JPH05227959A - New isoamylase, method for producing the same and method for producing saccharides with the same - Google Patents

Abstract

PURPOSE:To provide the subject new enzyme capable of being industrially used under a high temperature in a weak acidic to neutral region and useful for producing maltose, branched cyclodextrin, etc., from starch at a low cost in good productivity. CONSTITUTION:Isoamylase. The action: the isoamylase specifically hydrolyzes the alpha-1,6-glucoside bond of amylopectin, starch, etc.; substrate specificity: the isoamylase hydrolyzes the above-mentioned bond but does not hydrolyze pullulan; the optimum pH: 5.5-6; the stable pH: stable at 30 deg.C at a pH of 4.5-8 for 24hr; temperature suitable for actions: 45 deg.C; inactivation temperature: the isoamylase is perfectly inactivated at 45 deg.C at a pH of 4 and at a pH of 8 for 10hr; temperature stability: stable at a pH of 6 at a temperature up to 40 deg.C for 15min; inhibition, activation and stabilization: the isoamylase is inhibited with Cu, Hg, etc., and is stabilized with Ca; molecular weight (SDS-PAGE method): 88000; isoelectric point: 8.7. The enzyme is obtained by culturing an isoamylase- producing bacterium [preferably new Flavobacterium odoratum KU strain (FERN P-12711)] belonging to Flavobacterium odoratum and separating from the cultured product.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION The present invention relates to flavobacterium
The present invention relates to a novel isoamylase obtained by culturing Odratam KU strain ( Flavobacterium odoratum KU), a method for producing the same, and a method for producing a saccharide using the same.

[0002]

BACKGROUND OF THE INVENTION Isoamylase is an enzyme that hydrolyzes α-1,6-glucopyranoside bonds in starch, amylopectin, glycogen, and their partial hydrolysates. Isoamylase-producing bacteria include Pseudomonas amyloderamosa (Bi
ochim. Biophys. Acta., 212, 458, 1970, Cytophaga (FEBS LETTERS, 12, 96)
Paragraph, 1970), Flavobacteriu
m ), (Starch / Starke, 32 volumes, 132 items, 1980). Also, a method for producing high-purity maltose by using these isoamylases in combination with β-amylases of various origins has been reported. For example, a method using an isoamylase produced by Pseudomonas amyloderamosa (J. Jpn. Soc. Starch Sc
i., Vol. 31, Item 38, 1984) and Flavobacterium ( Fl.
a method using an isoamylase produced by the genus avobacterium (Starch / starke, Volume 32, Item 352, 1980).

However, these isoamylases are not necessarily advantageous because their optimum pH, optimum temperature, etc. do not match the various amylases used in combination. That is, although the production of starch sugar is generally carried out at a high temperature of 50 ° C. or higher and under a weakly acidic condition of pH 5.0 to 6.0, Pseudomonas amyloderramosa ( Pseudomonas)
The isoamylase produced by as amyloderamosa ) has an optimum pH of 3.0 to 4.0, which is biased toward the acidic region, and can be used in combination with acid-resistant soybean β-amylase.
-Amylase and bacterial β-amylase and α with weak acid resistance
-Combination with amylase was difficult. In addition, the genus Flavobacterium and Cytoferga
In a conventional isoamylase produced by the genus haga ),
The optimum temperature was as low as 40 ° C, and it was difficult to use industrially in terms of heat resistance.

On the other hand, starch, amylopectin, as pullulan and enzyme which hydrolyses alpha-1,6-glucopyranoside bonds of their partial hydrolysates, Klebsiella pneumoniae (Klebsiella pneumoniae) (Biochem.
Z., 334, p. 79, 1961) is known, and this enzyme is 20% (w / w) as used in industrial maltose production.
v) In the presence of a high concentration of substrate such as above, the reaction is reversible unlike isoamylase, and maltose is polymerized to form tetrasaccharide, and maltose is transferred to amylose. Unable to produce high purity maltose.

[0005]

As described above, conventional isoamylases have the following optimum pH and temperature.
Since it does not match with various amylases, it is disadvantageous in industrially producing high-purity sugars such as maltose in combination with amylase. Therefore, there has been a strong demand for the development of a novel isoamylase that can be industrially used at 50 ° C. or higher and in a weakly acidic to neutral range. By developing such an enzyme, it becomes possible to carry out production of maltooligosaccharides such as maltose from starch and branched CD with low cost and high productivity.

