JPH0820598A - New glucoprotein and its production - Google Patents

Abstract

PURPOSE:To obtain a glucoprotein having strong antitumor action, not directly only acting on tumor, but having also antitumor action due to increase in protecting function of a living body, being a substance excellent in heat resistance, acid resistance and alkali resistance and having high stability, capable of readily carrying out purification and preparation and expecting also extension of storage period. CONSTITUTION:This glycoprotein is a glycoprotein produced outside alga body by unicellular green algae of the genus Chlorella and contains polysaccharides containing >=70% galactose as constituent saccharide and a protein and has 60-80% polysaccharide content and 20-40% protein content and <=pH 4 isoelectric point and 265000 average molecular weight. The glycoprotein is obtained by highly purifying the supernatant of cultured products of Chlorella by various kinds of chromatography and has high purity.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a glycoprotein produced by a single cell green alga of the genus Chlorella outside the algal body, and particularly to a glycoprotein having an antitumor effect.

[0002]

2. Description of the Related Art Mushrooms and bacteria are known as glycoproteins or polysaccharides having antitumor activity. All of these have been found to exhibit antitumor effects by enhancing the defense function of the body against tumors, but their social utility value is not high at present. Therefore, there is a demand for an antitumor agent that exhibits an antitumor effect by highly enhancing biological defense.

On the other hand, regarding glycoproteins prepared from green algae such as unicellular green algae of the genus Chlorella (hereinafter sometimes referred to as chlorella), those having a molecular weight of 120,000 (JP-A-61-69728) are known. It has been reported. However,
It is difficult to prepare a large amount of this substance because it has a direct effect on cells and it takes time to extract and purify.

As an antitumor substance produced by Chlorella outside the algal body, a glycoprotein having a molecular weight of 200,000 (Japanese Patent Application No. 3-9
0002), but this substance has problems in purity, stability in acid / alkali solution, and heat resistance.

[0005]

As described above, as described above,
It is known that Chlorella produces polysaccharides and glycoproteins outside the algal cells, but these substances have little or no antitumor effect, or even if they have, they have heat resistance, acid resistance, and alkali resistance. However, it is a cause of complicatedness of sterilization and high cost for purification.

The present invention is a novel glycoprotein produced outside the algal cells by unicellular green algae of the genus Chlorella, which has an antitumor effect and is a highly unique sugar having high heat resistance, acid resistance and alkali resistance. It is an object to provide a protein and a method for producing the protein.

[0007]

[Means for Solving the Problems] In view of the above circumstances, the present inventors have as a result of earnest research, and as a result, chlorella is produced in a culture medium and has an antitumor activity under high temperature, acidic or alkaline conditions. A novel glycoprotein with high purity was found.

The glycoprotein according to the present invention is a glycoprotein produced outside the algal cells by unicellular green algae of the genus Chlorella, which contains a polysaccharide containing 70% or more of galactose as a constituent sugar and a protein. It is characterized by having a saccharide content of 60 to 80%, a protein content of 20 to 40%, an isoelectric point of pH 4 or less, and an average molecular weight of 265,000.

The method for producing a glycoprotein according to the present invention will be described below.

As a method for culturing Chlorella, any method of autotrophic (Autotrophic), heterotrophic (Heterotrophic) or mixed nutritional (Mixotrophic) may be used. It is preferable to culture under mixed nutrient conditions. At the time of culturing, it is necessary to pay sufficient attention to sterilization and handling of each device, culture solution, etc. so that microorganisms other than Chlorella do not mix.

For the culture, first, small-scale culture of the seed strain stored in the agar slant medium is carried out using a Sakaguchi flask. Then, using a general chlorella liquid culture medium, the cells are cultured for several days to several weeks while gradually scaling up. The obtained chlorella culture solution is centrifuged to separate it into a supernatant containing the target glycoprotein and a chlorella alga.

The polymer component is separated from the obtained supernatant by ultrafiltration, dialysis or gel filtration chromatography, and then purified by appropriately combining various affinity chromatography and gel filtration chromatography to obtain the desired sugar. A protein (hereinafter referred to as A3 R3) is obtained. Examples of the chromatography that can be used in the present invention include anion exchange chromatography, metal chelate chromatography, Con-A lectin fixed column chromatography, RCA lectin fixed column chromatography, and gel filtration chromatography. All of them may be used or only a part thereof may be used depending on the purpose. Also,
The order of performing each chromatography is arbitrary,
It is not particularly limited.

The obtained glycoprotein has an antitumor effect and can be used as a main component of an antitumor agent. In addition, the supernatant of the culture solution can be used as it is as an antitumor agent.

[0014]

【Example】

Example 1 Cultivation of chlorella and preparation of glycoprotein A seed strain (Chlorella Industrial in-house number CK-22) 1 stored in an agar slant medium (glucose medium) was inoculated into a Sakaguchi flask and shaken for 4 days. Cultured. After that,
It was transferred to a 10 liter Jafamentor and cultured for 3 days. After culturing this culture solution for another 4 days, the culture solution was harvested. The composition and culture conditions of the medium used here are as follows.

