JPH0576961B2 - - Google Patents

Description

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

[Conventional technology]

Currently, glucose and high fructose isomerized sugar 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 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 contains protease that destroys the target enzyme. Therefore, when producing useful substances using enzymatic reactions, it is necessary to remove these disadvantageous impurities. For this reason,
This places a significant burden on the subsequent separation and purification process of the reaction product. On the other hand, salting-out methods and liquid chromatography alone, which are known as general purification methods, have a limit to partial concentration, and in order to improve separation accuracy, these operating conditions, such as the type of precipitant, concentration, pH, and packing It is necessary to go through a complicated process that involves changing the type of agent, 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. Must be. From the above, these known purification methods are limited to research reagents and the like. In particular, 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 was found that the adsorption capacity was extremely low and it was not practical for glucoamylase.

[Problem that the invention seeks to solve]

An object of the present invention is to provide a method for producing polyglucan using an adsorbent that can selectively and efficiently adsorb only glucoamylase from a glucoamylase-containing liquid containing various impurities other than glucoamylase, such as a culture solution. It is in.

[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 hyperbranched polysaccharides, powders, or gels are added, they are not adsorbed, and the dissolved hyperbranched polysaccharide molecules and glucoamylase 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 present invention provides non-reducing glucose residues in which glucose is polymerized through α-1,4 bonds and branched from the main chain through α-1,6 bonds. A shortened polyglucan is obtained by treating a polyglucan formed by linking branched chains with an open end with amylase to shorten the branched chains, or a crosslinked polyglucan obtained by crosslinking glycogen intermolecularly or intramolecularly. Yes, preferably
The glucose residues corresponding to the branching points of the branched chain account for at least 5% or more of the total number of glucose residues in the molecule, and the number of glucose polymerizations in the branched chain is 2 or more and 6
The following are crosslinked polyglucans. In addition, the present invention provides non-reducing glucose in which glucose is polymerized through α-1,4 bonds and branched from the main chain through α-1,6 bonds. A method for producing a crosslinked polyglucan, in which a polyglucan formed by bonding branched chains with residues at open ends is crosslinked until the solubility in water becomes 0.01 or less at 60°C. The purpose of this method is to shorten the number of glucose polymerizations by adding amylase to polyglucan, and as amylase, one or both of β-amylase and glucoamylase are usually used.

Enzymes applicable to the polyglucan of the present invention are generally applicable to amylases that hydrolyze the non-reducing terminal side of α-1,4 glycosidic bonds, so-called glucoamylases (γ-amylases). The present enzyme is not particularly limited to its originating organism as long as it is glucoamylase. 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.

Highly branched glucan is used as the raw material glucan for producing the adsorbent of the present invention.
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. These glycogen and highly branched amylopectin are also not particularly limited to the originating organisms. For example, glycogen can be of animal or microbial origin.

Furthermore, as these glucans, not only glucans separated from the above-mentioned natural raw materials but also processed glucans in which these branches are shortened by enzymatic treatment may be used. As mentioned above, glucoamylase or β-amylase is used to shorten the glucan chains. The types of these enzymes are not particularly limited. The reaction conditions during the treatment are appropriately selected depending on the type and concentration of the substrate glucan, but the number of branched glucose polymerizations should be kept at 2 or more and 6 or less.
Adjustments will be made as appropriate.

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

The above-mentioned glucan cross-linking method is not particularly limited, but the degree of cross-linking is such that the solubility of the adsorbent of the present invention in 100 g of water is below 60°C, which is the temperature used to adsorb glucoamylase. Intermolecular crosslinking may be performed so that the weight becomes 0.01 g or less. Therefore, the adsorbent according to the invention prepared by crosslinking is a hydratable gel or solid.

The crosslinking reaction and conditions depend on the type and concentration of glucan,
It is selected as appropriate depending on the usage conditions during adsorption. Examples of the reaction include crosslinking between OH groups of glucose residues using epihalogenhydrin. Epichloralhydrin 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 polyglucan concentration in the raw material solution is at least 1
% or more is preferable. Strong alkalis such as caustic soda and caustic potash are used as alkali, but the concentration is
It is necessary to contain an alkali of 1N or more and an equivalent amount or more to the halogen of the epihalogen. temperature is 30
It is carried out above ℃. The time is appropriately selected depending on the temperature and alkali concentration, but 30 minutes or more is required at 60°C.

[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. and other types of enzymes, as shown in the examples of the present invention,
Can selectively adsorb glucoamylase. In particular, enzymes that are inconvenient for enzyme purification, such as proteolytic enzymes, can be removed. Furthermore, glucoamylase can be adsorbed at a higher density.

