JPH03244382A - Galactanase and microorganism capable of producing the same - Google Patents

Abstract

PURPOSE:To enable obtaining of an oligosaccharide in high yield with hardly any formation of monosaccharides even in hydrolyzing dietary fiber by using a galactanase, produced by a new microorganism belonging to the genus Bacillus and having specific enzymological properties. CONSTITUTION:A new strain Bacillus sp. S-1 (FERM P-11228), Bacillus sp. S-2 (FERM P-11229) or Bacillus sp. S-39 (FERM P-11230), having specific mycological properties such as negative to denitrifying reaction and belonging to the genus Bacillus is cultured using a culture medium containing soybean cake, etc., as a carbon source and an amino acid, etc., as a nitrogen source. Microbial cells are then removed by a means such as centrifugation and the resultant crude enzymic liquid is subsequently purified to afford galactanase having the following specific enzymological properties, reactive with soybean fiber, producing a galactooligosaccharide, hardly liberating monosaccharides (galactose), reactive with beta-1,4-galactan without reacting with beta-1,3-galactan, etc. The resultant galactanase is used to provide an oligosaccharide from dietary fiber in high yield.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to a novel galactanase and a novel microorganism that produces the same.

[Prior art]

Traditionally, food '$! Acid decomposition, alkaline decomposition, enzymatic decomposition, etc. have been used to decompose I fibers, etc., but these methods often produce single #M types, and it is necessary to modify dietary fibers (e.g., solubilization, lower molecular weight). etc.) or oligosaccharides, the disadvantage is that the yield is low.

Among dietary fibers, fibers derived from plants such as citrus fruits, potatoes, and soybeans have a main chain with β-1,4 galactoside bonds, and β-1,4 galactoside bonds are used as enzymes to hydrolyze these bonds. 1,4-galactanase is known, but the previously known β-1,4-galactanase is also a single I! (galactose).

On the other hand, microorganisms that produce β-1,4-galactanase include fungi (e.g. Penicillium citrt).
num origin), bacteria (e.g. Bacillus
(originating from the genus Lus) are known, but both β-
It hydrolyzes 14-galactoside bonds to produce single class Ii (galactose), and produces less galacto-oligosaccharides.

[Problem to be solved]

As mentioned above, when dietary fiber is hydrolyzed using acids, alkalis, enzymes, etc., there is a problem in that a large amount of monosaccharides are produced and the yield of oligosaccharides is reduced.

The present inventors have discovered that: Even when dietary fiber is hydrolyzed, the production of monosaccharides (galactose, etc.) is extremely small, and Oligo II can be used in high yields.
The aim of the present invention was to develop a new galactanase capable of obtaining (galactooligosaccharides, etc.) and (1) a new microorganism that produces such a galactanase.

[Means to solve the problem]

As a result of intensive research to achieve the above objective, the present inventors were able to screen microorganisms belonging to the genus Bacillus that produce enzymes suitable for the above objective from soil in the natural world (Table 1 shows the differences from Bacillus 5ubtilis). ), the target galactanase can be produced, isolated, and identified (Table 2 shows mold-derived and Bacillus-derived galtanases).
The present invention has been completed.

Among the properties of the screened bacteria, the following are known Bacillus
This is a table showing the main properties that are different from S. llus 5ubtilis.

(Left below) Table-1 Mycological properties *2 *3 *4 Anaerobic growth Voges-Proskauer test
+Gas from glucose hydrolysis of casein
+Utilization of citrate
+N1trate reduced to n1
trite Growth at pH 6,8 nutrient broth ++
+ + + + + + + ± However, *l is Bacillus 5ubtilis (quoted from the Bacillus manual) *2 is Bacillus sp, S-1*3 is Bacillus
illus sp, S-2*4 is Bacillus
sp, 5-39, and the following is Bacillus sp,
S-1, Bacillus sp, S2 and Bac
Among the enzymatic properties of galactanase obtained from Penicillium sp.
) derived from Bacillus 5ubtilis
Bacillus 5ubtil derived from K-50
is var amylosacchariticus
.

It is a comparison table with that derived from Bacillus 5ubtilis WT168.

