JPH0219392A - Galactosaminooligosaccahride and production thereof - Google Patents

Abstract

NEW MATERIAL:A compound shown by formula I (n is 0-10). EXAMPLE:A compound shown by formula II. USE:An antitumor agent. PREPARATION:alpha-1,4-Polygalactosamine shown by formula III is hydrolyzed with a hydrolysis enzyme to give a compound shown by formula I. The starting substance shown by formula III is produced by I-I [FERM P-3928 (FERM BP-1180)] belonging to the genus Paecilomyces. Hydrolysis enzyme of polygalactosamine is novel and is obtained by cultivating Pseudomonas sp H881 (FERM P-8955).

Description

DETAILED DESCRIPTION OF THE INVENTION (Field of Industrial Application) The present invention relates to amino-oligosaccharides, more particularly to novel galactosaminooligosaccharides, and methods for producing the same.

(Prior Art) In recent years, it has become known that polysaccharides or their oligosaccharides produced by microorganisms, plants, or animals have various physiological activities, and interest in polysaccharides and their oligosaccharides has increased.

For example, in polyglucosamine (chitosan), it has been discovered that chitin, chitosan, and their oligosaccharides have excellent physiological activity such as antitumor activity.

Furthermore, since polygalactosamine is a polysaccharide similar to the above-mentioned polyglucosamine, polygalactosamine is expected to have excellent physiological activity, and interest in polygalactosamine is increasing.

(However, n in formula 1 represents 0 to 10).

However, there are very few polygalactosamines (α-1,4-galactosaminogalactan) derived from microorganisms, such as PF-101 and PF derived from B. deuteromycetes.
-102 is only known (Tokuko Sho 56-1
Galactosaminooligosaccharides, which are oligosaccharides such as those used in the present invention, are novel and completely unknown compounds.

(Problems to be Solved by the Invention) The above-mentioned PF-101 and PF-102 have strong aggregation activity despite being relatively related polysaccharides to polyglucosamine, which is an aminopolysaccharide with physiological activity. The current state of the technology is that no other particular physiological activity has been confirmed.

(Means for Solving the Problems) The present invention was made in view of the current state of technology, and is aimed at developing a new physiologically active substance based on PF-101 or PF-102. It has been done.

As a result of extensive and deep research from various perspectives, we have focused on oligosaccharides, which are decomposition products of these polysaccharides.

As a result of intensive research on decomposition method 1 separation and purification method, we succeeded in isolating various oligosaccharides.
Moreover, by identifying each of them and confirming that all of them are new compounds that have not been described in literature, we have completed the present invention.

The galactosaminooligosaccharides according to the present invention are all new substances, and are strongly expected to have various beneficial physiological activities such as antitumor properties, either alone or in combination.

In carrying out the present invention, we focused on the above-mentioned PF-102 from various polygalactosamines as a starting material, from the viewpoint of having a clear chemical structure and ensuring a stable supply of mass production originating from microorganisms. .

In PF-102, D-galactosamine is mainly α-1,4
A basic polysaccharide with a molecular weight of 160,000 or more, α-1,4-
Polygalactosamine is a natural polysaccharide represented by the following formula. 1
It is obtained by further purifying a precipitate soluble in an acid aqueous solution precipitated by adding salts to a culture solution, and has the following physical and chemical properties.

(1) Agglomeration activity: Agglomerates suspended fine particles in extremely small amounts.

(2) Aggregation activity p)l range: Stably exhibits aggregation activity at pH 2 to 9.

(3) Temperature range for aggregation activity; aggregation activity is observed at 0-100°C.

(4) Coagulation active ionic strength; carbonic acid and Fe2 (SO4
) 3 inhibits aggregation activity, but other ions and ionic strength have no effect on aggregation activity, and Na
C1, K, and SO4 have no effect on IM at all.

(5) Elemental analysis; nitrogen 8.64%, carbon 42.80%,
Hydrogen 6.87% General formula: (Cj (□1NO4・SuH20)a (6) Color reaction; Ninhydrin reaction; It was confirmed as a single substance by gradient isofocus electrophoresis, with an isofocus (pI) of 8.5.

(8) Color of substance; light yellow (9) Distinction between basic, acidic, and neutral When suspended in water at 0.5% w/v, the pH is 7.5 (deionized water pH 5, 8) It is.

(10) Solubility in solvents ・Poorly soluble in hot water ・Poorly soluble in cold water ・Easily soluble in dilute acids ・Poorly soluble in dilute alkali ・Alcohols, acetone, chloroform.

Insoluble in benzene and n-pentane.

(11) Average molecular weight: 160,000 or more Examples of the acid salt of PF-102 mentioned above include phosphate, hydrochloride, acetate, lactate, and citrate.

The flocculating active substance PF-102 described above is produced, for example, by Deuteromycosis 1-1, which the present inventors isolated from a humus layer in the Wakayama region. Deuteromyces [1] was recognized as belonging to the genus Paecilo [1yces] and was named Benicillomyces [1.
0).

Next, Paecilomyces I-1 (Paecilomyces I-1)
es r -1).

[a] Observation under and above the microscope This fungus lacks conidiophores, and conidia are individual phialides that grow directly from vegetative hyphae or vegetative hyphal bundles.
) is derived in a long chain at the tip. Phialide is translucent and has a length of 20 to 45μ, with a slightly thick base (1.0 to 1.5μ) and a slightly tapered tip (0.5 to 1.5μ).
1.0μ), and some have a straight or slightly curved tip. The conidia are cigar-shaped (
or rod type), and its size is 4 to 6 x 1.0
~1.4μ.

Conidia usually form a chain of 25 to 35 conidia, but in rare cases, long-chain conidia are also observed. This chain of conidia is extremely fragile and can be easily broken by a severe shock.

[b] Growth status in each medium (25°C flat culture) (1
) The colonies on Czapek agar medium grew well and reached a diameter of about 45 mm on the 14th day. The flora is white, velvety to woolly, with a tuft-like bulge in the center, and a circular area around the colony. Water drops/
No wrinkles. The underside of the colony is white in the early stage of culture, and the central part is pale yellow in the later stage of culture.

No pigment production was observed on the agar.

(2) Colonies on malt agar medium grew well, with a diameter of about 54+ nm on the 14th day.
The area around the colony is not circular but resembles a plum grove.