In addition, the polyglucan of the present invention has so-called intermolecular and/or intramolecular three-dimensional crosslinking,
Therefore, it is insoluble in water, and can be easily purified from the culture filtrate by simply desorbing the adsorbed glucoamylase, greatly simplifying the purification process.

〔Example〕

Example 1 Escherichia coli glycogen (estimated molecular weight 4×10 ⁵ ,
95 ml of 0.05 M sodium acetate buffer (PH 5.0) was added to 5 g of dry powder with an estimated degree of branching of 25%) and dissolved by heating to 70° C. with stirring. This was cooled to 50°C, 30ml of which was taken, 2ml of a glucoamylase solution containing 10 units of glucoamylase originating from Aspergillus orizea IFO-4M6 was added thereto, and the mixture was maintained at 40°C.

As a result of measuring the amount of glucose produced after 2 hours,
This amount reached approximately 30% of the number of glucose residues in the sample glycogen molecule. The molecular weight at that point was 6×10 ⁴ .

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

To this was added 10 ml of water, and while stirring, the mixture was heated to 70°C to dissolve and form a viscous liquid. Next, 33 ml of 6N caustic soda was added and placed in a 200 ml reaction flask equipped with a stirrer and reflux condenser. Next, 100 ml of epichlorohydrin was added and 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 gelatinous material in the aqueous layer at the bottom was filtered off. 50 ml of ethanol was added to this gel-like material, stirred, and then filtered to recover the gel-like material. The recovered gel was resuspended in 50 ml 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,
Dispersed in 50ml of ethanol. This was filtered, and the gel-like material was dried and crushed to obtain 0.7 g of white powder.

The solubility of this powder in water at 60℃ is 100g of water.
However, it was 0.001g. In addition, 60% of this powder
The weight ratio of the hydrated gel to the present powder after being immersed in ℃ water for one day was 13.2. The average degree of polymerization of branched chains was found to be 4.2 by measuring the non-reducing end groups of this powder. Next, in order to examine the adsorption of glucoamylase to this powder, the following experiment was conducted.

Culture solution of Aspergillus oryzae IFO-4176 40
ml (containing 0.50 units/ml of α-amylase and 1.30 units/ml of glucoamylase) was cooled to 6°C, 0.2 g of the above adsorbent powder was added, and the mixture was mixed and contacted at 5 rpm 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 0.50 units/ml, and the glucoamylase was 0.01 units/ml. 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% (1.29 units) of glucoamylase was 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 40 ml of culture solution of Aspergillus oryzae IFO-4176 from the same lot as in Example (α-amylase 0.50
units/ml, containing 1.30 units/ml of glucoamylase) was cooled to 6°C, 0.2 g of amylose powder was added, and mixed and contacted at 5 rpm 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 0.02 units/ml, 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/ml) of α-amylase present in the culture filtration was adsorbed, and 1% (0.01 units/ml) of glucoamylase was adsorbed.
ml) and was virtually not adsorbed.

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

Example 2 Escherichia coli glycogen (estimated molecular weight 4×10 ⁵ ,
90 ml of water was added to 10 g of dry powder with an estimated degree of branching of 25%), and while stirring, the mixture was heated to 70°C to dissolve and form a viscous liquid. Add 33ml of 6N caustic soda to this, and mix this into 500ml with a stirrer and reflux condenser.
into a reaction flask. Then add 140 ml of epichlorohydrin and stir at 20 rpm for 50 min.
Heated at ℃ 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. 50 ml of ethanol was added to this gel-like material, stirred, and then filtered to recover the gel-like material. The recovered gel was resuspended in 50 ml 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,
Dispersed in 50ml of ethanol. This was filtered, and the gel-like material was dried and crushed to obtain 9.2 g of white powder.

The solubility of this powder in water at 60℃ is 100g of water.
However, it was less than 0.002g. 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 oryzae IFO−4176
40 ml (containing 0.50 units/ml of α-amylase and 1.30 units/ml of glucoamylase) was cooled to 6°C, 0.2 g of the above adsorbent powder was added, and the mixture was mixed and contacted at 5 rpm for 2 minutes. This is filtered, and the α-
Amylase activity and glucoamylase activity were measured.

The α-amylase in the supernatant was 0.50 units/ml, and the glucoamylase was 0.01 units/ml. 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 3 Escherichia coli glycogen (estimated molecular weight 4×10 ⁵ ,
80 ml of water was added to 20 g of dry powder with an estimated degree of branching of 25%), 40 ml of 10N caustic soda was added with stirring, and the mixture was placed in a 500 ml reaction flask equipped with a stirrer and a reflux condenser. Next, add 200 ml of epichlorohydrin and heat to 70°C while stirring at 20 rpm.
It was heated for 48 hours.

