JPS5930721B2 - β-1,3-glucan derivative - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to highly reactive β-1,3-glucan derivatives for use in the preparation of water-soluble enzymes or columns for affinity chromatography.

β-1,3-glucan derivatives are, for example, water-insoluble β-
1,3-glucan and cyanogen halide in the presence of water,
It can be produced by adding an alkali to raise the pH so that the glucan does not dissolve, and causing the reaction.

In general, methods for activating polysaccharides with cyanogen halides to lead to derivatives that easily form covalent bonds with substances having primary or secondary amino groups include, for example, Natur
e, 214, 1302 (1967) or Tokuko Sho 45
-38543, etc., and there are too many documents related to methods for preparing water-insoluble enzymes and the like according to this method.

However, there are no examples of using β-1,3-glucan as a carrier in these documents.

The activation reaction with cyanogen halide is usually carried out in an alkaline pH around 11, but since β-1,3-glucan has the property of dissolving under this pH condition, this method can be used directly for β-1,3-glucan. Even if applied to one glucan, it is impossible to obtain activated water-insoluble β-1,3-glucan.

The water-insoluble β-1,3-glucan used to produce the β-1,3-glucan derivative of the present invention can be powdered or shaped into various forms by utilizing its physical properties. Water-insoluble β-1,3-glucan is shaped into small inverted shapes, fibers, films, or cores so that it can be used as a carrier for insolubilizing enzymes or affinity chromatography. A material coated on the surface of a substance and shaped into microcapsules can be used, and this can be reacted with cyanogen halide to lead to an activated derivative while retaining its shape. Water-insoluble β-1,3-glucans include, for example, polysaccharides produced by bacteria of the genus Alcaligenes or Agrobacterium, specifically Alcaligenes fuecalis, Barr.
Polysaccharides produced by Myxogenes strain 10C3K (
AgriculturalBiOlOgicalChe
mistryOl. 3O. pagel96 (1966)
), mutant strain NTK-u of Alcaligenes faecalis var myxogenes strain 10C3K (IFOl3l4
Polysaccharides produced by O) (Special Publication No. 48-32673
) (hereinafter referred to as PS-1), Agrobacterium radiobacterium 1 (IFOl3l27) and its mutant strain U
-19 (IFOl3l26)
Special Publication No. 48-32674) (hereinafter referred to as PS-2),
Pachyman, a component of the herbal medicine Karei, laminaran, a component of brown algae, and water-insoluble beta-β-, a component of yeast cell walls.
Examples include 1,3-glucan.

Further, as a method for shaping these water-insoluble β-1,3-glucans, all methods commonly used in this field can be applied. For example, to shape it into a fiber, water-insoluble β-1,3-glucan is dissolved in an aqueous sodium hydroxide solution, the resulting solution is extruded into a fiber through a nozzle into an aqueous hydrochloric acid solution, and neutralized. To form a film, for example, water-insoluble β-1,3 glucan is dissolved in an aqueous potassium hydroxide solution, and the solution is spread on a glass plate to a uniform thickness. After coating with water, it is immersed in an aqueous hydrochloric acid solution to neutralize it and form a gel (Japanese Patent Publication No. 48-44865). In order to shape it into small spheres, for example, a method containing water-insoluble β-1,3-glucan is used. A method of heating a liquid and discharging, dropping or spraying it into oil to form a gel (Japanese Unexamined Patent Publication No. 48-52953), water-insoluble β-1,
A method in which 3-glucan is dissolved in an aqueous sodium hydroxide solution and the resulting solution is dropped into an aqueous hydrochloric acid solution through a nozzle to neutralize it and form a gel (Japanese Patent Publication No. 48-44865), or a method in which water-insoluble β1,3-glucan is A method in which an alkaline aqueous solution is dispersed in an organic solvent that is not freely miscible with water and an organic acid is added thereto, or a core substance is added to an alkaline aqueous solution of water-insoluble β-1,3-glucan and this is mixed freely with water. For example, a method can be used in which the organic acid is dispersed in an immiscible organic solvent and an organic acid is added thereto (Japanese Patent Application No. 50127196). As the cyanogen halide used as an activator, it is usually possible to use bromine, chloro or iodo compounds, or optionally mixtures thereof.

