JP5105278B2 - Magnetic chitosan-based enzyme immobilization carrier and method for producing the same - Google Patents

Description

  The present invention relates to a chitosan-based enzyme immobilization carrier having magnetism that can be collected by magnetic force while maintaining the performance as an enzyme immobilization carrier and a method for producing the same.

  Conventionally, when an enzyme is used in a bioreactor or biosensor, an immobilization technique for making the solvent insoluble is used so that the enzyme can be repeatedly used. As immobilization methods, there are methods of directly cross-linking and insolubilizing enzymes, but they are combined with enzyme immobilization carriers such as glass, alumina, silica gel, cellulose and derivatives thereof, chitosan and derivatives thereof, and synthetic ion exchange resins. The system is mainstream. In particular, chitosan and its derivatives have various functional groups in the molecule, so that the enzyme is easily adsorbed and immobilized electrostatically or physically, and it is difficult to denature or deactivate. Is increasing. Among them, the granular porous and cross-linked chitosan-based carrier disclosed by the present applicants (see Patent Document 1) is widely used as a substance having excellent substance diffusibility and physical strength in the particles. in use.

On the other hand, there is a demand for quick and easy separation and recovery of immobilized enzyme and reaction products from the viewpoint of ease of use in scale-up and practical application. Enzyme immobilization with magnetism to recover by magnetic force A method using a carrier for use is disclosed (see Patent Documents 2 and 3). For the method of imparting magnetism to chitosan and using it as an enzyme immobilization carrier, chitosan is dissolved in an acidic aqueous solution, magnetic particles are dispersed in this chitosan solution, and this dispersion is neutralized with a basic solution. Several proposals have been made (see Patent Documents 4 and 5).
Japanese Examined Patent Publication No. 63-54285 Japanese Patent Laid-Open No. 5-268611 JP-A-10-75780 Japanese Examined Patent Publication No. 6-51114 Japanese Patent Laid-Open No. 17-296942

In the above background art, the chitosan-based enzyme immobilization support formed by dispersing magnetic particles in a chitosan solution and neutralizing this dispersion with a basic solution is such that the magnetic particles are embedded inside the support. There is a drawback that it is difficult to improve the collection performance. However, increasing the blending amount of magnetic particles causes problems that the exposed particles are detached or chitosan is peeled off, and further, the enzyme immobilization sites are decreased and the activity is decreased accordingly. There is.
Accordingly, an object of the present invention is to provide a chitosan-based enzyme immobilization carrier capable of being collected by magnetic force while maintaining the performance as an enzyme immobilization carrier and the like, and a method for efficiently producing the same. And

In order to solve the above problems, the present inventor has intensively studied, and as a result, the granular porous crosslinked chitosan is subjected to alkali treatment in the presence of ferrous ions and ferric ions, thereby supporting the enzyme immobilization carrier. The present inventors have found that a carrier for immobilizing a chitosan-based enzyme having magnetism that can be collected by magnetic force while maintaining the performance as described above has been obtained, and the present invention has been achieved.
That is, the present invention provides a granular porous cross-linked chitosan with an iron ion solution in which the ratio of ferrous ions to the total number of moles of ferrous ions and ferric ions is in the range of 30 mol% to 80 mol%. After bringing the total number of moles of iron salt into a range of 0.4 × 10 ⁻² mol to 3.0 × 10 ⁻² mol with respect to 25 mL of cross-linked chitosan, treatment with an alkaline solution and ferrous ions and Immobilization of magnetized chitosan-based enzyme having magnetized particles formed in granular porous crosslinked chitosan so that the increase in the ash content of magnetized particles comprising ferric ions is in the range of 2% to 23%. The present invention relates to a carrier for use and a method for producing the same.

  Since the present invention uses granular porous cross-linked chitosan having excellent immobilized enzyme activity as a base, a good enzyme reaction can be obtained, and magnetism is imparted by post-treatment. Since it is uniformly and finely dispersed throughout the system and can exhibit excellent performance in terms of magnetic collection capability, it can be expected to be applied to various bioreactors and biosensors.