After the reaction was completed, the contents were cooled to room temperature, and the gelatinous material in the aqueous layer at the bottom was filtered off. 50 ml of ethanol was added to this gel-like material, stirred, and then filtered to recover the gel-like material. The recovered gel was resuspended in 50 ml 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 ml of ethanol. This was filtered, and the gel-like material was dried and crushed to obtain 18.3 g of white powder.

The solubility of this powder in water at 60℃ is 100g of water.
However, it was less than 0.001g. Furthermore, after immersing this powder in 60°C water for 1 day, the weight ratio of the hydrated gel to this powder was 9.6.

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

Culture solution of Aspergillus oryzae IFO-4176
40 ml (containing 0.50 units/ml of α-amylase and 1.30 units/ml of glucoamylase) was cooled to 6°C, 0.2 g of the above adsorbent powder was added, and the mixture was mixed and contacted at 5 rpm for 2 minutes. This is filtered, and the α-
Amylase activity and glucoamylase activity were measured.

The α-amylase and glucoamylase concentrations in the supernatant were 0.51 unit/ml and 0.01 unit/ml, respectively. 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 4×10 ⁵ ,
70 ml of water was added to 30 g of dry powder with an estimated degree of branching of 25%), 40 ml of 10N caustic soda was added with stirring, and the mixture was placed in a 500 ml reaction flask equipped with a stirrer and a reflux condenser. Next, add 40 ml of epichlorohydrin and heat at 80°C while stirring at 20 rpm.
Heated for 48 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. 150 ml of ethanol was added to this gel-like material, stirred, and then filtered to recover the gel-like material. The recovered gel was resuspended in 50 ml of ethanol and filtered to recover a gel-like substance. This operation was repeated three more times. Next, it was suspended in 150 ml of distilled water and filtered. After repeating this operation three times, the mixture was again dispersed in 150 ml of ethanol. Filter this and
After drying the gel-like material, it was crushed to obtain 25 g of white powder.

The solubility of this powder in water at 60℃ is 100g of water.
However, it was less than 0.001g. Furthermore, after immersing this powder in 60°C water for one day, the weight ratio of the hydrated gel to this powder was 7.9.

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

Culture solution of Aspergillus oryzae IFO−4176
40 ml (containing 0.50 units/ml of α-amylase and 1.30 units/ml of glucoamylase) was cooled to 6°C, 0.2 g of the above adsorbent powder was added, and the mixture was mixed and contacted at 5 rpm for 2 minutes. This is filtered, and the α-
Amylase activity and glucoamylase activity were measured.

The α-amylase in the supernatant was 0.51 units/ml, and the glucoamylase was 0.02 units/ml. 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 85 ml of water was added to 15 g of dry powder of large intestine glycogen (estimated molecular weight 4 x 10 ⁵ , estimated degree of branching 25%), and while stirring, 33 ml of 6N caustic soda was added, and this was cooled with a stirrer and reflux. into a 500 ml reaction flask with a container. Next, add 150 ml of epichlorohydrin and heat to 75°C while stirring at 15 rpm.
It was heated for 36 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. 100 ml of ethanol was added to this gel-like material, stirred, and then filtered to recover the gel-like material. The recovered gel was resuspended in 50 ml 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 150 ml of ethanol. Filter this and
After drying the gel-like material, it was crushed to obtain 14 g of white powder.

The solubility of this powder in water at 60℃ is 100g of water.
However, it was less than 0.001g. 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 oryzae IFO-4176
40 ml (containing 0.50 units/ml of α-amylase and 1.30 units/ml of glucoamylase) was cooled to 6°C, 0.2 g of the above adsorbent powder was added, and the mixture was mixed and contacted at 5 rpm for 2 minutes. This is filtered, and the α-
Amylase activity and glucoamylase activity were measured.

The α-amylase in the supernatant was 0.50 units/ml, and the glucoamylase was 0.01 units/ml. 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% (1.29 units) of glucoamylase was adsorbed.

Example 6 Bovine liver glycogen (estimated molecular weight 3.5×10 ⁵ ,
90 ml of water was added to 10 g of dry powder with an estimated degree of branching (estimated degree of branching of 27%), and the mixture was heated to 70° C. with stirring to dissolve it to obtain a viscous liquid. Add 33ml of 4N caustic potash to this,
This was placed in a 500 ml reaction flask equipped with a stirrer and reflux condenser. Next, add 140 ml of epichlorohydrin and heat at 50°C for 5 minutes while stirring at 20 rpm.
heated for an hour.

