JPS63219390A - Production of chitin oligosaccharide - Google Patents

Abstract

PURPOSE:To produce chitin oligosaccharide in high yield of hydrolysis, by treating chitin such as colloidal chitin with a culture filtrate of ray fungus No.25 (FERM P-9208) and biologically hydrolyzing. CONSTITUTION:Chitin is treated with a supernatant liquid obtained by cultivating ray fungus No.25 (FERM P-9208) in a liquid medium containing chitin as only one carbon source to produce produce chitin oligosaccharide. Colloidal chitin is preferably as the chitin and the hydrolysis reaction temperature of chitin is preferably 30-70 deg.C, more preferably 40-60 deg.C. To be more concrete, hydrolysis reaction of chitin can be carried out in a buffer solution in a neutral range, such as phosphoric acid buffer solution at pH 7, at 30-70 deg.C. Colloidal chitin previously made into a swollen state is used as substrate chitin.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to a method for biologically degrading chitin to produce physiologically active chitin oligosaccharides.

More specifically, the present invention relates to a method for producing chitin oligosaccharide with improved decomposition yield by biologically decomposing chitin using a culture filtrate of a new strain of Streptomyces nu 25.

[Prior art] Chitin is the β-1 form of N-acetyl-D-glucosamine, which was discovered and named from beetles by A.
It is a type of mucopolysaccharide consisting of 4 bonds, and its official name is (1
→4) -2-acetamido-2-deoxy-D-glucan. Its chemical structure is shown in the following formula, and it is a glucan in which the hydroxyl group at the 2-position of the glucose structural unit is substituted with an acetamido group.

Chitin polysaccharide and its water-soluble or sparingly soluble lower homologue N-acetyl-chito-oligosaccharide (hereinafter referred to as chitin oligosaccharide) are known to have an immune function-promoting effect (March 1985). The 2nd Chitin and Chitosan Symposium sponsored by the Chitin and Chitosan Research Group held on the 22nd and 23rd, and the collection of lecture abstracts, pages 3 to 6). That is,
Chitin oligosaccharides, especially their tetrasaccharides to heptasaccharides (chitotetraose, chitopentaose, chitohexaose, chitoheptaose), cause a significant increase in blood polymorphonuclear leukocyte (PMN) and peritoneal exudate cell (PEC) numbers. , these induced PMNs and PECs have strong active oxygen producing ability and antimicrobial activity, especially candidacidal activity. Thus, chitin oligosaccharides have a physiologically active action that can contribute to early biological defense through activation of phagocytes.

One of the conventionally well-known methods for producing chitin oligosaccharides is a method of hydrolyzing chitin polysaccharides with concentrated acid or concentrated alkali.

Biochit Biophys, Acta, 83
(1964) 245-255 decompose chitin in concentrated hydrochloric acid and fractionate the oligosaccharides produced using an activated carbon-Celite column, but most of the decomposed sugars obtained by this method are monosaccharides. Occupied by N-7 cedyl glucosimine,
Only a small amount of chitobiose and chitotriose are produced, and the amount of oligosaccharides larger than tetraose, which have the above-mentioned useful physiological activities, produced is extremely small.

Furthermore, a method of biologically decomposing chitin, that is, decomposing chitin into its oligosaccharides using chitinase, a chitin degrading enzyme, is also known.

J. Riot, Chew, Vol. 254.
No, 11. pp4901-4907゜1979 and tFJ, Biol, Chcg+, Vol, 2
57. No, 3. pp1392-1397.198
2 is the production of new chitin (chitosan and UDP-GIC) by endochitinase collected from wheat malt and yeast, respectively.
Chitin synthesized using MAC and chitin synthase)
It is described that chitotetraose and chitopentaose are produced from However, the amount of chitotetraose and chitopentaose produced by these biological degradation methods is only at a level that can be detected by analytical methods using radioactive isotopes, and they cannot be isolated and purified. It cannot be used effectively.

