JPS6357601A - Three-dimensionally crosslinked polymeric sub-stance and its production - Google Patents

Abstract

PURPOSE:To obtain the title polymeric substance useful as an adsorbent for selectively adsorbing beta- and gamma-amylases, by intermolecularly crosslinking an alpha-1,4-homooligomer of glucose. CONSTITUTION:An alpha-1,4-homooligomer of glucose, which is an oligosaccharide composed of 2-6 glucose molecules linearly polymerized through an alpha-1,4-bond is intermolecularly crosslinked with an epihalohydrin (e.g., epichlorohydrin) as a crosslinking agent so that the solubility in 100g of water at the freezing point to 60 deg.C may be 0.001g. In this way, a three-dimensional matrix can be formed.

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to a novel three-dimensionally crosslinked polymeric material, and particularly to a three-dimensionally crosslinked polymeric material useful as an adsorbent for selectively adsorbing β-amylase and γ-amylase. This invention relates to an original crosslinked polymer material and a method for producing the same.

[Conventional technology]

Currently, high fructose sugar and maltose contain starch with α-amylase and γ-amylase.
It is industrially produced by hydrolysis using amylase or β-amylase.

As described above, β-amylase and T-amylase are extremely useful in the industrial production of useful low-molecular-weight sweeteners from starch.

Generally, enzymes are produced by liquid culturing enzyme-producing bacteria.

Enzymes used as research reagents are partially or highly purified through complex processes that combine salting out, ion exchange chromatography, electrophoresis, and the like.

On the other hand, industrial enzyme preparations can be obtained as a concentrate obtained by concentrating the culture filtrate as it is, as a dried powder, or as a concentrate obtained by separation and purification or as a dry powder thereof. However, concentrated liquids and crudely dried culture filtrates contain large amounts of unpleasant odor components and coloring components contained in the culture solution, so they must be removed when separating and purifying the desired reaction product. It becomes necessary. Furthermore, the culture filtrate often also contains proteolytic enzymes (proteases). For crude enzyme solutions such as culture filtrates that contain proteolytic enzymes other than amylase, during purification or when the desired reaction is progressing,
Amylase is often degraded and inactivated by the action of proteolytic enzymes. Therefore, when producing useful substances using enzymatic reactions, it is necessary to remove these disadvantageous impurities.

This places a significant burden on the subsequent separation and purification process of the reaction product. On the other hand, the salting-out method and liquid chromatography, which are known as general purification methods, have a limit of partial concentration, and in order to improve the separation accuracy, these operating conditions, such as the type and concentration of precipitant, the pH 1 packing material, etc. It is necessary to go through a complicated process that involves changing the type, type of adsorption/desorption liquid, etc., and combining these.

In addition, a large amount of salt is added as a precipitant and an eluent during the operation, and a desalting operation is also necessary when moving on to the next step. Furthermore, BOD wastewater with a high salt concentration of several tens of percent is not easy to treat.

From the above, these known purification methods are naturally limited to research reagents and the like.

Among these, β-amylase and T
- If an adsorbent that can selectively and efficiently adsorb amylase is developed, both enzymes can be concentrated and separated from the culture filtrate in one step with high purity.

The inventors discovered that amylose, a linear polymer of glucose (molecular weight 10-405, glucose polymerization number (5
We focused on an adsorption method using adsorbents (X10'' to 2X10') and applied this to β-amylase and T-amylase.

However, it was found that the adsorption capacity was extremely low, less than 1/103 of that of α-amylase, making adsorption separation virtually impossible.

[Problem that the invention seeks to solve]

An object of the present invention is to provide a novel three-dimensionally crosslinked polymeric substance formed by intermolecularly crosslinking α-1,4 homooligomers of glucose, and a method for producing the same. It can be used as an adsorbent that can selectively and efficiently adsorb and separate the enzyme from a solution containing β-amylase, γ-amylase, or both, which contains a wide variety of impurities in addition to β-amylase and γ-amylase. be.

