JP2894292B2 - Galactanase S-2 and Bacillus sp. S-2 producing the same - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a novel galactanase and a novel microorganism producing the same.

[0002]

2. Description of the Related Art Conventionally, dietary fiber and the like have been decomposed by acid decomposition, alkali decomposition or enzymatic decomposition. However, in these methods, monosaccharides are often generated, and dietary fiber or the like is modified (for example, However, in the case of using oligosaccharides for solubilization, low molecular weight, etc.), the yield is reduced.

Among dietary fibers, fibers derived from plants such as citrus fruits, potatoes, and soybeans have a main chain of β-1,4 galactoside bonds, and β is an enzyme that hydrolyzes the β-1,4 galactoside bonds. Although -1,4 galactanase is known, conventionally known β-1,4 galactanase also degrades to monosaccharide (galactose).

On the other hand, fungi (for example, those originating from Penicillium citrinum) and bacteria (for example, originating from Bacillus genus) are known as microorganisms that produce β-1,4 galactanase. It hydrolyzes 4 galactoside bonds to produce monosaccharides (galactose) and produces less galacto-oligosaccharides.

[0005]

As described above, when dietary fiber is hydrolyzed with an acid, an alkali, an enzyme, or the like, a large amount of monosaccharides is produced, and the yield of oligosaccharides is reduced.

[0006] The present inventors (1) have produced a very small amount of monosaccharides (such as galactose) even when hydrolyzing dietary fiber, and are able to obtain oligosaccharides (such as galacto-oligosaccharides) in high yield. And (2) a novel microorganism producing such galactanase.

[0007]

Means for Solving the Problems As a result of intensive studies to achieve the above object, the present inventors have been able to screen microorganisms belonging to the genus Bacillus that produce enzymes suitable for the above purpose from soil in nature (see Table 1). Bacillus subtilis is shown), and the desired galactanase can be produced, separated, and identified (Table 2 shows mold-derived and Bacillus s.
ubtilis-derived galactanase), which has led to the completion of the present invention.

That is, the present invention relates to galactanase S-2 having the following enzymatic properties. a. Action: Acts on soybean fiber to produce galactooligosaccharides and monosaccharides (galactose)
Is hardly released. b. Substrate specificity: acts on β-1,4 galactan but not β-1,3 galactan.
c. Working pH is 9-12. d. Working temperature is 30-60 ° C. e. The optimum pH is 10.0. f. pH stability is 4-12. g. Temperature stability is less than 50 ° C. h. The optimal temperature is 50 ° C. i. Molecular weight is 4
Ten thousand.

The present invention also relates to Bacillus sp. S-2 (Microtechnical Laboratories No. 11229) which belongs to the genus Bacillus and produces galactanase and has the following mycological properties. a. Physiological properties, etc .: (1) Denitrification reaction is negative. (2) Positive gas generation from glucose. (3) The use of citric acid was negative for the Cosa medium. (4) The pH range for growth is 7.5-10.
b. Positive anaerobic growth, negative VP test, negative casein hydrolysis. c. Nitrate reduction is negative.

[0010]

BEST MODE FOR CARRYING OUT THE INVENTION The following is a table listing the main properties of the screened bacteria which differ from known Bacillus subtilis.

[0011]

[Table 1] Mycological properties However, * 1 is Bacillus subtilis (quoted from the Barges Manual) * 2 is Bacillus sp. S-2 and the following is a known mold (Penicillium) among the enzymatic properties of galactanase obtained from Bacillus sp. S-2 Origin, B
acillus subtilis derived from K-50, Bacillus subtilis
It is a comparison table with those derived from var amylosacchariticus and Bacillus subtilis WT168.

[0012]

[Table 2] Enzymatic properties The substrate specificity is β1,4 galactan. The reaction product of * 1 is Gal << Gal2 <Gal3, the reaction product of Gal4 * 2 is Gal, and the reaction product of Gal2 * 3 is Gal, Gal2 * 4. The reaction product is Gal-Gal4 * 5, and the reaction product is Gal-Gal4. * 1 is galactanase S-2 * 2 is mold (Penicillium) -derived galactanase (Agric.
Biol. Chem, 49, 3445 (1986).