The central part of the bacterial flora is white, but the peripheral part is pale yellow. The thickness of the flora is medium, and the central part is slightly concave. Water drops/
No wrinkles were observed, and the entire back surface of the colony was pale yellow. A pale yellow pigment was produced on the agar medium.

(3) Potato dextrose agar medium colony growth is very good <about 60 av in diameter on day 14
Reach +. It forms a white, velvety to woolly, fairly thick bacterial flora, with a slightly swollen central area, a slightly pale yellow flora in the center of the plate, and a relatively thick white flora around it. . There are no wrinkles on the surface, but a few light brown water droplets can be seen. There are several radial wrinkles on the back of the colony, and concentric yellow shading can be observed. There is diffusion of pale yellow pigment into the agar.

(4) YpSs agar medium (composition: starch 1.5%, yeast extract 0.4%, HPO40.1%, Mg
SO40.05%.

(Agar 2%) Colony growth was good and reached a diameter of about 50 mm on the 14th day. It is a fluffy, woolly, thick bacterial flora all over its white color. No water drops or wrinkles. The underside of the colony has no noteworthy features. No pigment production.

(5) MY2. Agar medium (composition: glucose 20%, polypeptone 0.5%, yeast extract 0.3%, malt extract 0.3%, agar 2%) Colony growth was not very good, with a diameter of about 30 mm on the 14th day.
+IIm. Aerial mycelium does not form much and there are many fine wrinkles, and the peripheral part is pale yellow and the central part is pale brown. The underside of the colony is pale yellow with fine wrinkles and no pigment production.

Based on the above morphological characteristics and culture properties, this bacterium is considered to be a monophialide deficient bacterium.
icspecies of Paecilomyces
(Agnes, H, S, 0nionsand G,
L, Barron; 1967, Mycology
cal papers No. 107, Co@monsa
alth Mycological In5titut
e.

Paecilomyces bacillisporus, described in Key, England).
bacillisporus).

That is, the morphology of the conidia, which is considered the most important characteristic of Classification 1 of Deuteromycota, is extremely similar to that of P. bacillisporus, and the morphology of the myalides is also very similar. However, on the other hand, there are some differences in culture characteristics in various media, and P
The growth rate of Bacillisporus is slower than that of this fungus, and the hyphae are white in the early stage and pinkish in the late stage of culture (
This bacterium differs in that it is initially white and depending on the culture medium, it becomes pale yellow in its later stages. However, even in the above-mentioned literature, P. bacillisporus strains vary in culture characteristics and conidial size. (Str
ains of P, bacillisporus s
how variation in cultural
Characteristics and in 5p
5ize), this bacterium is Paecilomyces bacillisp.
It is thought to be Benicillomyces orus or a related bacterium, but since there is no conclusive evidence, it was designated as Benicillomyces I-1.

Although this property is not considered very important as an indicator for the classification of Deuteromycota, the physiological properties of Honkan will be shown below.

[C] Physiological properties (1) Availability of carbon sources Using Czapek's medium as a basic medium, various carbon sources were added in place of sucrose, and the growth was observed. As a result, soluble starch, glycogen, trehalose, raffinose, cellobiose, It makes great use of maltose, sucrose, glucose, fructose, galactose, mannose, inositol, sorbitol, and glycerin.

Next, inulin, lactose, arabinose, ribose,
Mannitol, lactic acid, and succinic acid could be used fairly well.

The availability of xylose, rhamnose, and citric acid is low, and the availability of tartaric acid and oxalic acid is completely unavailable.

(2) Utilization of nitrogen source As a result of using Czapek's medium as a basic medium and changing the nitrogen source to see the growth rate, it was found that any of the ammonia, amino, and nitrate forms of nitrogen can be utilized well.

(3) Growth temperature The optimal growth temperature is 23 to 25°C, and growth is observed at 30'C, but not at 35°C.

(4) Growth PH G, Ye medium (composition glucose 2%, yeast extract 0.2
When the growth was observed in the pH range of 2 to 10 (%), it grew well at any pH.

Paecilomyces I-1 can be cultured by a conventional liquid culture method for filamentous fungi.

Spores or hyphae of Paecilomyces I-1 are inoculated into a liquid medium and cultured aerobically. Glucose as a carbon source,
Maltose, sucrose, R powder, blackstrap molasses, etc. can be used, but it is preferable to use glucose. As a nitrogen source, inorganic nitrogen such as ammonium sulfate and sodium nitrate, and organic nitrogen such as peptone and yeast extract can be used. .

Although the culture temperature can be changed as appropriate within the range in which the flocculating active substance-producing bacteria produce the flocculating active substance, it is usually preferable to culture at 20 to 25°C. The culture time varies depending on the culture conditions, but is usually about 4 to 5 days, and the culture may be terminated at an appropriate time by estimating the time when the agglutinating active substance reaches its maximum.

In the present invention, various salts are added to the culture filtrate or filtrate concentrate, and if precipitation does not occur, an alkali is added as necessary to adjust the pH to 7 to 9 to cause precipitation, and the precipitate is separated and washed with water. Highly purified PF-102 can be obtained by dissolving this in a dilute acid aqueous solution, adding a salt again, or adjusting the pH to 7 to 9 by adding an alkali or the like, and precipitating it.

The salt added to the solution containing PF-102 is one or more salts including the following exemplified salts.

Namely, hydrochlorides such as potassium chloride, sodium chloride, calcium chloride, and ammonia chloride, nitrates such as potassium nitrate and sodium nitrate, acetates such as sodium acetate, dipotassium sulfate,
Sulfates such as ammonium sulfate, calcium sulfate, copper sulfate, phosphoric acid 2
Examples include phosphates such as potash, monopotassium phosphate, disodium phosphate, and monosodium phosphate.

Any salt can be added as long as it is dissolved, but
The preferred amount is 0.5 to 50% based on the PF-102 containing liquid.
, more preferably about 2 to 40%.

Depending on the type of salt added, pl+ may be 7 or more, so in this case, PF-10 can be obtained without adjusting the pH.
2 precipitates, so it is enough to separate the precipitate 1a If no precipitation occurs even after adding salt, use an alkali such as caustic soda to adjust the pH to 7 to 9, preferably 8 to 5, which is the isoelectric point. The pH may be adjusted nearby.