[Means for solving problems]

As a result of intensive studies on adsorbents for β-amylase and γ-amylase, the present inventors found that glucose
, 4-linked oligosaccharide molecules form a complex molecule with β-amylase or T-amylase molecules. However, since these complexes with oligosaccharides are water-soluble, recovery of the enzyme is not easy. Therefore, as a result of various studies, it was discovered that a new crosslinked polymer substance obtained by intermolecularly crosslinking the oligosaccharide can be made insoluble in water without impairing its adsorption ability to the enzymes, The present invention has now been completed.

A first feature of the invention is that preferably the glucose molecule is
A polymeric substance capable of selectively adsorbing α-amylase and T-amylase, which is formed by chemically intermolecularly crosslinking linearly polymerized oligomers with ~6 molecules of α-1,4 bonds to form a three-dimensional matrix. This is the manufacturing method.

Examples of typical structural units of the substance when epichlorohydrin is used as a crosslinking agent are represented by the following formulas (1) and (I
I).

(Margins below this page) In the formula c, 1 is a non-reducing end, 2 is a reducing end, and 3 is a crosslinked chain.] The enzyme to which the three-dimensionally crosslinked polymeric substance of the present invention can be applied as an adsorbent is β- amylase and γ-amylase. The originating organism is not particularly limited as long as it falls within the classification of this enzyme.

For example, β-amylase originating from filamentous fungi such as Aspergillus and Rhizopus, yeast such as Satucharomyces, and bacteria such as Bacillus can be used. Furthermore, T-amylases originating from filamentous fungi such as Aspergillus and Rhizopus, yeasts such as Satucharomyces, and bacteria such as Bacillus and Clothuridium can also be used.

The oligosaccharide used as a raw material for producing the crosslinked polymeric substance of the present invention is an oligosaccharide having a polymerization number of 2 to 6, in which gluco-fila molecules are linearly polymerized with α-1°4 bonds. These oligosaccharides may be a single component or a mixture. However, if one or both of β-amylase and T-amylase is desired to be absorbed relatively selectively, the type of sugar is appropriately selected as described above.゛The method for crosslinking the oligosaccharides described above is not particularly limited, but
The degree of crosslinking is 6°C above the freezing point, which is the temperature used to adsorb β-amylase or T-amylase.
Intermolecular crosslinking may be performed at 0° C. or below so that the solubility of the crosslinked polymer substance obtained per 100 g of water is 0.01 g or less.

Therefore, the crosslinked polymeric materials of the present invention prepared by the crosslinking process are hydratable gels or solids.

The crosslinking reaction and conditions are appropriately selected depending on the type and concentration of the oligosaccharide and the conditions used during enzyme adsorption. Examples of the reaction include crosslinking between OH groups of glucose residues using epichlorohydrin. Epichlorohydrin is the most practical epihalogen. In the case of epihalogen, the amount added is preferably 0.5 to 3 times the amount of the raw material polyglucan solution. The concentration of polyglucan in the raw material solution is preferably at least 1%. As the alkali, a strong alkali such as caustic soda or caustic potash is used. It is necessary to include The temperature is 30°C or higher. Although the time is appropriately selected depending on the temperature and alkali concentration, 30 minutes or more is required at 60°C. Stirring is effective for contact between the epihalogen layer and the water layer during the reaction and for increasing the viscosity of the liquid due to gel formation.

〔Example〕

Hereinafter, the present invention will be explained in more detail using examples.

Example 1 90 g of water was added to 10 g of maltose and dissolved by heating to 50°C. Add 3N caustic soda 33- to this,
This was placed in a 500-mm reaction flask equipped with a stirrer and a reflux condenser. Next, 140-chlorohydrin was added, and the mixture was heated at 70° C. for 15 hours while stirring at 2 Qrpm.

After the reaction was completed, the contents were cooled to room temperature, and the gel-like substance in the lower aqueous layer was filtered off. Add 50ml of ethanol to this gel.
was added, stirred and filtered to recover the gel-like substance.The recovered gel was re-suspended in 50% ethanol and filtered to recover the gel-like substance.The fabrication process was repeated three more times. , suspended in 50ml of distilled water, filtered, and repeated 3 times, and then dispersed again in 50ml of ethanol. Filter this, dry the gel-like substance, and then crush it to form a white polymer powder 9
．． 4g was obtained.

