JP2676453B2 - Alkaline isoamylase, microorganism producing the same, and method for producing the alkaline isoamylase - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a novel alkaline isoamylase useful as a detergent component, a microorganism producing the same, and a method for producing the alkaline isoamylase.

[0002]

BACKGROUND OF THE INVENTION Isoamylase is an enzyme that cleaves only α-1,6 glucoside bonds in a starch-based polysaccharide molecule having a branched structure such as glycogen and amylopectin. And Kobayashi [ N
Ature , 167 , 606 (1951)], it was first discovered in yeast cells. After that, by Harada et al., Pseudomonas amilo de la Mosa ( Pse
udomonas amyloderamosa SB
-15) [Biochim. Biophys. Act
a, 212,458 (1970)], the isoamylase produced was isolated and purified, and its properties were clarified.

Further, flavobacterium ( Flavova)
cterium sp. ) [ Staerke 32, 1
32 (1980)], Cytophage ( Cytophag
e sp. ) [ FEBS lett . , 12, 96 (19
70)], Bacillus amyloliquefaciens ( Bac
illus amyloliquefaciens )
[ FEBS lett . , 57, 1 (1975)], Escherichia Intermedia ( Escherich
ia intermedia ) [ Appl . Micro
biol . , 15, 492 (1967)], yeast Lipomyces cononenkoe ( Lipomyces kono).
nekoe ) [ Appl . Environ . Micr
obiol . , 44, 1253 (1982)] and the like have been reported to produce isoamylase.

It is known that isoamylase has hydrolytic activity not only for glycogen and amylopectin but also for α-1,6 glucoside bonds in starch, pullulan and branched oligosaccharides formed by partial decomposition thereof. ,
It is called "debranching enzyme". In addition, isoamylase is used for the study of the fine structure of glycogen and amylopectin, and since this enzyme is extremely powerful compared to other debranching enzymes, it has a strong reverse reaction and can produce various branched cyclodextrins. It is being used industrially because it can be easily synthesized ["Starch Science", 33, 119 (198).
6)].

It has been found that the detergency mainly against starch stains is dramatically improved by blending such a debranching enzyme containing isoamylase with a amylase in a detergent (JP-A-1-132192). .

However, the isoamylase conventionally found in the natural world has a pH of 3.5 to 6.5 and exhibits maximum and stable enzyme activity in the neutral or acidic region, so-called neutral or acidic. Isoamylase, which exhibits maximum activity in the alkaline region required as a composition for dishwashing detergents and detergents for clothing, or has alkali resistance, so-called alkali isoamylase and alkali-resistant isoamylase have not yet been found. It has not been issued.

In the present specification, the alkaline isoamylase means that the optimum pH is in the alkaline region,
Alkali-tolerant isoamylase refers to an isoamylase having an optimum pH in the neutral to acidic range, but having an activity similar to that at the optimum pH and maintaining stability even in the alkaline range. Further, the neutral means a pH range of 6 to 8, and the alkaline means a pH range exceeding it.

Therefore, it is suitable as a cleaning composition component,
It has been desired to develop an isoamylase having an optimum pH in the alkaline region.

[0009]

DISCLOSURE OF THE INVENTION The inventors of the present invention have made extensive studies in view of such circumstances, and as a result, have found that a specific Bacillus ( Baci)
by culturing a microorganism belonging to the llus) genus,
It was found that an isoamylase having an optimum pH in the alkaline region can be obtained, and the present invention has been completed.

That is, the present invention provides an alkaline isoamylase having the following enzymatic properties (1) to (6).

(1) Action: Hydrolyzes α-1,6 glucoside bond of glycogen and amylopectin. In addition, it does not hydrolyze the α-1,6 glucoside bond of amylose and pullulan. (2) Working pH and optimum pH, acting in the pH range of pH 5 to 11.5, and having optimum pH around 9. (3) pH stability, extremely stable in the range of pH 7 to 10, pH 6 to 10.
The activity of about 50% or more is maintained even at 5 (40 ° C,
Processing for 10 minutes). (4) Working temperature range and optimum working temperature, acting in the range of 40 to 60 ° C., optimum working temperature is about 55 ° C.
It is. (5) Temperature stability, extremely stable up to 50 ° C (glycine at pH 9.0-
Treatment in table salt-sodium hydroxide buffer for 30 minutes). (6) Molecular weight, molecular weight by sodium dodecyl sulfate (SDS) electrophoresis is about 65000 ± 1000.