The present invention is described in detail below.
The granular porous cross-linked chitosan in the present invention is obtained by, for example, dissolving a granular porous chitosan obtained by dissolving chitosan in an acidic aqueous solution and then dropping the solution into a basic solution with an epoxy-based or isocyanate-based crosslinking agent. There is no particular limitation as long as it is cross-linked. Examples of the crosslinking agent used here include hexamethylene bis- (2,3-epoxypropyldimethylammonium chloride), hexamethylene bis- (2,3-epoxypropyldiethylammonium chloride), and propylene bis- (2,3 -Epoxypropyldimethylammonium chloride), propylene bis- (2,3-epoxypropyldiethylammonium chloride), ethylene glycol diglycidyl ether, diethylene glycol diglycidyl ether, triethylene glycol diglycidyl ether, trimethylene glycol diglycidyl ether, tetramethylene Epoxy crosslinking agents such as glycol diglycidyl ether, hexamethylene glycol diglycidyl ether, epichlorohydrin, hexamethy Diisocyanate, 4,4′-diphenylmethane diisocyanate, 2,4-tolylene diisocyanate, naphthalene diisocyanate, 1,4-cyclohexane diisocyanate, 1,4-phenylene diisocyanate, xylylene diisocyanate, isophorone diisocyanate and other isocyanate-based crosslinking agents, Etc.

Further, the particle diameter of the granular porous crosslinked chitosan is not particularly limited, but if it is too large, the surface area becomes small, so that the efficiency of the enzyme reaction is deteriorated. If it is too small, the treatment becomes difficult. A particle diameter of ˜3000 μm is preferable, and 120 to 1200 μm is particularly preferable.
If the pore size is too large, the surface area becomes small and the efficiency of the enzyme reaction deteriorates.If it is too small, the diffusion rate of the substance in the porous reaction in the enzyme reaction decreases, and the apparent enzyme activity decreases. 100 to 7500 angstroms are preferable, and 1000 to 2000 angstroms are particularly preferable.

As an ion source of ferrous ions and ferric ions in the iron ion solution in the present invention, there are general-purpose and commercially-available products corresponding to hydrochloride, sulfate, citrate, acetate, etc., respectively. Any salt can be used, but from the viewpoint of cost, availability, and utilization of waste (generated in the acid cleaning step of the steel sheet), each chloride is preferable.
The iron ion solution in the present invention is a mixed aqueous solution of ferrous ions and ferric ions, and the ratio of ferrous ions to ferric ions is the total number of moles of ferrous ions and ferric ions. It is preferable that the ratio of the ferrous ion to is in the range of 30 mol% to 80 mol%, and more preferably in the range of 45 mol% to 80 mol%. If the ferrous ion is less than 30 mol%, a sufficient magnetic force cannot be obtained, and it cannot be collected even if it is close to a magnet. On the other hand, if the ratio of ferrous ions is greater than 80 mol%, the amount of immobilized enzyme will decrease even if it can be collected with a magnet.

The charged amount of the mixture of ferrous chloride ion and ferric chloride ion in the present invention is in the range of 0.4 × 10 ⁻² mol to 3 × 10 ⁻² mol with respect to 25 mL of granular porous crosslinked chitosan. In addition, the ash content must be in the range of 2% to 23%. If the total molar amount of iron ions is less than 0.4 × 10 ^-2 mol, or if the increment of ash is less than 2%, there is no problem with the amount of immobilized enzyme or the activity of the immobilized enzyme. Even if it approaches, the performance to collect becomes insufficient. Further, when the total molar amount of iron ions exceeds 3 × 10 ⁻² mol, or when the increment of ash exceeds 23%, it is not preferable because the magnetic substance is likely to be detached due to generation spots of the magnetic substance. . The ash content in the present invention refers to the weight of the carrier for immobilizing chitosan-based enzyme on which magnetized particles that have been completely dried as described later are formed, and the residue after firing of the carrier for immobilizing the chitosan-based enzyme. It is a value obtained by measuring the weight.

