JPH04300898A - New glycoprotein conjugate and production thereof - Google Patents

Abstract

PURPOSE:To obtain the subject new glycoprotein containing polysaccharides abundantly containing galactose and a protein and having an antineoplastic effect in an industrially advantageous manner by culturing a unicellular green algae belonging to Chlorella genus and separating a sepcified molecular weight product fraction from the culture solution. CONSTITUTION:A platinum loop of seed strain of a unicellular green algae belonging to Chlorella genus stored on an agar slant medium (glucose medium) is inoculated in a liquid medium and then subjected to shaking culture for 4 days. The culture solution is subsequently transferred to a jar fermenter, cultured for 3 days and then transferred to a tank followed by 4 days culture. After completion of culture, the culture solution is centrifuged to remove chlorella cell bodies and a supernatant is collected, which is ultrafiltered and then purified by hydrophobic chromatography, anion-exchange chromatography, etc., thus giving the objective glycoprotein conjugate composed of polysaccharides containing >=50wt.% galactose and a protein respectively in an amount of 50-70wt.% and 30-50wt.% and having 200000 average molecular weight.

Description

[Detailed description of the invention]

0001

FIELD OF INDUSTRIAL APPLICATION This invention relates to glycoprotein complexes produced outside the algae by unicellular green algae of the genus Chlorella, and particularly to glycoprotein complexes having antitumor activity.

0002

BACKGROUND OF THE INVENTION Conventionally, proteins or polysaccharides derived from mushrooms or bacteria have been known to exhibit antitumor effects. All of these have been found to exhibit antitumor effects by increasing the body's defense function against tumors.

On the other hand, regarding green algae such as unicellular green algae of the genus Chlorella (hereinafter sometimes referred to as Chlorella),
Extraction and separation of an antitumor glycoprotein, which has a molecular weight of 120,000 and is composed of several types of sugars, has been reported (Japanese Patent Application Laid-open No. 69728/1983). The antitumor effect of this substance is enhanced by hydrolysis treatment using amylase.
Shows direct cell killing effect. However, since this substance is an intracellular substance, it contains various components and is not secreted outside the algae, making fractional purification and large-scale collection difficult.

[0004] Substances produced by chlorella outside the algae include mucilage polysaccharide (Japanese Patent Publication No. 61-96992) and Moo
re,B. G. , Tischer, R. G. polysaccharides (
Science, 145:586-587, 19
64), but these substances have not been found to have antitumor effects.

[0005]

[Problem to be solved by the invention] As mentioned above, conventionally,
Although it is known that Chlorella produces polysaccharides outside the algae, these substances have not been found to have antitumor effects. Furthermore, the glycoprotein complex that Chlorella produces outside the algae is also unknown.

[0006] The object of the present invention is to provide a novel glycoprotein complex that is produced extracellularly by unicellular green algae of the genus Chlorella and has an antitumor effect, and a method for producing the same.

[0007]

[Means for Solving the Problems] In view of the above-mentioned circumstances, the present inventors have conducted intensive research and have discovered a novel glycoprotein complex produced by Chlorella in the culture solution, which has an antitumor effect.

The glycoprotein complex according to the present invention is a glycoprotein complex produced outside the algal body by a unicellular green alga of the genus Chlorella, and contains a polysaccharide containing 50% or more of galactose as a constituent sugar and a protein, It is characterized in that the polysaccharide content is 50 to 70%, the protein content is 30 to 50%, and the average molecular weight is 200,000.

[0009] The method for producing a glycoprotein complex according to the present invention will be explained below.

[0010] As a cultivation method for Chlorella, autotrophic (
Autotrophic), Hetertrophic
otrophic) or mixotrophic (Mixot
rophic) may be used, but in consideration of efficiency, it is preferable to culture under heterotrophic conditions or mixotrophic conditions. During cultivation, sufficient care must be taken to sterilize and handle each equipment, culture solution, etc. to prevent contamination with microorganisms other than chlorella.

[0011] For culturing, first, a small-scale culture of the seed stock stored in an agar slant medium is carried out using a Sakaguchi flask. Next, using a common liquid culture medium for chlorella, the culture is gradually increased in scale for several days to several weeks. The obtained chlorella culture solution is separated into a supernatant (abbreviated as CVS) containing the target glycoprotein complex and chlorella algae by centrifugation.

The obtained CVS is subjected to ultrafiltration, dialysis or gel filtration chromatography to remove polymer components (CVS).
(abbreviated as SU) is separated and then purified using hydrophobic chromatography, anion chromatography, chelate chromatography, etc. to obtain the desired glycoprotein complex.

The obtained glycoprotein complex has an antitumor effect and can be used as a main component of an antitumor agent. Note that CVS, which is a supernatant of the culture solution, and CVSU-U, which is a crude product, can also be used as an antitumor agent as they are.