Table-2 Enzymatic properties *1 *2 *3 *4 *5 *5
*7 Optimum pH 10.0 Same as left 4.0 4.56.07.0
6.0 Optimal temperature 'C40504055556050 Temperature stability'C<50 <45 <60 <55 <50
<60 <50 Isoelectric point pH 7, 14, 28, 46, 9 Molecular weight (10,000) 4.0 3.6 3.73.73
．． 54.0 substrate specificity is β1,4 galactan, the reaction product of is Gal<<Galz ~ the reaction product of Ga1a is Gal<<Gal. <Ga13. The reaction product of Ga14 is Gal<<Ga1. , the reaction product of Ga1z is Gal, the reaction product of Ga1z is Gal, and the reaction product of Ga1z is Gal -Gal.

The reaction product is Gal-Gal.

*1 is galactanase S-1 *2 is galactanase S-2 *3 is galactanase 5-39 *4 is galactanase derived from fungus (Penicillium) (Agric, Biol, Chem, 49°34
45 (1986) cited.

*5 is Bacillus 5ubtilis K-5
Galactanase derived from 0 (Agric, Biol, Ch
em, 351891 (1971) cited.

*6 is Bacillus 5ubtilis var.
Galactanase from amylosaccharium (Agric, Biol, Chem, 36.194
6 (1972) cited.

*7 is Bacillus 5ubtilis WT16
Galactanase J from B, Bio1. Chem,,2
51°5904 (1976).

First, the galactanase of the present invention will be explained.

The enzymatic properties of the galactanase of the present invention are as follows.

(a) The action is to act on soybean fiber to produce galactooligosaccharide and hardly release monosaccharide (galactose).

(b) Substrate specificity affects β-1,4 galactan but not β-1,3 galactan.

(c) The optimum temperature is 40°C.

For example, ■Galactanase S-1. Galactanase S-
2 is (a) optimal pH is 10.0, pH stability is 4-12
, the temperature stability is less than 50℃, among which galactanase S
The molecular weight of -2 is 40,000. Furthermore, among these, the reaction product of galactanase S-1 contains very little (very little) Gal and mainly produces Ga1z to Ga14, and the reaction product of galactanase S-2 contains very little Gal. (
), Ga12 than Ga1. , Ga14 are mainly produced. Also, ■galactanase 5-39 is (a)
The optimum pH is 4.0. pH stability is 4-10.5, temperature stability is less than 60℃, isoelectric point pH is 7.1, molecular weight is about 3
, 60,000 (according to SDS polyacrylamide gel electrophoresis). In addition, the reaction product contains very little Gal (
It is impossible), Ga1z, Ga1. Mainly generated.

Also, even between pH 3.5 and 10.0, it is about 50% of the optimum pH.
It has a relative activity of about 50% or more at an optimum temperature of 30 to 60°C.

The method for measuring enzyme activity and the buffer used therefor are as follows.

[Method for measuring enzyme activity] Mix 0.1 mf of the enzyme solution with 0.5 ml of the 0.1 M buffer shown in the following table containing 1% purified soybean fiber, and incubate at 40°C for 6 hours.
The reaction was allowed to proceed for 0 minutes. Centrifugation after reaction M (15,00 OR
, P, M, x 1 minute) and measure the total sugar content in the supernatant obtained by the phenol-sulfuric acid method. Under these conditions, the amount of enzyme that produces sugar equivalent to 1 μmol of galactose per minute is defined as 1 unit.

[Buffer]

pH range buffer 3-4 Citrate buffer 4-6 Acetate buffer 6-8 Phosphate buffer 8.5-10.5 Glycine-NaC1-NaO
H buffer 11=12 KCL-NaO) 1 buffer Next, the microorganism producing galactanase of the present invention will be explained.

The mycological properties of this microorganism are as follows.

(1) According to microscopic observation of the morphology, it is a bacillus. For example, the size of Bacillus sp. S-1 is 0.5 to 0.
．． 7 tt m X2. O~3.0 μm, Bacillus sp. S-2 is 0.5~0.7 μm×1.5~3
．． 0 am, Bacillus sp. 5-39 is 0.5 to 0
．． It is 6 μm×3.0 to 4.0 am. One end of the bacterial body is an oval endospore (0.5~1.0 um x 1.0~
There are also 0-simultaneous hairs that produce 2.0 μm) and are motile.