In the present invention, the above-mentioned granular porous crosslinked chitosan is added and mixed in an iron ion solution containing ferrous ions and ferric ions, but the method of addition mixing is not particularly limited, and iron ions are sufficient. What is necessary is just to make it osmose | permeate the inside of a granular porous bridge | crosslinking chitosan. For example, the treatment may be performed for about 1 to 6 hours while shaking at 100 to 300 strokes / minute at about 30 to 80 ° C. In this process, dextran or the like that plays a role as a protective colloid may be added to the system in order to suppress precipitation associated with coagulation of coprecipitated iron.
Next, in the present invention, after the above addition and mixing treatment, an alkaline aqueous solution is added to form magnetized particles in the granular porous crosslinked chitosan. As the alkaline aqueous solution used at this time, any conventional aqueous solution of a basic substance can be used, but among them, aqueous ammonia or aqueous sodium hydroxide is preferably used. The amount of the aqueous alkali solution may be appropriately selected so that the pH of the reaction system is 8 or more. However, if it is used excessively, the elimination of the cross-linking groups and modifying groups of the granular porous cross-linked chitosan, and the pores. This is not preferable because the amount of enzyme immobilization may be reduced.
The treatment after adding the alkaline aqueous solution may also be conducted for about 1 to 6 hours while shaking at 100 to 300 strokes at about 30 to 80 ° C. as described above.

  EXAMPLES Hereinafter, the present invention will be specifically described with reference to examples, but the present invention is not limited to these examples. In Examples and Comparative Examples, reaction spots, magnetism, chitosan enzyme immobilization carrier form, ash content, protein adsorption rate, and immobilized enzyme immobilization rate, expression titer, and expression rate Was measured or evaluated by the following method.

(1) Reaction spots (appearance evaluation)
The color tone of the carrier for immobilizing chitosan-based enzyme prepared by the method shown in Examples was visually observed, and reaction spots were evaluated from the color spots. When there was no color spot and excellent homogeneity, it was evaluated as ◯, when there was a color spot but within the range acceptable in quality, Δ, and when color spot was large and quality was unacceptable, it was evaluated as x.
(2) Adhesion to magnet (appearance evaluation)
The chitosan-based enzyme immobilization support prepared by the method shown in the Examples was transferred to a glass petri dish, and RO water was added until the chitosan-based enzyme immobilization support was submerged. (As One Co., Ltd.) was inserted into the system, and the state of adhesion to the magnet and the state of being attracted were evaluated by visual observation. The evaluation results are indicated by ○ when attached, and × when not attached or cannot be taken out of the system.
(3) Scanning electron microscope (SEM) observation
The carrier for immobilizing chitosan-based enzyme prepared by the method shown in the Examples was washed with RO water, methanol, ethanol, and t-butyl alcohol in this order, solvent exchanged, and lyophilized. After freeze-drying, gold was sputtered on the surface and observed at 5000 times using JSM 6060LV manufactured by JEOL.