Addition of salt to PF-102-containing solution and optionally p117
By performing the adjustment in steps 9 to 9, PF-102, which did not easily precipitate due to the interference of impurities, will start to precipitate, and will be separated from the impurities and precipitated. This precipitate can be separated by centrifugation or filtration with a filter cloth.

Even if the culture solution is subjected to isoelectric point treatment at pH 8.5, PF-102 remains
Considering that no precipitation of PF-102 occurred at all, it is extremely surprising that the addition of salt alone causes complete precipitation of PF-102.

Since the separated precipitate contains a large amount of salt, it is washed with water or a solvent to desalt it, and then dissolved in acid.

The acid may be any acid such as an organic salt such as acetic acid or an inorganic acid such as hydrochloric acid, and the concentration is preferably about 0.01 to 3 mol.

After dissolving the precipitate in acid, the precipitate is easily precipitated by only treatment near the isoelectric point of 2117-9, so add an alkali such as caustic soda and adjust the pH to 7-9, preferably pH 8.5. and adjust the pH to obtain a precipitate.

Furthermore, in order to purify this precipitate, wash it with water etc.
A precipitate can be obtained by dissolving in acid again and adjusting the pH to 7 to 9.

This purification process can be repeated any number of times, and when the purification is completed, the precipitate becomes almost pure and PF-102, an α-1,4-galactosaminogalactan having the chemical structure described above, is obtained. It is.

Polygalactosamine (PF-102) thus obtained
) can be hydrolyzed with acid, alkali or enzyme, and then isolated and purified to obtain the target galactosaminooligosaccharides singly or as a mixture of several types.

For example, in the case of acid hydrolysis, a commonly used acid solution such as hydrochloric acid is used, and acid hydrolysis is usually performed while heating.

Thereafter, hydrochloric acid is removed by concentration under reduced pressure, or by decolorizing the filtrate with activated carbon and treating it with an anion exchange resin. The thus obtained galactosaminooligosaccharide mixture is subjected to separation and purification treatment such as chroma 1-graphy,
Each fraction may be collected and each galactosaminooligosaccharide may be isolated.

As described above, oligosaccharides can be obtained by hydrolyzing polygalactosamine with acid or alkali, but the yield of oligomers, especially those with a high degree of polymerization, is relatively low. As a result of random decomposition, the amount of oligosaccharides obtained is in the order of mono-galactosamine, di-galactosamine, tri-galactosamine, tetra-galactosamine, and penta-galactosamine, and the higher the degree of polymerization, the lower the yield. It turns out.

Therefore, in order to obtain oligosaccharides with relatively large degrees of polymerization by decomposing polygalactosamine, the chemical method described above has a limit in terms of yield. After reaching a point of view and considering it, we decided to focus on biological methods. In other words, the need for polygalactosamine-degrading enzymes that can degrade polygalactosamine and produce various oligosaccharides with different degrees of polymerization at high yields has been highlighted.

As a result of searching for polygalactosamine-degrading bacteria among a wide range of microorganisms, the present inventors discovered that bacteria belonging to the genus Pseudomonas produce a novel polygalactosamine-degrading enzyme. We succeeded in obtaining various oligosaccharides.

The physicochemical properties of this novel polygalactosamine-degrading enzyme are as follows: (1) Action and substrate specificity It acts on polygalactosamine (α-1,4 galactosaminogalactan) to produce oligogalactosamine.

Other polysaccharides such as polyhexose, chitin, starch (α-1,4 glucan), glycogen (α-1,4 glucan), pullulan (α-1,4 glucan), dextran (α-1,6 glucan) , laminaran (β~1.3
glucan), carboxylcellulose (β-1,4-glucan), chitosan (β-1,4-gelcosaminoglucan), ethylene glycol chitin (β-1,4N-acetylgelcosaminoglucan), N of Pssudomonas solanacearum -Acetylgalactosaminogalactan (β-1,3N
-acetylgalactosaminogalactan) (Y, Aki
yama,, at, al,, Agric.

Biol, Ches., 50(3)747.1986
) etc. have no effect at all.

(2) Optimal pH and stable pH range When using sodium citrate phosphate buffer, the optimal pH
H is 4.5 to 7.0, and the stable range is pH 4.5 to 7.0.
It is 8.0.

(3) Enzyme activity measurement method Enzyme activity was measured using Paecilomyees I-1 as a substrate.
Using PF-11 or PF-102 (the main constituent sugar is α-1,4 galactosaminogalactan) produced by the fungus,
This 0.5% 10.1M acetate buffer pH 6,0 solution 0
, add 0.5 m12 of enzyme solution to 5 m12, and heat at 37°C.
, react for 10 minutes, and reduce the resulting reducing power with Somogyi-
Measured by Nelson method. Note that one unit of the enzyme was defined as an activity that increases the reducing power equivalent to 1 μmol of galactosamine per minute.

(4) Range of suitable temperature and temperature stability for action As a result of measurements in the range of 20 to 70°C, the optimum temperature for this enzyme is 55°C, and the temperature decreases rapidly above this temperature.

Next, we looked at temperature stability. When the residual activity was observed when kept at each temperature for 0 to 80 minutes under the condition of pHl1i, 0, 70% of the activity remained after 1 hour at 50°C.

(5) Effects of metal ions, etc. Various metal ions and inhibitors 1 mM (PCMB only 0
．． After standing in a solution containing 1mM) at 37°C for 1 hour, the residual enzyme activity was measured and expressed as a relative value. (Table-1) Table-1
, metals, etc. Inhibitor Residual activity (%) Inhibitor Residual activity (%
) Additive-free 100 KCI 96 NaClCaC
1,98LiCl BaCl299 MnCl2CoCl288
NiC1゜CdCl298 5n
C12 FeC1,5FeC1゜ZnC1,92HgC12 Pb(C1l, C00)295 N)14C
1(NH4)-SO4,100CuS04tris l
) 97 SDS 2) NBS 3
) 88 EDTA 4) AIA
5) 100 PCMB 6)1)
Tris(hydroxyl)aminomethane 2) Sodium dodecyl sulfate 3) N-bromosuccinimide 4) Disodium ethylenediaminetetraacetate 5) Monoiodo vinegar 6) Mercuric paraoxybenzoate Based on the above results, this polygalactosamine degrading enzyme is , iron, copper, inorganic mercury and SO8.

(6) Enzyme purification method Isolation and purification of this enzyme can be performed according to conventional methods.