(2) Durham staining is positive. (3) It has no anti-acid properties. (b
) The growth status, etc. (the following numbers are weight %) are as follows.

■Meat juice agar plate medium (Difco Nutrient
brothO08, agar 1.5, NazCO31,0
, PH 10, 0), the growth condition is normal. For example, the colony shape of Bacillus sp. S-1 is irregular. The surface is rough and the margin is filiform. The color tone is 7 or white, opaque, and has no luster. Bacillus sp. S-2 is circular. The surface is smooth and the edges are smooth. The color is pale ocher, transparent, and glossy, and Bacillus sp. 5-39 has a round or irregular surface with a rough surface and an irregular or wavy edge. The color is slightly white and opaque, and the luster is slightly dull.

■Growth on broth agar slant medium (same size as broth agar plate medium) is normal; for example, Bacillus sp. S-1 is feather-like, lacks luster, and has a slightly white opaque color; Bacillus sp. 2 is filamentous, glossy, and transparent with a pale ocher color, while Bacillus sp. 5-39 is spread-like, with a slightly dull luster, and white and opaque.

■ Broth liquid medium (Difco Nutrient br
Bacillus sp.
39 grows slightly on the upper surface.

Next, (c) physiological properties etc. are as follows.

(1) Medium (KNO30, 1, Difco Nutri
ent brothO08, NazCO:, 1.0,
The reduction of nitrate by pH, 10, 0) is positive for Bacillus sp. S-1, but for Bacillus sp. S-2
and Bacillus sp. 5-39 are negative.

Denitrification reaction using the same medium was negative.

(2) Medium (peptone 1.0, glucose 0.5, Na2
The VP test using Bacillus sp. C031, 01 pH 10,0) was negative for Bacillus sp. S-1 and B. sp. S-2, but positive for B. sp. 5-39.

(3) Medium (peptone 1.0, NaCLO, 5,
Indole production by NazCOz 1.0, pH 10.0) was negative.

(4) Medium (Difco Nutrient brot)
h O, 8, Na, CO.

1.0, pH 10,0) and medium (Difco SI
M medium indicated amount, NazCO: + 1.0.
Both the lead acetate paper method (pH 10, 0) and the production of hydrogen sulfide in 51M medium were negative.

(5) Medium (soluble starch 1.0, polypeptone 0.5,
Yeast extract 0.5, K2HP0.0.1, MgSO4
・7H200,02, agar 1.5, NazCO31,
0, pH 10,0) was positive for starch decomposition.

(6) Medium (Difco Nutrient brot)
h O,8, gelatin 0.4, agar 1.5, NaOH
(adjusted to pH 10.0) gelatin decomposition activity was positive.

(7) Medium (skim milk 1.0, polypeptone 0.5
, yeast extract 0.5, K2HP0.0.1, MgSO4
・7H,00,02, agar 1.5, Na, CO:+
Casein degradation by Bacillus sp. S-1 and S-2 was negative for Bacillus sp.
is positive.

(8) Regarding the use of citric acid, ■Kosa medium CNHa
HzPO40.1, K2HPO40.1, citric acid 0.
2, NaCL0.5, MgSO447Hz00.0
2, NazCO* 1.0. PH10゜0) is negative.

However, ■Christensen medium (yeast extract 0.05,
Cystine hydrochloride 0.01. Sodium citrate 0.3
, NaCL O,5, Grape I! 0.02,KH,P
O.

0.1, agar 1.5, NazCOs 1.0, pH 1
0,0) is positive.

(9) Medium (Grape I!1.0, NaCLO,5
, Mg5O, 7H,00,02, K, HPO40,1
, N) 14H, PO, or KNo, 0.1, NaHC
Utilization of inorganic nitrogen sources by O+ 1.0, pH 9.2) was negative for both nitrates and ammonium salts.

