JPS6017513B2 - Immobilized enzyme reaction method - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for carrying out an enzymatic reaction using an enzyme or bacterial cells immobilized on a polyion complex.

Specifically, the present invention relates to a method of carrying out an enzyme reaction using a water-insoluble enzyme active substance in which an enzyme or a bacterial cell having enzyme activity is adsorbed and immobilized on a polyion complex formed from a polycationic polymer and a polyanionic polymer. Until now, there have been no known reports on enzyme immobilization methods using such polyion complexes. In recent years, with regard to the immobilization of enzymes or bacterial cells, we have developed immobilization techniques and enzyme reaction protocols using immobilized hydrophobic enzymatically active substances.

There is something self-aware about Seth's development. Conventional enzyme reactions use water-soluble enzymes, which are discarded after each use. However, in the immobilized enzyme process, enzymes can be used repeatedly for long periods of time, and the reaction method can be changed from a batch method to a continuous method. It has the advantage that the reaction can be carried out extremely efficiently and economically since it has become possible to convert it into a method. However, compared to the vast amount of previous reports on immobilized enzymes, only a few examples have actually been commercialized. The reasons for this are that the immobilization methods that have been reported so far are complicated and impractical, and that there is no advantage to immobilization because the lifespan is short despite the time and effort required for immobilization. had many problems. Furthermore, in many conventional methods, methods applicable to one enzyme system cannot be applied to other systems. Therefore, there is a long-awaited development of an immobilization technique that has a simple immobilization process and is versatile. The present inventor has arrived at the present invention as a result of intensive research into a new immobilization technology that meets the above requirements. The present invention relates to an enzyme reaction method characterized by using an enzyme or bacterial cells adsorbed and immobilized on a water-insoluble polyion complex formed from a polycationic polymer and a polyanionic polymer.

The interaction between polymer ions and enzyme proteins or bacterial cells has been well known.

For example, one example is the adsorption of enzymes onto an ion exchange resin, which is a polymer ion that does not fall off. It is also known that when water-soluble polymer ions and enzyme proteins form a complex, they become water-insoluble and precipitate, which can be used as a means of separating and purifying enzymes. It is also known to add polymer ions such as polyethyleneimine to a suspension of bacterial cells to cause the bacterial cells to coagulate and precipitate. However, even when enzymes or bacterial cells interact with polymer ions, they do not necessarily coagulate and precipitate, and even if they do precipitate, they are often in a form that cannot be used as an immobilized enzyme agent.

The method of adsorbing enzymes on ion exchanger lipids, which are water-insoluble cross-linked polymer ions, is an excellent method in some cases, but in many cases, the adsorbed enzymes are easily desorbed. There are drawbacks. The present inventor focused on the interaction between polymer ions and enzymes or microbial cells, and developed a new immobilization method that overcomes the deficiencies of the previously known methods described above. The present inventors have developed a method for immobilizing enzymes or bacterial cells having enzyme activity onto polyion complexes, and have completed the present invention.

Water-soluble polycationic and polyanionic polymers
It was known that a water-insoluble polyion complex is formed from aqueous solution, but the present inventor discovered that when a polyion complex is generated and precipitated from an aqueous solution, when enzymes or bacterial cells coexist, the polyion complex is formed. It has been found that enzymes or bacterial cells can be efficiently adsorbed and immobilized, and this product can be used as an immobilized enzyme agent. It is a novel and completely unknown method to carry out an enzyme reaction using an insoluble enzymatically active substance in which an enzyme or a bacterial cell having enzymatic activity is adsorbed and immobilized on such a polyion complex.

In some cases, the present inventors have also discovered that a polyion complex may be formed in advance from a polythionic polymer and a polyanionic polymer, and then brought into contact with an enzyme or a bacterial cell having enzyme activity in an aqueous medium. It was also discovered that bacterial cells can be adsorbed and immobilized on polyion complexes, and that this product can be used as an immobilized enzyme agent. The method of the present invention has the advantage that the immobilization operation is extremely simple, and one of its characteristics is that it is a versatile immobilization method that has an extremely wide range of applications. In the present invention, embodiments of enzymes or bacterial cells having enzyme activity to be immobilized include bacterial cell culture fluids, washed bacterial cells, crushed bacterial cells, crude enzyme extracts, crude enzymes, purified enzymes, and the like.