For example, precipitate with ethanol and Sephadex G-
50 column chromatography, side-Sephadex C
-25 column chromatography, phenyl-Sepharose 4B column chromatography, or a combination thereof.

(7) Molecular Weight The molecular weight of this enzyme is calculated to be 31,000 when measured by polyacrylamide gel slab electrophoresis.

(8) Polyacrylamide gel electrophoresis The purified enzyme was purified using a 7.5% polyacrylamide gel (pH 8) according to a conventional method.
, 6) When subjected to electrophoresis, a single band was observed.

(9) Isoelectric focusing of sucrose density gradient was performed using the conventional isofocus method. As a result, the isopoint of this enzyme is ρI=6.7.

This enzyme is a novel enzyme whose action and substrate specificity are completely unknown.

The polygalactosamine-degrading enzyme mentioned above is produced by, for example, Pseudomonas sp H881.

Pseudomonas sp t1881 is a strain that the present inventors isolated from soil, and its mycological properties are as follows.

(a) Morphological microscopic observation (cultured on broth agar medium at 30°C for 16 hours) (1) Cell size: 0.3-0.6X1. O~2.
0 micron bacilli (2) Cell pleomorphism: Not observed (3) Motility: Possesses polar flagella and is motile (4) Presence or absence of spores: Not formed (5) Durham staining: Negative ( 6) Acid-fastness: Negative (b) Growth status in various media (1) Broth agar plate culture: Colonies are light yellow-brown in color when grown at 30°C for 24 hours, with a smooth, glossy surface and semi- to opaque. There is no pigment formation.

(2) Broth agar slant culture: Grows well.

(3) Meat juice liquid culture: Grows in a thick film on the surface of the culture solution, moderate growth in the liquid 6 (4) Meat juice gelatin puncture culture Grows on the surface and liquefies in layers.

(5) Lidomus milk: alkaline, completely liquefied.

(c) Physiological properties (1) Nitrate reduction: Negative (2) Denitrification reaction: Negative (3) MR test: Positive (4) VP test: Negative (5) Indole production: Negative (6) Hydrogen sulfide Production: Negative (7) Starch hydrolysis: Negative (8) Use of citric acid: Used in both Koser and Christensen's media.

(9) Use of inorganic nitrogen: Nitrate and ammonia are also used.

(10) Pigment production: KingA i negative, King
B: produces a weak blue fluorescent dye, Fagar +, produces a weak blue fluorescent dye, Pagar: negative (11) urease: positive (12) oxidase: positive (13) catalase: positive (14) Growth range : Growth Pl+5-9, optimum temperature 30
~40℃ (15) Attitude towards oxygen: Aerobic (16) O-F Eggplant 1-: Oxidation (17) Casein degradation: Positive (18) DNA degradation: Positive (19) Salt tolerance: 2% salt; Positive , 5% salt; negative (2
0) Generation of acid and gas from sugars Generation of acid Generation of gas (1) L-arabinose + (2) D-xylose + (3) D-glucose + (4) D-mannose + (5) D-fructose ( 6) D-galactose + (7) maltose (8) sucrose + (9) lactose (10) trehalose + (+1) D-sorbitol (12) D-mannitol (13) inositol (14) glycerin ( 15) Starch (d) Other properties (1) Accumulates poly-β-hydroxybutyric acid ester (PH8) in bacterial cells in a nitrogen source-deficient medium.

(2) It grows using arginine and betaine as the sole carbon source and does not have arginine dehydrolase activity.

(3) Decompose fatty acids (Tween 80.60.20).

(4) Grows at 40℃. Unable to grow at 4°C.

The taxonomic properties of this bacterium, which has the ability to produce the above-mentioned novel polygalactosamine-degrading enzyme, were
Of Deterministic Bacteriology” 8th edition (
1974) and Burgess Manual of Systematic Bacteriology, Volume 1 (1984).
In contrast to the classification of 2010), this bacterium does not require a gross factor, accumulates PH8, grows using arginine and betaine as sole carbon sources, is negative for arginine dehydrolase, negative for denitrification, and is grown at 40°C. From viable to section 2 (or ItNA group 2) P, cepac
ia, P, gl, adioli.

It seems to be a related bacterium of P. marginate, but P.
cepacia differs in nitrate reduction positivity and carbon source assimilation ability in D(-)-trehalose, maltose, lactose, and maleic acid. P. gladioli
means 1st square, 1st round, maleic acid, m-
The results of assimilation of hydroxybutyric acid ester are different.

The results for z-hydroxybutyric acid ester are different from P, marginate. Also, P, cepac
ia, P.

marginate produces a non-fluorescent dye, but this bacterium produces KingB* Fagar and L-glutamic acid,
When L-arginine, threonine, and L-histidine are used as the sole carbon source, a weak fluorescent dye (blue-white fluorescence) is produced, but no non-fluorescent dye is observed under various medium conditions. From these results, this bacterium is classified as P.

cepacia, P. gladioli, P.
The physiological properties of this bacterium are different from the It is to produce acid. This property is similar to Pseudomonas
Genus, psychrotrophic P, fragi, p.

taetrolens (both section 5) P,
It is similar to P. 1undensis, but there are differences in growth temperature.

Based on the above results, this bacterium was recognized as a new species of Pseudomonas, and was named Pseudomonas sp H881.
It has been deposited as FERM P-8955.

As a culture medium for polygalactosamine-degrading enzyme-producing bacteria, either a synthetic medium or a natural medium can be used as long as it contains a suitable amount of carbon sources, nitrogen sources, inorganic substances, and other nutrients. Suitable examples of the culture medium include:
Polygalactosamine 0.25%, glucose 0.25%
, yeast extract 0.05%.

An example is polypeptone 0.05%, pH 7.0.

The culture temperature is in the range of 20 to 40°C, preferably 30 to 38°C, and the culture starting pH is 6 to 8, preferably around 7 and 35 to 38°C.
If the culture is performed with shaking or deep stirring for 72 hours, polygalactosamine-degrading enzyme can be obtained in the culture solution. Then, the borigalac 1-samine degrading enzyme is isolated and purified as necessary.

For example, the crude enzyme is separated from the culture filtrate by ethanol precipitation, dissolved in an aqueous medium, and Sephadex G-
50 gel filtration, CM-Sephadex C-25 ion exchange chromatography, phenyl-Sepharose C
A purified polygalactosamine degrading enzyme is obtained by treatment such as L-4B hydrophobic chromatography.

