JP3031534B2 - How to make arabinose from beet arabinan - 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 arabinose in a high yield by decomposing beet arabinan at a high decomposition limit.

[0002]

2. Description of the Related Art Arabinose is an elegant sweet saccharide having a sweetness of about 80% of that of sucrose (sugar), and is hardly absorbed from the mucous membrane of the intestinal tract. It is known that sucrose and maltose are digested by arabinose when excreted in the urine and taken up by metabolic system and do not become an energy source. It was found that absorption was hindered, and it merely acted on the taste buds as a sweet substance and did not become an energy source, that is, it had disaccharide hydrolase inhibitory activity.

[0003] For this reason, arabinose has attracted much attention as a carbohydrate food, and development of an industrial production method thereof has been desired.

[0004] L-arabinose is present in hemicellulose at high concentrations as arabinan, arabinoxylan, gum arabic and arabinogalactam.

[0005] On the other hand, pulp discharged after collecting sucrose from beet contains 12% arabinose per dry weight of the pulp.
Arabinose in these beet pulp exists as arabinan, which is a polysaccharide in which many of them are linked in a chain, although about 13% is present.

[0006] Of these arabinans, 30% of the arabinans have a main chain of 1,5 bonds of arabinose, and every other molecule has another 1,3 bonds of another arabinose, a so-called "comb tooth shape". L-arabinose having the shape of "", and the other 70% are unidentified arabinans.

[0007] As a method capable of recovering arabinose from beet pulp, a so-called "alkali extraction-acid decomposition method" in which a polysaccharide is extracted with an alkali and the extracted polysaccharide is decomposed with an acid, is called an alkali extraction of the polysaccharide. So-called "alkaline extraction-enzymatic decomposition method" in which the extracted polysaccharide is decomposed with an enzyme
Can be considered.

The "alkali extraction-acid decomposition method" is generally used for producing oligosaccharides and monosaccharides from polysaccharides for research purposes such as structural analysis.

Regarding the "alkaline extraction-enzymatic decomposition method", a study on the decomposition of arabinan in beet pulp with an enzyme is carried out by A. Kaji, H. Taki, A. Shimazaki, T. Shinkai (Tech. Bull. Fa.
c.Aga.Kagawa Univ., 15, 34 (1963) or Kusakabe, Yasui,
Kobayashi (Journal of the Japanese Society of Agricultural Chemistry, 49,295 (1975)), L.Weinstei
n, P. Albersheim (Plant Physiol 63, 425 (1979)).

Of the above two methods, the "alkali extraction-acid decomposition method" requires strong conditions such as extreme pH and high temperature, and it is difficult to set reaction conditions in terms of equipment or handling operation, and therefore, industrial scale There are difficulties in doing so.

There is also a problem that a large amount of the used acid or alkali remains in the recovered arabinan, which greatly increases the cost in the purification step.

On the other hand, the "alkali extraction-enzymatic decomposition method"
It is considered that it is most suitable for extracting arabinose from beet pulp as a raw material because H can perform a mild reaction in the vicinity of neutral and the temperature is around room temperature, the industrial conditions can be easily set, and the purification load is light. .

[0013]

In the "alkaline extraction-enzymatic decomposition method", enzymes obtained from various microorganisms are used.

For example, A. Kaji et al. (Tech. Bull. Fac. Aga. Kag
awa Univ., 15, 34 (1963)) is Aspergillus niger (Asp.
ergillus niger), but the degradation rate of beet arabinan is as low as 53%.

A. Kaji et al. (Biochica et Biophysica Ac
ta, 410, 354 (1945)) or S. Kaneko et al. (Appl.Environ.Mi
crobiol., 60, 3425 (1994)) is Bacillus subris
lussubtilis), the degradation rate of beet arabinan was 3.3% or 15%, respectively.
% And low.

On the other hand, Kusakabe et al. (Journal of the Japanese Society of Agricultural Chemistry, 49, 29)
5 (1975)) is Aspergillus nige
It has been reported that the 100% degradation limit was reached in the reaction using the enzyme of r).