Furthermore, the present invention can be applied to enzymatic reactions using any enzyme or microbial cells having enzymatic activity (bacteria, actinomycetes, yeast, mold, etc.). Examples of enzymes that can be used in the present invention include glucose isomerase, invertase, glucoamylase, Q-amylase, P-amylase, pullulanase, cellulase, chymotrypsin, papain, lipase, aminoacylase, cis-epoxysuccinate hydrolase, and fumarase. , as/glutase, as/glutase, urease, catalase, peroxidase, L-amino acid oxidase, glutathione reductase, and the like. Examples of microbial cells having enzyme activity include microbial cells belonging to genera such as Alcaligenes, Bacillus, Micrococcus, Brevibacterium, Corynebacterium, Arthrobacter, Nocardia, Streptomyces, Benicillium, and Satucharomyces. Polymer ions used in the present invention include strongly acidic water-soluble polyanions such as polystyrene sulfonic acid, polypynylsulfate, and ligninsulfonic acid; weakly acidic water-soluble polyanions such as polyglutamic acid, polyacrylic acid, polymethacrylic acid, and alginic acid. Examples include strong base-type water-soluble polycations such as polyanions, polyvinylbenzyltrimethylammonium salts, and polyvinyl-N-methylpyridinium salts, and weakly base-type water-soluble polycations such as polyvinylpyridine, polyethyleneimine, and polylysine.

Furthermore, ordinary cation exchange resins and anion exchange resins, which are water-insoluble crosslinked polymer ions, can also be used as one of the polymer ion components forming the polyion complex in the present invention. In addition to the above-mentioned organic polymer ions, it is also possible to use inorganic polyanionic polymers such as condensed silicates, and water glass is one example. In the present invention, it is a preferable condition for carrying out the method of the present invention that at least one of the combination of polycationic polymer and polyanionic polymer used is a water-repellent polymer ion.

The actual combination of polymer ions to be used is selected depending on the enzyme to be immobilized or the bacterial cell having enzyme activity, and depending on the conditions. For example, in the case of a combination of water-soluble polymer ions, generally When a strong acid type polyanion and a strong base type polycation are combined, there are advantages such as ease of handling of the resulting polyion complex having enzymatic activity. In the present invention, conditions for preparing a polyion complex containing an enzyme or a bacterial cell having enzyme activity will be described below.

Polymer ions may be added to an aqueous medium containing enzymes or microbial cells having enzyme activity, and after being concentrated, polymer ions having an opposite charge to the previously added polymer ions may be added. The order of addition of the polycationic polymer and polyanionic polymer varies depending on the enzyme to be immobilized or the microbial cell that has enzymatic activity, and the order of addition may affect the activity of the obtained fixed-term enzyme agent. It may be unrelated. A more preferable method is to bring an enzyme or a microbial cell having enzyme activity into contact with one of the polymer ions and then add an oppositely charged polymer ion as described above. It is also possible to adopt a method in which the enzyme or the bacterial cells are adsorbed and immobilized on the polyion complex by producing a polyion complex from the polyion complex and then contacting the polyion complex with an enzyme or bacterial cells having enzyme activity in an aqueous medium. .

The amount of polymer ions used in the present invention is desirably such that the weight of the polyion complex produced is the same or greater than the weight of the enzyme to be immobilized or the bacterial cells having enzyme activity.

Regarding the quantitative relationship between the polycationic polymer and polyanionic polymer used, in the case of water-resistant, strongly basic or strongly acidic polymer ions, the ion equivalent should be selected so that the ion equivalent is 1:1, but in general, In many cases, the ratio is not 1:1 depending on the combination. If one polymer ion is in excess, excess polymer ions will remain in the solution when the polyion complex is precipitated. You can eliminate waste caused by overuse. Temperature at which a polyion complex containing enzymes or bacterial cells with enzyme activity is generated, FH
Although it can be selected as appropriate within a range that does not reduce the enzyme activity, the temperature is preferably around room temperature, and the pH is preferably around the optimum pH for the enzyme.

