JP3064061B2 - Method for producing polysaccharide - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for producing a polysaccharide, and more particularly, to culturing a marine hydrogen assimilating bacterium in the presence of a culture gas to produce a polysaccharide, and culturing the obtained polysaccharide. The present invention relates to a method for producing a polysaccharide, which is separated from a liquid.

[0002]

BACKGROUND OF THE INVENTION Japanese Patent Application Laid-Open No. 53-11309
No. 2 includes Pseudomonas hydrogenogenora so. Belonging to the genus Pseudomonas isolated from paddy soil. no
v. Is cultivated in the presence of hydrogen, and a method for producing a polysaccharide that separates the polysaccharide from the culture is disclosed. However, the marine hydrogen assimilating bacterium Hydrogeno obtained from the marine environment is disclosed.
It is not known that vibriomarinus ( hydrogenobibrio marinus) can be cultured to produce polysaccharides, especially glucose polymers.

[0003] The present inventors have proposed Hydrogenovibrio ma, a hydrogen-assimilating bacterium already obtained from the marine environment.
As a result of various studies on the new use of rinus , it was found that by culturing the bacterium under specific conditions, it is possible to efficiently produce a polysaccharide, particularly a glucose polymer having glucose-like properties consisting only of glucose. The invention has been completed.

[0004]

In order to solve the problems of the present invention, Hydro
Genovibrio marine hydrogen assimilating bacterium H
hydrogenovrio marinus ( Hydogenovibrio marinus ) was converted to H ₂ , O ₂ and CO
It is intended to provide a method for producing a polysaccharide, comprising culturing in the presence of a culture gas containing ₂ as an essential component, and separating the polysaccharide from the obtained culture solution.

[0005] The marine hydrogen assimilating bacterium Hydrogenovibrio marinus used in the present invention.
(Hydrogenobibrio marinus) (hereinafter sometimes abbreviated as the present bacterium) has been deposited with the RIKEN Microbial Strain Preservation Facility under the Application No. 7688, and is easily available. The chemical properties are as follows. Gram stain - spore forming ability - motility + cell morphology comma shape size 0.2 to 0.5 × 1 to 2 [mu] m nutrients H ₂ and Na ₂ S ₂ O ₃ GC content 44.1 mol% cytochrome type b, c , O hydrogenase membrane-bound only

[0006] The culturing conditions of the present bacterium are a temperature of 25 to 45 ° C, preferably 32 to 42 ° C, and a pH of 5 to 8, preferably 6 to 42 ° C.
7, the salt concentration 0-1 mol, preferably 0.2 to 0.7 mol, H ₂ concentration of 70% by volume or more in the culture gas, preferably 78 vol% or more, O ₂ concentration from 5 to 40 volume%, preferably 5-20 volume%, CO ₂ concentration 7-15% by volume, preferably from 10 to 12 volume%, the optimal culture conditions are a temperature of 37
° C, pH 6.5, salt concentration 0.5M and O in culture gas.
_{2 The} concentration is 5 to 11% by volume.

The bacterium is the first bacteriophilic hydrogen bacterium to be isolated for the first time as an absolute autotroph, and is the first marine hydrogen-assimilating bacterium obtained from the marine environment.
This bacterium exhibits a very high growth rate as an autotroph with a specific growth rate of about 0.6 hr ^-1 in batch culture, and is good even at a partial pressure of O _{2 of} 40%, that is, under a concentration of 40% by volume in the culture gas. It is characterized by strong oxygen tolerance, which means that it grows well.

[0008] In obtaining preferable polysaccharide production conditions in the present invention, the reaction is carried out in a state in which some of the conditions for growing microorganisms are insufficient, that is, the rate-limiting reaction is carried out, whereby biological components such as sugars and proteins are obtained. In view of the fact that it is generally known to accumulate N, the O ₂ -limited, N-limited and Mg-limited were tried on the hydrogen-assimilating bacterium, and the following describes how the sugar content increases. explain. Here, O ₂ rate-limiting means that the growth curve becomes a stationary phase due to a decrease in dissolved oxygen concentration,
In other words, it means a state in which O ₂ is deficient in the stationary period. For example, culture gas H ₂ : O ₂ : CO ₂ volume ratio = 8: 1: 1
When the culture was started at 7: 2: 1 during the culture and continued during the culture, as shown in FIG. D. _540nm =
The O ₂ rate-limiting in about 30. The N rate control condition means that N is deficient in the stationary period, for example, (NH ₄ )
_The culture was started with 5 g / l of ₂ SO ₄ and the pH was adjusted with NH ₄
When the reaction was carried out with 6N NH ₄ OH until 10 ml of OH was introduced, and then switched to 6N NaOH, as shown in FIG. D. _At about _{540 nm} = 10, it becomes N rate limiting. In this case, Fe and Mg are added. The above Mg
The rate-limiting condition, when means that Mg is insufficient to stationary phase, for example, to start the cultured _{MgSO 4 · 7H 2 O 0.2g /} l, and the culture was continued without adding the way, FIG. 8 As shown in FIG. D. _At about _{540 nm} = 10, the rate becomes Mg-limited. In this case, Fe is added. Therefore, as the polysaccharide production conditions in the present invention, it is preferable to culture under the O ₂ rate-limiting condition in the stationary phase in that the amount of microorganisms is large and the production amount of polysaccharide is large.