The solubility of this powder in water at 60° C. was 0.002 g or less per 100 g of water. Furthermore, after immersing this powder in water at 60° C. for 1 day, the weight ratio of the hydrated gel to this powder was 14.0.

On the other hand, 20 d of the culture filtrate of Aspergillus oryzae IFO-4176 (containing 0.50 units of α-amylase, 0.98 units of β-amylase, and 1.30 units of γ-amylase) was cooled to 6°C, and the above 0.2 g of polymer powder was added and stirred at 5 rpm for 2 minutes. This was filtered to obtain a filtrate 20-. α-amylase activity, β-amylase activity, and γ-amylase activity in the filtrate were measured.

α-amylase in the filtrate is 0.50 units/-1β-amylase is 0.97 units/-1T-amylase is 0.01
The unit was /-.

The amount of enzyme adsorbed to the polymer powder was calculated as the value obtained by subtracting the enzyme concentration after contact from the enzyme concentration before contact with the polymer powder. As a result, α-amylase is θ units/−1β
- 0.015 units/d of amylase and 1.29 units/d of γ-amylase were adsorbed to the polymer powder. Therefore, α present in the culture filtrate
- 0% of amylase, 1% of β-amylase and 99% of T-amylase were adsorbed.

Next, the above enzyme-adsorbed polymer powder was placed in a 20 mf container, and a solution 4- adjusted to pH 8 with caustic soda was added thereto, and the mixture was stirred at 5 rpm and brought into contact for 2 minutes. This is 3
The mixture was centrifuged at 00 rpm for 5 minutes to separate the polymer powder and supernatant. Furthermore, water 2- was added to the polymer powder to suspend it, and the mixture was centrifuged again at 3000 rp+* for 5 minutes to separate solid and liquid into a washing liquid and the polymer powder.

The supernatant liquid obtained above and the washing liquid were combined to obtain enzyme desorption liquid 6-.

As a result of measuring the enzyme concentration of the above enzyme desorption solution, α-amylase θ units/-2 β-amylase 0.65 units/-1
γ-amylase was 4.25 units/@1.

Therefore, α-amylase 0 units (recovery rate 0%), β-amylase 3.9 units (2%), γ-amylase 25.
5 units (98%) were recovered.

Furthermore, no protease activity was detected in this liquid, and neither coloring nor odor was observed. Moreover, the amount of organic matter and the amount of inorganic matter in this solution are each 1.5% of the content in the stock solution.
%, decreased to 1.4%. By the above method, it was possible to selectively concentrate and purify the γ-amylase in the stock solution by about 3 times.

Example 2 80af of water was added to 20g of maltose and dissolved by heating to 50°C. Add 33 m of 4N caustic potash to this,
This was placed in a 5001 Bl reaction flask equipped with a stirrer and reflux condenser.

Next, add 140-m of Ebichlorohydrin and add 20-
It was heated at 80° C. for 6 hours while stirring at rpm.

After the reaction was completed, the contents were cooled to room temperature, and the gel-like substance in the lower aqueous layer was filtered off. 50% of ethanol was added to this gel-like material, stirred, and then filtered to recover the gel-like material.

The collected gel was again suspended in 50 d of ethanol, filtered, and a gel-like substance was collected. The 0-piece fabrication was repeated three more times. Next, the gel was suspended in 50 nl of distilled water and filtered.

After repeating this operation three times, the mixture was again dispersed in 50% of ethanol. This was filtered, and the gel-like material was dried and crushed to obtain 18.1 g of white polymer powder.

The amount of carbon dissolved in water at 60° C. of this powder was 0.003 g or less per 100 g of water. Furthermore, after immersing this powder in water at 60° C. for 1 day, the weight ratio of the hydrated gel to this powder was 15.2.