The present invention also includes a Bacillus .
The present invention provides a microorganism belonging to the genus s ) and having the above-mentioned alkaline isoamylase-producing ability.

The present invention further provides a method for producing alkaline isoamylase, which comprises culturing the above microorganism and collecting the above alkaline isoamylase from the culture.

The enzymatic properties of the alkaline isoamylase of the present invention will be described below. The enzyme activity was measured according to the measured pH according to the buffer solutions shown below (each 40
mM) was used and the following method was followed.

(Buffer type) pH 4 to 6; acetate buffer, pH 6 to 8; tris-maleic acid buffer, pH 8 to 11; glycine-salt-sodium hydroxide buffer, pH 11 to 12; potassium chloride-hydroxylation Sodium buffer,

(Method for measuring enzyme activity) (1) Measurement of isoamylase activity by DNS method, buffer solution in which oyster-derived glycogen (final concentration in the reaction system is 0.5%) is dissolved in various buffer solutions 0 To 9 ml,
0.1 ml of enzyme solution was added, and the mixture was reacted at 40 ° C. for 30 minutes. After the reaction, the reducing sugar was quantified by the 3,5-dinitrosalicylic acid (DNS) method. That is, the reaction solution 1.0
1.0 ml of DNS reagent was added to ml, and color was developed by heating at 100 ° C. for 5 minutes. After cooling, 4.0 ml of deionized water was added to dilute, and colorimetric determination was performed at a wavelength of 535 nm. The enzyme titer is 1
The amount of enzyme that produces a reducing sugar corresponding to 1 μmol of glucose per minute was defined as 1 unit (1 U).

(2) The measurement of isoamylase activity by the iodine coloring method and the activity measurement in the purification step were carried out under the following conditions. That is, 0.5 ml of the enzyme solution was added to 0.5 ml of 1% (w / v) potato-derived amylopectin, and the reaction was carried out at 40 ° C. for 30 minutes. After the reaction, 0.2 ml was withdrawn from the reaction solution, and iodine solution (0.005% I ₂ + 0.015%
KI) 2 ml was added, and deionized water was further added to 8.0 ml for dilution, and colorimetric determination was carried out at a wavelength of 600 nm. The enzyme titer was 1 unit (1 U) of the amount of enzyme required to increase the absorbance by 0.01 per minute.

(Enzymatic Properties) (1) Action: Hydrolyzes α-1,6 glucoside bond of glycogen and amylopectin. In addition, it does not hydrolyze the α-1,6 glucoside bond of amylose and pullulan. Table 1 shows the results of measuring the relative activity of alkaline isoamylase with respect to each substrate.

[0019]

[Table 1]

(2) Working pH and optimum pH, it works in the range of pH 5 to 11.5, and has optimum pH around 9. The isoamylase activity is 0.5% oyster-derived glycogen, 10 mM acetate buffer (pH 4 to 6), tris-maleic acid buffer (pH 6 to 8), glycine-salt-sodium hydroxide buffer (pH 8 to 11). 1 shows the relationship between the reaction pH and the relative activity measured by reacting at 40 ° C. for 15 minutes using a reaction system of potassium chloride-sodium hydroxide buffer (pH 11 to 12).

(3) pH stability, extremely stable at pH 7 to 10 and maintaining an activity of about 50% or more even at pH 6 to 10.5 (treatment at 40 ° C. for 10 minutes). The isoamylase activity at each pH was 0.
5% oyster-derived glycogen, 10 mM acetate buffer (pH 4-
6), Tris-maleic acid buffer (pH 6 to 8), glycine-saline-sodium hydroxide buffer (pH 8 to 11) and potassium chloride-sodium hydroxide buffer (pH 11 to 1)
FIG. 2 shows the relationship between the treatment pH and the residual activity measured by reacting at 40 ° C. for 10 minutes using the reaction system of 2).

(4) Working temperature range and optimum working temperature: it works in a wide range of 40 to 60 ° C., and the optimum working temperature is about 55.
Appears in ° C. The results of measuring the relationship between the reaction temperature and the relative activity when the reaction was carried out under the above conditions at pH 9.0 are shown in FIG.