Examples of the alkali that can be used include sodium hydroxide, potassium hydroxide, etc., and it is usually preferable to use it as a 1-5N aqueous solution. It is preferable to add the alkali until the pH value becomes 9 to 13, especially 11Cl.
Preferably, it is around l. Addition of alkali increases water-insoluble β-1,
The 3-glucan is added slowly so as not to dissolve, and the addition rate is generally about 0.2 to 0.5 pH units/min. A more specific example of activation of shaped water-insoluble β-1,3-glucan with cyanogen halide is as follows. Water-insoluble β-1 shaped into small spheres
, 1 volume of 3-glucan was suspended in 20 volumes of water, 20 volumes of water containing 0.1 to 3 volumes of cyanogen halide was added, and the pH of the reaction solution was adjusted at any temperature between O and 50°C while stirring. , PHll by dropwise addition of 2N sodium hydroxide solution
The activation reaction is completed by raising the globules (at a rate of about 0.5 pH units/min) without disintegration and keeping them in PHll for 15 minutes.

After the reaction is completed, P is removed from the solid and washed with 100 volumes of water to obtain small spherical activated β-1,3-glucan. The obtained activated β-1,3-glucan is insoluble in water and alkaline solutions, non-coagulable when heated, and hydrophilic, and has a size that allows a sufficient flow rate to be obtained when packed in a column and used. Because they are strong particles, it is possible to prepare carrier-ligand conjugates for water-insoluble enzymes or affinity chromatography with excellent properties, for example, by the following method. For the preparation of
can be used to advantage. The β-1,3-glucan derivative of the present invention is produced by reacting water-insoluble β-1,3-glucan with cyanogen halide as described above. , a polymeric compound with up to 490 glucose units (according to light scattering method), each unit being an almost linear 1,3-β
- Formed glucan bonds (Special Publication No. 48-3267
3 and 48-32674), and its structure is shown by the following formula. In addition, conventionally, for example, dextran cross-linked with shrimp chlorohydrin by bromsyanide,
The activation reaction of polysaccharides such as agarose is said to occur by the following mechanism [European Journal of Biochemistry 1].
OwnOfBiOchemistry))〇The β
The β-1,3-glucan derivative of the present invention obtained by reacting -1,3-glucan with cyanogen halide (as described in Reference Example 1 below, the presence of carboxylic acid amide and imidocarboxylic acid) This has been confirmed and can be expressed by the following equation.

[In the formula, in any one of R, Rl and R2, one C
The groups ONH2 and C=NH are introduced. ]
That is, the present invention provides a high molecular weight β1,3-glucan derivative having up to 490 glucose units (according to a light scattering method) consisting essentially of repeating units represented by the following formula.

[In the formula, CONH2 and C-NH groups are introduced in any of R, Rl and R2, and the number of nitrogen atoms per glucose residue (average value) (based on elemental analysis) is 0.1 ~1.1].

Preparation of carrier-ligand conjugates for water-insoluble enzymes or affinity chromatography (J) The reaction is carried out by reacting proteins, peptides, amino acids, enzyme substrates or inhibitors, antigens, antibodies, hormones, etc., preferably in a slightly alkaline aqueous solution at any temperature of about 0 to 50°C.

Below, with actual examples and reference examples, β-1,
The method for producing the 3-glucan derivative will be explained in more detail, but these are not intended to limit the present invention in any way.

Example 1 Take white powder 109 of PS-1 in a 500 ml beaker, add 200 TfL of distilled water and stir with a magnetic stirrer to sufficiently swell PS-1.
Add 200 ml of W(V) bromcyan aqueous solution and heat at 25°C.
While continuing to stir, 2N sodium hydroxide solution was added dropwise using an automatic titrator to gradually increase the pH at a rate of about 0.5 pH units/min until the pH reached 11.
The reaction was completed by maintaining the pH for about 15 minutes.

After the reaction was completed, PS-1 particles were removed from the reaction solution and further washed with distilled water 11 to obtain activated PS-1.

(Dry weight is 10.059). Practical example 2 Using the same method as in Example 1, 19 pieces of white powder of PS-1 were added to 2 ml of a 5% (W/) bromcyan aqueous solution.
, 40w11 and 80m1, respectively, to obtain activated PS-1.

Practical Example 3 10 g of white powder of PS-2 was activated with bromcyan using the same method as in Example 1, and activated PS-2 was obtained.
-2 was obtained (dry weight was 10.089).