[0014]

[Examples] Example 1 Cultivation of Chlorella and preparation of glycoprotein complex Seed stock (
Chlorella Industries internal number CK-22) 1 platinum loop was inoculated into a Sakaguchi flask and cultured with shaking for 4 days. Thereafter, the culture solution was transferred to a 10 liter jar fermenter and cultured for 3 days. This culture solution was further transferred to a 1 ton tank, and 4
After culturing for one day, the culture solution was harvested. The composition of the medium and culture conditions used here are as follows.

Medium composition (per liter of medium) Glucose 100
g-urea
7.5 g potassium phosphate
2.5 g magnesium sulfate 7
Hydrate 2.5 g Culture conditions pH 7.0 (adjusted with NaOH) 37°C The obtained Chlorella culture solution was centrifuged at 6000 rpm for 15 minutes, and the supernatant containing the desired glycoprotein complex (CVS ) and Chlorella algae. This CVS
PTHK membrane (Millipore Labora
After ultrafiltration using Tory Co., Ltd., 200 times), freeze-drying was performed to obtain a polymer component (CVSU-U). The yield of CVSU-U was 2 to 3 g per liter of culture solution. Example 2 Purification of Glycoprotein Complex 10 g of CVS-U obtained in Example 1 was sequentially fractionated while confirming the active fraction by the following method.

First, CVS-U was subjected to Phenyl-Sepharose CL-4B (hydrophobic chromatography) and eluted with 1/10M phosphate buffer (pH 6.81) to collect the highly hydrophilic first fraction ( (abbreviated as P1) was fractionated. Next, this fraction was subjected to anion exchange chromatography to collect a second fraction (abbreviated as P1Q2). This P1Q2 was further fractionated by chelate chromatography to obtain 2.2 g of the second eluted fraction with high antitumor activity (abbreviated as Q2C2).

The following measurements were carried out to investigate the chemical properties of the obtained antitumor active ingredient (Q2C2).

(a) General analysis The respective contents of sugar, protein, lipid, nucleic acid, phosphate group, and sulfate group were determined. At this time, total sugars are calculated as galactose using the phenol/sulfuric acid method.
Proteins were determined using the Lowry method in terms of bovine serum albumin, lipids were determined using the gravimetric method using chloroform methanol extraction and fatty acid detection was performed using gas chromatography, and phosphate groups were determined using Bar.
by the tlett method and for the sulfate group by Dod
It was determined by the gson-Price turbidimetry. The results are shown below.

[0019] Sugar 51.8
% Protein 36.3% Lipid Not detected Nucleic acid
Phosphate group not detected Sulfate group not detected When similar measurements were performed on 30 samples, the content of sugar and protein was 50 to 70% each.
, and within the range of 30-50%.

(b) Sugar composition The sugar composition was calculated using 2.5M trifluoroacetic acid.
After hydrolysis at 0°C for 7 hours, it was measured as a trifluoroacetic acid derivative by gas chromatography. The results are shown below. In addition, the content rate was shown in (w/w)%.

Rhamnose 14.4 Arabinose 1.0 Xylose 0.8 Mannose 4.8 Glucose Trace galactose 76.9 Glucosamine 2.1 (c) Amino acid composition The amino acid composition consists of 6N hydrochloric acid containing 0.05% β-mercaptoethanol. After hydrolysis at 110° C. for 24 hours, it was measured as a phenylthiohydantoin derivative by high performance liquid chromatography. The results are shown below. In addition, the content rate was shown in (w/w).

Aspartic acid 11.1
Glutamic acid 13.7 serine
6.2 Threonine
6.9 histidine
6.2 Glycine
5.6 alanine
9.5 Tyrosine 5.
2 Arginine 2.8 Methionine 0.9 Valine
7.0 proline
5.4 Phenylalanine
3.6 lysine
3.5 Isoleucine 3.0 Leucine 9.5 (d) Infrared absorption spectrum The infrared absorption spectrum of Q2C2 is shown in FIG.

(e) Elemental analysis The results of elemental analysis are as follows.

C 41.1%H
5.9% N 4.4% (f) Molecular weight Q2C2, 0.1M phosphate buffer (pH 6.81
) as the eluent, Sephacryl S-300
It was subjected to gel filtration chromatography using HR (10 x 460 mm). The results are shown in FIG. In Figure 2, the dashed line represents sugar, and the solid line represents protein (280 nm
UV absorption). In addition, the relationship between various proteins (molecular weight markers) and elution time is shown in Figure 2.
Measurements were made in the same manner as in the case of , and the results are shown in FIG. Figure 2
From FIG. 3, it was revealed that the average molecular weight of Q2C2 was 200,000.