00) Medium (soluble starch 1.0, polypeptone 0.5,
Yeast extract 0.5, K! ) IPo, 0.4, Mg5
O, 7 H, OO, 02, agar 1.5, NazCO
31,0, PH10,0) was negative.

01) Medium (KH2P040.1, NaJPO40,1
, urea 2.1, yeast extract 0.01, 0.02% phenol red solution 5.0, pH 7.0) was negative for urease.

02) Medium (soluble starch 1.0, polypeptone 0.5,
Yeast extract 0.5, K! HP0.0.1, MgSO4
・7 H,00,02, agar 1.5, Na, C0,1
, 0, pH 10, 0> oxidase is Bacillus
Bacillus sp. S-1 is negative, but Bacillus sp.
-2 and Bacillus sp. 5-39 are positive.

03) Medium (soluble starch 1.0, polypeptone 0.0.
5 yeast extract 0.5, KzHPO, O, L MgSO4
・7H, 00, 02, agar 1. 5, Na, COz 1
, 0, pH 10,0) was positive for catalase.

04) Growth range, etc. ■Growth temperature range is 20-45
The optimum temperature range for ℃1 growth is 25-40''C, for example, the growth temperature range for Bacillus sp. S-1 is 20-40''C.
1 The optimum temperature range for growth is 25-35°C, while the temperature range for Bacillus sp. S-2 is 20-45°C. 1 The optimum temperature range for growth is 25-4°C, Bacillus sp. 5-39.
The growth temperature range is 25-45℃ 1 The optimal growth temperature range is 3
0~40''C is suitable.

■The appropriate pH range for growth is pH 6 to 10, and the optimum pH range for growth is pH 7.5 to 9.5.
Growth of Bacillus sp. S-1 and Bacillus sp. S-2 p.
H range is 7.5-10, optimum pH range for growth is 8-9.5
is appropriate, and the growth pH of Bacillus sp.
The range is 6.0-9.5, the optimum pH range for growth is 7.5-9
．． 5 is appropriate.

Also, 05) Table 3 shows the presence or absence of acid and gas generation from sugars.

Table-3 Sugars Acid production Gas production ■L-arabinose ■D-xylose ■D-glucose ■D-mannose ■D-fructose ■D-galactose ■maltose ■sucrose ■lactose [phase] Trehalose ■D-sorbit ±~10 ± ~10@D-Mannit + +@Innojit
* Glycerin * Starch 10 However, * Lactose acid production and gas generation by Bacillus sp. S-1 and Bacillus sp. S-2 are ±, but Bacillus sp. 5-39 is 10. Also, acid production and gas generation by Bacillus sp. S-1 and Bacillus sp. S-2 of D-sorbitol were 10, but Bacillus sp. 5-39 was poor.

06) Bacillus sp. S-1 grows in a salt-containing medium up to 10% salt, and Bacillus sp.
2 grows up to 15% salt, Bacillus sp. 5-3
9 grows up to 2% salt.

Formation of a’n dihydroxyacetone was negative.

Regarding the above mycological properties, please refer to the Bersey's Manual (B
ergy's Manual of Determin
As a result of comparison and search using the 8th edition of Active Bacteriology as a reference, these bacteria are recognized to be a type of spore-forming bacterium of the genus Bacillus, but there are no known strains of the genus Bacillus that match them. was found, and it was determined that this bacterium was a new strain. These strains were divided into Bacillus sp. S-1 and Bacillus sp.
They were named Bacillus sp. 2 and Bacillus sp. 5-39 and entrusted to the Microbial Technology Research Institute. 11228, respectively.
No. 11229 and No. 11230.

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

Any carbon source, nitrogen source, etc. used in the culture medium can be used as long as it can be recycled. For example, carbon sources include soybean meal, plant fibers such as wheat bran, blackstrap molasses, starch, and various I! Various amino acids, cornstarch liquor, soybean flour, peptides, yeast extract, urea, and other nitrogen-containing substances can be used as nitrogen sources.

Other minerals, vitamins, etc. may be used as appropriate.