(5) Temperature stability, extremely stable up to 50 ° C. (pH 9.0 glycine-
Treatment in table salt-sodium hydroxide buffer for 30 minutes). Further, it shows a residual activity of 40% or more even at 55 ° C. The relationship between the treatment temperature and the residual activity measured by the reaction under the above conditions is shown in FIG.

(6) Molecular weight, molecular weight measured by SDS electrophoresis is about 65,000.
± 1000.

The alkaline isoamylase of the present invention further has the following properties (7) to (9).

(7) Effect of metal ion, 1 mM Hg ²⁺ strongly inhibits the activity.

(8) Effects of surfactants, various surfactants (for example, sodium linear alkylbenzene sulfonate (LAS), sodium alkyl sulfate ester (AS), sodium salt of polyoxyethylene alkyl sulfate ester (ES), Even if treated with a 0.05% solution of sodium α-olefin sulfonate (AOS), sodium α-sulfonated fatty acid ester (α-SFE), sodium alkyl sulfonate (SAS), soap and softanol at 40 ° C. for 30 minutes. Almost no inhibition of activity.

(9) Effect of chelating agent, chelating agent EDTA (10 mM), citric acid (0.
(05%) and zeolite (0.05%) show little activity inhibition.

The microorganism capable of producing the alkaline isoamylase of the present invention is particularly limited as long as it belongs to the genus Bacillus and has the ability to produce the isoamylase having the above-mentioned enzymatic properties (1) to (6). However, for example, Bacillus sp. ( Bacillus sp.), Which is a kind of alkalophilic microorganism collected from soil in Mito City, Ibaraki Prefecture, is used.
p. ) KSM-3309.

Bacillus sp. KSM-3309, which is an example of the microorganism of the present invention, has the following mycological properties. In the following, the following 20 types of media were used for the classification of strains. Unless otherwise specified, separately sterilized sodium carbonate (Na ₂ C) was used.
O ₃₎ 0.5 wt% (hereinafter referred to simply as%) containing.

Composition of medium used (indication:%): Medium 1. Nutrient broth, 0.8; Agar powder (manufactured by Wako Pure Chemical Industries, Ltd.), 1.5. Medium 2. Nutrient broth, 0.8. Medium 3. Nutrient Broth, 0.8; Gelatin,
20.0; Agar powder (manufactured by Wako Pure Chemical Industries, Ltd.), 1.5. Medium 4. Bactoli Thomas Milk, 10.5. Medium 5. Nutrient Broth, 0.8; KNO ₃ ,
0.1. Medium 6. Bactopeptone, 0.7; NaCl, 0.5;
Glucose, 0.5. Medium 7. SIM agar medium (manufactured by Eiken Chemical Co., Ltd.), indicated amount. Medium 8. TSI agar medium (Eiken Chemical Co., Ltd.), indicated amount. Medium 9. Yeast extract, 0.5; Bactopeptone, 1.
5; K ₂ HPO ₄ , 0.1; MgSO ₄ .7H ₂ O, 0.
02: Soluble starch, 2.0: Agar powder (manufactured by Wako Pure Chemical Industries, Ltd.),
1.5. Medium 10. Coser medium (manufactured by Eiken Chemical Co., Ltd.), indicated amount.

Medium 11. (A) Yeast extract, 0.05; N
a ₂ SO ₄ , 0.1; KH ₂ PO ₄ , 0.1; glucose,
1.0. (B) Yeast extract, 0.05; Na ₂ SO ₄ , 0.1;
KH ₂ PO ₄ , 0.1; glucose, 1.0; CaCl ₂ ·
_{2H 2 O, 0.05; MnSO 4} · 4~6H 2 O, 0.0
1; FeSO ₄ .7H ₂ O, 0.001; MgSO ₄ .7
H ₂ O, 0.02. As the nitrogen sources, sodium nitrate, sodium nitrite, ammonium chloride and ammonium phosphate are 0.25%, 0.2025%,
The medium was used by adding it to the mediums (a) and (b) so that the concentrations became 0.158% and 0.195%. Medium 12. King A medium "Eiken" (Eiken Chemical Co., Ltd.), indicated amount. Medium 13. King B medium "Eiken" (Eiken Chemical Co., Ltd.), indicated amount. Medium 14. Urea medium "Eiken" (Eiken Chemical Co., Ltd.), indicated amount. Medium 15. Filter paper for cytochrome oxidase test (manufactured by Nissui Pharmaceutical Co., Ltd.). Medium 16.3% hydrogen peroxide solution. Medium 17. Bactopeptone, 0.5; yeast extract, 0.
5; K ₂ HPO ₄ , 0.1; glucose, 1.0; MgS
_{O 4 · 7H 2 O, 0.02} . Medium 18. Bactopeptone, 2.7; NaCl, 5.5;
K ₂ HPO ₄ , 0.3; glucose, 0.5; bromothymol blue, 0.06; agar powder (manufactured by Wako Pure Chemical Industries, Ltd.), 1.
5. Medium 19. (NH ₄ ) ₂ HPO ₄ , 0.1; KCl, 0.0
2; MgSO ₄ .7H ₂ O, 0.02; yeast extract, 0.
05; sugar, 1.0. Medium 20. Casein, 0.5; yeast extract, 0.5; glucose, 1.0; K ₂ HPO ₄ , 0.1; MgSO ₄ .7H
₂ O, 0.02; agar powder (manufactured by Wako Pure Chemical Industries, Ltd.), 1.5.