In the present invention, the enzymatic reaction is not particularly limited, but if the polyion complex containing enzymes or bacteria having enzyme activity is obtained in the form of powder, it may be suspended in a reactor partitioned with a filter. Enzyme reactions can be carried out using an azure tank type reactor. It is also possible to fix the enzyme as a cake on a filter and carry out the enzymatic reaction without passing the reaction solution through a furnace. Furthermore, it is also possible to mold polyion complexes containing enzymes or microbial cells with enzyme activity into granules, or to disperse them on granular porous carriers. In this case, a column reaction method in which these are packed into a column is used. can be taken. The present invention will be explained in more detail below with reference to Examples, but the present invention is not limited thereto.

The phosphate buffer used in the Examples was prepared from disodium monohydrogen phosphate and monosodium dihydrogen phosphate, and the acetate buffer was prepared from acetic acid and sodium acetate in a conventional manner. Among the polymer ions used in this example, commercially available sodium polyacrylate, polyethyleneimine, and potassium borivinyl sulfate were used. Poly(N-methyl-2-methyl-5-vinylpyridine) iodide (hereinafter abbreviated as PMVP) is a polymer with a molecular weight of approximately 40,000 obtained by polymerizing 2-methyl-5-vinylpyridine and quaternized with methyl iodide. was used. Further, as the potassium polystyrene sulfonate (hereinafter abbreviated as PSSK), a polymer having a molecular weight of about 100,000 obtained by polymerizing potassium styrene sulfonate was used. Examples 1 to 3 Glucose isomerase cells (glucose isomerase Nagase, manufactured by Nagase Sangyo ■) were suspended in a 0.09M phosphate buffer solution having a pH of 7.2.

The activity titer of this suspension was 2.58 U'. 5 skins each of this suspension (i.e. total active titer 12.9 U)
Put it in a beaker, add 10 drops of 2.1% PMVP aqueous solution, and stir. In each of them, 10% of 2.2% PSSK aqueous solution
(Example 1), 1.6% polypynylsulfate potassium aqueous solution (
Example 2) and 1.3% silicic acid aqueous solution (Example 3) were added thereto, and after stirring for about 5 minutes, the mixture was filtered in an oven and washed with water to obtain an immobilized glucosysomerase agent. The silicic acid aqueous solution used in Example 3 was diluted water glass with a Si02/Na20 molar ratio of 3.25 and neutralized to pH 7 to 8 with hydrochloric acid immediately before use. (The silicic acid aqueous solutions used in subsequent examples were prepared in the same manner.) Measurement of glucose isomerase activity was performed as follows. Glucose 0.2 as substrate solution
mole/so, MgS047 day 200.02 mole/
Prepare a 0.0 skin phosphate buffer solution (PH7.2) containing it. In the case of a bacterial cell suspension, add the above substrate solution 1' to bacterial cell suspension 1'. The immobilized bacterial cells of Examples 1 to 3 were prepared using substrate solution 5', 0.05M phosphate buffer (pH 7.2).
5. Suspend on the desk. After the mixture was kept at 60° C. for 1 hour to react, the amount of fructose produced was measured by the cysteine-carbazole sulfuric acid method. Activity titer IU
is defined as the amount of enzyme that produces fructose 1 mimic under the above conditions. Table 1 shows the results of measuring the activity of the immobilized enzyme preparation prepared as described above. Table 1 The activity ratio in the table is {(activity titer of immobilized enzyme agent)/
(Total activity titer of the bacterial cell suspension used)} Defined as 100.

Note that after the reaction, the immobilized glucosysomerase agent of Example 1 was separated from the furnace, washed with water, and used again for the reaction, but
No decrease in activity was observed even after the fifth test. Examples 4-6 Glucose isomerase Nagase at pH 7.2 0.0
Suspended in M phosphate buffer.