The polysaccharide produced in the present invention is a polymer of glucose, and can be separated and purified from a culture solution obtained by culturing, for example, by the following method.

[0010] The culture solution obtained by the culturing in the present invention is diluted after heat treatment, centrifuged, and the obtained precipitate is washed with water, suspended in an alkaline solution to obtain the original scale. Next, the mixture is homogenized under ice cooling, stirred under a nitrogen stream, neutralized with an acid, added with celite, stirred, and centrifuged. Next, TCA is added to the obtained supernatant under ice-cooling, followed by centrifugation after standing. Then, the obtained supernatant is neutralized with an alkali, and ethanol is added thereto under ice cooling to form a precipitate, followed by centrifugation. Then, after dissolving in the obtained precipitated water, dialyzing the water, filtering through celite, adding ethanol under ice cooling to form a precipitate, and centrifuging. Then, the obtained sediment was reduced to 50
%, 70%, 90% ethanol, absolute ethanol, and ethyl ether, and dried in vacuo to obtain a purified polysaccharide.

The polysaccharide obtained in the present invention was assayed for purity by UV scanning of 0.1% aqueous solution and gel filtration. FIG. 1 shows a UV scan of a 0.1% by weight aqueous solution of the polysaccharide obtained in the present invention. In FIG. 1, the vertical axis represents absorbance, and the horizontal axis represents wavelength. FIG. 1 shows that the polysaccharide is a pure polysaccharide containing no protein or the like. FIG.
2 shows the pattern of gel filtration of the polysaccharide of the present invention, that is, the molecular weight distribution. FIG. 2 shows that the polysaccharide of the present invention gives a single peak having an average molecular weight of about 5 × 10 ⁶ . 2, the vertical axis represents the absorbance, and the horizontal axis represents the fraction number. Here, the gel filtration conditions are as follows: Column Sehalose CL-4B Eluate 0.1 M phosphate buffer Flow rate 34 ml / h Column calibration Various dextrans (manufactured by Sigma) Sample concentration 0.9 wt% (4 ml) FIG. It can be seen that the polysaccharide has an average molecular weight of about 5 × 10 ⁶ .

The polysaccharide obtained by the method of the present invention has the following characteristics with respect to the constituent components of the polysaccharide described in “Experimental Method for Starch and Related Carbohydrates”, published by Gakkai Shuppan Center (1986), pp. 105 and 139. have. (%) Rabbit liver (%) ^* β-amylase degradation rate 62 49 Average unit chain length 36 18 Average external chain length 24 11 Average internal chain length 1 16 * Agricurtural and Biological Chemirtry, 45 , 2
Rabbit liver polysaccharide analysis method described on pages 09-216 (1981).

[0013] Characteristics of the polysaccharide obtained by the present invention (Char
The actuation is as follows. (1) The detection of amino acids and amino sugars by ninhydrin coloring is negative. (2) As a result of analysis by TLC, most of the color development considered to be glucose is found, and all other spots are trace amounts. (3) The analysis results by HPLC are shown in FIG. In FIG. 3, the vertical axis represents the refractive index, and the horizontal axis represents time. FIG.
This shows that glucose is the only constituent sugar of the glucose polymer.

The polysaccharides obtained in the present invention are advantageous for production when the growth rate is high, and are advantageous for isolated production when they are glucose polymers. On the other hand, although it is a glucose polymer, its production is limited, but it is conceivable that breeding is performed so that the polysaccharide is dissolved in the culture solution. Conversely, since it is a glucose polymer, it can be expected to have some physiological activity.

[0015]

According to the present invention, the cultivation of marine hydrogen assimilating bacteria having a high growth rate is advantageous for isolation and production, and is a glucose polymer having the characteristics of glucose homopolymer. Sugars can be easily and industrially advantageously produced.

[0016]

The present invention will be described in more detail with reference to the following examples.