[Objective of the present invention] As mentioned above, as long as the conventional method for producing chitin oligosaccharides is used, chitin oligosaccharides of tetraose or higher, which have a useful physiological effect of promoting immune function, cannot be used for laboratory-scale bioactivity research. Although it is possible to obtain a usable amount, it cannot be expected to be stably supplied to the extent that it can be used effectively to actually exhibit its physiological activity.

Therefore, an object of the present invention is to ensure a stable supply of chitin oligosaccharides of tetraose or higher. In addition, in the method of biologically decomposing chitin, the decomposition yield is improved so that chitin oligosaccharides of tetraose or more are decomposition products and the resulting layer can be isolated and purified for effective use. The purpose is to improve In addition, it is also an object of the present invention to provide microorganisms that are optimal for such decomposition.

"Structure of the present invention" The present invention is based on the discovery that the culture filtrate of 25 strains of actinomycetes isolated and fixed from the soil of the San'in region promotes efficient decomposition of chitin into constituent oligosaccharides. Based on (
.

The method for producing chitin oligosaccharides of the present invention using this Actinomycete No. 25 is based on Actinomycetes No. 25. It consists of allowing the culture filtrate of No. 25 to act on chitin.

[Detailed description of the invention] The present invention will be specifically explained below.

Actinomycetes No. 25 The means for creating Actinomycetes No. 25 used in the method of the present invention and its general mycological properties are as follows.

(a) Creation Method Several soil samples were collected on April 4, 1986 from paddy soil near Miyazu City, Kyoto Prefecture, and were screened, cultured, and stored as follows.

Screening: ■ Suspend 1 scoop of soil supertil in 10 d of sterile water,
After standing still for about 30 seconds, the supernatant liquid O, 5 was transferred to a square bottle containing 10 d of culture medium (same medium as described in Example 1 described below. The same applies hereinafter), sealed with a silicone stopper, and heated at 50°C. It was statically cultured for 1 day.

■ 5 cc of the culture solution obtained in ■ was added to the medium 1011d.
Transfer to a square bottle containing 100 ml of water, add a silicone stopper, and heat at 50°C.
The cells were cultured with shaking for days (reciprocating shaking at 705 strokes/min).

0.5 cC of the culture solution obtained in 2) was transferred to a square bottle containing 10 ae of medium, a silicone stopper was applied, and the bottle was cultured with shaking at 50°C for 5 days (705 strokes/5 inches).

■ The culture solution obtained in ■ - Platinum loop was mixed with colloidal chitin (
It was spread on a plate solidified with 2% agar containing colloidal chitin (prepared in Example 3 described later) and heated at 50°C.
It was statically cultured for 1 day.

■ Isolate the colony that appeared in ■ and add it to the medium 10 Ill.
! Transfer to a square bottle containing 51 (70
Strokes/win) L/win was used as a storage culture solution.

Strain homogeneity is determined by using a Bennett agar plate.
◇Culture method: Chitin medium (same medium as described in Example 1 described later)
Transfer 1 cc of the stock culture solution to a 500-capacity Erlenmeyer flask containing 200 rtdl, and culture at 50°C for 5 days (1 cc).
00 rpm, one rotary speed.

Storage method: The bacterial cells obtained by the above culture method were freeze-dried in a vial according to a conventional method, sealed and stored at 4°C.

(b) Growth status in various media (C) Physiological properties■ Growth temperature range: 25-55℃ (5σ℃ is optimal)■
Optimum pH for growth: Around near ■ Liquefaction of gelatin: Liquefaction.

■Hydrolysis and bicomposition of starch.

■ Production of melanin-like pigment: Produced.

(d) Assimilation of various carbon sources (expressed as 10÷, +1±, -).

: ■ L-arabinose − ■ D-xylose − ■ D-glucose ++ ■ D-fructose − ■ Sucrose ± ■ Inositol + ■ L-rhamnose ± ■ Raffinose ~ ■ D-Mannitol ++ Actinobacteria 25 of the present invention One of its characteristics is that its growth temperature is relatively high (25 to 55 degrees Celsius, especially 30 to 55 degrees Celsius, especially 55 degrees Celsius).
It is a thermophilic bacterium that is optimal after 0″Cm).