The activity titer of this suspension was 3.6 U/device. To this suspension 5M (total activity titer 18U), add 2.1% PMVP.
aqueous solution low [, 2.2% PSSK aqueous solution 10],
In Example 4, PMVP→PSSK was added in the order, and in Example 5, the reverse order was added to prepare an insoluble glucose isomerase agent immobilized on the polyion complex. In Example 6, 10 parts of a 2.1% PMVP aqueous solution and 10 parts of a 2.2% PSSK aqueous solution were mixed to generate a polyion complex, and to this, bacterial cells and suspension 5 parts were added. An insoluble glucose isomerase agent was prepared. The activity was measured in the same manner as in Examples 1-3. The results are shown in Table 2. In addition, the insoluble glucose isomerase recovered by washing the reactor with water after the reaction was repeatedly used.
No decrease in activity was observed even after the fifth test. Table 2 Examples 7 to 9 Glucose isomerase crude enzyme extracts were prepared by ultrasonication using Glucose Isomerase Nagase.

The activity titer of this glucose isomerase extract was 5.
The secretion was 9U'. This extract 5 [(total activity titer 29
．． 5 U), 2.1% PMVP aqueous solution 10', 2.2
% PSSK aqueous solution 10', in Example 7, PMVP→
in the order of PSSK, and in the reverse order in Example 8,
A permanent glucose isomerase agent immobilized on a polyion complex was prepared. In Example 9, 10 2.1% PMVP aqueous solutions and 10 2.2% PSSK aqueous solutions were mixed to form a polyion complex, and 5' of the crude enzyme extract was added to this to dissolve insoluble glucose. An isomerase agent was prepared. The activity was measured in the same manner as in Examples 1-3. The results are shown in Table 3. The insoluble glucose isomerase recovered by washing the reactor with water after the reaction was used repeatedly, but no decrease in activity was observed even after the fifth use. Table 3 Example 10 A 2.1% PMVP aqueous solution 30 skin was added to a 1.3% silicic acid aqueous solution (neutralized to pH 7), and the resulting polyion complex was separated in a furnace, washed with water, and dried.

Examples 7 to 9 of this polyion complex 0.5 minutes
In addition to the crude enzyme extract of glucose lysomerase used in 5 volumes (overall titer 29.5 U), the mixture was flocculated, separated in a furnace, and washed with water to obtain an insoluble glucose lysomerase agent. When an enzymatic reaction was carried out using the thus obtained glucose isomerase agent, its activity titer was 29 U (activity ratio 9
8%). After the reaction, the Okasadai Giza enzyme was recovered by separate furnaces and water washing, and was used three times, but almost no decrease in activity was observed. Comparative Example 1 For comparison with Example 10, Amberlite IRA-904 (S0
4 type) was used for 0.5 days in the same manner as in Example 10, the glucose isomerase crude enzyme extract 5 was obtained with an overall titer of 29.
5U) was used to prepare an insoluble glucose isomerase agent.

The activity of this non-stable glucose isomerase agent was measured and found to be 21 U (activity ratio 71%). After the reaction, the immobilized glucose isomerase agent was recovered by separate furnace and washing with water, and when reused, the activity decreased by 12 U'. Examples 11-13 Nocardia tartaricans
taricans) nov. sp. ES31T strain (Shikoken Bacteria No. 3374) was mixed with 1% propylene glycol, 0.3% urea, 0.2% KH2P04, and Na2H.
P040.2%, MgS047day 200.1%
, FeS047 ratio 00.001%, CaC122
Also 00.003%, MnS044day 200.0004%
, yeast extract 0.05%, surfactant Pluronic L-
A medium (pH 7.0) with a composition of 610.1% was inoculated and cultured in 20 jar fermenters containing 10 soybeans, and 0.5% disodium cisepoxysuccinate was added during the logarithmic growth phase.
was added and cultured for 2 hours at 30:00 to obtain bacterial cells having cisepoxysuccinate hydrolase activity.

The bacterial cell concentration of the obtained culture solution was 4.6 U/s, and the activity titer of cisepoxysuccinate hydrolase was 89.5 U/s.
It was secretion. Take 5' of this culture solution in a beaker and
．． Add 10ml of 1M phosphate buffer (pH 8.0) and mix. In Example 11, 1 part of a 5% aqueous polyethyleneimine solution (neutralized with hydrochloric acid to pH 8) was added, and then 1 part' of a 2.5% sodium polyacrylate aqueous solution was added. In Example 12, 2.2% PSS
10 of the K aqueous solution [then a 2.1% PMVP aqueous solution was added.