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 ml of ethanol was added to this gel-like material, stirred, and then filtered to recover the gel-like material. The recovered gel was resuspended in 50 ml 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 ml of ethanol. This was filtered, and the gel-like material was dried and crushed to obtain 9.3 g of white powder.

The solubility of this powder in water at 60℃ is 100g of water.
However, it was 0.003g. In addition, 60% of this powder
The weight ratio of the hydrated gel to the present powder after being immersed in ℃ water for one day was 12.3.

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

Culture solution of Aspergillus oryzae IFO-4176 40
ml (containing 0.50 units/ml of α-amylase and 1.30 units/ml of glucoamylase) was cooled to 6°C, 0.2 g of the above adsorbent powder was added, and the mixture was mixed and contacted 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 0.50 units/ml, and the glucoamylase was 0.02 units/ml. 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 7 Bovine liver glycogen (estimated molecular weight 3.5×10 ⁵ ,
80 ml 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 ml of 6N caustic soda, which was placed in a 500 ml reaction flask equipped with a stirrer and reflux condenser. Next, 180 ml of epichlorohydrin was added and 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. 80 ml 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 80 ml of ethanol and filtered to recover a gel-like substance. This operation was repeated three more times. Next, suspend in 50ml of distilled water,
It was filtered. After repeating this operation three times, the mixture was again dispersed in 80 ml of ethanol. This was filtered, and the gel-like material was dried and crushed to obtain 9.3 g of white powder.

The solubility of this powder in water at 60℃ is 100g of water.
However, it was 0.001g. In addition, 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.

Culture solution of Aspergillus oryzae IFO-4176 40
ml (containing 0.50 units/ml of α-amylase and 1.30 units/ml of glucoamylase) was cooled to 6°C, 0.2 g of the above adsorbent powder was added, and the mixture was mixed and contacted at 5 rpm 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 0.50 units/ml, and the glucoamylase was 0.02 units/ml. 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 substantially adsorbed, and 98% (1.28 units) of glucoamylase was adsorbed.

Example 8 Oyster shell glycogen (estimated molecular weight 4×10 ⁵ ,
90 ml of water was added to 10 g of dry powder with an estimated degree of branching of 25%), and heated to 70° C. with stirring to dissolve and form a viscous liquid.

To this was added 33 ml of 6N caustic soda, which was placed in a 500 ml reaction flask equipped with a stirrer and reflux condenser. Next, 140 ml of epichlorohydrin was added and heated at 50° C. for 5 hours while stirring at 15 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 ml of ethanol was added to this gel-like material, stirred, and then filtered to recover the gel-like material. The recovered gel was resuspended in 50 ml of ethanol and filtered to recover a gel-like substance.

This operation was repeated three more times. Next, distilled water
It was suspended in 50 ml and filtered. 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, use 50ml of ethanol again.
dispersed into

This was filtered, and the gel-like material was dried and crushed to obtain 9.1 g of white powder.

The solubility of this powder in water at 60℃ is 100g of water.
However, it was less than 0.001g. Furthermore, after immersing this powder in 60°C water for one day, the weight ratio of hydrated gel to powder was 10.8.

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

Culture solution of Aspergillus oryzae IFO-4176 40
ml (containing 0.50 units/ml of α-amylase and 1.30 units/ml of glucoamylase) was cooled to 6°C, 0.2 g of the above adsorbent powder was added, and the mixture was mixed and contacted at 5 rpm 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 0.50 units/ml, and the glucoamylase was 0.02 units/ml. 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, glucoamylase can be selectively adsorbed from a liquid containing a wide variety of organic and inorganic impurities and other types of enzymes, such as a culture filtrate of enzymatic producing bacteria. Moreover, an adsorbent having such a function can be easily produced.

[Brief explanation of the drawing]

FIG. 1 shows a typical structural unit of a substance corresponding to claim 1 of the present invention using epihalogenhydrin as a crosslinking agent. FIG. 2 shows a representative unit of a substance according to claim 1 of the present invention using epihalogenhydrin as a crosslinking agent. 1... Non-reducing end, 2... Branched chain, 3... Crosslinked chain, 4
...main chain, 5...branch point.

Claims (1)

[Claims] 1. Branching with an open end at a non-reducing glucose residue branched from the main chain by α-1,6 bonds, in which glucose is polymerized by α-1,4 bonds and branched from the main chain by α-1,6 bonds. A polyglucan in which the branched chains are shortened by treating a polyglucan formed by linking chains with amylase, or a crosslinked polyglucan characterized in that glycogen is crosslinked until its solubility in water becomes 0.01 or less at 60°C. Method for producing glucan.