JPH0568546A - Novel cyclomaltodextrinase, method for producing the same, and microorganism producing the enzyme - Google Patents

Abstract

(57) [Summary] [Objective] A novel cyclomaltodextrinase, which is an enzyme produced extracellularly with a high suitable temperature for action, excellent temperature stability, weakly optimum pH for action, and a method for producing the same. A microorganism producing the enzyme is provided. [Structure] Bacillus stearothermophilus No. K
The H-17 strain (Microtechnology Research Institute No. 12481) is cultured, cyclomaltodextrinase is collected from the culture supernatant, and purified if necessary. This cyclomaltodextrinase hydrolyzes and transfers the α-1,4-glucopyranoside bond in the maltooligosaccharide having a degree of polymerization of maltotriose or higher to produce a series of maltooligosaccharides. Further, it acts on cyclodextrins and produces maltooligosaccharides derived from the degree of polymerization of the cyclodextrins as the main component.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a novel cyclomaltodextrinase (EC, 3, 2, 1, 1) which produces a series of maltooligosaccharides from maltooligosaccharides and cyclodextrins (hereinafter abbreviated as CDs). 54), a method for producing the same, and a microorganism that produces the enzyme. More specifically, it is obtained by culturing a novel thermophilic bacterium belonging to Bacillus stearothermophilus, acts on maltooligosaccharides having a degree of polymerization of maltotriose or higher, and produces a series of maltooligo containing maltose as a main component. The present invention relates to cyclomaltodextrinase that produces saccharides and acts on CDs to produce maltooligosaccharides derived from the degree of polymerization of CD as a main component, a method for producing the same, and a microorganism that produces the enzyme.

[0002]

2. Description of the Related Art There have been very few reports on cyclomaltodextrinase. For example, Bacillus macerans , Biochem., 7: 121-124, 19
68), Bacillus coagula
ns, Agric. Biol. Chem., 47, 1441-1447, 1983.
Year), Bacillus sphaericus ,
Japanese Patent Application No. 1-152257) , Xanthomonas campestris (JP-A-3-83582), and Pseudomonas amilo de la Mosa ( Pesudomonas am)
yloderamosa , Biochim. Biophys. Acta, 391, 96-108
Page, 1975), etc. are only known.

All of these enzymes decompose CD well, but since they are intracellular enzymes, not only the enzyme preparation is complicated, but also the productivity is limited. Furthermore, since it is inferior in temperature stability and the optimum temperature of the enzyme reaction is low, there is a problem in using it as an industrial enzyme for producing starch sugar.

[0004]

Generally, when starch sugar is produced using an enzyme, an enzyme having a high suitable temperature for action and high stability at the temperature is desired. This is because the enzyme reaction at low to medium temperature cannot prevent microbial contamination in the reaction system,
Not only unnecessary by-products are generated by a secondary reaction by contaminating microorganisms, but also a large amount of an alkaline agent is required for pH adjustment due to a decrease in pH during the reaction. It is not economical because it requires a large-scale purification equipment to remove

The optimum pH for the action of the enzyme is preferably weak acidity of about pH 4-6. This is because when the enzymatic reaction is carried out under neutral to alkaline conditions, the starch sugar produced is isomerized, resulting in a decrease in the required purity of the starch sugar.

Further, when the enzyme used is produced by a microorganism, it is preferable that the enzyme is produced and accumulated outside the cells (in the medium). This is because, in the case of the intracellular enzyme, the productivity of the enzyme is limited and not only economically problematic, but also a complicated step is required for extraction and purification of the enzyme from the bacterial cell.

Therefore, an object of the present invention is to provide a novel cyclomaltodextrinase, which is an enzyme produced outside the cells, which has a high suitable temperature for action, excellent temperature stability, a weakly optimum pH for action and a weak acidity. It is to provide a production method and a microorganism that produces the enzyme.

[0008]

Means for Solving the Problems The present inventors have found that maltotrix, which is an enzyme produced outside the bacterial cell, has a high optimum temperature for action, excellent temperature stability, weakly optimum pH for action, and weak acidity. Cyclomaltodextrinase having the action of more strongly decomposing maltooligosaccharides and CDs having a degree of polymerization of ose or more, particularly α-CD (cyclohexaamylose), as compared with other CDs to produce maltooligosaccharides As a result of broadly searching the microorganisms that produce the enzyme from the natural world, it was found that one strain belonging to Bacillus stearothermophilus (KH-17 strain) isolated from the soil strongly produced the enzyme, and completed the present invention. did.