Generally, thermophilic bacteria are few among the tIi bacteria.

The second characteristic of Actinobacteria 25 of the present invention is expressed by the thermal stability of its spores. That is, 5 Id of the culture solution grown in chitin medium was aseptically aliquoted into test tubes, sealed with silicone rubber stoppers, and incubated in a heating bath for a certain period of time (0, 5, 10, 2
(0, 30, 60, 90 minutes) heated to 95°C, cooled in cold water immediately after heating, inoculated onto a plate of 5ennet medium, and cultured at 50°C, no colonies were observed from any of the treated bacterial solutions. appeared. As described above, the spores of Actinobacterium sp. 25 exhibit resistance to heat treatment at 95° C. for 90 minutes.

In addition, although it is generally known that actinomycetes have a relatively slow growth rate, actinomycetes No. 1 of the present invention. The specific growth rate μlaX of 25 is 0.82 (Tryptic Soy B
td (roth), and the td (doub
ling time) is 0.84 hours, so it is characterized as a fairly fast-growing actinomycete.

In order to identify the taxonomic position of the actinomycete Nα25 of the present invention, amino acid and diamino acid analyzes of its cell wall were performed according to conventional methods.
It was revealed that the cell wall composition was I. Furthermore, when the sugar in the bacterial cells of the same isolated strain No. 25 was identified according to a conventional method, the sugar pattern was determined to be B or C. From these analysis results, actinomycete NCL25 is considered to be a Streptomyces-related bacterium.

Therefore, when we investigated the differences between this strain and the previously known thermophilic strains of the genus Streptomyces, we found the following significant differences.

Streptomyces casei (5trel) tolyCe
s Ca5ei): Actinobacteria Nα25 hydrolyzes starch, but Streptomyces casei does not hydrolyze starch.

Streptomyces rectus (5treptosyce)
Streptomyces rectus): Streptomyces rectus 25 liquefies gelatin and peptonizes skimmed milk, whereas Streptomyces rectus does not liquefy gelatin and does not peptonize skimmed milk.

Str Leptomyces thermosiastateichus (Str
eptomyces thermodiastatic
us): Actinobacterium Nα25 peptonizes skim milk, but Streptomyces thermosiastateicus does not peptonize skim milk.

Streptomyces thermofuscus (5trepto-
1yCeS thermorusctls): radial radiation 1
The growth temperature for order 25 is 55°C at most, but Streptomyces thermofuscus grows even at 65°C.

Streptomyces thermoviolaceus (Strep
toIyces thergiov+o+aceus)
: Actinobacterium NG25 is positive for nitrate reduction, but Streptomyces thermoviolaceus is negative for nitrate reduction.

Streptomyces thermovulgaris (Strepto
lyccs thermovulgaris): Actinobacteria no. The growth temperature of Streptomyces thermovulgaris is 55°C at most, but Streptomyces thermovulgaris can grow at 60°C.

Based on the above data, the actinomycete Nf125 of the present invention was determined to be a new strain.

In addition, actinomycete 1se 25 of the present invention was submitted to the Institute of Microbial Technology, Agency of Industrial Science and Technology on February 20, 1985 (Accession Number), Microbiological Research Institute No. 9208 (FEIIHP-9208).
It has been deposited with.

Culture method and filtrate preparation When culturing Actinomycete 25, the medium used must contain chitin as a carbon source. Preferably, it is a completely synthetic medium containing only chitin as the sole carbon source. By using a chitin medium, the culture filtrate of Streptomyces 81125 will contain chitinase, which is a chitin-degrading enzyme, as described below.

In addition, the customary phosphates, nitrates and other inorganic salts can be present in the culture medium.

The optimal pl+ of the culture medium is adjusted to approximately 7.

Examples of preferred medium compositions are as follows.

