JPS6356501A - Cellulose gel having biochemical affinity and production thereof - Google Patents

Abstract

PURPOSE:To obtain the titled gel having epsilon-polylysine bonded through a functional group to a spherical cellulose particle, by reacting the spherical cellulose particle containing an introduced functional group to be reacted with epsilon-polylysine with the epsilon-polylysine. CONSTITUTION:A spherial cellulose particles to which a functional group (e.g. formly group, epoxy group, carboxyl group, etc.) to be reacted with epsilon-polylysine is reacted with the epsilon-polylysine and optionally subjected to aftertreatment to give the aimed gel. An epsilon-polylysine having 20-30 polymerization degree obtained by cultivating obtained by cultivating Streptomyces Albulus in a culture containing glycerol, ammonium sulfate, yeast essence, etc., followed by separation and purification is preferable as the epsilon-polylysine. Hard gel having high mechanical strength, useful for affinity chromatography showing high speed of flow and good purification efficiency free from release of ligand is obtained.

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] More specifically, the present invention relates to an insoluble carrier in which 8-polylysine is bonded to a spherical cellulose gel.

[Conventional technology]

In recent years, with the progress of biotechnology, the importance of separation and purification techniques for minute amounts of physiologically active substances produced by cell culture, genetic manipulation, etc. has increased. Combinations of gel filtration, ion exchange, centrifugation, etc. have been used as separation and purification methods. These operations were problematic in that they took a long time and resulted in loss of the target substance. In contrast, affinity chromatography, which separates substances using biological affinity, has recently become widely used.

[Problem that the invention seeks to solve]

Most conventional Affinitei chromatography agents have been manufactured using the polysaccharide agarose as a base and binding the ligand after activation with brom cyanide. Antibodies, antigens, enzymes, amino acids, peptides, hormones, nucleic acids, etc. are used as ligands. However, because it is based on agarose, conventional Affinity gel is soft.
Due to its weak mechanical strength, it was difficult to achieve a high flow rate when packed in a column for chromatography, making it difficult to use industrially. On the other hand, among ligands, lysine or polylysine, which is a basic amino acid used for purification of plasminogen, has recently attracted attention, and an affinity gel in which these are bound to agarose has been developed. However, since these gels are bonded using the bromcyan method, they have drawbacks such as detachment of the ligand, low flow rate, and poor purification efficiency.

The object of the present invention is to have hardness, strong mechanical strength, high flow rate when performing chromatography, and to provide functional groups to spherical cellulose gel by using the Ide-polylysine of the present invention as a ligand. It concerns a new gel that is bonded via .

Polylysine, which is made by polymerizing the amino acid lysine, has conventionally been made from α-lysine, in which the amino group at the α-position of lysine is condensed with a carboxyl group.
-Polylysine was a synthetic product, but the BoIJ I7 gin used in the present invention is a microorganism Streptomyces albulus (5trepto-myces albulus).
It is possible to use e-polylysine in which the e-amino group produced by US) is condensed. Its structural formula is shown below.

Its manufacturing method is described in Japanese Patent Publication No. 59-20359. That is, after culturing Streptomyces albulus in a culture solution containing glycerol, ammonium sulfate, yeast extract, etc., polylysine is obtained by separating and purifying the culture. The degree of polymerization of this polylysine is 20-30. Also, according to another document by the same inventors (Fermentation and Kogyo Vol. 43, p. 902, Agr.
.

(Biol, Chem, Vol. 45, p. 2503) This polylysine differs from conventional polylysine in that the amino group at the e-position of lysine is condensed with the carboxyl group at the α-position, so-called e-lysine.
It has also been revealed that it is polylysine.

The cellulose to which e-Bo IJ IJ-Jin is bound is a true spherical particle, and the following examples are examples of its production method.

(1) By the method described in JP-A No. 53-86749,
A method of dissolving cellulose acetate in an organic solvent, suspending this solution in an aqueous medium, spheroidizing it, evaporating the organic solvent to obtain cellulose ester particles, and saponifying the particles to obtain cellulose particles.

(21 (The method of JP-A-56-24429, in which the porosity is adjusted by adding an aliphatic higher alcohol etc. to a solution of cellulose acetate as an application of method 11).