When the novel polygalactosamine-degrading enzyme thus obtained is allowed to act on polygalactosamine, various galactosaminooligosaccharides can be effectively obtained. This treatment may be carried out according to a conventional method of hydrolysis using an enzyme, and examples thereof include the following method.

First, polygalactosamine is dissolved in a low concentration of acid. As the acid, in addition to organic acids such as acetic acid and formic acid, a wide range of inorganic acids other than sulfuric acid can be used.

After adjusting the pH of the polysaccharide solution obtained in this way, the polygalactosamine degrading enzyme prepared above was added,
Enzymatic decomposition is performed at an appropriate temperature of around 37°C.

The low-molecular decomposition reaction product is removed from the reaction solution, adsorbed onto an ion exchange resin, and then eluted with a solvent with an appropriate concentration gradient to obtain various galactosaminooligosaccharide components, which are then purified. The desired oligosaccharides are then obtained.

The target galactosaminooligosaccharide can be obtained alone or as a mixture by acid or alkaline hydrolysis or enzymatic decomposition as described above, alone or in an appropriate combination. That is, by hydrolyzing polygalactosamine as described above, each of galactosamine oligo-disaccharides to dodecaccharides can be obtained extremely effectively, and if necessary, an appropriate mixture of each oligosaccharide can also be freely obtained. This is possible.

The oligosaccharide obtained in this way was analyzed by IIPLC, TLC.
It can be used as a standard product such as chitin and chitosan oligomers, and is expected to have similar or different physiological activity to chitin and chitosan oligomers.
For example, since it has particularly promising antitumor activity, it can be used as a variety of pharmaceuticals or as raw materials or intermediates thereof.

Antitumor activity is expected not only for each oligosaccharide alone, but also for oligosaccharide mixtures (e.g., trisaccharides, tetrasaccharides).

In some cases, even stronger antitumor activity can be expected if the oligosaccharide is a mixture of pentasaccharides), and in any case, the oligosaccharide according to the present invention can be used as an antitumor agent. Also,
It can also be used as a food additive, nutritional supplement, health agent, agricultural chemical, and industrial chemical.

Next, examples of the present invention will be shown.

Example 1 Preparation of polygalactosamine 600 g of glucose, 60 g of polypeptone, 1□ CaC
・2t (20125g dissolved in 17Q tap water, 'aN
After adjusting to ρ117.0 with aol+ solution, it was transferred to a 30Q jar fermenter.

Pressurize this medium solution by injecting steam.

Heat sterilization (121°C, 20 minutes) was performed. Add 150+nR to the cooled medium (final liquid volume 20Q) in a 500n+12 Erlenmeyer flask (3% glucose, 0.3% polypeptone, 17.0% CaCl, 0.5%, p)
Benicillomyces I − was cultured with shaking at 26°C for 4 days.
IF[ERM P-3928 (FERMBP-118
0) was aseptically inoculated at a volume ratio of approximately 10%. 27 days after vaccination
The cells were cultured for 5 days under the following conditions: °C, aeration rate of 0.5 VVM, and agitation number of 20 ORPM.

After the culture is completed, culture filtrate i is obtained by filtering the culture with a filter cloth.
I got n. This culture filtrate was heated to 50°C to 60°C and passed through an ultrafiltration membrane with a molecular weight cutoff of 160,000 (UF membrane tubular module F type manufactured by Mitsubishi Rayon Engineering Co., Ltd.) to remove the low molecular fraction. It was concentrated until the volume of the removed liquid was about 30%. Furthermore, about 1.4000XG
Cell residue and heat-denatured proteins were removed by centrifugation.

After centrifugation, approximately 750 g of common salt (approximately 25% concentration) was added to supernatant fraction 3Q, stirred, dissolved, and plipped with concentrated NaOH.
+ was adjusted to 7.0 to 8.5. - The salted-out material was left to stand overnight, and the salted-out material was collected on Saran cloth (copolymer of vinylidene chloride and vinyl chloride).

Furthermore, a large amount of slightly alkaline water (pH
7.0 or more) to wash away excess common salt, neutral sugars, and other impurities mixed in the culture solution at the same time.

Next, approximately three times the volume of 0.1M hydrochloric acid solution was added to the salted out product after washing with water and dissolved. A concentrated NaOH solution was added to this lysate to adjust it to pl+8.5, which is the isoelectric point of polygalactosamine. - After leaving it overnight to precipitate the ten-analyte precipitate, the precipitate was collected on a saran cloth in the same manner as above and washed with a large amount of tap water. This washed product was dissolved once again in 0.1M hydrochloric acid, subjected to isoelectric precipitation, and purified by repeated washing with water.

The purified precipitate was sterilized at 121° C. for 15 minutes and then freeze-dried to obtain 7 g of purified powder of PF-102 containing polygalactosamine as a main component (about 99% purity as polygalactosamine).

Depending on the purpose, a part of the above purified powder is dissolved in 0.1M hydrochloric acid and fractionated using an ultrafiltration membrane with a molecular weight cutoff of 300,000 (Molecular Sieve Membrane Type XM 300 manufactured by Amicon), with an average molecular weight of 16
It can also be fractionated into those with an average molecular weight of ~300,000 and those with an average molecular weight of 300,000 or more.

Example 2 Preparation of galactosaminooligosaccharide 100 g of purified poly-galactosamine (PF102) was dispersed in 4N hydrochloric acid, 2Q, and hydrochloric acid hydrolyzed at 80° C. for 8 hours in an Erlenmeyer flask equipped with a cooling tube.

After decomposition, the hydrochloric acid solution was filtered through a filter paper to remove undecomposed residues, and about 100 g of activated carbon was added to decolorize the solution. next,
This solution was passed through a column (8X 75c+++) packed with anion exchange resin AG3X4A (manufactured by Bio-Rad, USA) to remove hydrochloric acid.

Next, the obtained galactosaminooligosaccharide mixture is activated and packed into a column using Sephadex C-25 (2
, 5X 100cI11) and after thorough washing with water, O ~
It was eluted with a linear concentration gradient of 2.5M NaCl, resulting in the fractionation of 12 peaks (Figure 1). In this case, ΔOD=2 nm is the measurement result of galactosamine by the indole-hydrochloric acid method.