Therefore, a first object of the present invention is to provide a novel isoamylase satisfying the above requirements.

A second object of the present invention is to provide a method for producing the above novel isoamylase simply and in high yield.

A third object of the present invention is to provide a method for obtaining sugars from starch or its decomposition products in good yield using a novel isoamylase satisfying the above requirements.

[0009]

Means for Solving the Problems In the industrial use, the inventors of the present invention have conducted an intensive search to obtain a microorganism capable of saccharifying isoamylase at 50 ° C. or higher and weakly acidic side, and as a result, found that Kagoshima prefecture Bacteria collected from the above soil are extremely useful for achieving the above purpose, and by aerobically culturing them, a novel isoamylase satisfying the above requirements is produced and accumulated in the culture in high yield. The present invention has been completed and the present invention has been completed.

That is, the novel isoamylase of the present invention is characterized by having the following physicochemical properties. (A) Action The α-1,6-glucopyranoside bond in amylopectin, starch, glycogen and their partial hydrolysates is specifically hydrolyzed. (B) Substrate specificity α-1, in amylopectin, starch and glycogen,
It hydrolyzes 6-glucopyranoside bonds and not pullulan. (C) Optimum pH 5.5 to 6.0 (d) Stable pH It is stable at pH 4.5 to 8.0 under the conditions of 30 ° C. and 24 hours. (E) Suitable temperature for action 45 ° C. (f) Deactivation temperature, pH 45 ° C. Under conditions of 10 minutes, it is completely deactivated at pH 4 and pH 8, and under conditions of pH 6.0 and 15 minutes, 50.
Deactivates completely at ℃. (G) Temperature stability Under conditions of pH 6 and 15 minutes, it is stable up to 40 ° C. (H) Inhibition, activation and stabilization This enzyme is inhibited by copper, mercury and zinc and stabilized by calcium. (I) Molecular weight (SDS-PAGE method) 88,000 (nu) Isoelectric point (Isoelectric Focusing method) 8.7

The isoamylase is preferably an extracellular secretory enzyme.

Further, the method for producing isoamylase of the present invention comprises the steps of flavobacterium odoratum ( Flavobacterium
It is characterized by culturing an isoamylase-producing bacterium belonging to odoratum ) and collecting isoamylase from the culture.

As the above-mentioned isoamylase-producing bacterium, flavobacterium odoratum KU strain ( Flavobacterium odotam) , which was newly searched and isolated from the natural world by the present inventors, was used.
ratum KU, and Microorganism Research Institute, No. 12711) are preferably used. The mycological properties of this microorganism are as shown below.

(1) Morphological characteristics The vegetative cells are rod-shaped bacteria of 0.8 to 1.0 × 1.5 to 2.0 μm and do not form spores and have no motility.

(2) Physiological properties As shown in Table 1.

[0016]

[Table 1]

Regarding the above-mentioned mycological properties, Bergy's Mannual of Systematic Bacteriology
With reference to Volume 1, this bacterium was designated as a Flavobacterium odoratum KU strain ( Fravobacterium odoratum KU). In addition, this bacterium was approved by the Institute of Microbial Science and Technology of the Institute of Industrial Technology in 1992.
It was deposited on January 18, 2014, and the deposit number is Microindustrial Research Institute No. 12711 (FERM P-12711).

The method for producing isoamylase of the present invention will be described in more detail. The isoamylase-producing bacterium belonging to Flavobacterium odoratum as described above was ingested in an appropriate medium, and the growth temperature, preferably Is 25 to 40 ° C., more preferably 30
By aerobically culturing at ~ 37 ° C, preferably for 24 to 96 hours, as an extracellular exocrine enzyme in the culture solution,
The isoamylase is produced and accumulated.