However, although this enzyme has a high decomposition limit, the enzyme activity is 8 mg arabinose / 5 ml enzyme solution / 30 min reaction, and the amount of the enzyme that produces 1 micromol of arabinose per minute is defined as 0.3 unit. Very low, converted to units / ml.

As described above, the conventionally known enzymes are not satisfactory in terms of decomposition limit or enzyme activity for industrially recovering arabinose from total arabinan using beet pulp as a raw material.

On the other hand, the present inventor has conducted a broad search from nature for strains producing beet arabinan-degrading enzyme with a high titer, which degrades beet arabinan at a high decomposition limit. Bacillus subtilis (Bacil subtilis) isolated from soil collected from the primeval forest in the Tsugenmoto area
lus subtilis) H-4 and a Bacillus subtilis sp. strain belonging to homologous and homologous taxonomic classification were found to be suitable.

[0020]

Accordingly, the present invention has been developed based on the above findings by culturing a Bacillus subtilis spp. That produces a high titer of a beet arabinanase that degrades beet arabinan at a high decomposition limit. The present invention proposes a method of obtaining an arabinan-decomposing enzyme and degrading beet arabinone using the enzyme to obtain arabinose.

The H-4 strain, which is a representative example of the Bacillus subtilis species used in the present invention, has the following mycological properties. The test and classification of mycological properties are described in "Bergey's Manual of Systematic Bacteriology".
Bacteriology) (1986) ".

A. Form 1. 1. Cell shape Bacillus 2. Cell size 0.8 × 1.9 μm Mobility +4. Presence of spores +5. Gram stain +

B. Physiological properties Casein degradability +2. Starch degradability +3. DNAse degradability -4. Urea decomposability -5. Gelatin +

C. Acid generation Dextrose +2. Cellobiose +3. Galactose-4. Mannose +5. Melibiose + 6. Raffinose-7. Salicin + 8. Xylose + 9. ONPG +

D. Growth in 10% saline -E. Growth under anaerobic conditions -F. Voges-Proskauer test + G. Oxidation reaction -H. Growth on NGKG medium-I. Decomposition of hippurate + J. Growth at 50 ℃ +

K. Carbohydrate metabolism Glycerin + 2. Erythritol-3. D-arabinose-4. L-arabinose + 5. Ribose + 6. D-xylose + 7. L-lyxose-8. Adonitol-9. β-methyl-D-xyloside-10. Galactose-11. Glucose +12. Fructose + 13. Mannose + 14. Sorbose-15. Rhamnose-16. Darcitol-17. Inositol +18. Mannitol + 19. Sorbitol + 20. α-methyl-D-mannoside-21. α-methyl-D-glucoside + 22. N-acetylglucosamine-23. Amygdalin + 24. Arbutin + 25. Esculin + 26. Salicin + 27. Cellobiose + 28. Maltose + 29. Lactose-30. Melibiose +31. Sucrose +32. Trehalose + 33. Inulin +34. Meletitose-35. Raffinose + 36. Starch +37. Glycogen + 38. Xylitol-39. Gentiobiose + 40. D-Turanose + 41. D-lyxose-42. D-tagatose-43. D-fucose -44. L-fucose-45. D-arabitol-46. L-arabitol -47. Gluconsan Salt-48.2-Keto-Gluconsan Salt-49.5-Keto-Gluconsan Salt-

Biological properties β-galactosidase +2. Arginine dihydrolase-3. Lysine decarboxylase-4. Ornithine decarboxylase-5. Hydrogen sulfide productivity-6. Urease-7. Tryptophan deaminase-8. Indole productivity-9. VP reaction +

Based on the above bacteriological properties, the above strain was identified as Bacillus subtilis H-4, and this strain was deposited with the National Institute of Bioscience and Human-Technology, National Institute of Advanced Industrial Science and Technology. Subtilis H-4 (FER
M P-15482).