Actual example 4 Small spherical (particle size 50-200μ) PS-1125m1 (
(equivalent to approximately 59 PS-1 as dry weight) to 100 ml of water.
and 100ml of 5% (W/) bromcyan aqueous solution,
Using an automatic titrator under stirring at 25°C, 2N sodium hydroxide solution was added dropwise at a rate of about 0.5 PH units/min to gradually increase the pH to 11, and The reaction was completed by maintaining the pH for about 15 minutes.

After the reaction is complete, remove the solid matter from the reaction solution, and add 5 ml of distilled water.
Activated small spherical PS-1 by washing with 00wL1
(Dry weight: 5.19). Practical Example 5 Using the same method as in Practical Example 4, add each small spherical (particle size 50 to 200 μ) PS-119 (as dry weight) to 2 d, 40 m of a 5010 (W/V) bromcyan aqueous solution.
1 and 80 d, respectively, to obtain activated small spherical PS-1.

Actual fiber example 6 5 f! of PS-2 in fibrous form (thickness about 0.2 to 0.3 m7!L, length about 10 (1-J mo V!)) was taken and treated in the same manner as in Example 4. , activated fibrous PS-2
(Dry weight: 5.159).

Example 7 59 pieces of PS-1 in the form of a film (about 0.2 mm thick) were taken and treated in the same manner as in Example 4 to obtain activated PS-1 in the form of a film (dry weight: 4, 95 f). ).

Reference Example 1 Powdered PS-1 and small spherical PS-1 activated with cyanogen halide obtained in Regretful Examples 1, 2, and 4 to 7
, fibrous PS-2 and film PS-1 exhibited the following properties.

(1) Shape All shapes were almost unchanged from those before activation treatment.

(2) Thermocoagulability Unlike those before activation, none of them had any thermocoagulability, and no jelly formation was observed even when heated in boiling water for 15 minutes.

(3) Unlike the one before activation, both are strong alkaline aqueous solution,
Dimethyl sulfoxide or concentrated urea solution (8M)
It became completely insoluble.

(4) Quantification of N by elemental analysis When N was quantified by elemental analysis, the number of N atoms (average value) introduced per glucose residue by the activation treatment was as shown in Table 1.

(5) Infrared absorption spectrum (KBr method) The infrared absorption spectrum of untreated powdered PS-1 is as shown in Figure 1, and the infrared absorption spectrum of activated powdered PS-1 is as shown in Figure 2. It is.

Comparing the spectra of the two, activated PS-1VC has
1730cm-1,1625? -1,780? A new absorption band was observed at -1. These are 1730CTIL-
1 is carboxylic acid amide, 1625? -1 and 780cT
It is considered that n-1 is an absorption band derived from imidocarboxylic acid. Small spherical PS-1, fibrous PS-2, and film PS-1 all have the ability to absorb carboxylic acid amide (
1730 (1-J mo V1-1) and imidocarboxylic acid absorption (1625CrfL-1, 7800f1L-1). (6) Stainability Nakanishi et al.'s method [CarbOhydra
teResearch, To, 47-52 (1974)
], the stainability with water-soluble dyes was investigated and the results are shown in Table 2.

Reference Example 2 Make a suspension of activated PS-1 19 (dry weight) obtained in Practical Example 1 in 20 ml of distilled water, add 10 ml of 0.2 M carbonate buffer (PH 8.5) to 11 pronase (Kaken Chemical Co., Ltd.). Co., Ltd.) 20~/T! 2ml of Ll solution and ? Add 8 ml of distilled water and stir at 5°C and pH 8.5 for 2 hours.
The reaction was allowed to proceed for 0 hours.

After the reaction, the PS-1 gel was filtered with a glass filter and 0
．． Washed sequentially with 80wL of 2M glycine solution, 80ml of 0.5M sodium chloride solution, and 40ml of distilled water,
A water-insoluble enzyme of pronase was obtained. Synthetic substrate P-
Using toluenesulfonyl-L-arginine methyl ester at 25°C, pH 8. When pronase activity was measured using O, it was found that 6% of the pronase activity used in the coupling reaction
It was found that 3010 is a water-insoluble enzyme.
In addition, the total protein amount in the above washing solution was measured by the Rolling method (0.H.
L0wryeta1: J. BiOl. Chem. The water insolubilization rate of the protein was calculated to be 77% by quantifying the amount of pronase (Udanryo, 265 (1951)) and subtracting it from the protein amount of the pronase used initially.