As the above medium, various known materials that can be obtained at low cost can be used. For example,
As the nitrogen source, corn steep liquor, polypeptone, soybean meal, bran, meat extract, yeast extract, amino acid liquid, etc. are used, and as the carbon source, starch syrup, maltose, various starches, soluble starch, starch liquefaction liquid, Dextrin, cyclodextrin, etc. are used. In addition to these nitrogen sources and carbon sources, various salts, for example, inorganic salts such as magnesium salts, potassium salts, phosphates, and various vitamins are added as necessary. Examples of preferred medium include, for example, 0.5% soluble starch, 0.1% polypeptone, 0.1% yeast extract, 0.1%.
1% NH ₄ NO ₃ , 0.14% KH ₂ PO ₄ , 0.02
A liquid medium containing% MgSO ₄ .7H ₂ O and 0.01% sodium L-glutamate can be mentioned.

Since the above-mentioned isoamylase-producing bacterium produces isoamylase outside the microbial cells, after culturing, the microbial cells are removed from the culture supernatant by centrifugation to remove the crude enzyme of isoamylase of the present invention. A liquid can be obtained. Industrially, this crude enzyme solution can be used as it is,
Economical and advantageous. However, it can be further purified before use. As the purification method, for example, salting out with ammonium sulfate, a solvent precipitation method with ethanol, acetone, isopropanol, etc., a starch adsorption method, an ultrafiltration method, a general enzyme purification method with an ion exchange resin or the like can be adopted. ..

The preferred specific examples of the method for producing isoamylase of the present invention are as follows. Flavobacterium odoratum KU strain ( Flavobacterium odoratum KU,
MICRO LAB. No. 12711), 2% soluble starch,
1% soy protein (trade name "Sorupi K", Nisshin Oil Mills Co., _{Ltd.), 0.1% NH 4 NO 3} , 0.14% KH 2
PO ₄ , 0.1% CaCl ₂ · 2H ₂ O, 0.02% M
The cells are inoculated into a medium containing gSO ₄ .7H ₂ O and 0.01% L-sodium glutamate, and cultured aerobically at 30 ° C. for 96 hours. The culture broth thus obtained is 10,000 ×
g, the cells are centrifuged at 4 ° C. to remove bacterial cells, and a supernatant (crude enzyme solution) of about 1.3 L (liter) is obtained. Next, 13 g of cornstarch is added to this supernatant and left at 4 ° C. for 2 hours to adsorb isoamylase to starch. Then, the starch to which the isoamylase was adsorbed was
Collect by centrifugation at 0xg, 4 ° C. 10 of this starch
It is dissolved in 0 mL (milliliter) of 1% (w / v) maltotetraose solution (pH 6.0) and left for 2 hours with stirring at room temperature to elute isoamylase from starch. The eluate was centrifuged at 10,000 × g at room temperature to remove starch, and the resulting supernatant was added to 0.2M Na
10 mM Tris / HCl buffer (pH 8.0) containing Cl
Against dialysis overnight at 4 ° C. This enzyme solution was concentrated using an ultrafiltration membrane having a molecular weight cut off of 10,000, and then Sephacryl S-30 equilibrated with the above-mentioned Tris-hydrochloric acid buffer solution.
Gel filtration is performed using a 0HR column and the active fractions are collected. The purified enzyme thus obtained was confirmed to have a single band by polyacrylamide gel disc electrophoresis, and the activity yield was about 23%.

The novel isoamylase of the present invention thus obtained has the physicochemical properties described above.

The isoamylase activity measuring method and activity indicating method in the present invention are as follows. That is,
0.5M to 0.35mL of 0.5% glutinous rice starch solution
0.1 mL of the acetate buffer solution (pH 6.0) is mixed with 0.1 mL of the enzyme solution diluted appropriately, and the mixture is reacted at 45 ° C. for 15 minutes. Thereafter, 0.5 mL of an iodine solution (0.5 M potassium iodide solution containing 0.05 M iodine) diluted 5 times with 0.1 N HCl was added to stop the enzymatic reaction, and 10 m
After adding L water and stirring sufficiently, it measures at 610 nm using a spectrophotometer. As the unit of enzyme activity, 1 unit was the amount of enzyme that increased 0.01 absorbance per minute under the above conditions.

Next, in the method for producing a saccharide of the present invention, the novel isoamylase described above is used in combination with at least one enzyme selected from the group consisting of α-amylase, β-amylase and glucoamylase, It is characterized in that it acts on starch or its hydrolyzate.