* 3 indicates galactanase derived from Bacillus subtilis K-50 (cited from Agric. Biol. Chem, 35, 1891 (1971)).

* 4: Bacillus subtilis var amylosacc
harilium-derived galactanase (Agric. Biol. Chem, 36, 1
946 (1972) quoted.

* 5 is galactanase derived from Bacillus subtilis WT168, J. Biol. Chem., 251, 5904 (1976).

First, the galactanase of the present invention will be described. The enzymatic properties of the galactanase of the present invention are as described above.

The reaction product of galactanase S-2 is
Gal is extremely low (almost no), Gal3 than Gal2,
It mainly produces Gal4.

The method for measuring the enzyme activity and the buffer used for the method are as follows. [Method of measuring enzyme activity] 0.1 ml of the enzyme solution was mixed with 0.5 ml of a 0.1 M buffer solution containing 1% purified soybean fiber as shown in the following table, and reacted at 40 ° C for 60 minutes. After the reaction, centrifuge (1
The total amount of sugar in the supernatant obtained by 5,000 RPM × 1 minute) is measured by the phenol sulfate method. One minute per minute under these conditions
The amount of the enzyme that produces a sugar corresponding to μmol galactose is defined as one unit.

[Buffer] ------------------------------------------- -pH range buffer -------------------------------------------- 3 -4 Citrate buffer 4-6 Acetate buffer 6-8 Phosphate buffer 8.5-10.5 Glycine-NaCl-NaOH buffer 11-12 KCL-NaOH buffer ------------- ------------------------------- Next, a microorganism that produces (2) galactanase of the present invention will be described.

The microbiological properties of this microorganism are as follows. 1. Microscopic observation of the morphology indicates bacilli. The size of the cells is 0.5-0.7 μm × 1.5-3.0 μm. One end of the cells is an ovoid endogenous spore (0.5-1.0 μm × 1.0-2.
0 μm). It also has periflagellate and has motility.

2. Gram stainability is positive. 3. No acid resistance.
b. The growth state and the like (hereinafter the numerical values are% by weight) are as follows.

(1) Gravy agar plate medium (Difco Nutrient br
Growth on oth0.8, agar 1.5, Na2CO3 1.0, pH 10.0) is normal. The shape of the colony is circular. The surface is smooth,
The periphery is smooth. The color is light ocher transparent and shiny,
(2) Growth on gravy agar slant medium (same composition as gravy agar plate medium) is normal, and Bacillus sp.
It is glossy and the color tone is light ocher transparent.

(3) Liquid broth liquid medium (Difco Nutrient broth
It grows at 0.8Na2CO3 1.0, pH 10.0) but has no surface growth. Next, c physiological properties and the like are as follows.
1 medium (KNO30.1, Difco Nutrient broth 0.8, Na
Reduction of nitrate by 2CO3 1.0, pH 10.0) is negative.

The denitrification reaction using the same medium was negative. 2. Medium (peptone 1.0, glucose 0.5, Na2CO3 1.
0, pH 10.0) is negative for Bacillus sp. S-2.

3. Medium (peptone 1.0, NaCL 0.5, Na2CO3
1.0, pH 10.0) negative for indole formation.

4. Medium (Difco Nutrient broth 0.8, Na2C
O31.0, pH 10.0) and medium (indicated amount of Difco SIM medium, Na2CO3 1.0, pH 10.0) with lead acetate paper method, and SI
Production of hydrogen sulfide in M medium was both negative.

5. Medium (soluble starch 1.0, polypeptone 0.
5, Yeast extract 0.5, K2HPO4 0.1, MgSO4.7H2O 0.0
2. Decomposition of starch by agar 1.5, Na2CO3 1.0, pH 10.0) is positive.