The obtained galactosaminooligosaccharide of each peak was decolorized again with activated carbon, and if the degree of polymerization n < 4, it was decolorized using an electrodialysis machine and Micro Asylyzer G-1100 (manufactured by Asahi Kasei Corporation).
After desalting with
<n, desalt with an ultrafiltration membrane (Ul+-05 Ultrafilter Atovanchik 1-Yo Co., Ltd.),
It was concentrated and freeze-dried to obtain both gala-91 saminooligosaccharides. At this time, the recovered amounts of each of the two portions obtained are shown in Table 1.

In addition, when the optical rotations of each of the obtained galactosaminooligosaccharides were measured, the relationship shown in Figure 2 was established between the optical rotations and the degree of polymerization. It was found that they were eluted sequentially in ascending order of size. That is, FIG. 2 is a graph showing the relationship between the molecular optical rotation and the degree of polymerization of galactosaminooligosaccharides, where the product of the specific optical rotation [α]D and the molecular weight is the molecular optical rotation CM)n. .

(M)n/n and (n-1)/n have a linear relationship (n represents the degree of polymerization).

Both parts of Table 1 Dissolved salt concentration (mol) Galactosaminooligosaccharide (g) 1 0.27 11
．． 92 0.48 9.73
0.73 6.24
0.94 5.75 1
．． 16 3.66 1.3
3 3.07 1.58
2.38 1.73
2.19 1.86
1.610 1.99
1.111 2.10
1.012 2.22
0.8 Example 3 Preparation of polygalactosamine degrading enzyme Pseudomonas sp 8881. FERM P-89
55 in a 500 m 12 Erlenmeyer flask with 0.5 g of glucose.
5%, yeast extract 0.05%, polypeptone 0.05%
Inoculate 100 mρ of seed medium with the composition of
Incubate for 0 hours.

The obtained seed culture solution was placed in a 30Ω jar fermentor and added to an enzyme production medium containing 0.25% polygalactosamine (PF-102), 0.25% glucose, 0.05% yeast extract, and 0.05% polypeptone. Inoculated on 18Q and heated to 30℃
The cells were cultured for 48 hours at an aeration rate of IVVM and a stirring rate of 20 ORPM.

Centrifuge the obtained culture solution (14, OOOrpm) I
, remove the bacterial cells, add cooled ethanol to the obtained culture filtrate to a concentration of 60%, and precipitate the protein.
The precipitated protein is separated from the solution by centrifugation. The obtained protein was dissolved in 0.1 molar acetate buffer (pH 5,0
) equilibrated with a C tosephadex C-25 column (2
, 5×60 cm) and eluted using the same buffer with a gradient of O-0.5 molar saline.

The eluted enzyme activity fraction is collected and concentrated using an ultrafiltration device (molecular weight cut off: 10,000). Next, 0 containing 2 molar salt
．． A Sephadex G-50 column (5X 9
0 cm) chromatography. Next.

A phenyl-Sepharose CL-4B column equilibrated with the same solution with the salt concentration in the active section increased to 4 molar (
2,5X 20cm) and eluted with 0.1M acetate buffer with an inverse concentration gradient of 1 NaCl to purified polygalactosamine decomposed acid! 50 mg (yield 23.1%, specific activity 5
2 μg galN/min/mg protein).

Example 4 Preparation of galactosaminooligosaccharides (enzymatic degradation - Sephadex C-25 chromatography) 25 g of purified polygalactosamine was dissolved in 0.1 molar acetic acid of about 4.8 Q, and then adjusted to pH 6.0 with sodium hydroxide. The total liquid volume was adjusted to 5Q by adding water.

Using this polygalactosamine solution as a substrate, 5 mg (approximately 500 units) of purified polygalactosamine degrading enzyme (book 1)
The unit was enzymatically decomposed at 37°C for 1 hour by adding an enzyme titer capable of purifying 1 μmol of galactosamine per minute.

After decomposition, heat at 100℃ for 10 minutes to stop the enzyme reaction.
Insoluble matter was removed by centrifugation. Then, the pH of the obtained solution
was adjusted to 5.0 using acetic acid and adsorbed onto a weak cation exchanger C Tosephadex C-25 column (2X 40co+).

After washing the column with 0.1 molar acetate buffer (pH 5.0), it was eluted with a linear concentration gradient of 0 to 2.5 molar sodium chloride, and both isolated fractions were collected.

Both portions were desalted using an electrodialysis machine, Micro Acylyzer G3 (manufactured by Asahi Kasei Corporation), and freeze-dried to obtain purified galactosaminooligosaccharides.

At this time, the amounts recovered for each portion are shown in Table 2.

Table 2 (Galactosaminooligosaccharide) (g) Galactosamine 0.18 Galactosaminoligo 29 0.36 Galactosaminoligo 39 1.80 Galactosaminooligo-tetrasaccharide 1
．． 65 galactosaminoligo pentasaccharide 1.20 galactosaminoligo hexasaccharide 0.84 galactosaminoligo heptasaccharide 0.60 galactosaminoligo octasaccharide
0.48 Galactosaminoligo 9-saccharide 0.39 Galactosaminoligo-10saccharide 0.24 Galactosaminoligo 11&lf O,18 Galactosaminoligo-
12-saccharide 0.12 Example 5 Preparation of galactosamino-oligosaccharides (enzymatic degradation - Dowex 50W It was adjusted to ρ116.0, and the total liquid volume was 5Q. Using this polygalactosamine solution as a substrate, 10 mg of purified polygalactosamine-degrading enzyme (approximately 1000 units *1 unit is the enzyme titer that produces 1 μmol of galactosamine per minute)
was added and enzymatically decomposed at 37°C.

Decomposition reaction products with a molecular weight of 3000 or less are continuously removed from the reaction solution using a hollow fiber HIP-3 (manufactured by Amicon Far East Ltd., DC-2 type hollow fiber) and directly transferred to a cation exchange resin DAPEX 501i1. It was adsorbed onto X 8 (2.5X 50cm).

Elution of galactosaminooligosaccharides from DAPEX 50W x 8 was performed with a 0-4 molar hydrochloric acid concentration gradient.

Next, each of the resulting galactosaminooligosaccharide solutions was treated with an anion exchange resin CDR2O (manufactured by Mitsubishi Kasei) to remove hydrochloric acid. This solution was freeze-dried to obtain a purified galactosaminooligosaccharide. The amounts of each galactosaminooligosaccharide obtained are shown in Table 3.