Examples of the α-amylase include bacterial liquefaction-type thermostable α-amylase (trade name “Crystase T-5”, manufactured by Daiwa Kasei Co., Ltd.), maltotriose of Bacillus origin. A synthase such as a maltotetraose synthase originating from Pseudomonas stutzeri can be used. Further, as β-amylase, for example, β-amylase derived from soybean (manufactured by Nagase Biochemical Co., Ltd.), β-amylase derived from malt (trade name “Ultrazyme”, Enzyme
Biosystems) and the like can be used. Furthermore,
Examples of glucoamylase include Rhizops ( Rhiz
opus ) Origin glucoamylase (trade name "Sumiteam", manufactured by Shin Nippon Kagaku Co., Ltd.), Aspergillus spp ( As
pergillus ) -derived glucoamylase (trade name “ Amyloglucosidase ”, manufactured by Novo Co.) and the like can be used. In the present invention, pullulanase and the like may be used in combination with the above-mentioned enzyme, if necessary.

In the present invention, the starch hydrolyzate includes a starch liquefaction solution obtained by liquefying starch with liquefied α-amylase, various maltooligosaccharides such as maltose, cyclodextrin, and mixtures thereof. means. The type of enzyme used in combination with isoamylase and the type of substrate are determined according to the desired hydrolyzate.

[0027]

The novel isoamylase of the present invention hydrolyzes α-1,6-glucopyranoside bonds in amylopectin, starch and glycogen. It is known that the starch sugar production reaction suffers various adverse effects due to microbial contamination when carried out at low temperature and under weakly acidic conditions. Therefore, it is desirable to carry out the enzymatic hydrolysis reaction of this starch under conditions of as high temperature as possible, and under weakly acidic to neutral pH conditions.

However, the isoamylases isolated so far are inferior in thermostability, and most of them show an optimum temperature of 40 ° C. or lower. Further, the isoamylase produced by Pseudomonas amyloderamosa has a high optimum temperature of about 50 ° C., but since the optimum pH is as low as 3.0 to 4.0 as described above, There is a problem that the enzymes that can be used together are limited.

On the other hand, the novel isoamylase of the present invention has an optimum pH of 5.5 to 6.0 and an optimum temperature of action as high as 45 ° C., so that most of α used in industry is used. −
It can be used in combination with amylase, β-amylase, and glucoamylase, and the starch hydrolysis reaction can be carried out at 55 ° C. or higher and in the vicinity of a weakly acidic region. This means that microbial contamination is surely prevented, and it is not necessary to add an alkaline reagent for the purpose of compensating for pH decrease due to contamination, which means that a large reduction in manufacturing cost can be expected. Furthermore, since the reaction can be carried out at high temperature, the reaction rate is expected to be improved, and the productivity of decomposition products can be increased.

When the isoamylase of the present invention is used in combination with β-amylase to act on starch or its hydrolyzate, maltose can be produced in high yield. Further, if the isoamylase of the present invention is used in combination with maltriose-forming enzyme, maltotetraose-forming enzyme, etc., to act on starch or its hydrolyzate,
Malto-oligosaccharides such as maltotriose and maltotetraose can be produced in high yield. Furthermore, when the isoamylase of the present invention is allowed to act on a sugar solution containing a maltooligosaccharide such as maltotriose and cyclodextrin, a branched cyclodextrin can be obtained by a reverse synthesis reaction of isoamylase.

[0031]

【Example】

Example 1 Flavobacterium odoratum KU strain ( Flavobacterium
odoratum KU, Mikken Kenkyubo No. 12711), 2% maltose, 1% soy protein (trade name "Solpy K",
Nisshin Oil Co., Ltd.), 0.1% NH ₄ NO ₃ , 0.1
4% KH ₂ PO ₄ , 0.1% CaCl ₂ · 2H ₂ O,
_{0.02% MgSO 4 · 7H 2 O} , medium containing 0.01% L-glutamate sodium (pH 6.0) 100
Bacteria were inoculated into 15 flasks, each of which was dispensed in mL, at 9
The cells were cultivated by shaking at 180 rpm for 6 hours. After completion of the culture, the culture solution was centrifuged at 10,000 × g at 4 ° C. for 30 minutes to remove the cells, and 8.3 units / mL of the crude enzyme solution 1,3
00 mL was obtained.