6. The gelatin decomposition activity by the medium (Difco Nutrient broth 0.8, gelatin 0.4, agar 1.5, adjusted to pH 10.0 with NaOH) is positive.

7. Medium (Skim milk 1.0, polypeptone
0.5, yeast extract 0.5, K2HPO4 0.1, MgSO4 ・ 7H2O 0.0
2. Casein degradation by agar 1.5, Na2CO3 1.0 pH 10.0) is negative.

8. Regarding the use of citric acid, (1) Cosa medium (NH4H2PO4 0.1, K2HPO4 0.1, citric acid 0.2, NaCL
0.5, MgSO4.7H2O 0.02, Na2CO3 1.0, pH 10.0) are negative. However, (2) Christensen medium (yeast extract 0.0
5, cysteine hydrochloride 0.01, sodium citrate 0.3, N
aCL 0.5, glucose 0.02, KH2PO4 0.1, agar 1.5, Na2CO
3 1.0, pH 10.0) is positive.

9. Medium (glucose 1.0, NaCL 0.5, MgSO4
・ 7H2O 0.02, K2HPO4 0.1, NH4H2PO4 or KNO3 0.1, Na
HCO3 1.0, pH 9.2) uses inorganic nitrogen sources for nitrates,
Negative for both ammonium salts.

10. Medium (soluble starch 1.0, polypeptone
0.5, yeast extract 0.5, K2HPO4 0.4, MgSO4.7H2O
02, agar 1.5, Na2CO3 1.0, pH 10.0) negative for pigment formation.

11. Medium (KH2PO4 0.1, Na2HPO4 0.1, urea 2.1, yeast extract 0.01, 0.02% phenol red solution
5.0, pH 7.0).

12. Medium (soluble starch 1.0, polypeptone
0.5, yeast extract 0.5, K2HPO4 0.1, MgSO4.7H2O 0.
02, agar 1.5, Na2CO3 1.0, pH 10.0) is positive for Bacillus sp. S-2.

13. Medium (soluble starch 1.0, polypeptone 0.5 yeast extract 0.5, K2HPO40.1, MgSO4.7
H2O 0.02, agar 1.5, Na2CO3 1.0, pH10.
Catalase by 0) is positive.

14. A suitable growth temperature range is 20 to 45 ° C, and an optimum growth temperature range is 25 to 40 ° C. (2) The growth pH range is suitably 7.5 to 10, and the optimal growth pH range is 8 to 9.5.

15. Table 3 shows the presence / absence of acid and gas generation from saccharides.

[0038]

[Table 3] -------------------------------------------- Saccharic acid Generated 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 ++ (11) D-sorbitol ++ (12) D-mannitol ++ (13) inosit ± ± (14) glycerin ± ± (15 ) Starch + + -------------------------------------------- 16. The growth in the medium containing salt grows up to 15% of salt. 17. Production of dihydroxyacetone is negative.

Regarding the above mycological properties, the Bergy's Manual of Determinative Bacterio
The results of comparison and search with reference to the 8th edition indicate that these bacteria are a member of the genus Bacillus, a spore-forming bacillus, but some of the known Bacillus strains are identical. No, this strain was determined to be a new strain. This strain was named Bacillus sp. S-2 and was entrusted to the Research Institute of Microbial Industry. No. 11229.

The microorganism of the genus Bacillus of the present invention can be cultured in a medium to produce galactanase.

As the carbon source, nitrogen source and the like used in the medium, any can be used as long as they can be assimilated. For example, as a carbon source, soybean meal, plant fiber such as wheat bran, waste molasses, starch,
Various sugars and the like, and nitrogen-containing substances such as various amino acids, corn steep liquor, soybean flour, peptides, yeast extract, urea and the like can be used as a nitrogen source.

In addition, minerals, vitamins and the like can be used as needed. After culturing the microorganism of the genus Bacillus of the present invention, the cells can be removed by known means such as centrifugation and filtration to obtain a crude enzyme solution. Further, a crude enzyme can be obtained by drying or the like using means such as salting out, dialysis, and ultrafiltration. It can be further purified to obtain a purified enzyme.