(3) Unexamined Japanese Patent Publication No. 5, 1987, in which cellulose is dissolved in a mixed solvent of paraformaldehyde and dimethyl sulfoxide and granulated.
7-159801, the method of Japanese Patent Publication No. 57-159802.

(4) The method of JP-A-52-11237, in which cellulose is dissolved in concentrated ammonia water containing cupric hydroxide and cuprous chloride and granulated.

(5) The method disclosed in JP-A-51-5361, in which viscose is dispersed in transformer oil and granulated.

(6) The method disclosed in JP-A-55-44312, in which cellulose is dissolved in a calcium thiocyanate salt solution and granulated.

(7) A method disclosed in JP-A-48-60754, in which purified linter is dissolved in a copper ammonia solution and granulated.

Next, in order to bond these spherical cellulose particles with e-polylysine, a reactive functional group is introduced into the cellulose.
Thereafter, it is reacted with e-polylysine. The following methods are available for this purpose.

(1) A method in which a formyl group is introduced into cellulose, and then this is reacted with 8-polylysine to form a thick base and reduced. In this case, K may be used to introduce formyl groups into cellulose, for example, by the following method.

■ Cellulose + (JCH, CH-CH, →ゝ.' (Cellulose) -0-CH, -CH-CH, -NH.

H (→Tulerose)-0-CH, -CH-CH, -NH-
CH, -(C)I, ), -CH0H H (2) A method in which cellulose is reacted with bisoxirane to introduce an epoxy group, and this epoxy group is reacted with ε-polylysine.

(3) A method in which cellulose is reacted with ω-aminoalkylamine to introduce an amino group, and this terminal amino group is condensed with the carboxyl group of e-polylysine.

(4) A method in which cellulose is epoxidized with epichlorohydrin, then aminated and reacted with succinic anhydride to introduce a carboxyl group, and this terminal carboxyl group is condensed with the amino group of e-polylysine.

(5)' The carboxyl group obtained by the method (4) is activated by reacting with N-hydroxysuccinimide, (6) a bonding method using a diazonium derivative, and (7) a bonding method using a hydrazide derivative.

Recently, spherical particles of cellulose such as aminated-7rulofine (Cellulofine is a trademark. The same applies hereinafter) and formyl-cellulofine have been used as affinity gels, and it is convenient to use these.

For example, these bonding methods can be expressed as a reaction formula as follows.

(l) (Cellulose) -CHO+NH2- (Polylysine) -(Cellulose) -CN=N- (Polylysine) 11 Toki (Cellulose) CHt NH (Polylysine) (
2) (cellulose) -0-CH, -CH-CH, -0-
(CH,)4-0-CH, -PCH -CH-CHz + NHt- (polylysine) → (
→Tulerose)-0-CHt-CH-CHt-0-(C
) It) 4-0-CHt-Hatsu-CH-CH, -NH-(polylysine)CH (3) (→Turellose')-NH-(CH,)-NH,
+ HOOC- (polylysine) → (→turerose)
-ki■(-(CH,)-NHCO-(polylysine) (4
) (7vt嘱) -〇 -CH, -CH-CH, -NH
-CO-CH, -CH, -CH -COOH+ NH2- (polylysine) → (7rw-
x) -0-CH, -CH-CH, -NH-Co-CH,
-CH, -CH -CONH- (polylysine) CH (cellulose) -0-CI(, -CH-CH, -NH-
CO-CH, -CH, -CH -co-NH- (Polylysine) [Effects of the Invention] The e-polylysine-bonded cellulose of the present invention can be used as an unprecedented specific separation and purification agent.

A similar material is agarose gel (manufactured by Sigma) that has α-polylysine bound to it, but the polylysine is α-polylysine and the amino group at the α-position is condensed, so the free amino group is at the e-position. . Since the amino group at the ε-position of e-polylysine is condensed, the amino group at the α-position is free, and there are many free amino groups in e-polylysine-cellulose. It has different and unique properties and is very useful as a new separation and purification agent. Not only is polylysine different from conventional α-polylysine, but a major feature is that the carrier gel is cellulose. Agarose is a polysaccharide, but its structure has the disadvantage of being soft and having low mechanical strength. Therefore, when such a gel is scaled up and used as a chromatographic agent on an industrial scale, there are drawbacks such as the inability to maintain a high flow rate. The ε-polylysine-cellulose of the present invention has high mechanical strength), can obtain high flow rates, can be used on an industrial scale, and can be expected to be used as a new separation system.

