inter-polymer complexes result in a decrease in enzyme activity. Furthermore, as with other immobilization methods, depending on the substrate, it may not be able to contact the entrapped enzyme and exhibit no enzyme activity. In short, is that all there is to it? Considering various points j1! The first step is to select the optimal enzyme-polymer complex combination. ! It is.

Next, examples of the present invention will be shown and explained in detail.

Example 1 Add 10F of common salt to 100% of an aqueous solution of polymethacrylic acid (PMAA) having a molecular weight of 780,000, and use commercially available Ivertase.
In addition to i, the others are 5j! When prepared in the same manner as in Example 16, an interpolymer complex was obtained with a yield of about 10%. When the inverter (inclusion rate) was examined using the method of Example 1, it was approximately ao%.

Next, using this immobilized enzyme a2p, its activity was examined in various gauze H ranges, and the results shown in Table 2 were obtained.

Table 2 The results in Table 2 were approximately so, -ss% of the activity of invertase in the free case. When the immobilized enzyme obtained in this way was used repeatedly, the enzyme deviation occurred during the first 16 days.
Approximately 3 Chi were introduced in H1s, but Shi H? -0, no deviation of the enzyme was observed. Further, at pH 6 or above, dissolution of the complex occurred.
Add 10s1jt of invertase aqueous solution to the wt% aqueous solution, and slowly add 511s aqueous solution of polyhinylbenzyltrimethylammonium chloride 9(de (molecular weight 200,000)) to this solution while stirring.

In addition, we created an interpolymer complex based on ionic bonds. This complex was washed with water and then dried separately to obtain a white powdery polymeric complex with a yield of approximately 100%. The immobilized enzyme thus obtained was mixed with acetone and sodium bromide. When the mixture was dissolved in water and subjected to ultraviolet spectroscopic analysis in the same manner as in Example 1, it was found that about 80 invertases were contained. The activity of this immobilized enzyme was measured using 2F at various pH values using a 101 amount tisyosaccharide solution as a substrate. was exhibited in a wide PH range.Also, enzyme deviations were also observed at s PH2
, -PH was as low as about 2% within one day in the range of 13.

Example 5 Published Amerin, Cu, an inhibitor of invertase.

) (When the immobilized enzyme prepared in Example 1 and 3.4 in the presence of 1-9M Shirape was examined for the preservation effect of the inter-polymer complex against the enzyme, the results shown in Table 4 were obtained.

As mentioned above, the protective effect of the enzyme due to the inter-polymer complex was observed in all the inhibitors.2+ was particularly remarkable in the case of A = IJ and HP.Example 6 Example 4 In the same manner as in Example 4, the enzyme was immobilized using 20 ml of a 20 ims aqueous solution of filamentous fungal glucose oxidase.The enzyme coverage rate of the obtained immobilized enzyme was examined in the same manner as in Example 4, and was found to be approximately 45 It was Chi. When this immobilized enzyme IP was used and the enzyme activity was examined by ultraviolet absorption spectrum using a 10% glucose aqueous solution at GIH45, % a x lo M/MI
It became clear that the enzyme was oxidized with N, indicating that an immobilized enzyme with high activity was obtained.

Example 7 Five volumes of the immobilized enzyme prepared in Example 4 were packed into a column with a diameter of 1° and thoroughly washed with a 19H4L6 buffer solution.
It was allowed to flow down from the top of the column at a flow rate of /min. When the solution that passed through the column was analyzed by optical rotation method, 82
It was found that chi was hydrolyzed.

that's all Patent applicant Head 1) Yoshihito

Claims (1)

[Scope of Claims] (1) An immobilized enzyme enclosed by an intermolecular complex generated by so-called secondary intermolecular binding forces such as ionic bonds, hydrogen bonds, and charge transfer interaction forces between polymer chains. Production method. (2) A patent relating to the production method of (1) characterized in that the ionic strength, solvent, temperature, etc. are changed to control the enzyme inclusion rate in the polymer complex and the reaction activity of the produced immobilized enzyme. Manufacturing method according to claim (1). (2)) Regarding the immobilized enzyme produced by the production method of (l), the enzyme can be protected from various enzyme activity inhibition factors such as sDHs temperature, ionic strength, metals, organic and inorganic reagents, etc. using a polymer complex. Claims characterized in (
1) Manufacturing method. (4) Regarding the immobilized enzyme produced by the production method in (1), the presence of a polymer complex changes the optimum conditions that the enzyme originally has in a free state, such as pH/H1 temperature, ionic species, and ionic strength. Claims characterized in that (a method for producing υ)