13 g of corn starch was added to this crude enzyme solution, and the mixture was allowed to stand at 4 ° C. for 2 hours to adsorb isoamylase on starch. Then, the starch having adsorbed isoamylase was collected by centrifugation at 10,000 × g and 4 ° C. This starch was suspended in 100 mL of a 1% (w / v) maltotetraose solution (pH 6.0) and allowed to stand for 2 hours with stirring at room temperature to elute isoamylase from the starch. The eluate was centrifuged at 10,000 xg at room temperature to remove starch, and the resulting supernatant was added to 0.2MN.
10 mM Tris / HCl buffer (pH 8.
0) was dialyzed overnight at 4 ° C. After concentrating this enzyme solution using an ultrafiltration membrane with a molecular weight cut off of 10,000,
Sephacryl S- equilibrated with the above Tris / HCl buffer
Gel filtration was performed on a 300 HR column to collect active fractions. The purified enzyme thus obtained was confirmed to have a single band by polyacrylamide gel disc electrophoresis, and the activity yield was about 23%.

Example 2 The physicochemical properties of the purified isoamylase prepared in Example 1 were examined for the following items.

(1) Specificity To 5 mL of a 2% solution containing amylopectin, glycogen and pullulan, 300 units of the above purified isoamylase was added to 1 g of the substrate, and the mixture was reacted at 45 ° C. for 24 hours.
When the reaction product was measured by the HPLC method, amylopectin and glycogen were clearly decomposed, but pullulan was not decomposed.

(2) Optimum pH 0.2M acetate buffer (pH 4.0, 5.0, 5.5,
6.0), 0.2M phosphate buffer (pH 6.5, 7.
0, 7.5, 8.0), or 0.45 mL of a 0.5% (w / v) glutinous rice starch solution dissolved in a 0.2 M glycine buffer (pH 9.0), and the purified isoamylase was added to the solution. 0.1 mL of an isoamylase-containing solution previously dialyzed against water containing 1 mM EDTA was mixed, and the isoamylase activity was measured by the method described above. The result is shown in FIG. The optimum pH of isoamylase is pH 6.
The relative activity is shown with the enzyme activity at 0 being 100.

(3) Stable pH The isoamylase-containing solution obtained by treating in the same manner as in (2) was treated at pH 4 to 9.5 at 30 ° C. for 24 hours and 4 hours.
Relative activity was determined by treating residual enzyme activity as 100 when treated at 5 ° C for 10 minutes and treated at pH 6.0. The result is shown in FIG. In the figure, ∘-∘ indicates the result of treatment at 30 ° C. for 24 hours, and ● − ● indicates the result of treatment at 45 ° C. for 10 minutes. The buffer used was acetate buffer (pH
4-6), phosphate buffer (pH 6.5-8.0) and glycine-NaOH-NaCl buffer (pH 8.5-9.
5).

(4) Optimum temperature of action The activity of the isoamylase-containing solution obtained by treating in the same manner as in (2) was measured at each temperature, and the relative activity was determined with the enzyme activity measured at 45 ° C. being 100. It was The result is shown in FIG.

(5) Inactivation condition and temperature stability The purified isoamylase obtained in (1) was treated in the same manner as in (2), and then in the presence and absence of 10 mM CaCl ₂ .2H ₂ O. 35 ° C, 40 ° C, 45 ° C, 50 ° C and 5
It was treated at 5 ° C. for 15 minutes to make the remaining activity. The result is shown in FIG. In the figure, ○-○ indicates CaCl ₂ ·
As a result of not adding 2H ₂ O, ●-● indicates CaCl
Shows the results upon addition of ₂ · 2H ₂ O. Thus, it can be seen that the addition of Ca ²⁺ ions improves the temperature stability and maintains the activity even under the temperature condition of 50 ° C. or higher.

(6) Inhibition Into the isoamylase-containing solution treated in the same manner as in (2), various metal hydrochlorides or chemicals were added so that the final concentration was 1 mM (however, mercury was 0.1 mM and 1 mM). Reagents were added and activity was measured. And the activity without addition is 100
Table 2 shows the results of determining the relative activity.

[0040]

[Table 2]

(7) Molecular Weight The molecular weight of the isoamylase of the present invention determined by electrophoresis using SDS-polyacrylamide is 88,000.
It was 0. FIG. 3 shows the experimental result of the electrophoresis method.