On the other hand, 20 mZ of culture filtrate of Aspergillus oryzae IFO-4176 (α-amylase 0.50 units, β-amylase 0.98 units, γ-amylase 1.30 units/
) was cooled to 6℃, and 0.2g of the above polymer powder was added.
was added and stirred for 2 minutes at 5 rpm. This is filtered, α-amylase activity, β-amylase activity in the filtrate,
T-amylase activity was measured.

Each enzyme activity in the filtrate was measured. In the filtrate, α-amylase was 0.50 units/-1β-amylase was 0.88 units/-1γ-amylase was 0.01 units/ml.

The amount of enzyme adsorbed to the polymer powder was calculated as the value obtained by subtracting the enzyme concentration after contact from the enzyme concentration before contact with the polymer powder. As a result, α-amylase has O units/−1β
-amylase is 0.10 units/-1T-amylase is 1
．． Only 29 units/d of culture filtrate were adsorbed onto the polymer powder.

Therefore, α-amylase present in the culture filtrate was not adsorbed at all, but 10% of β-amylase and 99% of γ-amylase were adsorbed.

Next, the polymer powder adsorbed with the above enzyme was placed in a 20- container, and a solution 4- adjusted to pH 8 with caustic potassium was added.
Contact was performed for 2 minutes with stirring at 5 rps.

This was centrifuged at 300 rpm for 5 minutes to separate the polymer powder and supernatant. Furthermore, water 2-
was added and suspended, and centrifuged again at 3,000 rpm for 5 minutes to separate solid and liquid into a washing solution and polymer powder. Combine the supernatant liquid and washing liquid obtained above, and add 6ml of enzyme desorption liquid.
I got l.

As a result of measuring the enzyme concentration of the above enzyme desorption solution, α-amylase 0 units/-1 β-amylase 0.35 units/-2
T-amylase was 4.14 units/-.

Therefore, α-amylase 0 units (recovery rate 0%), β-amylase 2.1 units (10.7%), T-amylase 2
4.8 units (95%) were recovered.

Furthermore, no protease activity was detected in this liquid, and neither coloring nor odor was observed. In addition, the amount of organic matter and the amount of inorganic matter in this solution are each 1.4 relative to the content in the stock solution.
%, decreased to 1.2%. By the above method, T-amylase in the stock solution could be selectively concentrated and purified by about 3.2 times.

Example 3 80 g of water was added to 10 g of maltotriose and dissolved by heating to 50°C. To this was added 33 cm of 4N caustic soda, and the mixture was placed in a 500-cm reaction flask equipped with a stirrer and a reflux condenser.

Then add 14011i of Ebichlorohydrin,
The mixture was heated at 80° C. for 6 hours while stirring at 20 rpra.

After the reaction was completed, the contents were cooled to room temperature, and the gel-like substance in the lower aqueous layer was filtered off. Add 50 yards of ethanol to this gel.
was added, stirred, and then filtered to collect a gel-like substance.

The collected gel was suspended again in 50% of ethanol and filtered to collect the gel-like material. This process was repeated three more times. Next, the gel was suspended in 50% of distilled water and filtered.

After repeating this operation three times, it was again dispersed in 50 d of ethanol. This was filtered, and the gel-like material was dried and crushed to obtain 9.7 g of white polymer powder.

The solubility of this powder in water at 60°C was less than 0.002g per 100g of water.
The weight ratio of the hydrated gel to the present powder after being immersed in water for one day was 14.8.

On the other hand, culture filtrate of Aspergillus oryzae IMP-4076 20-(α-amylase 0.50 unit, β-amylase 0.98 unit, T-amylase 1.30 unit/lB
1) was cooled to 6°C, and 0.2 g of the above polymer powder was added.
was added and stirred for 2 minutes at 5 rpm. This is filtered, α-amylase activity, β-amylase activity in the filtrate,
T-amylase activity was measured.

Each enzyme activity in the filtrate was measured. The α-amylase in the filtrate was 0.50 units/-1β-amylase was 0.64 units/-1T-amylase was 0.03 units/d.

The amount of enzyme adsorbed to the polymer powder was calculated as the value obtained by subtracting the enzyme concentration after contact from the enzyme concentration before contact with the polymer powder.