Example 1 The following composition: (NH ₄ ) ₂ SO ₄ 2.0 g K ₂ HPO ₄ 2.5 g KH ₂ PO ₄ 0.5 g MgSO ₄ .7H ₂ O 0.2 g NaCl 29.3 g CaCl ₂ 10. _{0mg FeSO 4 · 7H 2 O 10.0mg} NiSO 4 · 7H 2 O 0.6mg trace component solution 2ml deionized water 1000 ml pH 7.0 trace component solution _{MoO 3 1.0mg ZnSO 4 · 7H 2} O 7.0mg CuSO 4 · 5H ₂ O 0.5mg H in _{2 BO 3 1.0mg MnSO 4 · 5H} 2 O 1.0mg CoCl medium 1000ml made by culture with ₂ · 6H ₂ O 1.0mg deionized water 1000ml, marine hydrogen Assimilating microorganisms Hydrogenovibrio
marinus (hydrogenobibrio marinus ) bacterium was inoculated by 10% of the reactor medium, and H ₂ : O ₂ :
CO ₂ volume ratio = 8: 1: 1 mixed gas (1 liter / m
in), and shaking culture was started at 37 ° C. under a stirring speed of 1000 rpm while adjusting the pH of the medium to 6.5 with 6N ammonia. In reactor culture (N
H ₄₎ using _{2 SO 4 5.0 (g / l} ). Was added _{0.2g / l FeSO 4 · 7H 2} O after the culture after 50 minutes, 2 g after culture initiation 6 hours / l MgSO ₄ · 7H ₂
O was added, and 11 hours after the start of cultivation, D.
_{When the 540 nm} value becomes about 8, the composition of the mixed gas is changed to H
_The volume ratio of ₂ : O ₂ : CO ₂ was changed to 7: 2: 1. Start of culture 10, 12, 15, 18, 22, 26,
6N after 30, 34, 40, 44, 48 and 53 hours
NH ₄ OH was converted to 3,15,23,35,45,57,6
The pH was adjusted by adding 8, 78, 90, 97, 105 and 113 ml, and a culture experiment was carried out for 53 hours. Bacterial growth is O. D. Detection was performed at _{540 nm} , and the obtained polysaccharide was quantified by the phenol sulfate method. FIG. 4 shows the time-dependent changes in the growth of the bacterial cells and the production amount of the polysaccharide. FIG. 4 shows that the polysaccharide content continues to increase with time even when the growth of the bacteria reaches the stationary phase. The culture results were as follows. Culture time 53 h Liquid volume after culture 1.6 l OD ₅₄₀ 70 Dry cell weight 33 g / l Cell wall component (about 10%) 33 x 0.1 = 3.3 g / l Sugar concentration of culture solution 9.87 g / l Polysaccharide concentration 9.87-3.3 = 6.57 g / l Total polysaccharide content 6.57 × 1.6 = 10.51 g Polysaccharide content per cell 9.87 / 33 = 0.30 g / gdcw Sugar in supernatant 0.57g

The obtained polysaccharide was separated and purified according to Shemcl using a glucose polymer. The details are as follows. After completion of the culture, the cell culture solution is heated at 90 ° C. for 10 minutes, diluted with distilled water,
The supernatant was removed by centrifugation at 7000 rpm for 10 minutes. Then, the separated sediment was washed with water and 1N Na
Suspend in OH to make the original scale, homogenize under ice-cooling, stir at room temperature under nitrogen stream for 3 hours, neutralize with HCl, add celite, stir for a few minutes, and centrifuge at 9000 rpm for 20 minutes. Was removed. Then, TCA was added to the separated supernatant under ice-cooling to a concentration of 5%.
The mixture was kept at 4 ° C. overnight, and the precipitate was removed by centrifugation at 9000 rpm for 30 minutes. Then, the separated supernatant was added to 6
The mixture was neutralized with N NaOH, and precipitated by adding 50% ethanol under ice-cooling, and the supernatant was removed by centrifugation at 6000 rpm for 10 minutes. Next, the separated precipitate is dissolved in water to make a solution of about 1%, dialyzed against water, filtered through celite, precipitated with 40% ethanol under ice cooling, and centrifuged at 6000 rpm for 10 minutes to remove the supernatant. Was removed. Next, the separated sediment was reduced to 50%, 70%.
%, 90% ethanol, absolute ethanol, and ethyl ether, and dried under vacuum to obtain 2.53 g of purified polysaccharide. The initial amount of cells was 35 g. The obtained purified polysaccharide was confirmed by the above-mentioned gel filtration and UV scanning.