After culturing the Bacillus microorganism of the present invention, the bacterial cells can be removed by known means such as centrifugation and filtration to obtain a crude enzyme solution. Further, the crude enzyme can also be obtained by drying, etc. using means such as salting out, dialysis, and ultrafiltration. It can also be further purified to obtain a purified enzyme.

〔Example〕

Embodiments of the present invention will be described below with reference to Examples.

Example 1 Soil collected from near Izumisano City, Osaka Prefecture, approximately 0. Suspend 5g in sterile water, apply it on an agar medium for isolation (*1),
C. for 1 to 2 days to form colonies. We selected those that formed a pellucid zone (halo) around the colony due to the decomposition of galactan, and obtained galactanase-producing bacteria. The mycological properties were as described in the detailed description of the invention.

Next, these acquired bacteria were further inoculated into a liquid medium (*2), cultured with shaking at 37°C for 2 days, and centrifuged (10,000
OR, P, M,

Bacteria with strong galactanase activity were screened using the above method, and one of them, Bacillus sp. S39, was obtained.

*L (agar medium)...pH 10.0 Okara (Fuji Oil Co., Ltd. "Proplus SAJ") 1% yeast extract 0.5% 2 HP
O40,1% M g S Oa ・7H200,02% Na2 C
O31% agar 1. 5%*2
(Liquid medium)...PH10.0 Okara (Fuji Oil Co., Ltd. "Proplus SA,") 2% polypeptone 0.5% yeast extract
0.5% NaC10.5% to 2 HPO40.1% M g S Oa ・7H200.02% Na2
C031% *3 (Measurement method for galactanase activity is the enzyme activity measurement method described above) Example 2 Bacillus sp. 5-39 strain obtained in Example 1 was placed in a liquid medium (I# same as *2 in Example 1). The cells were inoculated and cultured with shaking at 37°C for 40 days, and the supernatant obtained by centrifuging the bacterial cells was used as crude enzyme galactanase 5-39. Galactanase activity at pH 4 (according to method *3 in Example 1) was 1 unit/ml.

Next, Okara (Proplus SAJ, manufactured by Fuji Oil ■)
Add 25 units of the crude enzyme galactanase 5-39 to Ig, pH 4.0 (5 mM acetate buffer, reaction volume is 50
mj2) for 6 hours.

After the reaction, heat at 100℃ for 3 minutes to inactivate the enzyme, and use 150mj of ethanol! and the precipitated fraction was centrifuged (6000
R, P, M,
Galactooligosaccharides were obtained with a yield of 6%.

When the obtained galactooligosaccharide was analyzed by HPLC (high performance liquid chromatography) under the following conditions, galactose was detected with 28% galactobiose, 54% galactotriose, 15% galactotetraose, and 3% galactopentaose. I couldn't.

(HPLC conditions) Column: Senshu Pack (NH2-125IN) t4
．． 6X250mm Column temperature: room temperature Eluent Niacetonitrile/water = 2/l (■/■) Flow rate:
1 mj2/min Detector: Differential refractometer Example 3 Bacillus sp. S-1 strain and Bacillus sp. S-2 strain were obtained by screening in the same manner as in Example 1 using soil collected from near Yonago City, Tottori Prefecture.

The mycological properties were as described in the detailed description of the invention.

Example 4 The Bacillus sp. S-1 strain obtained in Example 3 was cultured in the same manner as in Example 2, and galactanase S-1 was prepared as a crude enzyme solution.
1 to 1.1 units/m/l! (Actually measured value at pH 10) was obtained.

25 units of galactanase S-1 were added to 1 g of soybean okara and reacted at pH 10, 0, and 40° C. for 6 hours, and in the same manner as in Example 2, an oligosaccharide was obtained with a yield of 41%.

The results of HPLC analysis are shown in Table 4 below.

Table 4 Galactose Galactobiose Galactotriose Galactotetraose Galactopentaose 6% 8% 33% 35% 18% Example 5 The Bacillus sp. S strain obtained in Example 3 was used in Example 2
It was cultured in the same manner as above to obtain 0.9 units/ml of galactanase S-2 (actual value at pH 10) as a crude enzyme solution.

25 units of galactanase S-2 were added to 1 g of soybean okara and reacted at pH 10, 0, and 40° C. for 6 hours, and in the same manner as in Example 2, an oligosaccharide was obtained with a yield of 39%.

