JPS6335605A - Crosslinked polyglucan and its production - Google Patents

Abstract

PURPOSE:To obtain a crosslinked polyglucan which can purify glucoamylase, by highly selective adsorption, by crosslinking a specified polyglucan having a nonreducing glucose residue as an open terminal. CONSTITUTION:The purpose crosslinked polyglucan is obtained by crosslinking a polyglucan formed by bonding branches having a nonreducing glucose residue as an open terminal and branched off from a main chain though alpha-1,6 bonds to the main chain composed of glucose units bonded through alpha-1,4 bonds until its solubility in water at 60 deg.C is decreased to 0.01 or below. Preferably, the number of glucose units polymerized is decreased by adding amylase to the polyglucan before it is crosslinked. As said amylase, at least either of beta-amylase and glucoamylase is usually used.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to polyglucan, and particularly to a crosslinked polyglucan that can highly selectively adsorb and purify glucoamylase, and a method for producing the same.

[Prior Art] Currently, glucose and high fructose corn syrup are industrially produced by hydrolyzing starch using α-amylase and glucoamylase. As described above, amylases (starch hydrolase group) are extremely useful in the industrial production of useful low-molecular substances from starch.

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

Industrial enzyme preparations can be obtained as a concentrated solution obtained by concentrating the culture filtrate as it is, as a dried powder, or as a concentrated solution obtained by separation and purification or as a dried powder thereof. However, concentrated liquids and crudely dried culture filtrates contain a large amount of unpleasant odor components and coloring components contained in the culture solution, so they cannot be used for many purposes.Therefore, useful substances can be extracted using enzyme reactions. During production, 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 alone, 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 of precipitant, concentration *PHe
It is necessary to go through a complicated process that involves changing the type of filler, the type of adsorption/desorption liquid, etc., and combining these. Not only that, for example, as described in JP-A-61-12284, a large amount of salt is added as a precipitant and eluent during the operation, and a salting operation is also performed when moving to the next step. For one or more reasons, these known purification methods are limited to research reagents and the like. Among them,
If an adsorbent that can selectively and efficiently adsorb glucoamylase, which is difficult to purify, is developed, it will be possible to concentrate and purify the enzyme from culture filtrate to high purity in one step.

The inventors focused on an adsorption method using amylose (a linear polymer of glucose) as an adsorbent, which has recently begun to be studied as a method for purifying α-amylase, and applied this to glucoamylase. However, it has been found that the adsorption capacity is extremely low and it is not practical for glucoamylase.6 [Problems to be solved by the invention] The purpose of the present invention is to absorb a wide variety of impurities other than glucoamylase such as culture fluid. From a glucoamylase-containing solution containing
The object of the present invention is to provide lipolyglucan as an adsorbent that can selectively and efficiently adsorb only the enzyme and a method for producing the same.

[Means for solving problems]

The present inventors focused on a conventionally known adsorption method using amylose (a linear polymer of glucose) as an adsorbent as a method for purifying α-amylase, and applied this to glucoamylase. However, the adsorption capacity was found to be extremely low and impractical. As a result of searching for an adsorbent for glucoamylase, we discovered that it is effectively adsorbed to glycogen, which has a high degree of branching, and that it is also relatively easily adsorbed to amylopectin.

However, we learned that these highly branched polysaccharides are water-soluble and extremely difficult to manipulate as adsorbents.

That is, even if a hyperbranched polysaccharide, powder, or gel is added, it is not adsorbed by these, and the dissolved hyperbranched polysaccharide molecules and gel coalase molecules form a complex and remain dissolved. As a result of intensive research, the present inventors solved the above problem by chemically crosslinking glycogen to make it insolubilized, and thus arrived at the present invention.