Example 2 The same procedure as in Example 1 was repeated except that the culture gas composition H ₂ : O ₂ : CO ₂ volume ratio = 8: 1: 1 was not changed on the way.
A 3-hour culture experiment was performed. FIG. 5 shows the time course of the growth of the bacterial cells and the production amount of the polysaccharide. 1.9 (g / l) of purified polysaccharide was obtained. From FIG. 5, it can be seen that, under these culture conditions, in the stationary phase, the sugar content also became steady and did not increase, and it was found that the amount of bacteria increased during growth, so that the sugar also increased. . According to the culture results, O. D. _{540 nm} 20 and sugar concentration 1.90 (g /
l).

[0020] Example 3 cultures 5 hours after the initiation of _{0.2g / l FeSO 4 · 7H 2} O
10 g after the start of the culture, 2 g / l MgSO _4.
7H ₂ O was added, and 8, 10, and 12 hours after the start of the culture, 3 ml, 8 ml, and 1 ml of 6N NH ₄ OH were added, respectively.
A 30-hour culture experiment was performed in the same manner as in Example 1 except that 0 ml was added, and 15 hours and 30 hours after the start of the culture, 23 ml of 6N NaOH was added to adjust the pH. FIG. 6 shows the time course of the growth of the bacterial cells and the production amount of the polysaccharide. 1.75 (g / l) of purified polysaccharide was obtained. From FIG. 6, it can be seen that the sugar content is not large as compared with the case of O ₂ rate-limiting, and that in the stationary period, the increase in sugar becomes steady, and that no sugar is accumulated as in the case of O ₂ rate-limiting. The results obtained were as follows. Culture time 30 h OD ₅₄₀ 18.3 Dry cell weight 8.6 g / l Cell wall component (about 10%) 8.6 × 0.1 = 0.86 g / l Sugar concentration in culture 1.75 g / l Polysaccharide concentration 1.75-0.86 = 0.89 g / l Amount of polysaccharide per cell 0.89 / 8.5 = 0.10 g / gdcw

Example 4 0.2 g / l of Example 1
_{FeSO 4 · 7H 2 O, 0.1g} / l FeSO 4 ·
Instead of adding 7H ₂ O and 2 g / l MgSO ₄ .7H ₂ O, 0.1 g / l Fe
It was added SO ₄ · 7H ₂ O, culture initiation 11,13,1
After 6,20,24,28,38 and 57 hours, respectively 6NNH ₄ OH 8,11,14,18,20,2
A 57-hour culture experiment was performed in the same manner as in Example 1, except that 2, 24, 26 and 26 ml were added to adjust the pH, and the culture gas composition was not changed. 1.9 (g / l)
Was obtained. FIG. 8 shows the time course of the growth of the bacterial cells and the production amount of the polysaccharide. FIG. 8 shows that the sugar content is not so large as compared with the O ₂ rate-limiting. The results obtained were as follows. Incubation time 40 h OD ₅₄₀ 20 Dry cell weight 9.4 g / l Cell wall component (about 10%) 9.4 × 0.1 = 0.94 g / l Sugar concentration of culture solution 1.90 g / l Polysaccharide concentration 1. 90-0.94 = 0.96 g / l Amount of polysaccharide per cell 0.96 / 9.4 = 0.10 g / gdcw

[Brief description of the drawings]

FIG. 1 is a graph of a UV scan of a 0.1% aqueous solution of a polysaccharide of the present invention.

FIG. 2 is a graph of a gel filtration pattern of the polysaccharide of the present invention.

FIG. 3 is a graph showing the results of HPLC analysis of the polysaccharide of the present invention.

FIG. 4 is a graph showing the time-dependent changes in bacterial growth and polysaccharide production in the present invention.

FIG. 5 is a graph showing changes over time in the growth of bacteria, the production amount of polysaccharides, and dissolved oxygen in the present invention.

FIG. 6 is a graph showing the time-dependent changes in bacterial growth and polysaccharide production in the present invention.

FIG. 7 is a graph showing changes over time in the growth of bacteria and the production of polysaccharides in the present invention.

────────────────────────────────────────────────── ─── Continued on the front page (58) Fields surveyed (Int. Cl. ⁷ , DB name) C12P 19/00-19/64 BIOSIS (DIALOG) WPI (DIALOG)

Claims (2)

(57) [Claims] 1. A belonging to Hydrogenovibrio genus marine hydrogen utilizing bacteria Hydrogenovibri
o marinus (hydrogenobibrio marinus)
Is cultured in the presence of a culture gas containing H ₂ , O ₂ and CO ₂ as essential components, and the polysaccharide is separated from the obtained culture solution. 2. The method for producing a polysaccharide according to claim 1, wherein the polysaccharide is a glucose polymer.