(Mycological properties) (a) As a result of microscopic observation, the size of the bacterial cells is 0.8 to 1.2 μm × 2.8 to 4.0.
It is a rod-shaped bacterium having a size of μm and produces an oval endospore (0.8 to 1.0 μm × 2.0 to 2.5 μm) in the center of the bacterium.
Motile with flagella. Gram staining is positive. No acid resistance.

(B) Growth condition in various media, (a) Meat agar plate culture (medium 1) Growth condition is good. The shape of the settlement is irregular, the surface is rough and the periphery is arborescent. The color of the settlement is pale yellow and semi-transparent. (B) Meat broth agar slope culture (medium 1) Grow. (C) Broth liquid culture (medium 2) Growth condition is poor. (D) Meat broth gelatin stab culture (medium 3) The growth condition is good. Liquefaction of gelatin is observed. (E) Litmus milk medium (medium 4) No milk coagulation or peptone formation is observed. Discoloration of litmus cannot be determined because the medium is alkaline.

(C) Physiological properties, (a) Nitrate reduction and denitrification reaction (medium 5) Nitrate reduction is positive. Denitrification reaction is negative. (B) MR test (medium 6) The medium cannot be judged because it is alkaline. (C) VP test (medium 6) Negative. (D) Formation of indole (medium 7) Negative. (E) Production of hydrogen sulfide (medium 8) Negative. (F) Starch hydrolysis (medium 9) Positive. (G) Use of citric acid Positive in Coser medium (medium 10).

(H) Use of Inorganic Nitrogen Source (Medium 11) Nitrate, nitrite and ammonium salts are used together. (I) Formation of pigment (medium 12, 13) Negative. (Nu) Urease (medium 14) Negative. (L) Oxidase (medium 15) Negative. (Wo) Catalase (medium 16) positive. (Wa) Range of growth (medium 17) The temperature range for growth is 12 to 45 ° C, and the optimum temperature range for growth is 1
It was 8 to 42 ° C. The pH range for growth is pH 8.5-10.
5. The optimum pH for growth was pH 9.5. (F) Attitude toward oxygen Aerobic.

(Vi) OF test (medium 18) Discoloration cannot be determined due to its alkaline nature. Only grows in aerobic conditions. (T) Utilization of sugar (medium 19) D-xylose, D-glucose, D-mannose, D
-Fructose, maltose, sucrose, trehalose, D-
Mannitol, D-inositol, D-sorbitol,
Utilizes glycerin, starch and dextrin. (H) Growth in salt-containing medium (medium 1 was modified) Grow up to 7% salt concentration. Poor growth with a salt concentration of 10%. (So) Casein degradation (medium 20) Positive.

Based on the above-mentioned mycological properties,
Manual of Systematic Bacteriology (Bergey's Manual of System)
E. Bacteriology) Volume 2 and Z. Genus. Bacillus ("The Genus Ba
Cillus "RE Gordon et al., Agricu
lture Handbook No. 427, Agr
iculturalResearch Service
e, U. S. Department of Agric
ultra Washington D.C. C. , (1
As a result of a comparative search with reference to 973)), this strain is recognized as a kind of Bacillus genus which is a spore bacillus. Since this strain cannot grow in the neutral region and shows good growth exclusively in the highly alkaline region, recently, Horikoshi and Akiba ("Alka
lophilic Microorganism ”, J
apan Scientific Society P
It is distinguished from so-called Alkalophilic microorganisms (bacilli belonging to the genus Bacillus that grow in a neutral pH range), which is claimed by less (Tokyo), 1982.