(g) Solubility Q2C2 is easily soluble in water. Example 3 Antitumor Effect of Each Component First, Meth-A fibrosarcoma was subcutaneously implanted into CDF1 mice at a rate of about 3 x 106 cells per mouse. 5 mg/PBS (phosphate buffered saline) per mouse at a time on days 2, 4, and 6 after transplantation.
0.2 ml of CVSU-U was administered intravenously, subcutaneously, intraperitoneally, or by intratumoral injection.

[0026] The tumor size of these mice was measured during the breeding period, the volume was calculated, and the antitumor effect was determined by comparison with the control group. Note that each sample (CVS-U) was sterilized in boiling water for 30 minutes. Moreover, as a control group, mice to which only PBS was administered were used. The results are shown in Table 1.

[0027]

[Table 1] As shown in Table 1, CVS-U showed remarkable tumor suppressive effects not only by intratumoral injection but also by intravenous injection. Furthermore, clear antitumor effects were also observed by intraperitoneal and subcutaneous injections.

[0028] Therefore, we conducted a more detailed study on an intratumoral injection system that is easy to administer and provides the most stable and strong effects.

First, Meth-A tumors were subcutaneously implanted into CDF1 mice at approximately 3 x 106 cells per mouse. Then 1 or 5 mg/PBS 0 per animal at a time on days 2, 4, and 6 after transplantation.
．． 2 ml CVS-U or Q2C2, or PBS
(control) was administered intratumorally.

[0030] The tumor size of these mice was measured during the breeding period, the volume was calculated, and the antitumor effect was determined by comparison with the control group. Note that each sample was sterilized in boiling water for 30 minutes. The results are shown in Table 2.

[0031]

[Table 2] As shown in Table 2, Q2C2 clearly showed a stronger antitumor effect compared to the raw material CVS-U, suppressing tumor growth by about 90%. That is, antitumor activity is localized to Q2C2, and Q2C2 can be considered to be an antitumor glycoprotein complex. Example 4 Enhancement of defense function against tumors in vivo C
The following experiment was conducted to examine whether the antitumor effect of VS-U is mediated by biological defense.

[0032] In a system in which Meth-A tumors were transplanted into BALB/C mice, an experiment was conducted under the same conditions as in Example 1 except for the following conditions. BALB/C mice have normal n
u/+ mice (thymus-bearing mice) and nu/n
U mice (mouses that lack T lymphocytes due to a thymus deficiency and do not perform normal immune reactions) were used. The results are shown in Table 3. Note that the values in the table are those obtained 14 days after tumor transplantation.

[0033]

[Table 3] As shown in Table 3, CVS-U showed approximately 70% tumor growth inhibition rate in normal nu/+ mice, but nu/+ mice, which lack T lymphocytes and have a reduced body defense function, were No effect was observed in nu mice. These results revealed that the antitumor effect of CVS-U involves biological defense reactions, particularly T lymphocytes.

[0034]

Effects of the Invention As described above, the novel glycoprotein complex according to the present invention is produced outside the algae by unicellular green algae of the genus Chlorella. It has a chemical composition that is significantly different from that of proteins: galactose-rich polysaccharides and proteins. This glycoprotein complex is
Since it is produced outside the algal body, that is, in the culture solution, it is easy to separate and purify, and the productivity is also very high, so it can be produced at low cost. In addition, since the chlorella culture solution has traditionally been discarded, it is also an effective use of industrial waste.

Furthermore, this glycoprotein complex has an antitumor effect by increasing the defense function of the living body.

[Brief explanation of the drawing]

FIG. 1 is a characteristic diagram showing the infrared absorption spectrum of the glycoprotein complex of the present invention.

FIG. 2 is a characteristic diagram showing the gel filtration chromatography pattern of the glycoprotein complex of the present invention.

FIG. 3 is a correlation diagram showing the relationship between proteins of various molecular weights and elution time in gel filtration chromatography.

Claims (4)

[Claims] Claim 1: A glycoprotein complex produced outside the algae by unicellular green algae of the genus Chlorella, comprising 50% of the constituent sugars.
A glycoprotein complex comprising the above galactose-containing polysaccharide and protein, the content of the polysaccharide is 50 to 70%, the content of the protein is 30 to 50%, and the average molecular weight is 200,000. 2. An antitumor agent containing the glycoprotein complex according to claim 1. 3. Claim 1, characterized in that a unicellular green alga of the genus Chlorella is cultured, the culture solution is centrifuged to separate a supernatant, and this supernatant is purified to obtain a fraction with an average molecular weight of 200,000. Method for producing the described glycoprotein complex. 4. The production method according to claim 3, wherein the purification is first carried out by ultrafiltration, dialysis or gel filtration chromatography, and then carried out by hydrophobic chromatography, anion chromatography or chelate chromatography. .