As a result, α-amylase was adsorbed at 0 unit/d, β-amylase at 0.34 unit/2T-amylase at 1.27 unit/wl, and the culture filtrate and bamboo polymer powder were adsorbed.

Therefore, α-amylase present in the culture filtrate was not adsorbed at all, but 35% of β-amylase and 98% of γ-amylase were adsorbed.

Next, the above-mentioned enzyme-adsorbed polymer powder was placed in a 20-mm container, 4 mf of a solution adjusted to pH 8 with caustic soda was added, and the mixture was stirred at 5 rpm and brought into contact for 2 minutes. This is 3
The mixture was centrifuged at 00 rpm for 5 minutes to separate the polymer powder and supernatant. Furthermore, water 2- was added to the polymer powder to suspend it, and the mixture was centrifuged again at 3,000 rpm for 5 minutes to separate solid and liquid into a washing liquid and the polymer powder. The supernatant liquid obtained above and the washing liquid were combined to obtain enzyme desorption liquid 6-.

As a result of measuring the enzyme concentration of the above enzyme desorption solution, α-amylase θ units/-1β-amylase 1.05 units/Td
, T-amylase 4.16 units/d.

Therefore, α-amylase 0 units (recovery rate 0%), β-amylase 6.27 units (32%), γ-amylase 25
．． 0 units (96%) were completely recovered.

Furthermore, no protease activity was detected in this liquid, and neither coloring nor odor was observed. Moreover, the amount of organic matter and the amount of inorganic matter in this solution are each 1.7 compared to the content in the stock solution.
%, decreased to 1.2%. By the above method, it was possible to selectively concentrate and purify γ-amylase in the stock solution by about 3.2 times.

Example 4 8-g of water was added to 1 g of maltotetraose and dissolved by heating to 50°C. To this was added 33 ml of 4N caustic soda, and the mixture was placed in a 200-cm reaction flask equipped with a stirrer and a reflux condenser.

Next, add 15-chlorohydrin and add 20r
The mixture was heated at 80°C for 6 hours while stirring at p-.

After the reaction was completed, the contents were cooled to room temperature, and the gel-like substance in the lower aqueous layer was filtered off. 50% of ethanol was added to this gel-like material, stirred, and then filtered to recover the gel-like material.

The recovered gel was suspended again in 50% of ethanol and filtered to recover the gel-like material. This process was repeated three more times. Next, it was filtered into 50% of distilled water.

After repeating this operation three times, the mixture was again dispersed in 50% of ethanol. This was filtered, and the gel-like material was dried and pulverized to obtain 0.90 g of white polymer powder.

The solubility of this powder in water at 60° C. was 0.001 g or less per 100 g of water. Furthermore, this powder was heated at 60°C.
The weight ratio of the hydrated gel to the present powder after being immersed in water for one day was 13.4.

On the other hand, 20 ml of culture filtrate of Aspergillus oryzae IMP-4076 (α-amylase 0.50 units, β-amylase 0.98 units, T-amylase 1.30 units/-
) was cooled to 6° C., 0.2 g of the above polymer powder was added, and the mixture was stirred at 5 rpm for 2 minutes.

This was filtered, and α-amylase activity, β-amylase activity, and T-amylase activity in the filtrate were measured.

The α-amylase in the filtrate was 0.50 units/-1 β-amylase was 0.29 units/rIi, and the γ-amylase was 0.03 units/d.

The amount of enzyme adsorbed to the polymer powder was calculated as the value obtained by subtracting the enzyme concentration after contact from the enzyme concentration before contact with the polymer powder.

As a result, only O units/d of α-amylase and 0.69 units/d of β-amylase and 1°27 units/d of T-amylase were adsorbed to the polymer powder of the culture filtrate.

Therefore, α-amylase present in the culture filtrate was not adsorbed at all, but 70% of β-amylase and 98% of T-amylase were adsorbed.

Next, the polymer powder adsorbed with the above enzyme was placed in a 20w1 container, and the pH was adjusted to 8.2 with caustic soda.
was added and allowed to contact for 2 minutes with stirring at 5 rpm. This was centrifuged at 300 rpm for 5 minutes to separate the polymer powder and supernatant. Furthermore, water 2- was added to the polymer powder to suspend it, and the mixture was centrifuged again at 3 QOO rpm for 5 minutes to separate the washing liquid and the polymer powder into solid and liquid.