The first feature of the present invention is that glucose is α-1,4
Glucose was polymerized into the main chain through α-1,4 bonds and branched from the main chain through α-1,6 bonds. A crosslinked polyglucan is obtained by crosslinking a polyglucan formed by linking branched chains with non-reducing glucose residues at open ends within one molecule, and preferably corresponds to the branching point of the branched chain. The cross-linked polyglucan has glucose residues accounting for at least 5% of the number of nine molecules of raw gold glucose residues, and the number of branched glucose polymerizations is 2 or more and 6 or less. Further, the feature of the second invention is that glucose is present in the main chain polymerized by μm1,4 bonds.
A polyglucan that is polymerized by -1,4 bonds, branched from the main chain by α-1,6 bonds, and has branched chains with non-reducing glucose residues at open ends has a solubility in water of 60°C. The method for producing a crosslinked polyglucan involves crosslinking the polyglucan before crosslinking to 0.01 or less, preferably by adding amylase to the polyglucan before crosslinking to shorten the number of glucose polymerization. As such, either one or both of β-amylase and taglucoamylase are usually used.

The enzyme number applicable to the polyglucan of the present invention is α-
This enzyme can be applied to all amylases that hydrolyze the non-reducing end of the 1,4 glycond bond, so-called glucoamylase (γ-amylase).As long as the enzyme is glucoamylase, it is not particularly limited to its source organism. For example, glucoamylase originating from filamentous fungi such as Aspergillus and Rhizopus, and glucoamylase originating from bacteria such as Bacillus and Clostridium can also be applied.

The glucans used as raw materials for making the adsorbent of the present invention include:
A highly branched glucan is used.

The degree of branching can be used as long as the glucose residues corresponding to the branching points account for 5% or more of the total number of glucose residues in the molecule. Glucans containing 15% or more branch points are even more preferred. Therefore, examples of glucan include glycogen and highly branched amylopectin. The source organisms of glycogen and highly branched amylopectin are not particularly limited; for example, glycogen of animal origin or microbial origin can be used.

In addition, as these glucans, not only glucan separated from the natural raw materials mentioned above, but also gelcan whose branches are shortened by enzymatic treatment may be used. Glucoamylase or β-amylase is used. The types of these enzymes are not particularly limited, and the reaction conditions during treatment are appropriately selected depending on the type and concentration of the substrate glucan, and are appropriately adjusted so that the number of branched chain glucose polymerizations is 2 or more and 6 or less. .

When the degree of branching of glucan is less than 5%, the glucoamylase adsorption capacity decreases, making it impractical.

The above-mentioned glucan crosslinking method is not particularly limited, but the degree of crosslinking is determined by dissolving the adsorbent of the present invention in 100g of water at 60°C or lower, which is the temperature used to adsorb glucoamylase. is 0. Intermolecular crosslinking may be performed so that the molecular weight becomes less than or equal to Olg. Therefore, the adsorbent according to the invention prepared by a crosslinking process is a hydratable gel or solid.

The crosslinking reaction and conditions include the type and concentration of glucan.

One reaction that is appropriately selected depending on the usage conditions during adsorption includes, for example, crosslinking between OH groups of glucose residues using shrimp halogenhydrin. Epichlorohydrin is the most practical shrimp halogen. In the case of shrimp halogen, the amount added is 0.5 to 0.5 to the raw material polyglucan solution.
Three times is preferred. [The polyglucan concentration in the preparation solution is preferably at least 1% or more. As the alkali, a strong alkali such as caustic soda or caustic potash is used, but the concentration is IN
In the above, it is necessary to contain an amount of alkali equal to or more than the halogen of the shrimp halogen. The temperature for 1 hour at 30°C or higher is appropriately selected depending on the temperature and alkali concentration, but 30 minutes or more at 60°C is required.

[Effect]

The polyglucan of the present invention has a branched chain length of 2 to 6 glucose, which is about the same length as the glucoamylase molecular chain, so it is free from a wide variety of organic and inorganic impurities such as culture filtrate of enzyme-producing bacteria. As shown in the Examples of the present invention, glucoamylase can be selectively adsorbed from liquids containing other types of enzymes. It's especially eggy.