Na HPO・12H2o 1-5g/I2KH2P
O41~3 〃 NH4NO31~5 〃 Mg5o ・7H200,3~2 〃 Fe50 -7820 0~0.5 ”CaC1・
2H2o O~0.5 Chitin
1~10n Trace gold 1"O・~1.Od Distilled water or tap water 1000 dpH6~
8 Examples of Ningwei5 metal salts Hoe34 Rg ZnSO4zu820 28N CuSO-5HO2// H3B034 N HnSO-5tl O4II CoCj22-6H204u Distilled water 1000 m Cultured under aerobic conditions F with stirring, as it is a thermophilic bacterium It is carried out at a temperature of 45-52°C.

Culture medium used in the method for producing chitin oligosaccharide of the present invention! The filtrate is a supernatant after culturing actinomycete Nα25 in this manner and removing solid components such as chitin and bacterial cells by centrifugation or the like. Actinobacteria no. No. 25 assimilates the carbon source chitin during the above culture and releases chitinase, a chitin-degrading enzyme, outside the bacterial cells, which is accumulated in the culture filtrate. The obtained supernatant is preferably concentrated by ultrafiltration and used in the next chitin decomposition reaction.

Production of chitin - The culture filtrate prepared as described above is applied to chitin. Chitin is biodegraded into its constituent sugars by chitinase, an extracellular enzyme derived from actinomycete 25, contained in the culture filtrate, but the yield of the decomposition products is small, especially oligosaccharides of tetrasaccharides or higher, especially pentasaccharides. many. The chitinase produced by Streptomyces NG25 has high activity in producing oligosaccharides of tetrasaccharide or higher, and in this respect it is clearly different from the chitinases derived from conventional wheat malt, yeast, etc. It is possible to distinguish between

The decomposition reaction of chitin is carried out in a neutral buffer solution, for example, p11.
7 in phosphate buffer at a temperature of 30-70°C, preferably 4
It can be carried out at 0 to 60°C. The substrate chitin concentration is preferably 0.1-1% (w/w) in the buffer solution.

As the substrate chitin, colloidal chitin which has been brought into a wB wet state in advance may be used. Colloidal chitin is obtained by dissolving chitin in concentrated acid and dispersing it in cold water or cold aqueous ethanol. In particular, when using a culture filtrate in the growth induction phase, which is before the logarithmic growth phase of actigenesis, it is preferable to use colloidal chitin.

[Effects of the present invention] According to the method of the present invention, it is possible to improve the decomposition yield of chitin oligosaccharides of tetrasaccharide or higher having useful physiological activities as described above, and to recover the chitin oligosaccharides by isolating, purifying, and recovering them. By doing so, the inherent physiological activity of the chitin oligosaccharide can be fully utilized.

[Example] Non-limiting examples of the invention are provided below.

11 - Actinomycetes Nα25 stored at 4℃
208), 5m of preserved bacteria was added to 50ml containing 200d of culture medium.
Aseptically inoculate a 0ag volume triangular flange with a navel at 50°C.
, 100 rpl for about 72 hours.

The composition of the medium was as follows.

N a2HPO4・12H2049/flKH2P04
3 llNHNO31' MGSo ・7H200,5# Fe50 -7H20o, ol tt CaC1-2H200,01N Chitin 3 n Trace metal salt*
0.5m! Distilled water
1000 at! pH7.0 slightly suspended metal salt Hoe34 q ZnSO-7tl 0 28 〃CuS04-5
H202n t13BO34n HnSO-511204n C0C1' 2 ・6112o 4 N Distilled water
1000 m The thus obtained preculture solution is aseptically inoculated into a 2 g jar fermenter containing 3 of the same medium composition as above. The main culture was carried out at a temperature of 50°C and with stirring at 500 rpm.

The test was carried out under the condition of ventilation fjk 11/main.

Sampling was performed over time during the main culture, and the sampling solution was allowed to stand for 5 minutes to precipitate chitin.

The absorbance (540 ni) of each supernatant thus obtained was measured, and the absorbance (540 ni) of each supernatant obtained was determined. When the growth curve of 25 was determined, it was as shown in Fig. 1 (logarithmic growth curve).

115-U Centrifuge the culture solution obtained at point A in Figure 1 (10,OO
Oxg, 20 l1lin) L/, and 2d of the supernatant (culture filtrate) was used for the chitin decomposition reaction.