The supernatant liquid obtained above and the washing liquid were combined to obtain enzyme desorption liquid 6-.

As a result of measuring the enzyme concentration of the above enzyme desorption solution, α-amylase was 0 units/ai, β-amylase was 1.31 units/m
l,r-amylase was 2.06 units/rnl.

Therefore, 0 units of α-amylase (recovery rate 0%), 7.84 units of β-amylase (40.0%), and 12.35 units of T-amylase (95%) were recovered.

Furthermore, no protease activity was detected in this liquid, and neither coloring nor odor was observed. Moreover, the amount of organic matter and the amount of inorganic matter in this solution are each 1.7 compared to the content in the stock solution.
%, decreased to 1.3%. As a result of the above, it was possible to selectively concentrate and purify γ-amylase in the stock solution by about 9.5 times.

Example 5 90 ml of water was added to 10 g of maltose hexaose,
It was heated to 50°C to dissolve it. Add 3 4N caustic soda to this
3- was added, and this was heated to 20°C with a stirrer and a reflux condenser.
0d into a reaction flask.

Then add 150ml of Ebichlorohydrin and add 2
The mixture was heated at 80° C. for 6 hours while stirring at 0 rpm+*.

After the reaction was completed, the contents were cooled to room temperature, and the gel-like substance in the lower aqueous layer was filtered off. 50% of ethanol was added to this gel-like material, stirred, and then filtered to recover the gel-like material.

The recovered gel was suspended again in 50% of ethanol and filtered to recover a gel-like substance. This operation was repeated three more times. Next, it was suspended in 50 ml of distilled water and filtered.

After repeating this operation three times, the mixture was again dispersed in 50% of ethanol. This was filtered, and the gel-like material was dried and crushed to obtain 9.7 g of white polymer powder.

The solubility of this powder in water at 60° C. was 0.001 g or less per 100 g of water. Furthermore, this powder was heated at 60°C.
The weight ratio of the hydrated gel to the present powder after being immersed in water for one day was 13.5.

On the other hand, culture of Aspergillus oryzae IMP-4076 t? ff120mZ (α-amylase 0.50 units,
β-amylase 0.98 units, γ-amylase 1.30 units
unit/Tn1) was cooled to 6°C and heated at 5 rpm for 2
Stir for a minute.

This was filtered, and α-amylase activity, β-amylase activity, and r-7 amylase activity in the filtrate were measured.

In the supernatant, α-amylase was 0.50 units/d, β-amylase was 0.02 units/ml, and T-amylase was 0.
03 units/d.

The amount of enzyme adsorbed to the polymer powder was calculated as the value obtained by subtracting the enzyme concentration after contact from the enzyme concentration before contact with the polymer powder. As a result, α-amylase was 0 units/-1
This means that 0.96 units/mZ of β-amylase and 1.27 units/mZ of γ-amylase were adsorbed to the polymer powder.

Therefore, α-amylase present in the culture filtrate was not adsorbed, but 90% of β-amylase and 98% of T-amylase were adsorbed.

Next, the above enzyme-adsorbed polymer powder was placed in a 20 mf container, and 4d of a solution adjusted to pH 8.2 with caustic soda was added thereto, and the mixture was stirred at 5 rps+ and brought into contact for 2 minutes. This was centrifuged at 300 rpm for 5 minutes to separate the polymer powder and supernatant. Furthermore, water 2- was added to the polymer powder to suspend it, and the mixture was centrifuged again at 3,000 rpm for 5 minutes to separate solid and liquid into a washing liquid and the polymer powder. The supernatant liquid obtained above and the washing liquid were combined to obtain enzyme desorption liquid 6-.

As a result of measuring the enzyme concentration of the above enzyme desorption solution, α-amylase O units/-1 β-amylase 25.5 units/-1
γ-amylase was 41.7 car position/wl.