Table 3 (Galactosaminooligosaccharide) (g) Galactosamine 0.8 Galactosaminoligo disaccharide 1.6 Galactosaminoligo 3
&! 7.0 Galactosaminooligotetrasaccharide 4.0 Galactosaminoligopentasaccharide 3.0 Galactosaminoligo-6
Sugar 1.2 Example 6 Preparation of galactosaminooligosaccharide <Hydrochloric acid decomposition - Dowex 501i1 X 8 chromatography>
25g of purified polygalac 1-samine in concentrated hydrochloric acid (12N)
It was dispersed in 250 mQ and hydrolyzed at 80°C for 4 hours.

Next, this solution was concentrated under reduced pressure to remove hydrochloric acid, and the galactosaminooligosaccharide was separated by column chromatography on Dapex 50W x 8 (2.5 x 50 cm) (eluted with a 0-5 molar hydrochloric acid concentration gradient). Purified.

Each purified galactosaminooligosaccharide was treated with AG3X4A to remove hydrochloric acid, and then lyophilized to obtain a purified galactosaminooligosaccharide.

The results are shown in Table 4.

Table 4 (Galactosaminoligo M) (g) Galactosamine 8.0 Galactosaminoligo disaccharide 6.0 Galactosaminoligo trisaccharide
4.0 Galactosaminoligotetrasaccharide 2.0 Galactosaminoligopentasaccharide 1.0 Galactosaminoligohexasaccharide 0.8 Galactosaminoligosaccharides were obtained in this way, but these are all new. The physical properties of galactosamine oligo-2l11Ili-12-12 are as shown below.

■, Name of substance: Galactosamine oligo-disaccharide 1) α-
Galactosamine disaccharides composed of only 1→4 bonds Ga1N1-+, Ga1N (however, Ga1N is a-D-
Indicates a galactosaminopyranosyl group. ) 2) Color and shape: Pale yellow amorphous powder 3) Taste: Sweet taste.

4) Solubility: Soluble in dilute acids (mineral acids such as hydrochloric acid or organic acids such as formic acid and acetic acid), aqueous solutions of salts such as sodium chloride, potassium chloride, and water, and common acids except dimethyl sulfoxide. It is sparingly soluble in organic solvents (methanol, ethanol, acetone, chloroform, etc.).

5) Show the following elemental analysis values.

C: 42.35, H: 7.06, N: 8.24
, O: 42.35 Expected molecular formula: C1□1(24
0'JN26) The molecular weight and structural formula are as follows.

Molecular weight: 340.2 7) Show the following color reaction.

Positive for indole-hydrochloric acid reaction, Elson-Morgan reaction, Somogyy-Nelson reaction, slightly positive for Morgan-Nilson reaction, Lowry-Forin reaction, negative for phenol-sulfuric acid reaction, and iodine reaction.

8) Optical rotation [α] et al’: +127.4 9) Melting point 164℃ 1.0) Figure 3 shows the ultraviolet absorption spectrum.

11) Figure 4 shows the infrared absorption spectrum.

2. Name of substance: Galactosamine oligo-trisaccharide 1) α
Galactosamine trisaccharide Ga1N1-)4GalN, -)4GalN (however, G
a1N represents a-D-galactosaminopyranosyl group.

) 2) Color and shape 2 ditto 3) Taste: Has a weak sweet taste.

4) Solubility 2 ditto 5) Show the following elemental analysis values.

C: 43.11. H: 6.99, N: 8.3
8, O: 41.52 Expected molecular formula: C1, H35
0□3Nff6) The molecular weight and structural formula are as follows.

Molecule lit: 501.3 7) Color reaction: same as above 8) Optical rotation [α]♂’: +158.3 9) Melting point It has a specific melting point. Carbonization begins at 160°C or higher.

10) Figure 5 shows the ultraviolet absorption spectrum.

11) Figure 6 shows the infrared absorption spectrum.

3. Name of substance: Galactosamine oligo-tetrasaccharide 1) α-
Galactosamine tetrasaccharide Ga1N1-+, Ga1N1-+4GalN1-+4G composed of only 1→4 bonds
alN (However, Ga1N represents an α-D-galactopyranosyl group.) 2) Color and shape: Same as above 3) Taste: Has a slight sweetness.

4) Solubility: Same as above 5) Show the following elemental analysis values.

C: 43.50, H: 6.95, N: 8.46
, O: 41.09 Expected molecular formula: C24H,,
0,□N46) The molecular weight and structural formula are as follows.

Molecular weight: 662.4 ■) Galactosamine pentasaccharide composed of only α-1 → 4 bonds Ga1N, -+4GalN1-+4GalN, -+40
a1. N□-+40alN (However, Ga IN is α-
Indicates 0-galactopyranosyl group. ) Color and shape Taste: Has a weak sweet taste.

Solubility: Same as above 7) Color reaction 2 ditto 8) Optical rotation [α]♂’: +174.1 9) Melting point: same as above 10) Figure 7 shows the ultraviolet absorption spectrum.

11) Figure 8 shows the infrared absorption spectrum.

4. Name of substance: Galactosamine oligo-pentasaccharide C: 4
3.74, H: 6.93. N: 8.51, O:
40.83 Expected molecular formula: C3o Hs 702
tNs6) The molecular weight and structural formula are as follows.

Molecule +ii: 823.4 7) Color reaction: same as above 8) Optical rotation [α]′D. : +183.8 Melting point: Same as above FIG. 9 shows the ultraviolet absorption spectrum.

Figure 10 shows the infrared absorption spectrum.

Name of substance: Galactosamine oligo-hexasaccharide α-1→4-linked galactosamine hexasaccharide Ga1N → 4calN → 4GaIN□ → 4GaIN
Engineering→4GaIN1→, Ga1N (However, Ga1N is a
-D-Galactopyranosyl group. ) Color and shape: Same as above Taste: Same as above Solubility: Same as above The following elemental analysis values are shown.

C: 43.90, H: 6.91. N: 8.5
4, ○: 40.65 Expected molecular formula' c, a
o68 o,,5 NG6) The molecular weight and structural formula are as follows.

Molecular weight: 984.6 7) Color reaction: same as above 8) Optical rotation [α] too’: +190.2 Melting point: Same as above FIG. 11 shows the ultraviolet absorption spectrum.