Further, since the mycological properties of this strain do not match any of the above-mentioned alkalophilic Bacillus, it was judged as a novel strain and Bacillus sp. KSM-330 was determined.
It was named 9 and was deposited at the Institute for Microbial Technology, Ministry of International Trade and Industry, Ministry of International Trade and Industry, as Microbiology Research Institute No. 13005.

In order to obtain the alkaline isoamylase of the present invention using the above strain, the medium may be inoculated with the strain and cultured according to a conventional method. It is preferable that a suitable amount of assimilable carbon source and nitrogen source be contained in the medium.

The carbon source and the nitrogen source are not particularly limited, but specific examples thereof include corn gluten meal, soybean flour, corn steep liquor, casamino acid, yeast extract, pharmamedia, meat extract, tryptone, and nitrogen source. Soyton, Hypro, Aji Power, Soy Bean Meal, Cottonseed meal, cultivator, adipron, organic nitrogen sources such as zest and inorganic nitrogen sources such as ammonium sulfate, ammonium nitrate, ammonium phosphate, ammonium carbonate, sodium nitrate, ammonium acetate,
The carbon source, soluble starch, insoluble starch, amylopectin, glycogen, in addition to branched oligosaccharides produced by pullulan and partial decomposition thereof, carbon sources that can be assimilated, for example, glucose, maltose, xylose, mannose, fructose, maltose, Examples thereof include sucrose, lactose, trehalose, mannitol, sorbit, glycerin, and assimilable organic acids such as acetic acid.

In addition, inorganic salts such as phosphates, magnesium salts, calcium salts, manganese salts, zinc salts, cobalt salts, sodium salts, potassium salts, and, if necessary, inorganic and organic micronutrient sources are appropriately added to the medium. It can also be added.

Collection and purification of the target substance, alkaline isoamylase, in the thus obtained culture can be carried out according to a general enzyme collection and purification means. That is, the bacterial cells can be removed from the culture solution by a usual solid-liquid separation means such as centrifugation or filtration to obtain a crude enzyme solution. This crude enzyme solution can be used as it is, but if necessary, a crude enzyme is obtained by a separation means such as a salting-out method, a precipitation method or an ultrafiltration method, and further purified and crystallized by a known method, It can also be used as a purified enzyme.

Hereinafter, examples of the method for producing and purifying the alkaline isoamylase of the present invention will be described. Alkaline Bacillus genus KSM-3309 strain with 1% glycogen (derived from oyster), 0.2% tryptone, 0.1% yeast extract,
0.03% KH ₂ PO ₄ , 0.1% (NH ₄ ) SO ₄ ,
_{0.02% MgSO 4 · 7H 2 O} , 0.02% CaCl 2
_{· 2H 2 O, 0.001% FeSO} 4 · 7H 2 O, 0.0
Aerobic shaking culture was carried out at 30 ° C. for 3 days in a medium containing 001% MnCl ₂ .4H ₂ O and 0.5% Na ₂ CO ₃ to remove bacterial cells from the resulting culture solution, and the supernatant was obtained. To get Then
Ammonium sulfate is added to the supernatant to 80% saturation, and the mixture is stirred at 5 ° C. for 12 hours to precipitate the protein. The precipitate is recovered by centrifugation, suspended in 10 mM Tris-hydrochloric acid buffer (pH 8.0), and dialyzed against the same buffer overnight.

DEAE-cellulose powder is added to the obtained dialysate to completely adsorb isoamylase on DEAE-cellulose. Then, the resin was washed with 10 mM Tris-hydrochloric acid buffer (pH 8.0), and 1 M sodium chloride was added.
The enzyme is eluted with 0 mM Tris-HCl buffer (pH 8.0). Furthermore, after concentration by dialysis against 10 mM Tris-hydrochloric acid buffer solution (pH 8.0), the solution was equilibrated with "DEAE-B".
io Gel A ”(manufactured by Bio-Rad),
0 to 1 using 10 mM Tris-HCl buffer (pH 8.0)
After elution with a concentration gradient of M sodium chloride, the active fractions were collected, concentrated using an ultrafiltration membrane with an average molecular weight cutoff of 10,000, and then concentrated in 10 mM tris-hydrochloric acid buffer (pH 8.0) containing 0.1 M sodium chloride. ) And dialyse overnight.