Furthermore, the polyglucan of the present invention has so-called intermolecular and/or intramolecular three-dimensional crosslinking, making it insoluble in water, and can be easily removed from the culture filtrate by simply desorbing the adsorbed glucoamylase. It can be purified in one step, and the purification process can be greatly simplified.

〔Example〕

Example 1 Escherichia coli glycogen (estimated molecular weight 4X10I!!)

95 mQ of 0.05 M sodium acetate buffer (pH 5,0) was added to 5 g of dry powder with estimated degree of branching (estimated degree of branching: 25%), and dissolved by heating to 70'C with stirring.This was cooled to 50°C. , take 30mm of it and find the Aspergillus genus (
Aspergillus orizea IF 0-
4M6) 2 mA of a glucoamylase solution containing 10 units of the original glucoamylase was added and maintained at 40°C.

As a result of measuring the amount of glucose produced after 2 hours, it reached approximately 30% of the number of glucose residues in the sample glycogen molecule. The molecular weight at that time was 6X104.

The aqueous solution was filled into a cellophane tube for dialysis, and dialyzed against water 5a at 10° C. for 10 hours. The contents were freeze-dried to obtain 0.9 g of white powder.

To this was added 10 mΩ of water, and heated to 70°C with stirring to dissolve it into a viscous liquid.Firstly, 33 mm of 6N caustic soda was added, and this was placed in a 200 mm reaction plastic vessel equipped with a stirrer and a reflux condenser. 0 then 100m
fl of epichlorohydrin was added, and the mixture was heated at 70° C. for 5 hours while stirring at 20 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. Add 50ml of ethanol to this gel.
2 was added, stirred and filtered to recover a gel-like substance. The recovered gel in 1 was resuspended in 50 mA of ethanol and filtered to recover a gel-like substance.

After repeating this operation three more times, distilled water 50mΩ
After repeating the 0 bottle operation 3 times,
Dispersed in 50 m m ethanol.

Filter this, dry the gel-like substance, and then crush it to form a white powder with 0
．． 7 g was obtained.

The solubility of this powder in water at 60°C was O,001g per 100g of water.
The weight ratio of the hydrated gel to the main powder after being immersed in water for one day was 13.2.The average degree of polymerization of the branched chains was 4.2 by measuring the non-reducing end groups of the six powders. The following experiment was conducted to examine the adsorption of glucoamylase to the powder.

Aspergillus oligo I FO-4176 culture solution 4
0 mΩ (containing α-amylase 0.50 units/mQ, glucoamylase 1.30 units/mΩ) was cooled to 6°C, 0.2 g of the above adsorbent powder was added, and 5 rp+*
for 2 minutes.

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

The α-amylase in the supernatant was calculated as the value obtained by subtracting the enzyme concentration after contact from the enzyme concentration before contact with 0.50 units/. As a result, α-amylase present in the culture filtrate was not substantially adsorbed, and 99% of glucoamylase (1,
29 units) were adsorbed.

Next, as a comparative example, adsorption of α-amylase and glucoamylase was carried out using amylose (derived from potato), which is known as an α-amylase adsorbent, instead of the adsorbent of the present invention.

Comparative Example 1 Aspergillus oligo IFO from the same lot as the example
-4176 culture solution 40 mA (a-amylase 0.50
units/m fl, glucoamylase 1.30 units/
m Q) was cooled to 6°C and amylose powder 0.
2 g was added and mixed for 2 minutes with a SRP roller to make contact. This was filtered, and α-amylase activity and glucoamylase activity in the filtrate were measured.

The α-amylase in the supernatant was 0.02 units/m·Ω, and the glucoamylase was 1.29 units.

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/m Q ) of α-amylase present in the culture filtration was adsorbed, and 1% (0.01 units/m Q ) of glucoamylase was adsorbed.
Ω) and things were not adsorbed on purchase.

The present invention will be explained in more detail below using an embodiment of the present invention.

Example 2 E. coli glycogen (estimated molecular weight 4X10B).