Therefore, α-amylase O units (recovery rate θ%), β-amylase 15.3 units (78.0%), and T-amylase 25.0 units (96.0%) were recovered.

Furthermore, no protease activity was detected in this liquid, and neither coloring nor odor was observed. In addition, the amount of organic matter and the amount of inorganic matter in this solution are each 1.8 compared to the content in the stock solution.
%, decreased to 1.9%. As described above, T-amylase in the stock solution was selectively concentrated and purified by about 19 degrees twice.

Next, adsorption experiments of α-amylase, β-amylase, and r-amylase were conducted as a comparative example using amylose (derived from potato), which is known as an α-amylase adsorbent, instead of the adsorbent of the present invention.

Comparative Example 1 Same Rondo Aspergillus oryzae IFO as in Example
4176 culture solution 40a! (α-amylase 0.50
unit, β-amylase 0.98 unit, T-amylase 1
．． (containing 30 units/-) was cooled to 6° C., 0.2 g of amylose powder was added and brought into contact with stirring at 5 rpm for 2 minutes. This is filtered, α-amylase activity in the filtrate,
β-amylase activity and γ-amylase activity were measured.

α-amylase in the filtrate is 0.02 units/d, β-amylase is 0.96 units/d, and γ-amylase is 1°29
The unit was /-. The amount of enzyme adsorbed to the amylose powder was calculated as the value obtained by subtracting the enzyme concentration after contact with amylose from the enzyme concentration before contact with amylose.

As a result, 96% (0.48 units/wl) of α-amylase present in the culture filtrate, 2% (0.02 units/-) of β-amylase, and 1% of T-amylase were adsorbed. (0.01 units/-), and virtually nothing other than α-amylase was adsorbed.

〔Effect of the invention〕

The present invention provides a novel three-dimensionally crosslinked polymeric substance formed by intermolecularly crosslinking α-1,4 homooligomers of glucose. This three-dimensionally cross-linked polymeric material is extracted from a liquid containing various organic and inorganic impurities and other enzymes, such as culture filtrate of enzyme-producing bacteria.
-Amylase can be selectively adsorbed and separated. In particular, enzymes that are inconvenient during enzyme purification, such as protease, can be removed.

Claims (1)

[Scope of Claims] 1. A three-dimensionally crosslinked polymeric material, characterized in that it is formed by intermolecularly crosslinking α-1,4 homooligomers of glucose. 2. The degree of polymerization of α-1,4 homooligomer of glucose is 2.
6. The three-dimensionally crosslinked polymeric material according to claim 1, wherein the three-dimensionally crosslinked polymeric material has a molecular weight of 6 to 6. 3. The degree of polymerization of α-1,4 homooligomer of glucose is 2.
The three-dimensionally crosslinked polymeric substance according to claim 2, which is characterized in that: 4. The degree of polymerization of α-1,4 homooligomer of glucose is 3
6. The three-dimensionally crosslinked polymeric material according to claim 2, wherein the three-dimensionally crosslinked polymeric substance has a molecular weight of 6 to 6. 5. 100g in the temperature range above freezing point and below 60℃
The three-dimensionally crosslinked polymeric substance according to any one of claims 1 to 4, wherein the solubility in water is 0.001 g or less. 6. A method for producing a three-dimensionally crosslinked polymeric substance, which comprises adding a crosslinking agent to α-1,4 homooligomer of glucose and causing the reaction to cause intermolecular crosslinking to form a three-dimensional matrix. 7. The method for producing a three-dimensionally crosslinked polymeric substance according to claim 6, wherein the crosslinking agent is an epihalogen. 8. The method for producing a three-dimensionally crosslinked polymeric substance according to claim 7, wherein the epihalogen is epichlorohydrin. 9. Due to the reaction with the addition of a crosslinking agent, the solubility in 100g of water is 0 in the temperature range from freezing point to 60℃.
．． 7. The method for producing a three-dimensionally crosslinked polymeric substance according to claim 6, characterized in that intermolecular crosslinking is carried out so that the weight of the three-dimensionally crosslinked polymeric substance is 0.001g or less.