Reference Example 3 1f1 of activated PS-1 obtained in Actual TM Example 1 was made into a suspension of 20ml with distilled water, and 10ml of 0.2M Tris-HCl buffer (PH8.O) and crystal α-
Add 10TrL1 of a 2.5W19/ml solution of amylase (manufactured by Sankyo Co., Ltd.) at 5°C, pH 8. After reacting with O for 4 hours, it was washed in the same manner as in Reference Example 2 to obtain water-insoluble α-amylase.

When α-amylase activity was measured using soluble starch as a substrate and reacted at pH 5.3 and 37°C, the rate of water insolubilization of the activity was 59%. The water insolubilization rate of protein was 650k).

Reference Example 4 Activated PS-1 19 obtained in Practical Example 1 was suspended in 20 ml of distilled water, and 10 Tn of 0.2 M phosphate buffer (PH8.O) and crystalline α-chymotrypsin (Sigma 2.5 instant/ml solution 10ml
was added, and a coupling reaction was carried out at 5° C. and pH 8.

The reaction was completed in 2 hours, and 78% of the initially added enzyme protein
% bound to PS-1. Using L-tyrosine ethyl ester as a substrate, 100k) at 27°C in the presence of ethanol.
When the esterase activity was measured at pH 7.8, the water insolubilization rate of the activity was 64%.

Reference Example 5 19 of activated PS-1 obtained in Practical Example 1 was added to 20T of distilled water!
1e, then suspended in 0.2M Tris-HCl buffer (PH8
．． O) 10 ml and 10 ml of a 2,5η/ml solution of wheat germ acid phosphatase (Seikagaku Corporation)
was added at 5°C, pH 8. After reacting for 18 hours while stirring with O, the mixture was washed in the same manner as in Reference Example 2 to obtain water-insoluble acid phosphatase.

using 0-carboxyphenyl phosphate as a substrate,
25℃, PH5. When the activity was measured in O, 52% of the initially added enzyme activity was insolubilized in water.

The water insolubilization rate of protein was 63,010. Reference example
6. Dilute activated PS-1 19 obtained in Example 1 with distilled water for 20 m
1 and add Acetobacter turbidans (AT) to this suspension.
CC9325) α-amino acid ester hydrolase extracted from bacterial cells and partially purified [For the preparation method and properties of this enzyme, see T. Takahashietal;BlOc
hem. J. U.K. K, 497 (1974)] 5T1Z
9 and 0.1M Tris-HCl buffer (PH8.O) 20
Add m1 and heat at 5°C, pH 8. The reaction was carried out under stirring at O for 4 hours.

After the reaction was completed, the PS-1 gel was removed and washed in the same manner as in Reference Example 2. The resulting water-insoluble enzyme was suspended in 40 ml of distilled water. The activity of this water-insoluble enzyme suspension was measured using D-phenylglycine methyl ester as a substrate.
It was 9.83 units/ml.

This indicates that 84% of the initially added enzyme activity was insolubilized. Furthermore, the protein insolubilization rate was 69%.

Using 25 ml of the above water-insoluble enzyme suspension, a bed volume of 5
A small column of m1 was prepared at 5°C with pH 7 containing D-phenylglycine methyl ester hydrochloride 15Tr19/Mll7-amino-3-deacetoxycephalosporanic acid 5η/d and methanol 10% [/V). When a substrate solution of .2 is passed through the column at a constant flow rate of 12 ml/Hr, the reaction solution flowing out from the column contains 7.50 mg/Hr.
ml of cephalexin.

Although this reaction was carried out continuously for 6 months, the cephalexin synthesis activity of this column did not decrease at all. Reference Example 7 Activated PS-1-19 obtained in the same manner as in Practical Example 1
was made into a 20 ml suspension with distilled water, and 20 ml of 0.1 M Tris-HCl buffer (PH8.O) containing 20 ml of L-leucylglycylglycine (manufactured by Mann Research Laboratories) was added thereto. After reacting at 5°C and pH 8 for 16 hours with gentle stirring, the PS-1 gel was
It was taken and washed in the same manner as in Reference Example 2.