90mM of water was added to the dry powder Log with an estimated degree of branching of 25%), and while stirring, it was heated to 70'C and dissolved to form a viscous liquid. To this was added 33 mQ of 6N caustic soda, and the mixture was placed in a 500 mm reaction flask equipped with a stirrer and a reflux condenser.

Then add 140 mjl of epichlorohydrin and add 2
The mixture was heated at 50° C. for 5 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. Add 50 m of ethanol to this gel.
Q was added, stirred, and then filtered to recover a gel-like substance. The recovered gel in 6 was resuspended in 50 mQ of ethanol and filtered to recover a gel-like substance.

After repeating this operation three more times, 50 m of distilled water was added.
After repeating the 0 operation of suspending in Q and filtering it 3 times, add 50 ml of ethanol again! dispersed into

This was filtered, and the gel-like substance was dried and ground, resulting in a white powder of 9.
．． 2 g was obtained.

The solubility of this powder in water at 60°C is 10.

g, it was 0.002g or less. Furthermore, after immersing this powder in 60°C water for 1 day, the weight ratio of the hydrated gel to this powder was 11.5.

Next, in order to examine the adsorption of glucoamylase to this powder, the following experiment was conducted.

Culture solution of Aspergillus oligo IFO-4176 40
m 41 (a-7m9-ze0.50 unit/m
n, containing 1.30 units of glucoamylase/ml)
was cooled to 6° C., 0.2 g of the above adsorbent powder was added and contacted by mixing at 5 rpm for 2 minutes.

This was filtered, and α-amylase activity and glucoamylase activity during filtration were measured.

The α-amylase in the supernatant was O,SO units/mα, and the glucoamylase was 0.01 units/mQ. The #adsorbent adsorbed on the adsorbent was calculated as the value obtained by subtracting the enzyme concentration after contact from the enzyme concentration before contact with the adsorbent. As a result, α-amylase present in the culture filtrate was not substantially adsorbed, and 99% (51.6 units) of glucoamylase was adsorbed.

Example 3 Escherichia coli glycogen (estimated molecular weight 4 x 10”).

8 mQ of water was added to 20 g of dry powder with an estimated degree of branching of 25%), 40 mj2 of ION caustic soda was added without stirring, and this was placed in a 500 mm reaction flask equipped with a stirrer and a reflux condenser. , 200 mΩ of epichlorohydrin was added, and the mixture was heated at 70° C. for 48 hours while stirring at 20 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. Add 50 m of ethanol to this gel.
Q was added, stirred, and then filtered to recover a gel-like substance. The recovered gel in 6 was resuspended in 50 mQ of ethanol and filtered to recover a gel-like substance.

After repeating this operation three more times, 50 mj of distilled water was added.
After repeating this procedure three times in which the suspension was suspended in 50 mfl of ethanol and filtered, it was again dispersed in 50 mfl of ethanol.

Filter this, dry the gel-like substance, and then crush it to form a white powder of 1
8.3 g was obtained.

The solubility of this powder in water at 60°C was 0.00°C or less per 100g of water. Furthermore, add 6 pieces of this powder.
The weight ratio of the hydrated gel to the present powder after being immersed in 0°C water for one day was 9.6.

Next, in order to examine the adsorption of glucoamylase to this powder, the following experiment was conducted.

Aspergillus oligo I FO-4176 culture solution 4
0 mm (containing 0.50 units/mΩ of a-amylase and 1.30 units/mQ of glucoamylase) was cooled to 6°C, 0.2 g of the above adsorbent powder was added, and mixed for 2 minutes with Srp■. I made contact with it.

This was filtered, and α-amylase activity and glucoamylase activity during filtration were measured.

α-amylase in the supernatant was 0.51 units/m Q ,
Glucoamylase was 0.01 units/mΩ. The amount of enzyme adsorbed on the adsorbent was calculated as the value obtained by subtracting the enzyme concentration after contact with the adsorbent from the enzyme concentration before contact with the adsorbent. As a result, α-amylase present in the culture filtrate was not substantially adsorbed, and 99% (51.6 units) of glucoamylase was adsorbed.