The results of the HPLC analysis were as shown in Table 5 below. Pen1cilli
U + S origin) and commercially available cellulase (Polyporus origin manufactured by Kyowa Hakko Kogyo ■)
Add 5 units each to okara in the same manner as in Example 3, and adjust the pH to 4.
5 for 6 hours to obtain oligosaccharides.

As a result of HPLC analysis, the yield of the former was 54%, and the sugar content was almost 100% galactose. The latter yield is 57%, sugars are galactose 44%, galactobiose 2
6%, galactotriose 24%, and galactotetraose 6%.

Example 6 Example of no effect on β-1,3 galactan.

Galactanase 5-39, S-1, and S-2 obtained in Example 2, Example 4, and Example 5 were mixed with β-1°3 galactan (
(manufactured by Sigma) under the conditions of Example 2, Example 4, and Example 5, but none of them decomposed.

Example 7 (Optimal pH and pH stability) According to the [enzyme activity measurement method], Example 2, Example 4,
Galactanase 5-39 obtained in Example 5, S-1,
The optimum pH of S-2 was investigated. The results are shown in FIGS. 1, 2, and 3.

Further, the results of examining pH stability by measuring the residual activity after holding each pH at 20° C. for 115 hours are shown in FIGS. 4, 5, and 6.

Example 8 (Optimum temperature and temperature stability) Optimal temperature and temperature stability of galactanases 5-39, S-1, and S-2 obtained in Example 2, Example 4, and Example 5 (measurement of residual activity after treatment for 15 minutes) was investigated by the above-mentioned [enzyme activity measurement method]. The results are shown in FIGS. 7, 8, 9, 10, 11, and 12.

Example 9 Galactanase 5-39 and galactanase S-2 obtained in the same manner as in Example 2 and Example 5 were purified by the following chromatography method, and electrophoresed together with a marker by SDS polyacrylic acid gel electrophoresis method. As a result, a single peak was observed as shown in FIGS. 13 and 14, respectively. This is regalactanase 5-39, which is placed in molecule placement piece 3.
60,000, and galactanase S-2 was found to have a molecular weight of approximately 40,000.

[Purification by chromatography method] ■For galactanase 5-39.

Ammonium sulfate was added to 500 ml of galactanase crude enzyme solution to make the ammonium sulfate 80% saturated, and the resulting precipitate was diluted with 8.0 OOR,
P, M, and XIO were collected by centrifugation and dissolved in 20 mj2 of water. After dialysis against 20mM phosphate buffer (pH 7.0) at 4°C for 24 hours, it was applied to a DEAE Toyopearl (manufactured by Tosoh) column (Φ1.6 x 21 cm) equilibrated with the same buffer. It eluted.

The non-adsorbed fractions were collected, concentrated using an ultrafiltration membrane (manufactured by Amicon) with a molecular weight cut-off of 10,000 or less, and added to a 20mM acetate buffer (pH
5,0) for 24 hours at 4°C. Thereafter, it was applied to a CM Toyopearl (manufactured by Tosoh) column (Φ1.6 x 21 cm) equilibrated with the same buffer solution for adsorption. After washing with the same buffer, elution was performed using the same buffer and sodium chloride at an ionic strength of 0 to 0.4 using the linear gradient method, and 1.8n+e fractions were collected, and the enzyme activity assay method was performed using the same buffer and sodium chloride. Enzyme activity was measured to obtain a galactanase active fraction. Add this enzyme solution to distilled water at 4℃ for 2 hours.
It was dialyzed for 4 hours and lyophilized to obtain about 2 mg of purified enzyme.

■In the case of galactanase S-2.

Ammonium sulfate was added to the galactanase crude enzyme solution 51 so that the ammonium sulfate saturation was 80%, and the resulting precipitate was mixed with 7,0 OOR, P, M×
It was collected by centrifugation for 20 minutes and dissolved in 100 ml of water. After dialysis against 20mM) Lis-HCl buffer (p)I 7.0) at 4°C for 24 hours, a DEAE Cellulofine (Seikagaku Corporation) column (Φ4.6) was equilibrated with the same buffer. x30 c+++) and adsorbed.