The above H-4 strain is most preferred as the Bacillus subtilis spp. That produces a high-titer beet arabinan-degrading enzyme that degrades beet arabinan at a high decomposition limit suitable for use in the present invention. IFO (fermentation research institute, same hereafter) 3007, IFO3108, IFO3134, IFO3335, IFO3336, IFO14140
Or ATCC (American Type Culture Collection, same hereafter) 6633, ATCC6984, ATCC7003, ATCC7058, ATCC
Bacillus subtilis strains such as 7060 and ATCC 7067 can be used.

The present invention also provides a Bacillus subtilis strain other than the above strain having the ability to produce an enzyme capable of degrading beet arabinan at a high decomposition limit with high activity, and a natural or artificial mutation of these strains. The resulting mutant strain can be used.

In culturing the above microorganisms, any of a commonly used solid medium and liquid medium may be used, but a liquid medium is preferred. The medium used for culturing the microorganism can use a carbon source that can be used by the microorganism as the carbon source, but is preferably araban, starch, sucrose, or glucose, and as the nitrogen source, yeast extract, casein, Natural nitrogen sources such as corn steep liquor, peptone, meat extract and the like and inorganic nitrogen compounds such as ammonium sulfate, ammonium salt, urea and sodium nitrate can be used.

The concentration of the carbon source is 1 to 20%, and the concentration of the nitrogen source is 1 to 5%.
The culture temperature is 20 to 35 ° C., the culture time is about 24 to 72 hours, and the culture can be carried out by aeration stirring culture or shaking culture.

Hereinafter, the present invention will be described in detail with reference to examples, but the present invention is not limited to these examples.

Example 1 (A) Preparation of total arabinan To 3.7 kg of beet pulp, 12 L of 2.5% calcium hydroxide solution was added, heated at 90-100 ° C. for 12 hours, cooled, and adjusted to pH 6.0 with acetic acid.
And then filter with a filter cloth. Ethanol was added to the obtained filtrate to generate a precipitate, and the precipitate was collected by filtration.
After dissolving in water, ethanol precipitation was performed again to obtain crude arabinan. Crude arabinan consists of cation exchange resin Diaion PK216 and anion exchange resin Diaion WA
After desalting using No. 30 and drying, 55 g of total arabinan was obtained.

(B) Culture medium Total arabinan 10 g Peptone 10 g Yeast extract 5 g Sodium nitrate 5 g Dipotassium phosphate 1 g Magnesium sulfate heptahydrate 0.5 g Tap water 1000 ml pH 7.0

In a 500 ml Erlenmeyer flask, 100 ml of the medium having the above composition was taken, and 2 ml of a culture solution of Bacillus subtilis H-4, which had been pre-cultured in the same medium, was inoculated at 30 ° C.
For 72 hours at 220 rpm. After cultivation,
The culture was centrifuged (1000 RPM) to remove the cells and used as an enzyme solution. Using 450 μl of a 2% total arabinan solution (pH 6.5, 0.1 M phosphate buffer) as a substrate, adding 50 μl of the enzyme solution to it, and performing a reaction at 40 ° C. for 10 minutes to measure the enzyme activity
It was 10 u / ml. Here, the enzyme activity 1u refers to the amount of the enzyme which decomposes the total arabinan of the substrate to produce 1 μM arabinose per minute.

(C) Decomposition example of arabinan 1 0.08 U of the enzyme was added to 1.0 ml of 2% total arabinan solution (arabinose purity 72%, 0.1 M phosphate buffer, pH 6.5), and the mixture was added at 40 ° C.
After the 18Hr reaction, 0.08U of the enzyme was added again, and the reaction was continued until 74Hr. When the reaction solution was analyzed by high performance liquid chromatography, 83.3% of the total arabinose was released.

(D) Decomposition example of arabinan 2 0.1 U of the enzyme was added to 1.0 ml of 2.5% total arabinan solution (arabinose purity 71.8%, 0.1 M phosphate buffer, pH 6.5) and the mixture was heated at 40 ° C.
After the reaction for 18 hours, the enzyme was further added again by 0.1 U, and the reaction was continued until 74 hours. Analysis of the reaction solution by high performance liquid chromatography revealed that 74.5% of the total arabinose was released.