For the dispersion containing the substrate chitin, chitin (purified chitin provided by Katokichi ■) was mixed with 50 mM phosphate buffer (
It was prepared by suspending it in pH 7) at a concentration of 0.32% (w/w).

Add 2 d of the above supernatant liquid to 2 ml of chitin dispersion liquid, and stir at 50°C for 1 hour.
The reaction was carried out by shaking for a time (1305 strokes/ml). The reaction solution was boiled for 5 minutes to stop the reaction, centrifuged (10,0OOX (]120 m1n), and the supernatant liquid was subjected to high performance liquid chromatography (IIPLc) to isolate and purify the produced chitin oligosaccharide. .

The analysis conditions for high-performance liquid chromatography are as follows, and 210 n for detection of chitin oligosaccharides.
m UV was used.

Column: ULTRON-NO3 (φ4.6X25) Eluate: On+in CH3CM/H20=7/3↓
Linear gradient 45+ein CC113C/It20-4/6↓Linear gradient 50+ein CH3CM/H20-7/301n Flow rate: 1 d/a+in Detection: UV 210 nm Column temperature:
40°C In this example, 1 g (dry weight) of starting chitin to 2. Chitopentaose of Qay was obtained.

As in Example 2, the culture filtrate at point A in FIG. 1 was used for the chitin decomposition reaction. However, colloidal chitin was used as the substrate chitin.

Colloidal chitin was prepared as follows. In other words, 10g of chitin (provided by Katokichi) was mixed with cold salt II.
500ae, filtered through a sintered filter, and the filtrate was suspended in 45001i! of cold deionized water to make the chitin into a colloid. Repeat centrifugation and washing with deionized water, prepare 5 ml of H with 28 NaOH, and centrifuge again. The final colloidal chitin weight is 0.
Suspend in 50 mM phosphate buffer y-(p)17) to give a concentration of 26% (W/W).

The above-mentioned supernatant liquid 2Id of point A was added to the colloidal chitin liquid 2- prepared in this way to carry out a chitin decomposition reaction. The decomposition reaction was carried out by shaking at a temperature of 50 °C (1305 tro
kes/win) I proceeded with L. After the decomposition reaction for 1 hour, the reaction solution was boiled for 5 minutes to stop the reaction, centrifuged (10,000 x g, 20 in) and the supernatant liquid was analyzed by HPLC under the same conditions as in Example 2. did.

In this example, starting chitin (colloidal chitin)
Chitopentaose was isolated and purified in an amount of 10.81119 per gram of dry amount ffi.

Friend-1 Colloidal chitin was decomposed in the same manner as in Example 3. However, the culture filtrate of actinomycete Nα25 used was B in Figure 1.
It was a supernatant liquid.

"Loidal chitin solution 2d prepared in the same manner as in Example 3"
2 m of the supernatant liquid from the above 8 points was added to the solution, and the decomposition reaction was allowed to proceed under the same conditions as in Example 3. The supernatant after a reaction time of 1 hour was analyzed by HPLC. The analytical conditions for IIPLC were the same as in Example 2.

In this example, starting chitin (colloidal chitin)
15 to 15 chitopentaose were produced per 19 dry amounts of Jl, an amount that could be sufficiently isolated and purified.

[Brief explanation of the drawing]

FIG. 1 shows the logarithmic growth curve of actino rNHQ25 according to the invention.

Claims (5)

[Claims] (1) Actinomycetes No. A method for producing chitin oligosaccharides, which comprises allowing the culture filtrate of No. 25 (Feikoken Kyoiku No. 9208) to act on chitin. (2) The culture filtrate is grown in a liquid medium containing chitin as the sole carbon source. 2. The method according to claim 1, wherein the method is a supernatant obtained by culturing No. 25. (3) The method according to claim 1 or 2, wherein the chitin is colloidal chitin. (4) Chitin decomposition reaction at 30-70°C, preferably at 40°C
4. A method according to any one of claims 1 to 3, characterized in that it is carried out at a temperature of ~60<0>C. (5) Actinomycetes No. 25 (Feikoken Bibori No. 9208).