Medium composition (per liter of medium) Glucose 100 g Urea 7.5 g Phosphate-potassium 2.5 g Magnesium sulfate heptahydrate 2.5 g Culture conditions pH 7.0 37 ° C. The obtained chlorella culture solution was 7000 rpm ( 6200G) for 30
After centrifugation for minutes, the supernatant containing the target glycoprotein and the Chlorella algal cells were separated. The polymer-side component (CVS-U) of this supernatant was concentrated and separated using an ultrafiltration membrane (molecular weight 10,000 membrane, manufactured by Japan Millipore) and freeze-dried. C
The yield of VS-U is 2 to 3 g per liter of culture solution.
Met. Example 2 Purification of Glycoprotein 10 g of CVS-U obtained in Example 1 was sequentially separated by the following method while confirming the active fraction.

First, CVS-U was subjected to strong anion exchange chromatography (Q-Sepharose Fast Flow, manufactured by Pharmacia Biotech), and a second fraction was obtained by gradient elution with NaCl. Next, this fraction was subjected to copper chelate affinity chromatography (manufactured by TOSOH), and the most active fraction was obtained by gradient elution with glycine.

The fraction thus obtained is still non-uniform in terms of electrophoresis, and its effect changes with changes in heat and pH. For this reason, the results of animal experiments using this fraction have relatively large variations and are difficult to use in practice. Therefore, further purification was performed by the following procedure.

First, the above-mentioned fraction was applied to a Con-A agarose column (manufactured by Seikagaku Corporation) and gradient elution was carried out using α-methylmannoside to collect the third fraction. Then, this fraction was applied to an RCA ₁₂₀ agarose column (manufactured by Seikagaku Corporation) and gradient elution was performed using lactose to collect the third fraction showing the highest activity. Then, this fraction was desalted through a gel filtration column and freeze-dried to obtain an active fraction (A3R3) (yield 50 mg).

The following measurements were carried out in order to investigate the chemical properties of the obtained active ingredient (A3R3).

The sugar and protein contents are measured by the phenol / sulfuric acid method (galactose conversion value) for the former and L for the latter.
Each was quantified by the owry method (BSA conversion value).

The molecular weight of the sample was measured by the gel filtration method (standard protein conversion value).

Electrophoresis for purity and molecular weight determination
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAG) using -20% gradient gel
E). The isoelectric point was measured by isoelectric focusing (IEF) using a 4% polyacrylamide gel (PAG) and an amphoteric carrier of pH 7-3.

For the infrared spectrum, FT-IR was used and K
It was measured by the Br tablet method. (A) Sugar / protein composition The results of analysis were 67.2% total sugar and 33.5% protein.

When the same measurement was carried out for 30 samples, the sugar and protein contents were 60% each.
Were in the range of ~ 80%, and 40-20%. (B) Sugar composition The sugar composition was measured by using 2.5 M trifluoroacetic acid at 100 ° C.
After hydrolyzing for a time, it was measured as a trifluoroacetic acid derivative by gas chromatography. The results are shown below. The content rate is shown in mol%.

Galactose 93.0 Mannose 5.6 Glucosamine 1.4 (c) Amino Acid Composition The amino acid composition is 0.05% β-mercaptoethanol and 0.1%.
After hydrolysis with 5.7 N hydrochloric acid containing 02% phenol at 110 ° C. for 24 hours, the phenylthiocarbamyl derivative was measured by high performance liquid chromatography. The results are shown below. The content rate is shown in mol%.

Aspartic acid 14.2 Glutamic acid 9.0 Serine 4.4 Threonine 9.4 Hydroxypurine 0.5 Histidine non-detection Glycine 9.0 Alanine 14.5 Tyrosine 1.8 Arginine 1.9 Methionine 2.1 Valine 10.5 Proline 3.2 Phenylalanine 3.3 Lysine 2.5 Isoleucine 3.7 Leucine 10.0 (d) Molecular weight The molecular weight was determined to be 0.1. N potassium phosphate buffer (pH
6.8) as the eluent and Sephacryl S-300HR column (φ1
0 × 460 mm, manufactured by Pharmacia Biotech Co., Ltd.). The result is shown in FIG. In the figure, the solid line indicates the protein content (absorption at 280 nm), and the broken line indicates the sugar content (absorption at 490 nm in the phenol / sulfuric acid method).
Are shown respectively. From this figure, the molecular weight of A3R3 is 26
It was revealed to be 5,000. (E) Electrophoresis FIG. 2 shows the molecular weight and the state of separation by SDS-PAGE.
The results of isoelectric focusing are shown in FIG. 3, respectively. In each figure, A shows the one by the protein staining (CBB-R250 staining) treatment, and B shows the one by the sugar staining (PAS staining) treatment. Lanes 1 to 3 show A3R3, lanes 4 to 6 show fractions before being applied to a Con-A agarose column, and lanes M, M1 and M2 show molecular weight markers. (F) Infrared absorption spectrum The measurement result by FT-IR is shown in FIG. (G) Solubility A3R3 is readily soluble in water. Example 3 Antitumor effect of each component (a) Comparison of antitumor effect by administration route of A3R3 First, BALB / c mice were treated with Meth-A fiber sarcoma at 5 × 10 ⁶ cells per mouse on the right flank. Subcutaneously transplanted. Two days, four days, and six days after transplantation, a total of 3 times, 1 mg of A3R3 per mouse was dissolved in phosphate buffered saline (PBS) and injected intravenously, subcutaneously, intraperitoneally or intratumorally. Was administered by. Then, 9 days after the first dose of Meth-A fibrosarcoma, an additional 5x per mouse
10 ⁶ cells of Meth-A fibrosarcoma were implanted subcutaneously on the left flank. By measuring the size of the sarcoma formed on the left side, not the direct action on the tumor cells but the protective function of the living body against the tumor can be more clearly understood.