(4) Ash content measurement
A sample which had been completely dried in a hot air circulating drier at 105 ° C. for 4 hours was placed in a ceramic crucible with a known tare weight, and then incinerated in an electric furnace at 600 ° C. for 5 hours. After ashing, it was immediately put into a desiccator, allowed to cool to room temperature in the desiccator, weighed, and determined the ash content from the following formula.
Ash content (%) = calcined residue weight (g) / absolute dry sample weight (g) × 100
(5) Albumin adsorption
Weigh out 5 ml of each enzyme immobilization carrier substituted with 0.01 M phosphate buffer (pH 7.4) prepared from 0.01 M disodium hydrogen phosphate and 0.01 M sodium dihydrogen phosphate into a plastic sealed container. Add 50 ml of 0.01 M phosphate buffer (pH 7.4) solution of 1% bovine serum albumin (MP Biomedicals, Inc.) and shake for 4 hours at 25 ° C. using a constant temperature rotary shaker. Speed: 125 rpm).
(6) Adsorption of hemoglobin
Weigh 5 ml of each enzyme immobilization carrier substituted with 0.01 M phosphate buffer (pH 7.4) in a plastic closed container, and add 0.1% of 1% bovine blood-derived hemoglobin (manufactured by Wako Pure Chemicals, reagent special grade). 50 ml of a 01M phosphate buffer (pH 7.4) solution was added, and the mixture was shaken at 25 ° C. for 4 hours (shaking speed: 125 rpm) using a constant temperature rotary shaker.
(7) Measurement of protein adsorption rate After adsorbing albumin or hemoglobin to each enzyme immobilization support for a predetermined time, dilute 5 mL of the reaction solution 10 times and spectrophotometer (UV-2450, manufactured by Shimadzu Corporation). The amount of adsorption was measured from the absorbance at 280 nm, and calculated based on the following formula 1.

[Example 1]
Weigh 25 mL of a carrier for immobilizing chitosan-based enzyme having a particle size of 840 to 1,190 μm and a specific surface area of 110 to 130 m ² / g (Fitobo Holdings Co., Ltd. Chitopearl BCW-2510) into a plastic sealed container, and use a dropper to remove moisture. Lightly removed. Here, a magnetized solution (ferrous chloride tetrahydrate 2.78 g (1.40 × 10 ⁻² mol), ferric chloride hexahydrate 4.17 g (1 .54 × 10 ⁻² mol) and a solution prepared by dissolving 1.81 g of dextran in 40.0 g of RO water were added and mixed well. Next, the temperature of the reaction system was raised to 60 ° C. using a constant temperature water bath, and the mixture was immersed and mixed at 125 strokes / minute for 2 hours. After a predetermined time, a solution obtained by diluting 5.75 g of 28% ammonia water (manufactured by Wako Pure Chemical Industries, Ltd.) with 43.61 g of RO water was added to the reaction system and mixed well. At this point, the system turned brown. Thereafter, the temperature of the reaction system was raised to 60 ° C. again using a shaking water bath, and the mixture was immersed and mixed at 125 strokes / minute for 1 hour. After a predetermined time, the system was separated by filtration and washed with water to obtain the desired product. The object was stored at 5 ° C. in RO water.

FIG. 1 shows a state of SEM observation of a carrier for immobilizing chitosan-based enzyme before treatment, and FIG. 2 shows a state of SEM observation of the obtained target product. As can be seen from the SEM photographs of FIGS. 1 and 2, the porous structure originally possessed by the support for immobilizing chitosan-based enzyme was maintained as it was even after the treatment. The target product was calcined and ashed, and the ash content was evaluated to be 22.4%. The ash content before magnetization was almost zero. FIG. 3 shows a state in which the remaining components of the crucible were observed by SEM. Although the pores contracted and disappeared by firing, the structure traced the shape of the original enzyme immobilization support. When the magnet was brought close to the moist chitosan-based enzyme immobilization support that had been magnetized, the carrier particles were attracted and adhered to the magnet as shown in FIG. Therefore, a carrier for immobilizing a chitosan-based enzyme having magnetism, which is one of the objects of the present application, was obtained.
When the adsorption rates of albumin and hemoglobin were evaluated, they were 46.9 mg / ml and 22.8 mg / ml, respectively, and the performance as a carrier for enzyme immobilization was sufficiently maintained. Therefore, it can be said that the object of the present application is the simultaneous provision of magnetism and enzyme immobilization. Table 1 summarizes the properties and evaluation results of the chitosan-based enzyme immobilization carriers after the magnetizing treatment in Examples 1 to 3 and Comparative Examples 1 to 3.