(8) Isoelectric point The isoamylase of the present invention obtained by the Isoelectric Focusing method had an isoelectric point of 8.7. FIG. 4 shows the measurement result of the isoelectric point.

Example 3 2% soluble starch, 1% soybean protein (trade name "Solpy K", manufactured by Nisshin Oil Co., Ltd.), 0.1% NH ₄ NO
₃ , 0.14% KH ₂ PO ₄ , 0.1% CaCl ₂・ 2
_{H 2 O, 0.02% MgSO 4} · 7H 2 O, 0.01%
Medium containing L-sodium glutamate (pH 6.0)
100 mL of each was placed in 20 500 mL Erlenmeyer flasks, and the Flavobacterium odoratum KU strain ( Flavob
Acterium odoratum KU and Microtechnology Research Institute, Microbiology Co., Ltd. No. 12711) were respectively inoculated and cultivated at 30 ° C. for 96 hours with rotary shaking at 180 rpm. 10,000 xg of this culture solution, 4
The cells were removed by centrifugation at 20 ° C. for 20 minutes to obtain 1850 mL of a crude enzyme solution of 10.5 unit / mL.

Next, 15 g of cornstarch was added to the crude enzyme solution, and the mixture was allowed to stand at 4 ° C. for 2 hours to adsorb isoamylase on starch. And 10,000 × g, 4 ℃
The starch to which isoamylase was adsorbed was collected by centrifugation at. 100 ml (1 ml) of this starch
% (W / v) maltotetraose solution (pH 6.0), and the suspension was allowed to stand for 2 hours with stirring at room temperature to elute isoamylase from starch. This eluate is 10,
Centrifugation at 000 × g at room temperature gave a supernatant without starch. The supernatant was concentrated using an ultrafiltration membrane having a molecular weight cut off of 10,000 to obtain about 5 mL of a concentrated crude enzyme solution of 2,500 units / mL.

Next, 10 L of a 25% (w / v) potato starch suspension was added to a bacterial liquefaction type thermostable α-amylase (trade name “Crystase T-5”, manufactured by Daiwa Kasei Co., Ltd.).
After liquefying at 105 ℃ using
After autoclaving for minutes, a starch liquefaction liquid with a DE (Dextrose Equivalent: ratio of total solids to direct reducing sugars) 2.7 was obtained. To 5 mL of this liquefied solution, 0.25% of β-amylase derived from soybean (10,000 U / g, manufactured by Nagase Biochemical Co., Ltd.) and 0.001% based on the weight of starch
The same bacterial liquefaction-type thermostable α-amylase as above,
Add 0 unit of the above crude solution of isoamylase concentrated enzyme, p
The reaction was carried out at H 6.0 and 55 ° C. for 72 hours.

0.2% (w
/ V) activated carbon was added, the mixture was heated in a boiling water bath for 5 minutes, and then filtered with a 0.45 μm pore size membrane filter. The sugar composition in the sugar solution thus obtained was measured by high performance liquid chromatography using a ULTRON PS-80N (trade name, manufactured by Affinity Engineering Co., Ltd.) column, and G _{1 was} 0.4%. , G ₂ : 89.3%,
G _3: 7.5%, oligosaccharide: 2.8%.

Example 4 The sugar solution obtained in Example 3 was treated with JP-A-2-1138.
The enzyme derived from Thermomonospora viridis TF-35 described in No. 86, 150 units per 1 g of starch,
The reaction was continued for 48 hours.

The obtained sugar solution was decolorized by the same method as in Example 3 and filtered, and then the sugar composition was measured by the same high performance liquid chromatography method as in Example 3, and the result was G ₁ : 3. 0
%, G ₂ : 94.0%, G ₃ : 2.5%, oligosaccharide:
It was 0.5%.

Example 5 Approximately 10 L of a 30% (w / v) corn starch suspension was mixed with a bacterial liquefaction type thermostable α-amylase (trade name “Crystase T-5”, manufactured by Daiwa Kasei Co., Ltd.). After being liquefied at 90 ° C., it was immediately autoclaved at 120 ° C. for 30 minutes to obtain a starch liquefied liquid of DE5.5.