Left and right tumor sizes were measured 12 days after implantation of each tumor. The size of the tumor was measured by measuring the diameter of the tumor and expressed as an area. Also, transplant the presence or absence of a tumor on the left side
It was measured after 15 days. The results are shown in Table 1 below.

[0028]

[Table 1] As shown in Table 1, A3R3 showed the strongest antitumor effect in intratumoral injection, but the effect was also observed in other administration routes. (B) Comparison of antitumor effect between A3R3 and a commercially available drug The effect of intratumoral injection in which A3R3 shows the strongest effect is shown by the commercially available antitumor immunostimulant picibanil (O
K432). The results are shown in Table 2 below.

[0029]

[Table 2] As shown in Table 2, the antitumor effect of A3R3 is clearly stronger than that of commercially available drugs that show similar effects. (C) Difference in antitumor effect between lots of A3R3 The difference in antitumor effect between lots of A3R3 was examined. Comparison of effects was performed using the tumor on the left side. The results are shown in Table 3 below.

[0030]

[Table 3] As shown in Table 3, all of the 5 tested lots showed a stable and strong antitumor effect on the tumor on the left side. (D) Effect of physical treatment on A3R3 on antitumor effect After treatment of A3R3 shown in Table 4 below, each treated product was administered at 1 mg per mouse, and its antitumor effect was measured. The results are also shown in Table 4.

[0031]

[Table 4] As shown in Table 4, A3R3 is a substance which shows stability due to oxidation treatment with sodium periodate (NaIO ₄ ), acidic conditions with hydrochloric acid, alkaline conditions with sodium hydroxide, and heat treatment, and has significantly increased stability. Is.

[0032]

INDUSTRIAL APPLICABILITY As described above, the novel glycoprotein according to the present invention has a strong re-implantation tumor suppressive effect that surpasses even a commercially available natural product-derived antitumor agent, and is extremely excellent in heat resistance, acid resistance and alkali resistance. It is a highly stable substance. Therefore, compared with similar glycoproteins, a simple purification method or sterilization method can be selected from a wider range according to the purpose, and further formulation is easy. In addition, storage conditions can be relaxed, and the storage period can be extended.

This glycoprotein exhibits an antitumor effect by enhancing the defense function of the living body rather than directly acting on the tumor.

The glycoprotein according to the present invention is produced from the culture supernatant conventionally treated as waste. Therefore, the high value-added of the unnecessary material is realized, and the industrial advantage is extremely large.

[Brief description of drawings]

FIG. 1 is a graph showing the results of gel filtration chromatography of glycoprotein A3R3 according to the present invention.

FIG. 2 is a photograph showing an electrophoresis pattern by SDS-PAGE method showing the purity and molecular weight distribution of glycoprotein A3R3 according to the present invention.

FIG. 3 is a photograph showing an electrophoretic pattern by the IEF method showing the isoelectric point of glycoprotein A3R3 according to the present invention.

FIG. 4 FT- of glycoprotein A3R3 according to the present invention
The graph which shows the infrared absorption spectrum by IR.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI Technical display location C12P 21/00 A 9282-4B // A61K 35/80 A 8217-4C (C12P 21/00 C12R 1 : 89)

Claims (4)

[Claims] 1. A glycoprotein produced by a single-celled green alga of the genus Chlorella outside the algal body, comprising a polysaccharide containing 70% or more of galactose as a constituent sugar and a protein, and the content of the polysaccharide is Glycoprotein having a protein content of 60 to 80%, a protein content of 20 to 40%, an isoelectric point of pH 4 or less, and an average molecular weight of 265,000. 2. An antitumor agent containing the glycoprotein according to claim 1. 3. A single cell green alga of the genus Chlorella is cultured, the culture solution is centrifuged to separate the supernatant, and the supernatant is purified to obtain a fraction having an average molecular weight of 265,000. Item 2. A method for producing a glycoprotein according to Item 1. 4. The method according to claim 3, wherein the purification is carried out by ultrafiltration, dialysis or gel filtration chromatography, and then by several steps of affinity chromatography and gel filtration chromatography.