The adsorption rate of albumin and hemoglobin was measured as follows.
To 5 ml of the carrier for immobilizing the magnetizing enzyme, 10 ml of an aqueous solution (acylase amano aqueous solution manufactured by Amano Enzyme Co., Ltd.) dissolved and prepared to have a titer of 150 U in RO water is added, and the acylase is shaken at room temperature for 2 hours. Was fixed. After weighing 5 ml of the reaction solution from the system and diluting it 10 times, the absorbance at 280 nm was measured. What carried out the same operation without putting the support | carrier for magnetizing enzyme immobilization was made into the blank, and the amount of immobilized acylases was calculated | required from the difference with a blank light absorbency.

Next, in order to examine the activity of the immobilized acylase, the following operation was performed.
The acylase-immobilized carrier prepared by the above operation was sufficiently washed until the absorbance at 280 nm was 0.025 or less to remove unreacted enzyme. 1 ml of the obtained magnetized carrier or non-magnetized carrier was put in a screw tube, 2 ml of citrate buffer (pH 5.0) and 1 ml of 0.5 mM cobalt chloride aqueous solution were added and reacted at 37 ° C. for 5 minutes. Subsequently, 1 ml of an N-acetyl-DL-methionine solution was added to the system as a substrate solution, stirred at a test tube mixer, and allowed to react at 37 ° C. for 15 minutes. After filtering the reaction mixture, 1 ml was collected from the filtrate, and the reaction was stopped by heating in a hot water bath at 80 ° C. for 3 minutes. After cooling the reaction solution with running water, 1 ml of a ninhydrin solution prepared by the method described later and 0.05 ml of stannous chloride solution were added and ignited in a 90 ° C. hot water bath for 20 minutes. After cooling with running water, 5 ml of 50% 2-propanol aqueous solution was added, and the absorbance at 570 nm was measured. A system in which the same operation was performed without adding the enzyme immobilization carrier was prepared as a blank, and the absorbance was measured. The N-acetyl-DL-methionine, ninhydrin solution, and aqueous stannous chloride solution used for the reaction were prepared as follows.
N-acetyl-DL-methionine solution: N-acetyl-DL-methionine (Wako Pure Chemical Industries, Ltd., reagent grade, MW = 191.25) weighed 0.478 g, RO water 10 ml and 1N sodium hydroxide solution 2 ml Was added and dissolved. Thereafter, N / 10-sodium hydroxide solution was added to the system to adjust the pH to 8.0, and RO water was added to make 25 ml.
Ninhydrin solution: 1.0 g of ninhydrin (for automatic amino acid analysis by Wako Pure Chemical Industries, Ltd., 8.14) is weighed and 25 ml of ethylene glycol monomethyl ether (reagent special grade manufactured by Wako Pure Chemical Industries, Ltd., MW = 76.1). After adding and dissolving, 25 ml of citrate-sodium hydroxide buffer (pH 5.0) was added and shaken.
Stannous chloride aqueous solution: stannous chloride (special grade, manufactured by Wako Pure Chemical Industries, Ltd., MW = 225.63) 0.1 g, 6.2 ml of citric acid-sodium hydroxide buffer (pH 5.0) In addition, it was dissolved.

[Example 2]
Ferric chloride tetrahydrate 1.39 g (0.70 × 10 ⁻² mol), ferric chloride hexahydrate 2.09 g (0.77 × 10 ⁻² mol), and dextran 0 The same operation as in Example 1 was performed except that a magnetized solution obtained by dissolving 90 g in 42.5 g RO water was used. As shown in Table 1, a carrier satisfying the object of the present invention was obtained.

Example 3
Ferrous chloride tetrahydrate 0.70 g (0.35 × 10 ⁻² mol), ferric chloride hexahydrate 1.04 g (0.38 × 10 ⁻² mol), and dextran 0 The same operation as in Example 1 was performed except that a magnetized solution obtained by dissolving 0.45 g in 43.8 g of RO water was used. As shown in Table 1, a carrier satisfying the object of the present invention was obtained.