Example 4 Escherichia coli glycogen (estimated molecular weight 4 x 10”).

Add 70 m+2 of water to 30 g of dry powder with an estimated degree of branching of 25%), add 40 mU of ION caustic soda without stirring, and transfer this to a 500 mff tank equipped with a stirrer and reflux condenser.
1 into a reaction flask. Then add 400 mQ of epichlorohydrin.

The mixture was heated at 80° C. for 48 hours while stirring in a 2-orp oven.

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 150m of ethanol to this gel-like substance.
A was added, stirred, and then filtered to recover a gel-like substance. The recovered gel was resuspended in 50 mA of ethanol and filtered to recover a gel-like substance.

After repeating this operation three more times, 150 m of distilled water
After repeating the procedure three times in which the suspension was suspended in 150 m m of ethanol and separated by filtration, it was again dispersed in 150 m m of ethanol. This was filtered, and the gel-like material was dried and crushed to obtain 25 g of white powder.

The solubility of this powder in water at 60°C was 0.001 g or less per 100 g of water. Furthermore, after immersing this powder in 60°C water for 1 day, the weight ratio of the hydrated gel to this powder was 7.9.

Next, to check the adsorption of glucoamylase to this powder,
The following experiment was conducted.

Culture solution of Aspergillus oligo IFO-4176 40
cooled to 6°C,
0.2 g of the above adsorbent powder was added and brought into contact at 5 rpm for 2 minutes.

This was filtered, and α-amylase activity and glucoamylase activity during filtration were measured.

The α-amylase in the supernatant was 0.51 units/mQ, and the glucoamylase was 0.02 units/mn. The amount of enzyme adsorbed on the adsorbent was calculated as the value obtained by subtracting the enzyme concentration after contact with the adsorbent from the enzyme concentration before contact with the adsorbent. As a result, α-amylase present in the culture filtrate was not substantially adsorbed, and 98% (1.28 units) of glucoamylase was adsorbed.

Example 5 Glycogen in the large intestine (estimated molecular weight 4X10B).

85 mQ of water was added to 15 g of dry powder with an estimated degree of branching of 25%), and while stirring, 33 mρ of 6N caustic soda was added, and this was placed in a 500 mA reaction flask equipped with a stirrer and a reflux condenser. of epichlorohydrin and stirred at 75°C for 3 minutes while stirring at 15 rpm.
Heated for 6 hours.

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 100% ethanol to this gel.
m II was added, and after stirring, the gel was collected by filtration. The recovered gel was resuspended in 50 m of ethanol, and the gel was collected by filtration. This procedure was repeated three more times. 0 Next, 1 was suspended in 50 mQ of distilled water and filtered.
After repeating this operation three times, add 150 m of ethanol again.
Dispersed in Q. This was filtered, and the gel-like material was dried and crushed to obtain 14 g of white powder.

The solubility of this powder in water at 60'C was 0.001 g or less per 100 g of water. Furthermore, after immersing this powder in 60° C. water for one day, the weight ratio of the hydrated gel to this powder was 8.1.

Next, in order to examine the adsorption of glucoamylase to this powder, the following experiment was conducted.

Culture solution of Aspergillus oligo IFO-4176 40
mjl (containing α-amylase 0.50 units/mQ, glucoamylase 1.30 units/mQ) was cooled to 6°C, 0.2 g of the above adsorbent powder was added, and 5
rpn+ for 2 minutes.

This was filtered, and α-amylase activity and glucoamylase activity during filtration were measured.

α-Amylase in the supernatant is 0.50 units/m II
, glucoamylase was 0.01 units/m Q. The amount of enzyme adsorbed on the adsorbent was calculated as the value obtained by subtracting the enzyme concentration after contact with the adsorbent from the enzyme concentration before contact with the adsorbent. As a result, α-amylase present in the culture filtrate was not substantially adsorbed, and 99% of glucoamylase (1,
29 units) were adsorbed.