The active fraction further contains 0.1 M sodium chloride.
Equilibrated with mM Tris-HCl buffer (pH 8.0)
Phacryl S-200 "(Pharmacia) column, and eluted with the same buffer containing 0.1 M sodium chloride,
The active fraction was collected. After concentrating this using an ultrafiltration membrane, it is dialyzed overnight using 10 mM Tris-hydrochloric acid buffer (pH 8.0).

The purified enzyme thus obtained was subjected to polyacrylamide gel electrophoresis (gel concentration 7.5%) and SDS.
Electrophoresis (gel concentration 15%) gives a single band with an activity yield of about 4%.

[0048]

EFFECTS OF THE INVENTION The alkaline isoamylase of the present invention has an optimum pH on the alkaline side unlike the conventionally obtained isoamylase, is extremely stable in a wide range of pH, and has an optimum temperature. Since 40% or more of the activity remains even at 55 ° C., it can be suitably used as a blending component of a detergent composition. Furthermore, the present invention, which can easily produce the enzyme using a microorganism capable of producing such an alkali isoamylase, has great industrial significance.

[0049]

EXAMPLES The present invention will be described below in more detail with reference to examples, but the present invention is not limited to these examples.

Example 1 A soil of Mito City, Ibaraki Prefecture, was suspended in a spoonful (about 0.5 g) of sterile physiological saline and heat-treated at 80 ° C. for 15 minutes. The supernatant of this heat treatment solution was appropriately diluted and applied to a separation agar medium (medium A). Then, this was cultured at 30 ° C. for 3 days to form a colony. The isoamylase-producing bacteria were obtained by selecting those that form a transparent zone due to the dissolution of glycogen (derived from oysters) around the settlement. Further, the obtained bacteria were inoculated into the liquid medium B, and cultured with shaking at 30 ° C. for 3 days.
The isoamylase activity of the supernatant after centrifugation was measured at pH 9.0 to screen the alkaline isoamylase-producing bacterium, and the alkaline isoamylase-producing bacterium Bacillus sp. KSM-3309 (F
ERM P-13005). The compositions of medium A and medium B are shown below.

[0051]

TABLE 2 Medium A: Composition (wt%) Glycogen (oyster-derived) 0.5 polypeptone 0.2 Yeast extract _{0.1 KH 2 PO 4 0.03 (NH} 4) 2 SO 4 0.1 MgSO 4 · 7H ₂ O 0.02 CaCl ₂ .2H ₂ O 0.02 FeSO ₄ .7H ₂ O 0.001 MnCl ₂ .4H ₂ O 0.0001 Agar 1.5 Na ₂ CO ₃ 0.5 Purified water Balance pH 9.2

[0052]

[Table 3] Medium B: Composition (% by weight) Glycogen (derived from oyster) 1 Tryptone 0.2 Yeast extract 0.1 KH ₂ PO ₄ 0.03 (NH ₄ ) ₂ SO ₄ 0.1 MgSO ₄ .7H ₂ O 0.02 CaCl ₂ .2H ₂ O 0.02 FeSO ₄ .7H ₂ O 0.001 MnCl ₂ .4H ₂ O 0.0001 Na ₂ CO ₃ 0.5 Purified water Balance pH 9.2

Example 2 Bacillus sp., An alkaline isoamylase-producing bacterium
KSM-3309 strain was inoculated into liquid medium B of Example 1,
The cells were cultured at 30 ° C. for 3 days with shaking. After culturing, the bacterial cells were removed by centrifugation to obtain a crude isoamylase enzyme solution. Further, by freeze-drying according to a usual method, crude enzyme preparations shown in Table 4 below were obtained (enzyme activity is a measured value at pH 9.0).