In Example 13, 2.1% PMVP aqueous solution 5' was added first, and then 1.3% silicic acid aqueous solution 10' was added.

The precipitate thus obtained was separated in a furnace and washed with water to obtain immobilized bacterial cells. The enzyme activity of the culture solution and immobilized bacterial cells was measured as follows.

2M disodium cisepoxysuccinate aqueous solution, 5% B
L-Han X aqueous solution (nonionic surfactant, manufactured by Nikko Chemicals), 0.1M phosphate buffer (pH 8), 3:0.
A mixed solution having a volume ratio of 6.8 is used as a substrate solution.

The above-mentioned immobilized bacterial cells are suspended in a mixed solution consisting of culture solution 11 and substrate solution 2', or a mixed solution consisting of substrate solution 10' and 0.1M phosphate buffer 5'. 40q
The mixture was allowed to undergo a shaking reaction for 1 hour at 30°C, and the tartaric acid produced was quantified by a colorimetric method using an ammonium metavanadate reagent.
The amount of cisepoxysuccinic acid hydrolase that produces 1 female tartrate under the above reaction conditions is defined as the active titer IU. Table 4 shows the results of enzymatic reactions performed using the immobilized bacterial cells prepared above.

Table 4 Example 14 A diluted aqueous solution of commercially available invertase (manufactured by Yeast, manufactured by Wako Pure Chemical Industries) was prepared, and its activity titer was 75.5 U'.

Add 10 minutes of 1.6% polyvinyl potassium sulfate aqueous solution to 5 sides of this invertase solution, stir, and then add 2.1% PM.
yP aqueous solution was added, and the precipitate was separated in a furnace and washed with water to obtain a hydrophobic invertase agent. Invertase activity was measured as follows. 1 invertase solution,
A mixture consisting of 1 part of 0.3M sucrose aqueous solution and 1 part of 0.03M acetate buffer (pH 5.6);
The water-insoluble invertase was suspended in a mixture consisting of 5 parts of a 3M sucrose aqueous solution, 5 parts of a 0.03 M acetate buffer (pH 5.6), and 5 parts of water.
After reaction for 20 minutes, the reducing sugar produced was quantified by the dinitrosalicylic acid method. The amount of enzyme that produces one reducing sugar under the above conditions is defined as IU.

Claims (1)

[Claims] 1. In a reaction using an immobilized enzyme, an enzyme adsorbed and immobilized on a water-insoluble polyion complex formed from a polycationic polymer and a polyanionic polymer or a bacterial cell having enzyme activity is used. An enzyme reaction method characterized by: 2. The method according to claim 1, wherein at least one of the polycationic polymer and the polyanionic polymer is a water-soluble polymer. 3 Enzyme or bacterial cells having enzyme activity immobilized on a polyion complex by adding reactively charged polymer ions to an aqueous medium containing enzyme or bacterial cells having enzyme activity and polycation polymer or polyanionic polymer. The method according to claim 1, characterized in that the method uses: 4. A polyion complex in which an enzyme or bacteria having enzyme activity is adsorbed and immobilized by contacting a polyion complex formed from a polycationic polymer and a polyanionic polymer with an enzyme or bacteria having enzyme activity in an aqueous medium. The method according to claim 1, characterized in that Tux is used. 5. The method according to claim 1, characterized in that a culture solution, bacterial cells, crushed bacterial cells, crude enzyme extract, crude enzyme, or purified enzyme is used as the enzyme or bacterial cells having enzyme activity. 6. The method according to claim 1, wherein glucose isomerase or a bacterial cell having glucose isomerase activity is used as the enzyme or the bacterial cell having enzyme activity. 7. The method according to claim 1, characterized in that cis-epoxysuccinate hydrolase or microbial cells having cis-epoxysuccinate hydrolase activity are used as the enzyme or microbial cells having enzyme activity. 8. The method according to claim 1, characterized in that invertase is used as the enzyme.