Example 6 90 m2 of water was added to dry powder Log of beef liver glycogen (estimated molecular weight 3.5×10I5°, estimated degree of branching 27%), and heated to 70'C with stirring to melt and obtain a viscous liquid. Ta. Add 33 mQ of 4N caustic potash to this,
This was placed in a 500 mA reaction flask equipped with a stirrer and a reflux condenser. Then, 140 mQ of epichlorohydrin was added.

The mixture was heated at 50° C. for 5 hours while stirring at 20 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. Add 50 mQ of ethanol to this gel-like substance.
was added, stirred and filtered to recover a gel-like substance. The recovered gel was resuspended in 50 mm of ethanol and filtered to recover a gel-like substance.

This operation was repeated three more times. Next, 50 m of distilled water
After repeating the procedure of suspending in 100ml and filtering it 3 times,
It was again dispersed in 50 mΩ of ethanol.

This was filtered, and the gel-like substance was dried and ground, resulting in a white powder of 9.
．． 3 g was obtained.

The solubility of this powder in water at 60°C was 0.003g per 100g of water.
The weight ratio of the hydrated gel to the present powder after being immersed in water for one day was 12.3.

Next, the following experiment was conducted to examine the adsorption of glucoamylase to the powder. '40 mA of culture solution of Aspergillus oligo IFO-4176 (a-amylase 0.50 units/m 12 , glucoamylase 1.30
unit/m Q) was cooled to 6 °C, 0.2 g of the above adsorbent powder was added and contacted by mixing at 5 rpm for 2 minutes.

This was filtered, and α-amylase activity and glucoamylase activity during filtration were measured.

α-amylase in the supernatant was 0.50 units/mfl,
Glucoamylase was 0.02 units/mQ. The amount of enzyme adsorbed on the adsorbent was calculated as the value obtained by subtracting the enzyme concentration after contact with the adsorbent from the enzyme concentration before contact with the adsorbent. As a result, α-amylase present in the culture filtrate was not adsorbed substantially, and 98% of glucoamylase (1,2
8 units) were adsorbed.

Example 7 Beef liver glucogen (estimated molecular weight 3.5X10').

80 mQ of water was added to 20 g of dry powder with an estimated degree of branching of 27%), and heated to 80° C. with stirring to form a paste. To this was added 66 mQ of 6N caustic soda, which was then placed in a 500 mΩ reaction flask equipped with a stirrer and a reflux condenser. Next, 180 mQ of epichlorohydrin was added, and the mixture was heated at 70° C. for 9 hours while stirring at 10 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. Add 80ml of ethanol to this gel-like substance.
2 was added, stirred, and filtered to collect a gel-like substance. The recovered gel was resuspended in ethanol 80rnQ, filtered, and a gel-like substance was recovered. This operation was repeated three more times. Next, the procedure of suspending in 50 mQ of distilled water and filtering was repeated three times, and then dispersed again in 80 mM of ethanol. This was filtered, and the gel-like material was dried and ground to a white powder9.
3 g was obtained.

The solubility of this powder in water at 60°C was 0.001 g per 100 g of water. Furthermore, 60% of this powder
The weight ratio of the hydrated gel to the present powder after being immersed in °C water for 1 day was 7.2.

Next, in order to examine the adsorption of glucoamylase to this powder, the following experiment was conducted.

Aspergillus oryzae FO-4176 culture solution 40
mΩ (containing α-amylase 0.50 units/mQ, glucoamylase 1.30 units/mQ) was cooled to 6 °C, 0.2 g of the above adsorbent powder was added, and the mixture was heated at 5 rpm.
for 2 minutes.

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

α-Amylase in the supernatant is 0.50 units/m Q
, glucoamylase was 0.02 units/m12. The amount of enzyme adsorbed to the above-mentioned adhesive was calculated as the value obtained by subtracting the enzyme concentration after contact with the adsorbent from the enzyme concentration before contact with the adsorbent. As a result, α-amylase present in the culture filtrate was not adsorbed substantially, and 98% of glucoamylase (1,2
8 units) were adsorbed.