Figure 12 shows the infrared number spectrum.

Name of substance: Galactosamine oligosaccharide consisting only of α-1→4 bonds GalN, -+4GalN1-+4GalN1-+,
Ga1N1→4GalN□-+, Ga1N, -+4G
alN 2) Color and shape 2) Same as above 3) Taste: Same as above 4) Solubility: Same as above 5) The following elemental analysis values are shown.

C: 44.02, H: 6.90, N: 8.56
, O: 40.52 Expected molecular formula 2C4□H7,0
□9N? 6) The molecular weight and structural formula are as follows.

Molecule ffi: 1145.7 GalN1+, Ga1N, →-, GalN2) Color and shape 2 Same as above 3) Taste: Same as above 4) Solubility: Same as above 5) The following elemental analysis values are shown.

C: 44.10, H: 6.89, N: 8.58
．． 0: 40.43 Expected molecular formula: C45Hs
oOizNs6) The molecular weight and structural formula are as follows.

Molecular weight: 1306.8 7) Color reaction: same as above 8) Optical rotation [α] too’: +194.9 melting point 2 ditto FIG. 13 shows the ultraviolet thermal absorption spectrum.

FIG. 14 shows the infrared absorption spectrum.

Name of substance: Galactosamine oligo-8 & fα-1→4
8M of galactosamine composed only of bonds 7) Color reaction 2 ditto above 8) Optical rotation [α] also ': +198.5 9) Melting point: ditto 10) Figure 15 shows the ultraviolet heat absorption spectrum.

11) Figure 16 shows the infrared absorption spectrum.

8. Name of substance: Galactosaminooligosaccharide-9 sugar 1) α
Galactosamine 9-saccharide Ga1N, ÷4GalN1→-p4GalN, composed of only -1→4 bonds Color and shape:
Taste: Same as above Solubility: Same as above C: 44.17, H: 6.88, N: 8.
59, O: 36.06 Expected molecular formula: C, 41
＋□. 1.037N96) The molecular weight and structural formula are as follows.

Molecular weight: 1467.9 Melting point: Same as above FIG. 17 shows the ultraviolet thermal absorption spectrum.

FIG. 18 shows an infrared absorption spectrum.

Name of substance: Galactosamine oligo-1decaccharide α-1→4
Galactosamine's 10-saccharide Ga1N, which is composed only of bonds, →4GaIN1→y*4GalN2) Color and shape 2) Same as above 3) Taste: Same as above 4) Solubility: Same as above 5) The following elemental analysis values are shown.

C: 44.23, H: 6.88, N: 8.60
, O: 40.23 Expected molecular formula: CG, H engineering 12
041N Eng.

6) The molecular weight and structural formula are as follows.

Molecular weight: 1629.0 7) Color reaction: same as above 8) Optical rotation [α] et al’: +201.2 7) Color reaction: same as above 8) Optical rotation [α] et’: +203.4 Melting point: Same as above FIG. 19 shows the ultraviolet absorption spectrum.

FIG. 20 shows the infrared absorption spectrum.

Name of substance: Galactosamine oligo-11 sugar α-1→4
11M Ga1N□+4GalN1-)7+4GalN of galactosamine composed only of bonds Color and shape 2 Same as above Taste: Same as above Solubility: Same as above The following elemental analysis values are shown.

C: 44.27, H: 6.88, N: 8.61
．． O: 40.25 Expected molecular formula: CG1. H
izzO4sNx, 6) The molecular weight and structural formula are as follows.

Molecular weight: 1790.1 7) Color reaction 2 ditto 8) Optical rotation [α] is also 0: +205.2 Melting point: Same as above FIG. 21 shows the ultraviolet absorption spectrum.

FIG. 22 shows an infrared absorption spectrum.

Name of substance: Galactosamine oligo-12 sugar α-1→4
The 12-saccharide Ga1N of galactosamine, which is composed only of bonds, -←4 G a l N □ Output 4 Ga
l N Color and shape 2 Same as above Taste: Same as above Solubility 2 Same as above 5) The following elemental analysis values are shown.

C: 44.31, H: 6.87, N: 8.62
, O: 40.21 Expected molecular formula: CtzHt3
404gNtz6) The molecular weight and structural formula are as follows.

Molecular weight: 1951.2 7) Color reaction: same as above 8) Optical rotation (a) 3’: +206.8 9) Melting point: same as above 10) Figure 23 shows the ultraviolet absorption spectrum.

11) Figure 24 shows the infrared absorption spectrum.

(Effects of the Invention) The galactosaminooligosaccharide according to the present invention is a new compound that has not yet been published in any literature, and can be used in medicines, agricultural chemicals, food additives,
It is a compound useful as industrial chemicals and their intermediates.

Specific applications of the novel compound according to the present invention include, for example, a flocculant, an antitumor agent, and the like.

[Brief explanation of the drawing]

Figure 1 shows the C-tocephadex C-25 column chromatography pattern of the galactosaminooligosaccharide fractionated in Example 1, and Figure 2 shows the pattern of the galactosaminooligosaccharide fractionated on a C-tocephadex C-25 column. 1 is a diagram illustrating the relationship between molecular optical rotation and degree of polymerization. Section 3.5.7.9.11.13.15.17.19.2
Figure 1.23 is a diagram showing the ultraviolet absorption spectra of galactosamine oligo-disaccharides to dodecaccharides, respectively. 4.6.8, 10.12.14.1G, ■8.20.
Figures 22 and 24 are diagrams showing the infrared absorption spectra of galactosamine oligo-disaccharides to decasaccharides, respectively. Agent Patent Attorney Door 1) Parent Male 0, D492 0-4 ~ tn O (JI OUI NaCR (M) (n-1)/n Procedural Amendment Export) August 15, 1988 6, Amendment Target drawing 7, details of correction (1) Figure 21 will be corrected as shown in the attached sheet.

Claims (1)

[Claims] 1. Galactosaminooligosaccharide represented by the following formula: ▲ Numerical formula, chemical formula, table, etc. ▼ (However, in the formula, n represents 0 to 10). 2. A method for producing galactosaminooligosaccharide represented by the formula below, which is characterized by decomposing α-1,4-polygalactosamine: ▲There are mathematical formulas, chemical formulas, tables, etc.▼ (However, in the formula, n is (represents 0 to 10).