5 mL of this starch liquefaction solution was taken, and 0.2% per 1 g of starch of malt-derived β-amylase (trade name "Ultrazyme", manufactured by Enzyme Biosystems) and 100 units of Example 3 were prepared. Isoaamylase concentrated crude enzyme solution was added, and the pH was 6.0 at 55 ° C.
The reaction was carried out for 72 hours.

The sugar solution thus obtained was decolorized by the same method as in Example 3 and filtered, and then the sugar composition was measured by the same high performance liquid chromatography method as in Example 3. As a result, G ₁ : 3. Two
%, G ₂ : 76.5%, G ₃ : 14.7%, oligosaccharide:
It was 5.6%.

Example 6 By weight ratio, maltose and α-CD (cyclodextrin) were in a ratio of 3: 1, maltose and β-CD were in a ratio of 5: 1, and maltose and γ-CD were in a ratio of 3: 1. For each sugar solution containing about 70% (w / v) concentration, the crude isoamylase enzyme solution prepared in Example 3 was added to the substrate 1.
7,500 units were added per g, pH 6.0, 55
The reaction was carried out at 72 ° C for 72 hours.

For each sugar solution after the reaction, Asahip
When the sugar composition was examined using ak GS-320 (trade name, manufactured by Asahi Kasei Co., Ltd.), branched α-CD 14.1%, branched β-CD 7.9%, and branched γ-CD 15.2% were produced, respectively. Was.

[0054]

As described above, according to the present invention,
It is possible to provide a novel isoamylase having an optimum pH in a weakly acidic to neutral range and a high suitable temperature for action. By using this isoamylase, it is possible to perform a saccharification reaction in a high temperature range of about 55 ° C, for example. It will be possible. Then, by using this isoamylase in combination with α-amylase, β-amylase, glucoamylase, etc., high-purity maltose not obtained by pullulanase, or malto-oligosaccharide having a polymerization degree higher than that, branched CD And the like can be industrially manufactured.

[Brief description of drawings]

FIG. 1 is a chart showing the results of measuring the pH dependence and temperature dependence of the relative activity of the isoamylase of the present invention.

FIG. 2 is a chart showing the results of measuring the relationship between the residual activity of the isoamylase of the present invention and pH and temperature.

FIG. 3 is a table showing the results of measuring the molecular weight of a protein containing isoamylase of the present invention by an electrophoresis method using SDS-polyacrylamide.

FIG. 4 is a chart showing the results of measuring the isoelectric point of the isoamylase of the present invention by the Isoelectric Focusing method.

Claims (5)

[Claims] 1. An isoamylase having the following physicochemical properties. (A) Action The α-1,6-glucopyranoside bond in amylopectin, starch, glycogen and their partial hydrolysates is specifically hydrolyzed. (B) Substrate specificity α-1, in amylopectin, starch and glycogen,
It hydrolyzes 6-glucopyranoside bonds and not pullulan. (C) Optimum pH 5.5 to 6.0 (d) Stable pH It is stable at pH 4.5 to 8.0 under the conditions of 30 ° C. and 24 hours. (E) Optimum temperature of action 45 ° C. (f) Deactivation temperature 45 ° C., complete inactivation at pH 4 and pH 8 under conditions of 10 minutes, 50 under conditions of pH 6.0 and 15 minutes.
Deactivates completely at ℃. (G) Temperature stability Under conditions of pH 6 and 15 minutes, it is stable up to 40 ° C. (H) Inhibition, activation and stabilization This enzyme is inhibited by copper, mercury and zinc and stabilized by calcium. (I) Molecular weight (SDS-PAGE method) 88,000 (nu) Isoelectric point (Isoelectric Focusing method) 8.7 2. The isoamylase according to claim 1, which is an extracellular secretory enzyme. 3. Flavobacterium odoratum ( Flav
A method for producing isoamylase, which comprises culturing an isoamylase-producing bacterium belonging to obacterium odoratum ) and collecting isoamylase from the culture. 4. An isoamylase-producing bacterium belonging to the Flavobacterium odoratum flavobacterium odoratum KU strain ( Fl
avobacterium odoratum KU, Microorganisms Research Institute No. 12711
No.) is used for producing the isoamylase. 5. The isoamylase according to claim 1, and α-
A method for producing a saccharide, which comprises acting in combination with at least one enzyme selected from the group consisting of amylase, β-amylase and glucoamylase, on starch or a hydrolyzate thereof.