After washing with the same buffer, elution was performed by the linear gradient end method using the same buffer and embodied sodium at an ionic strength of 0 to 0.7, and 12a+jl! The enzyme activity was measured by the enzyme activity measuring method described above to obtain an enzyme active fraction.

This fraction was concentrated using an ultrafiltration membrane (manufactured by Amicon) with a molecular weight cut-off of 10,000 or less, and this enzyme solution was equilibrated with the same buffer containing 0.1M NaCl using Cellulofine GC200m (
After washing with the same buffer solution, 1. Elution was performed with the same buffer containing OM NaCL to obtain a galactanase active fraction. This enzyme solution was dialyzed against distilled water at 4° C. for 24 hours and lyophilized to obtain about 2 mg of purified enzyme.

(Effects) As explained above, the present invention makes it possible to produce a new galactanase that acts on β-14 galactan to produce galactooligosaccharides in high yield with almost no galactose production, and This makes it possible to develop a new microorganism that produces enzymes.

[Brief explanation of drawings]

Figure 1 is a diagram showing the optimum pH of galactanase 5-39. Figure 2 is a diagram showing the optimum pH of galactanase S-1. Figure 3 is a diagram showing the optimum pH of galactanase S-2. Figure 4 shows the pH stability of galactanase 5-39. Figure 5 shows the pH stability of galactanase S-1. Figure 6 shows the pH stability of galactanase S-2. Figure 7 is a diagram showing the optimal temperature for galactanase 5-39. Figure 8 is a diagram showing the optimal temperature for galactanase S-1. Figure 9 is a diagram showing the optimal temperature for galactanase S-2. FIG. 10 is a drawing showing the temperature. FIG. 11 is a drawing showing the temperature stability of galactanase S-1. FIG. 12 is a drawing showing the temperature stability of galactanase S-1. FIG. 13, which is a drawing showing the temperature stability of No. 2, is a drawing showing the SDS polyacrylamide gel electrophoresis pattern of galactanase 5-39. FIG. 14 is a drawing showing the SDS polyacrylamide gel electrophoresis pattern of galactanase S-2.

Claims (10)

[Claims] (1) Galactanase (a) having the following enzymological properties
Action: Acts on soybean fiber to produce galactooligosaccharides and hardly releases monosaccharide (galactose). (b) Substrate specificity Acts on β-1,4 galactan but not β-1,3 galactan. (c) Working pH is 3-120 (d) Working temperature is 20-65°C. (2) The galactanase S-1 or galactanase S-2 according to claim (1), which has an optimum pH of 10.0, a pH stability of 4 to 12, and a temperature stability of less than 50°C. (3) The galactanase S-1 according to claim (2), wherein the optimum temperature is 40°C. (4) Claim in which the optimum temperature is 50°C and the molecular weight is 40,000 (
2) Galactanase S-2 as described. (5) Optimum pH is 4.0, pH stability is 4-10.5,
Temperature stability is less than 60℃, molecular weight is approximately 36,000 (SDS
The galactanase S-39 according to claim (1), which has a peak in (as determined by polyacrylamide gel electrophoresis). (6) A microorganism belonging to the genus Bacillus that produces galactanase and has the following mycological properties. (a) Physiological properties, etc. [1] Denitrification reaction is negative. [2] Gas generation from glucose was positive. [3] Cosa medium is negative in the utilization of citric acid. [4] The pH range for growth is pH 6.8 or higher. (7) The Bacillus microorganism according to claim (6), which is positive in anaerobic growth, negative in VP test, and negative in casein hydrolysis. (8) The Bacillus sp. S-1 according to claim (7), which is positive for nitrate reduction (Feikoken Bacterial Serial No. 11228). (9) The Bacillus sp. S-2 according to claim (7), which is negative in nitrate reduction (Feikoken Bacteria No. 11229). (10) Bacillus sp. S-39 (Feikoken Bacterial Serial No. 11230) according to claim (7), which is negative for nitrate reduction and does not grow in 5 to 10% salt.