In the ε-polylysine-cellulose of the present invention, free α-
Since there are many amino groups in the positions, when used as an affinity chromatography agent, it is possible to isolate ffg of enzymes, plasminogen, separation and purification of enzymes, separation of nucleic acids, separation of polysaccharides, etc. Unlike wood, it is efficient and -
Possible in large quantities at one time.

〔Example〕

As an example, a method for binding e-polylysine to cellulose spherical particles and an example of the use of the obtained carrier will be shown below, but the present invention is not limited to such examples.

Example 1 Formyl-cellulofine (manufactured by Chisso ■), a formylated cellulose commercially available as an affinity carrier, Sakun Yola dry product (suction filtration on a Puchner funnel) 50F (approximately 7011 Lt) and 0.59 e
-0.2M Nat HP Oa containing polylysine-
100 g of NaOH buffer (1) Hll, 0) was added and stirred at 30°C for 1 hour. Thereafter, 400 μ of sodium cyanoborohydride (5CBH) was added and stirred overnight. Furthermore, L-lysine 14,6N1SCBH4001n9
was added and stirred for 2 hours.

After filtration, washing with distilled water was repeated to obtain e-polylysine-cellulose gel. The immobilized e-polylysine was analyzed using a colorimetric method (J, Po
lym, Sci, Polym, Chem, Ed
, vol. 22, p. 1281, 1984) and was quantified at 95 cm per gel.

Example 2 Suction-dried cellulose gel 100I granulated by the method of Example 1 of JP-A No. 55-44312 was heated to lN-
Dissolve in NaOH solution for 80 minutes and then add Na B H
45JF and 12Wll of 1,4-bis-(2,3-epoxybiloxy)-butane were added and reacted at 25°C for 5 hours. After the reaction was completed, it was filtered and thoroughly washed with water. Suction-dried epoxy-activated cellulose gel thus obtained 100. F to 0.2 M Na, CO
1.2 y of ε-polylysine suspended in 130 d of 3 solution
was added and reacted at 4°C for 15 hours. 1.0 after completion of reaction
Washed with MNa Cl. Add 100 d of 5M hydroxyamine hydrochloride under neutral conditions to remove excess epoxy groups.
The mixture was added, stirred, and then filtered. The immobilized B-polylysine had a ratio of 1 xrl to 4.

Example 3 Suction-dried cellulose gel granulated by the method of Example 1 of JP-A No. 56-24429.
130r of 4M KIO4 solution! Lt was added and stirred at room temperature for 1 hour. After thoroughly washing with water, 150 g of 1.0M hexamethylenecyamine was added and stirred for 6 hours. After the reaction was completed, it was washed with water. The thus obtained aminated cellulose gel 100I contained 73.0.9"i of e-borylide, 150 ml of 0.1 M sodium carbonate, and 1-ethyl-3.
-(3-dimethylaminopropyl)carbodiimide 1.
09 was added and stirred at room temperature for 3 hours. After the reaction is completed, filter
After washing with water, 150ml of 0.1M sodium carbonate and 2.0.9ml of L-lysine were added, and after reacting for 1 hour, it was washed with water. The amount of immobilized e-polylysine was 910 μl per gel 11R.

Example 4 Aminated cellulose commercially available as an affinity carrier - Aminated Seven Rulofine (manufactured by Chisso ■)
Suction dry product 100I of 0.1 M NaC1
After washing with water, it was suspended in 0.1 M NaCl for 150 d. To this was added 12N succinic anhydride little by little. Meanwhile, 20% NaOH was added and the mixture was stirred at 30° C. for 6 hours while keeping the pH at 6.0. Filter the gel to 0.1 M
-Suspended in NaOH and stirred at room temperature for 30 minutes. Thereafter, this gel was washed with water to obtain a succinylaminocellulose gel. This gel 1001 contains 0.2 M Na2HP04-NaOH buff 7a (pH 11,0) containing 1.51 ε-polylysine and 1-ethyl-3.
-(3-dimethylaminopropyl)carbodiimide hydrochloride (1.51 g) was added, and the mixture was stirred at room temperature for 8 hours.