[0043]

The embodiments of the present invention will be described below with reference to examples. Example 1 About 0.5 g of soil collected from the vicinity of Yonago City, Tottori Prefecture, was suspended in sterile water, applied to an agar medium for separation (* 1), and cultured at 37 ° C. for 1 to 2 days to form colonies. Those that formed a clear zone (halo) based on the decomposition of galactan around the colonies were selected, and galactanase-producing bacteria were obtained. Mycological properties were as described in the detailed description section of the invention.

Next, these obtained bacteria were further added to a liquid medium (*
2), and cultured with shaking at 37 ° C. for 2 days. The supernatant obtained by centrifugation (10,000 RPM × 10 minutes) was measured for galactanase activity by the following method (* 3).

By the above method, bacteria having a strong galactase activity were screened, and one of them was Bacillus sp.
2 was obtained.

* 1 (agar medium) pH 10.0 Okara [Proplus SA manufactured by Fuji Oil Co., Ltd.] 1% Yeast extract 0.5% K2 HPO4 0.1% MgSO4.7H2O 0.02% Na2 CO3 1% Agar 1.5% * 2 (liquid medium) pH 10.0 Okara ("Proplus SA" manufactured by Fuji Oil Co., Ltd.) 2% Polypeptone 0.5% Yeast extract 0.5% NaCl 5% K2 HPO4 0.1% MgSO4.7H2 O 0.02% Na2 CO3 1% * 3 (The method for measuring galactanase activity is the above-mentioned enzyme activity measuring method) Example 2 Bacillus sp S- obtained in Example 1 The two strains were inoculated into a liquid medium (same as * 2 in Example 1), cultured with shaking at 37 ° C. for 40 days, and the supernatant obtained by centrifuging the cells was used as crude enzyme galactanase S-2. . The galactanase activity at pH 10 (by the method * 3 in Example 1) was 0.9 units / ml.

Next, 2 g of the above-mentioned crude enzyme galactanase S-2 was added to 1 g of okara (“Proplus SA”, manufactured by Fuji Oil Co., Ltd.).
Five units were added and reacted at pH 10.0 and 40 ° C. for 6 hours.

After the reaction, the enzyme was inactivated by heating at 100 ° C. for 3 minutes, 150 ml of ethanol was added, the precipitated fraction was removed by centrifugation (6000 RPM × 10 minutes), and the supernatant was concentrated under reduced pressure. Galacto-oligosaccharides were obtained in a% yield.

When the obtained galactooligosaccharide was analyzed by HPLC (high performance liquid chromatography) under the following conditions, galactobiose 9%, galactotriose 3
Galactose was 3%, galactotetraose 38%, galactopentaose 16% and galactose 4%.

(HPLC conditions) Column: Sensepak (NH2-1251N)
4.6 × 250 mm Column temperature: room temperature Eluent: acetonitrile / water = 2/1 (V / V) Flow rate: 1 ml / min Detector: differential refractometer Comparative Example 1 Commercially available cellulase (Sigma, originated from Penicillium genus) ) And commercially available cellulase (Polyporus manufactured by Kyowa Hakko Kogyo Co., Ltd.)
Genus) was added to okara in the same manner as in Example 2 in 25 units each, and reacted at pH 4.5 for 6 hours.
An oligosaccharide was obtained.

As a result of analysis by HPLC, the former yield was 54%, and galactose as sugar was almost 100%. The latter had a yield of 57% and sugars of 44% galactose, 26% galactobiose, 24% galactotriose and 6% galactotetraose. Example 3 An example in which it does not act on β-1,3 galactan.

Galactanase S-2 obtained in Example 1
Was reacted with β-1,3 galactan (manufactured by Sigma) under the conditions of Example 2, but none of them decomposed. Example 4 (Optimal pH and pH stability) The optimal pH of galactanase S-2 obtained in Example 1 was examined by the above [enzyme activity measurement method]. The results are shown in FIG.