[0054]

[Table 4]

Example 3 The crude enzyme solution obtained in Example 2 was purified according to the following procedure to obtain alkaline isoamylase. That is, ammonium sulfate was added to the supernatant of the crude enzyme solution with stirring so as to be 80% saturated, and then stored at 5 ° C for 12 hours to precipitate the protein. The precipitate was recovered by centrifugation and 10 mM
After suspending in Tris-hydrochloric acid buffer solution (pH 8.0), it is dialyzed against the same buffer solution overnight. Next, DEAE-cellulose powder was added to the dialysate to completely remove the isoamylase.
Adsorb on cellulose. Then, after washing with 10 mM Tris-hydrochloric acid buffer (pH 8.0), 1 M sodium chloride was added.
The enzyme is eluted with 0 mM Tris-HCl buffer (pH 8.0). Furthermore, after concentration by dialysis against 10 mM Tris-hydrochloric acid buffer solution (pH 8.0), the solution was equilibrated with "DEAE-B".
io Gel A "and adsorbed on 10 mM Tris-HCl buffer (pH 8.0) with a concentration gradient of 0 to 1 M sodium chloride, and the active fractions were collected to obtain an average molecular weight cutoff of 10
After concentrating with 000 ultrafiltration membranes, it is dialyzed against 10 mM Tris-hydrochloric acid buffer (pH 8.0) containing 0.1 M sodium chloride overnight. 10 containing the above active fraction in 0.1 M sodium chloride
Equilibrated with mM Tris-HCl buffer (pH 8.0)
Phacryl S-200 "column, 0.1.
Elution was performed with the same buffer containing M sodium chloride, and the active fractions were collected. This was concentrated using an ultrafiltration membrane and then dialyzed against 10 mM Tris-HCl buffer (pH 8.0) overnight. About the obtained isoamylase, as shown in FIG. 5, Davis (Ann.NY.Acad.Sci., 121, 4
04 (1964)) and then stained with Comassie Brilliant Blue to confirm that it gave a single band.

Example 4 The isoamylase obtained in Example 3 was subjected to SDS electrophoresis according to a conventional method. FIG. 6 shows the results. From this, it was found that the molecular weight of this enzyme was 65,000 ± 1000.

[Brief description of the drawings]

FIG. 1 is a drawing showing the relationship between reaction pH and relative activity of alkaline isoamylase of the present invention.

FIG. 2 is a drawing showing the relationship between the treatment pH of alkaline isoamylase of the present invention and the residual activity.

FIG. 3 is a drawing showing the relationship between the reaction temperature (pH 9.0) and the relative activity of the alkaline isoamylase of the present invention.

FIG. 4 is a drawing showing the relationship between the treatment temperature (pH 9.0) and the residual activity of the alkaline isoamylase of the present invention.

FIG. 5 is a drawing showing the results of electrophoresis of the alkaline isoamylase of the present invention according to the method of Davis.

FIG. 6 is a drawing showing the results of SDS electrophoresis of alkaline isoamylase of the present invention.

Continuation of front page (51) Int.Cl. ⁶ Identification number Office reference number FI technical display area C12R 1:07) (C12N 9/44 C12R 1:07)

Claims (5)

(57) [Claims] 1. An alkaline isoamylase having the following enzymatic properties. (1) Action: Hydrolyzes α-1,6 glucoside bond of glycogen and amylopectin. In addition, it does not hydrolyze the α-1,6 glucoside bond of amylose and pullulan. (2) Working pH and optimum pH, acting in the pH range of pH 5 to 11.5, and having optimum pH around 9. (3) pH stability, extremely stable in the range of pH 7 to 10, pH 6 to 10.
The activity of about 50% or more is maintained even at 5 (40 ° C,
Processing for 10 minutes). (4) Working temperature range and optimum working temperature, acting in the range of 40 to 60 ° C., optimum working temperature is about 55 ° C.
It is. (5) Temperature stability, extremely stable up to 50 ° C (glycine at pH 9.0-
Treatment in table salt-sodium hydroxide buffer for 30 minutes). (6) Molecular weight, molecular weight by sodium dodecyl sulfate (SDS) electrophoresis is about 65000 ± 1000. 2. A microorganism belonging to the genus Bacillus and having the alkaline isoamylase-producing ability according to claim 1. 3. A Bacillus sp. (Bacillus
sp. ) Named KSM-3309, Microbiology Research Institute
The microorganism having an alkaline isoamylase-producing ability according to claim 1, which has been deposited as No. 3005. 4. A method for producing alkaline isoamylase, which comprises culturing the microorganism according to claim 2 and collecting alkaline isoamylase from the culture. 5. A method for producing alkaline isoamylase, which comprises culturing the microorganism according to claim 3 and collecting alkaline isoamylase from the culture.