Furthermore, 5.0 g of L-lysine was added and stirred for 2 hours. After the reaction was completed, it was thoroughly washed with water. The immobilized ε-polylysine was 4.01 n9/gel.

Example 5 The succinylaminocellulose gel obtained in Example 4 was thoroughly washed in dioxane, dehydrated, and then suspended in 300 ytl of dioxane. N-hydroxysuccinimide and dicyclohexylcarbodiimide were added to a final concentration of 0.1M each. Stir for 60 minutes. 70
0 ml dioxane, 500 m methanol, 400 m
Washed with rttl dioxane. In this way, an N-hydroxysuccinimide esterified cellulose gel was obtained. 1% N in this gel 10.9 (suction dry product)
0.01 M NaHCOs (pH 7,
5) was added with 5Qm and ε-polylysine o, sy
was added and stirred at room temperature for 20 hours. KO, IM −) lithium hydrochloric acid (pH 9,0) to block remaining active groups.
The mixture was stirred at room temperature for 1 hour. Then 0,5 M-NaC
It was washed with 0.05 M borate buffer (pH 4,0) containing 1. In this way, a cellulose gel in which ε-polylysine was bonded via a functional group was obtained. e-bo IJ
The amount of IJ resin bound was 1 gel per rn9.

Example 6 (Purification of Plasminogen) ε-Polylysine-cellulose starting buffer prepared in Example 1. )
Equilibrated with. 200 d of human serum was applied to this column at a flow rate of 22
Add at ml/br, absorbance of eluate (2801m)
The cells were washed with starting buffer until the value was 0.05 or less.

The column was further washed with 59 ml of starting buffer containing 0.5 M NaCl to remove weak non-specifically adsorbed substances.

Then, 70 d of 0.2 M 8-aminocarbtetrahydric acid solution was poured to elute the plasminogen. 121 with the above operations
n9 plasminogen was recovered.

This was confirmed to be a pure product by SDS polyacrylamide gradient electrophoresis. The recovery rate per unit volume of gel is 1
．． It was 21R9/m.

Comparative Example 1 The same procedure as in Example 6 was carried out except that α-polylysine-agarose was used in place of the e-polylysine-cellulose gel, and 6R9 plasminogen was recovered. The recovery rate per unit volume of gel was 0.6 In9/G.

From a comparison of Example 6 and Comparative Example 1, it is clear that the purification efficiency of the gel of the present invention is much better than that of the conventional gel.

Example 7 (Separation and purification of T4 phage) 1 ml of e-po IJ IJ resin cellulose gel prepared in Example 2
was packed into a glass column (diameter 0.8 an
That's what it means. ) and equilibrated with 10 ml. Initiation buffer 1d containing 1.5 x 10'' T4 phage/1 tl was added to this, and the mixture was run at a flow rate of 2 rul/hr. It was further washed with initiation buffer IQ+a/.

At this time, the liquid eluted from the column was collected and the whole was designated as 12-. This was used as a flow-through liquid. Next KO, 5M -NaC
1 containing 0.05M glycine hydrochloride buff 7a (pH 3
, 0) 5 m/ at a flow rate of 4 d/hr. This was used as the eluate. As a control, the same experiment was carried out using cellulose gel granulated by the method of Example 1 of JP-A No. 55-44312 to which e-vo IJ 17 Gin was not bound, and the permeate solution and eluate were collected respectively. did. The number of phages in the above samples was calculated using Escherichia Co11.
) was assayed using an agar double layer. The results are shown in Table 1 below, and the e-polylysine-cellulose gel was T4
It has been shown that it adsorbs phages, and is useful for phage separation and purification.