Example 4
As an iron ion source, 1.27 g (0.64 × 10 ⁻² mol) of ferrous chloride / tetrahydrate and 0.43 g (0.16 × 10 ⁻² mol) of ferric chloride / hexahydrate were used. mol) was used, and the same operation as in Example 1 was performed except that a magnetized solution in which 0.45 g of dextran was dissolved in 42.9 g of RO water was obtained to obtain a carrier satisfying the object of the present invention. Table 2 summarizes the properties and evaluation results of the chitosan-based enzyme immobilization carriers after the magnetizing treatments in Examples 4 to 6 and Comparative Examples 4 and 5.

Example 5
As an iron ion source, 0.85 g (0.43 × 10 ⁻² mol) of ferrous chloride tetrahydrate and 0.85 g (1.27 × 10 ⁻² mol) of ferric chloride hexahydrate. mol), and the same operation as in Example 1 was performed except that a magnetized solution in which 0.45 g of dextran was dissolved in 42.9 g of RO water was used. As shown in Table 2, a carrier satisfying the object of the present invention was obtained.

Example 6
As an iron ion source, 0.43 g (0.22 × 10 ⁻² mol) of ferrous chloride / tetrahydrate and 1.27 g (0.47 × 10 ⁻² mol) of ferric chloride / hexahydrate were used. mol), and the same operation as in Example 1 was performed except that a magnetized solution in which 0.45 g of dextran was dissolved in 42.9 g of RO water was used. As shown in Table 2, a carrier satisfying the object of the present invention was obtained.

Example 7
Implementation was performed except that a chitosan enzyme immobilization carrier (chitopearl BCW-3010 manufactured by Fujibo Holdings Co., Ltd.) having a particle size of 840 to 1,190 μm and a specific surface area of 120 to 150 m ² / g was used as the chitosan enzyme immobilization carrier. A carrier for immobilizing a magnetizing enzyme was produced in the same manner as in Example 1.

  Regarding the activity of immobilized acylase measured by the carrier for immobilizing chitosan-based enzyme obtained by the method of Example 7 and Example 8 above, the immobilization rate of the carrier sample, the immobilization titer based on the following formula And expression enhancement, and the expression rate was calculated by Equations 2-4. As a result, as is apparent from Table 3, it was shown that the magnetized carrier according to the present invention has the same performance as that of the non-magnetized carrier and there is no performance degradation due to magnetization.

The various factors in the above formula are as follows.
330.94: L-methionine amount that gives an absorbance difference of 1.000 determined from a calibration curve prepared with L-methionine 149: Weight (μg) relative to 1 μmol of L-methionine
16: Sample dilution multiple x unit conversion factor W: amount of immobilized enzyme carrier used for activity measurement (g)

Example 8
A chitosan enzyme immobilization carrier (chitopearl BCW-3510 manufactured by Fujibo Holdings Co., Ltd.) having a particle size of 840 to 1,190 μm and a specific surface area of 150 to ²⁰⁰ m ² / g was used as the chitosan enzyme immobilization carrier. The same operations as in Example 7 were performed to evaluate the performance of the magnetized acylase-immobilized support. As a result, as is apparent from Table 3, it was shown that the magnetized carrier according to the present invention has the same performance as that of the non-magnetized carrier and there is no performance degradation due to magnetization.

[Comparative Example 1]
Ferrous chloride tetrahydrate 11.1 g (5.58 × 10 ⁻² mol), ferric chloride hexahydrate 16.7 g (6.18 × 10 ⁻² mol), and dextran 7 The same operation as in Example 1 was performed, except that a magnetized solution in which 23 g was dissolved in 25.0 g of RO water was used. As a result, as shown in Table 1, adhesion to the magnet was not observed, and a carrier satisfying the object of the present invention was not obtained.