Example 8 Glucogen from oyster shellfish (estimated molecular weight 4 x 10”).

90 mA of water was added to the dry powder Log with an estimated degree of branching of 25%), and the mixture was heated to 70° C. with stirring to dissolve and form a viscous liquid.

To this was added 33 mg of 6N caustic soda, which was then placed in a 500 m12 reaction flask equipped with a stirrer and a reflux condenser.Next, 140 mm of epichlorohydrin was added and stirred at 15 rpm.

Heated at 50°C for 5 hours.

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 mA of ethanol to this gel.
was added, stirred and filtered to recover a gel-like substance.The recovered gel was resuspended in 50 mQ of ethanol and filtered to recover a gel-like substance.

After repeating this operation three more times, distilled water 50mΩ
The operation of suspending and filtering was repeated three more times.
Next, the suspension in 50 mA of distilled water and filtration were repeated three times, and then dispersed again in 50 mA of ethanol.

This was filtered, and the gel-like substance was dried and ground, resulting in a white powder of 9.
．． 1 g was obtained.

The solubility of this powder in water at 60° C. was 0.001 g or less per 100 g of water. Furthermore, add 6 pieces of this powder.
The weight ratio of the hydrated gel to the present powder after being immersed in 0°C water for one day was 10.8.

Next, in order to examine the adsorption of glucoamylase to this powder, the following experiment was conducted.

Culture solution of Aspergillus oligo IFO-4176 40
mm (containing α-amylase O, SO units/m 1!, glucoamylase 1.30 units/m 11) was cooled to 6 °C, 0.2 g of the above adsorbent powder was added, and 5
Mix and contact for 2 minutes at rpm.

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

The α-amylase in the supernatant was 0.50 units/mQ, and the glucoamylase was 0.02 units/mjl. The amount of enzyme adsorbed on the adsorbent was calculated as the value obtained by subtracting the enzyme concentration after contact with the adsorbent from the enzyme concentration before contact with the adsorbent.

As a result, α-amylase present in the culture filtrate was not substantially adsorbed, and 98% (1.28 units) of glucoamylase was adsorbed.

〔Effect of the invention〕

According to the present invention, from a culture filtrate of enzymatic progenitor bacteria and a liquid containing various organic and S-organic impurities and other types of enzymes,
Can selectively adsorb glucoamylase. Moreover, an adsorbent having such a function can be easily produced.

[Brief explanation of the drawing]

Figure 1 shows a typical structural unit of a substance corresponding to claim 1 of the present invention using shrimp halogenhydrin as a crosslinking agent. Figure 2 shows the present invention using shrimp halogenhydrin as a crosslinking agent. Representative units of substances corresponding to claim 1 are shown below.

Claims (1)

[Scope of Claims] 1. Glucose is polymerized through α-1,4 bonds in the main chain in which glucose is polymerized through α-1,4 bonds, and α-1,
A crosslinked polyglucan is obtained by intermolecularly and intramolecularly crosslinking polyglucan formed by branched chains branched from the main chain by 6 bonds and having non-reducing glucose residues at open ends. 2. The crosslinked polyglucan according to claim 1, wherein glucose residues corresponding to branch points of the branched chain account for at least 5% or more of the total number of glucose residues in the molecule. 3. The crosslinked polyglucan according to claim 1 or 2, wherein the number of branched glucose polymerizations is 2 or more and 6 or less. 4. Glucose is polymerized through α-1,4 bonds to the main chain in which glucose is polymerized through α-1,4 bonds, and α-1,
Crosslinking in which a polyglucan consisting of a branched chain branched from the main chain by 6 bonds and having an open end with a non-reducing glucose residue is crosslinked until the solubility in water becomes 0.01 or less at 60°C. Method for producing polyglucan. 5. The method for producing a crosslinked polyglucan according to claim 4, wherein amylase is added to the polyglucan before crosslinking. 6. The method for producing a crosslinked polyglucan according to claim 4 or 5, wherein one or both of β-amylase and glucoamylase is used as the amylase.