FIG. 2 shows the results obtained by measuring the residual activity after maintaining at each pH for 15 hours at 20 ° C. and examining the pH stability. Example 5 (Optimum temperature and temperature stability) The optimum temperature and temperature stability of galactanase S-2 obtained in Example 1 (at each temperature)
The residual activity after treatment for 15 minutes was measured) according to the above [enzymatic activity measurement method]. The results are shown in FIGS. Example 6 Galactanase S-2 obtained in the same manner as in Example 1 was purified by the following chromatography method, and electrophoresed together with a marker by SDS polyacrylamide gel electrophoresis. As shown in FIG. One peak was shown. This indicates that galactanase S-2 has a molecular weight of about 40,000. [Purification by Chromatography] Ammonium sulfate was added to 5 l of galactanase crude enzyme solution so as to be 80% saturated with ammonium sulfate, and the resulting precipitate was collected by centrifugation at 7,000 RPM × 20 minutes, and collected by centrifugation.
dissolved in 1 l of water. After dialysis against 20 mM Tris-HCl buffer (pH 7.0) at 4 ° C for 24 hours, a DEAE Cellulofine (Seikagaku Corporation) column (Φ4.
(6 × 30 cm).

After washing with the same buffer solution, elution was carried out by a linear gradient method using the same buffer solution and sodium chloride at an ionic strength of 0 to 0.7. Was measured to obtain an enzyme active fraction.

This fraction was concentrated with an ultrafiltration membrane having a molecular weight cut off of 10,000 or less (manufactured by Amicon).
Cellulofine GC 200m equilibrated with the same buffer containing L
After applying to a column (manufactured by Seikagaku Corporation) with a buffer (Φ2.0 × 70 cm) and washing with the same buffer, the column was eluted with the same buffer containing 1.0 M NaCL to obtain a galactanase active fraction. This enzyme solution was dialyzed against distilled water at 4 ° C. for 24 hours and freeze-dried to obtain about 2 mg of purified enzyme.

[0056]

As described above, according to the present invention,
(1) A novel galactanase that acts on β-1,4 galactan to produce galacto-oligosaccharides with high yield without producing much galactose, and (2) a novel microorganism that produces such galactanase Is made possible.

[Brief description of the drawings]

 "

FIG. 1 is a drawing showing the optimal pH of galactanase S-2.

FIG. 2 is a drawing showing the pH stability of galactanase S-2.

FIG. 3 is a drawing showing the optimal temperature of galactanase S-2.

FIG. 4 is a drawing showing the temperature stability of galactanase S-2.

FIG. 5 is a drawing showing an SDS polyacrylamide gel electrophoresis pattern of galactanase S-2.

──────────────────────────────────────────────────の Continued on front page (51) Int.Cl. ⁶ Identification code FI (C12N 9/42 C12R 1:07)

Claims (2)

(57) [Claims] 1. Galactanase S-2 having the following enzymatic properties: a. Action It acts on soybean fiber to produce galactooligosaccharides and hardly releases monosaccharides (galactose). b. Substrate specificity It acts on β-1,4 galactan but not β-1,3 galactan. c. Working pH is 9-12. d. Working temperature is 30-60 ° C. e. The optimum pH is 10.0. f. pH stability is 4-12. g. Temperature stability is less than 50 ° C. h. The optimal temperature is 50 ° C. i. The molecular weight is 40,000. 2. Bacillus sp. S-2 belonging to the genus Bacillus and producing galactanase and having the following mycological properties (Microtechnical Laboratories No. 11229). a. Physiological properties (1) Denitrification reaction is negative. (2) Positive gas generation from glucose. (3) The use of citric acid was negative for the Cosa medium. (4) The pH range for growth is 7.5-10. b. Positive anaerobic growth, negative VP test, negative casein hydrolysis. c. Nitrate reduction is negative.