[Comparative Example 2]
Ferrous chloride tetrahydrate 5.56 g (2.80 × 10 ⁻² mol), ferric chloride hexahydrate 8.35 g (3.09 × 10 ⁻² mol), and dextran 3 The same operation as in Example 1 was performed except that a magnetized solution obtained by dissolving 0.62 g in 35.0 g of RO water was used. As a result, as shown in Table 1, adhesion to the magnet was not observed, and a carrier satisfying the object of the present invention was not obtained.

[Comparative Example 3]
Ferrous chloride tetrahydrate 0.35 g (0.18 × 10 ⁻² mol), ferric chloride hexahydrate 0.50 g (0.18 × 10 ⁻² mol), and dextran 0 The same operation as in Example 1 was performed except that a magnetized solution obtained by dissolving 0.23 g in 44.4 g of RO water was used. As a result, as shown in Table 1, adhesion to the magnet was not observed, and a carrier satisfying the object of the present invention was not obtained.

[Comparative Example 4]
As an iron ion source, a magnetized solution in which only 1.70 g (0.86 × 10 ⁻² mol) of ferrous chloride tetrahydrate was used and 0.45 g of dextran was dissolved in 42.9 g of RO water was used. The same operation as in Example 1 was performed except that it was used. As a result, as shown in Table 2, although magnetization was possible, the adsorption rate of hemoglobin decreased and a carrier satisfying the object of the present invention could not be obtained.

[Comparative Example 5]
As an iron ion source, a magnetized solution in which only 1.70 g (0.63 × 10 ⁻² mol) of ferric chloride hexahydrate was used and 0.45 g of dextran was dissolved in 42.9 g of RO water was used. The same operation as in Example 1 was performed except that it was used. As a result, as shown in Table 2, adhesion to the magnet was not observed, and a carrier satisfying the object of the present invention was not obtained.

  Since various functional groups can be introduced into the chitosan-based enzyme immobilization support, it is possible to cope with individual enzymes having different properties. In addition, the enzyme immobilization method can be selected from among the adsorption method, crosslinking method, and activation method as appropriate, and a highly stable immobilization method can be selected for immobilization of carbohydrates and protein-related enzymes. It is. The carrier obtained by the present invention has the above-described enzyme-immobilizing ability of chitosan, can be easily separated from the reaction product by magnetic force, and the immobilized enzyme can be easily recovered. Use and application can be expected for applications.

It is a scanning electron micrograph which shows the cross section of the support | carrier for chitosan type enzyme immobilization before a process. 2 is a scanning electron micrograph showing a cross section of a carrier for immobilizing a chitosan enzyme according to the present invention. 2 is a scanning electron micrograph of a crucible remaining component after calcining and ashing the chitosan enzyme immobilization support according to the present invention. 2 is a photograph showing a state in which a support for immobilizing chitosan-based enzyme according to the present invention is attracted to and adhered to a magnet.

Claims (2)

An iron ion solution in which the ratio of ferrous ions to the total number of moles of ferrous ions and ferric ions is in the range of 30 mol% to 80 mol% is added to granular porous crosslinked chitosan with respect to 25 mL of granular porous crosslinked chitosan. The total number of moles of iron salt is in the range of 0.4 × 10 ⁻² mol to 3.0 × 10 ⁻² mol. A carrier for immobilizing a chitosan-based enzyme having magnetism with an increase in ash content in the range of 2% to 23%. An iron ion solution in which the ratio of ferrous ions to the total number of moles of ferrous ions and ferric ions is in the range of 30 mol% to 80 mol% is added to granular porous crosslinked chitosan with respect to 25 mL of granular porous crosslinked chitosan. The total number of moles of iron salt is in the range of 0.4 × 10 ⁻² mol to 3.0 × 10 ⁻² mol, and then treated with an alkaline solution to obtain ferrous ions and ferric ions. A magnetized chitosan-based enzyme immobilization carrier comprising: a porous porous cross-linked chitosan that forms magnetized particles such that the increase in the ash content of the magnetized particles is in the range of 2% to 23%. Production method.