When calculated backward from the content of L-leucylglycylglycine recovered in the washing solution, the amount of L-leucylglycylglycine bound to PS-1 was 12.6Tn9. In addition, in the above method, when reactions were carried out in the same manner as in the above method using human serum gamma globulin (Fraction, manufactured by Sigma) and insulin (manufactured by Sigma) in place of L-leucylglycylglycine, PS -1K
The amount of bound gamma globulin is 15,1η,
The amount of insulin was 11.0~.

Reference Example 8 Small spherical activated PS-1 obtained in Examples 4, 6 and 7,
Fiber-like activated PS-2 and film-like activated PS
Take 19 (as dry weight) of each of -1 and add 207 rL of distilled water, 10 ml of 1% (W/V) P-aminophenethyl alcohol aqueous solution and 0.04 M
While stirring 10 ml of phosphoric acid buffer (PH6) again, the reaction was carried out at 5° C. and PH6 for 24 hours.

The reaction solution was filtered through P, and the solid matter was further removed by 200ml (1f) 0.5M
After washing with NaCl, the bound amount was determined by back calculation from the P-aminophenethyl alcohol recovered in the P solution. 47η for small spherical PS-1i1C and 33η for fibrous PS-2.
24 η of P-aminophenethyl alcohol (both per 1 g of carrier) was bound to the film PS-1. This value is based on the active group introduced into the carrier by the activation reaction (
This is thought to correspond to the amount of imidocarboxylic acid groups). Reference Example 9 1 g (dry weight) of the small spherical activated PS-1 obtained in Actual Example 4 was suspended in 40 ml of distilled water, and 0.2
5 ml of a 20-l solution of 10WLI1 pronase (manufactured by Kaken Kagaku Co., Ltd.) in M carbonate buffer (PH 8.5) and 5 ml of distilled water were added, and the mixture was reacted at 5° C. and pH 8.5 for 6 hours with stirring.

After the reaction, the small spherical PS-1 gel was filtered through a glass filter.
The solution was taken and washed sequentially with 120 ml of 0.2M glycine solution, 120 ml of 0.5M sodium chloride solution and 80 ml of distilled water to obtain a water-insoluble pronase enzyme. Using the synthetic substrate P-toluenesulfonyl-L-arginine methyl ester at 25°C and pH 8. When the pronase activity was measured using O, it was found that 60% of the pronase activity used in the coupling reaction was a water-insoluble enzyme. In addition, the total protein amount in the above washing solution was determined by the rolling method (0
．． H. L0wryeta1: J. BiOl. Chem.
193, 265 (1951)) and subtracted from the protein amount of pronase used initially, the water insolubilization rate of the protein was calculated to be 72%.

Reference Example 10 19 of the fibrous activated PS-2 obtained in Example 6 was made into a suspension of 20 grams in distilled water, and 10 ml of 0.2M phosphoric acid buffer (PH8.O) and crystal α- Add 2.5 to 10 grams of chymotrypsin (manufactured by Sigma) / 10 grams of solution,
The coupling reaction was carried out at 0.degree. C. and pH 8.

The reaction was completed in 4 hours, and 65% of the enzyme protein added at the beginning was
% bound to the carrier. L-tyrosine ethyl ester was used as a substrate at 27°C, pH 7.0 in the presence of 10% ethanol.
When the esterase activity was measured in Example 8, the water insolubilization rate of the activity was 54%.

Reference Example 11 Add 20 ml of distilled water and urease from rapeseed (Wako Pure Chemical KK) to the film-like activated PS-119 obtained in Actual Foam Example 7.
) and 0.04M phosphoric acid buffer (P
By adding H7.5) 5g and reacting at 5°C for 18 hours with stirring, 35% of the initially added enzyme activity was insolubilized.

The protein insolubilization rate was 44%.

[Brief explanation of drawings]

FIG. 1 shows the infrared absorption spectrum of powdered PS-1, and FIG. 2 shows the infrared absorption spectrum of activated powdered PS-1.

Claims (1)

[Scope of Claims] 1. A high molecular weight β-1,3-glucan derivative with 490 lucose units (according to light scattering method) consisting essentially of repeating units represented by the following formula. ▲There are mathematical formulas, chemical formulas, tables, etc.▼ [In either formula R, R_1 or R_2, -CON
H_2 and ▲There are mathematical formulas, chemical formulas, tables, etc. ▼ groups have been introduced, and the number (average value) of nitrogen atoms per glucose residue (based on elemental analysis) is 0.1 to 1.1] .