Example 2 75% of the culture supernatant obtained by culturing and removing bacteria in the same manner as in Example 1
Ammonium sulfate was added to reach saturation, and the mixture was allowed to stand at 4 ° C. overnight, and the resulting precipitate was collected by centrifugation. This precipitate fraction is
It was dissolved in a phosphate buffer of pH 6.5 and 0.1 M and further dialyzed against the same buffer to obtain a crude enzyme solution. The crude enzyme solution was diluted and adjusted with the same buffer so that the enzyme activity became 15 u / ml.

(E) Decomposition example 3 of arabinan Crude enzyme prepared in Example 2 in 1.0 ml of 2% total arabinan solution (arabinose purity 72%, 0.1 M phosphate buffer, pH 6.5) was added.
After adding 08 U and reacting at 40 ° C. for 18 hours, the enzyme was further added again with 0.08 U to continue the reaction until 74 hours. When the reaction solution was analyzed by high performance liquid chromatography, 80.2% of arabinose in total arabinan was liberated and produced.

Comparative Example 1 (G) Preparation of Enzyme Solution The IF03007 strain purchased from the Fermentation Research Institute was cultured by the same method as in (B) of Example 1, and an enzyme solution was prepared. When the enzyme activity was measured, 5.3u /
ml.

(H) Example 4 of decomposition of arabinan 0.07 u of the enzyme was added to 450 μL of 2% arabinan solution (arabinose purity 72%, 0.1 M phosphate buffer, pH 6.5), and the reaction was carried out for 45 hours. Analysis by liquid chromatography revealed that 82% of the total arabinose was released.

Comparative Example 2 (I) Preparation of Enzyme Solution An enzyme solution was prepared from the IF03108 strain purchased from the Fermentation Research Institute, in the same manner as in (G) of Comparative Example 1. The enzyme activity was 4.7u / ml.

(J) Decomposition example 5 of arabinan As in (H) of Comparative Example 1 , the enzyme was added to 450 μL of a 2% arabinan solution.
After adding 0.06 u and reacting for 45 hours, the reaction mixture was analyzed by high performance liquid chromatography to find that 81% of total arabinose was present.
% Was liberated.

Comparative Example 3 (I) Preparation of Enzyme Solution An enzyme solution was prepared from the IF014140 strain purchased from the Fermentation Research Institute, in the same manner as in (G) of Comparative Example 1 . The enzyme activity was 1.8 u / m.

(J) Decomposition of Arabinan 5 In the same manner as in (H) of Comparative Example 1 , the enzyme was added to 450 μL of a 2% arabinan solution.
After reacting for 68 hours after adding 0.06 u, the reaction mixture was analyzed by high performance liquid chromatography to find that the total arabinose was 81%.
% Was liberated.

Comparative Example 4 (I) Preparation of Enzyme Solution AT purchased from American Type Culture Collection
An enzyme solution was prepared for the CC6633 strain in the same manner as in (G) of Comparative Example 1 .
The enzyme activity was 1.7u / ml.

(J) Decomposition of arabinan 5 In the same manner as in (H) of Comparative Example 1 , the enzyme was added to 450 μL of a 2% arabinan solution.
After reacting for 72 hours after adding 0.06 u, the reaction mixture was analyzed by high performance liquid chromatography to find that 82% of the total arabinose was present.
% Was liberated.

[0049]

In summary, the present invention is to decompose total arabinan in beet pulp at a high decomposition limit using beet arabinan degrading enzyme obtained from Bacillus subtilis spp. Is big.

──────────────────────────────────────────────────続 き Continued on the front page (56) References Agric. Biol. Chem. 54 [4] (1990) p. 879-889 Journal of the Japanese Society of Agricultural Chemistry 54 [7] (1980) p. 561-567 (58) Fields surveyed (Int. Cl. ⁷ , DB name) C12P 19/14

Claims (1)

(57) [Claims] 1. Bacillus subtilius (Bacillus subtilius)
tilis) H-4 (FERM P-15482) is cultured to obtain beet arabinan-degrading enzyme, and the enzyme is used in beet pulp.
A method for producing arabinose from beet arabinan, comprising decomposing total beet arabinan to obtain arabinose.