Table 1 Example 8 (Purification of hyaluronic acid) L/7') Strep
to-coccus Zooepidemicus)
FERM BP-878 bacteria with peptone 1.5%,
Yeast extract 0.5%, bovine blood groove 0.5, monopotassium phosphate 0.3%, dipotassium phosphate 0.2%, glucose 2%
The cells were cultured in an aqueous solution (pH 7.0) containing 0.002% sodium thiosulfate, 0.01% sodium thiosulfate, 0.002% magnesium sulfate, and 0.01% magnesium sulfate. (For fermentation methods, see 1961 Society of Agricultural Chemistry Abstracts, page 510.)
The culture solution was fermented at 32° C. for 30 hours.

After completion of the heat treatment, the bacterial cells were isolated. Approximately 509 mA of ethanol in the liquid! was added to precipitate crystals.

The precipitated crystals were dissolved in 0.05 M) lithium buffer (pH 7.5), and the e-polylysine-cellulose gel 2 and 21 (diameter 8
cIrL×40c! IL) in addition to the column packed with 2,0
Elution was performed using a gradient method in which M NaCl was added.

Flow rate (600 ml/hr) The eluate was collected in 25 ml portions, and hyaluronic acid was quantified using Bitter (Anal).

The method for measuring uronic acid described in Biochem 4330 (1962) was used. The results are shown in FIG.

Fraction with high analysis value of uronic acid /1620~/f
Collect up to 640 ml of hyaluronic acid, desalt it by dialysis, concentrate and freeze-dry it to make a hyaluronic acid product. I got Ol. The hyaluronic acid obtained here had the following properties and was of high quality.

Molecular weight: 960,000 (viscosity method) Hyaluronic acid 289% Water: 10-factor Protein: (0,1-factor Nucleic acid: (0,5%) Gelcosaminoglucan sulfate: (0,01%) Example 9
(Measurement of flow rate of gel) The flow rate of the gel of the present invention produced in Example 4 and commercially available α-polylysine-agarose (manufactured by Sigma) was measured under the following conditions.

Column: 1.6x20cm elution
Solution: 55-phosphate buffer (pH 7.0) The results are shown in FIG. From this figure, it is clear that the gel of the present invention has a higher flow rate than conventional agarose-based gels without any problems, and is very advantageous when used industrially in large quantities.

[Brief explanation of the drawing]

Figure 1 is a diagram showing the elution curve of hyaluronic acid obtained by affinity chromatography performed in Example 8, and Figure 2 is a diagram showing the elution curve of hyaluronic acid obtained by affinity chromatography. Nine columns packed with IJ lysine agarose (C) K 50mM phosphate buffer (pH 7,0
) is a diagram showing the flow velocity when flowing. that's all

Claims (9)

[Claims] (1) A gel with biochemical affinity in which ε-polylysine is bound to spherical cellulose particles via a functional group. (2) The gel according to item (1), wherein the ε-polylysine is obtained from fermentation of Streptomyces albulus and has a degree of polymerization of 20 to 30. (3) Spherical cellulose particles into which a functional group that reacts with ε-polylysine has been introduced are reacted with ε-polylysine, and post-treatment is performed as necessary. Method for producing gels with bound biochemical affinities. (4) Item (3), wherein the spherical cellulose particles into which a functional group that reacts with ε-polylysine is introduced are spherical cellulose particles into which a formyl group is introduced, and the post-treatment is reduction. Method for producing gel. (5) The method for producing a gel according to item (3), wherein the spherical cellulose particles into which a functional group that reacts with ε-polylysine is introduced are spherical cellulose particles into which an epoxy group is introduced. (6) The spherical cellulose particles introduced with a functional group that reacts with ε-polylysine are a reaction product of spherical cellulose particles and a terminal amine of ω-alkylamine. Method of manufacturing gel. (7) Item (3) characterized in that the spherical cellulose particles into which a functional group that reacts with ε-polylysine is introduced are spherical cellulose particles into which a carboxyl group is introduced.
Method for producing the gel described in Section 1. (8) The spherical cellulose particles into which a functional group that reacts with ε-polylysine is introduced are obtained by introducing carboxyl groups into spherical cellulose particles and esterifying the carboxyl groups with N-hydroxysuccinimide. A method for producing a gel according to item (3). (9) Any one of items (3) to (8), wherein the ε-polylysine is obtained from fermentation of Streptomyces Albulus and has a degree of polymerization of 20 to 30. The method for producing the gel described in .