[0009] That is, the present invention provides a cyclomaltodextrinase having novel properties and belongs to Bacillus stearothermophilus and produces cyclomaltodextrinase extracellularly (in a medium). A strain having the ability (KH-17 strain), and a method for producing the novel cyclomaltodextrinase, which comprises culturing the strain and collecting the novel cyclomaltodextrinase from the culture. Is.

The physicochemical properties of the cyclomaltodextrinase of the present invention will be described below.

(A) Action The α-1,4-glucopyranoside bond in the maltooligosaccharide having a degree of polymerization of maltotriose or higher is hydrolyzed and rearranged to produce a series of maltooligosaccharides. It also acts on CDs and produces maltooligosaccharides derived from the degree of polymerization of the CDs as a main component. (B) Substrate specificity Malto-oligosaccharides and CDs having a degree of polymerization of maltotriose or higher are decomposed. The hydrolysis rate of α-CD is higher than that of other CDs and linear maltooligosaccharides having the same degree of polymerization. Starch, amylopectin, glycogen,
It hardly decomposes panose and isopanose.

(C) Optimum pH It works in the range of pH 4 to 8, and the optimum pH is around 5.5. (D) Stable pH range Stable at pH 6-9 when stored at 5 ° C for 24 hours. (E) Optimum temperature of action It works in the temperature range of 40 to 75 ° C, and the optimum temperature is around 60 ° C. (F) Inactivation condition About 90% of pH 4 is inactivated under storage conditions of 4 ° C and 24 hours, and completely inactivated at 80 ° C under conditions of pH 7 and 2 hours.

(G) Temperature stability pH 7 is stable up to 40 ° C. under the condition of 1 hour, 50
The residual activities at 90 ° C, 60 ° C, 70 ° C and 80 ° C are 90%, 70%, 30% and 13%, respectively. (H) Inhibition and activation It is inhibited by divalent cadmium, zinc, mercury, manganese, copper, nickel and pCMB (parachloromercury benzoate). (I) Molecular weight The molecular weight is about 106,000 (gel filtration method). (G) Isoelectric point The isoelectric point is about 4.7 (isoelectric focusing method).

Table 1 shows the difference in physicochemical properties between the enzyme of the present invention and the conventionally known cyclomaltodextrinase.

[0015]

[Table 1]

As is clear from Table 1, the enzyme of the present invention not only acts at high temperature but also outside the cells (in the medium) as compared with the conventionally known cyclomaltodextrinase.
It was different in that it was generated and accumulated in. To date, no enzyme having the above-mentioned properties has been known, so the cyclomaltodextrinase of the present invention was determined to be a novel enzyme.

The cyclomaltodextrinase of the present invention is a Bacillus stearothermophilus.
arothermophilus ), which is obtained by culturing a microorganism having the ability to produce the enzyme and collecting the enzyme from the culture.

As a microorganism belonging to Bacillus stearothermophilus used in the present invention and producing cyclomaltodextrinase, Bacillus stearothermophilus No. KH-17 strain ( Ba
Cillus stearothermophilus No. KH-17) is preferably used.

A. Morphological features (1) Cell shape and size: bacillus, size (0.6 ~
1.0) × (2.5 to 3.0) μm (microns) (2) Cell polymorphism: None (3) Motility: Motile, with periflagellates (4) Presence or absence of spores: Ascospore: Swelling slightly Spore shape: Elliptical Position of spore: Slightly on the terminal side (5) Gram stainability: Positive (6) Anti-acidity: Negative

B. Growth on various media (1) Meat broth agar plate culture It is white and forms circular colonies. The center is convex or convex. The rim is full-edged. No pigment production is observed. (2) Slop culture of broth agar Grows in a spread pattern. (3) Broth liquid culture The growth is weak, and a slight turbidity and sediment are formed, and an imperfect ring is formed on the surface. (4) Meat broth gelatin stab culture Only the surface was grown and no liquefaction of gelatin was observed. (5) Litmus milk No coagulation and no acid or alkali production. (6) No anaerobic growth with glucose / broth liquid medium is observed. (7) Generation of anaerobic gas from nitrate No gas is generated.

C. Physiological properties (1) Reduction of nitrate Reduction (2) Denitrification reaction Negative (3) VP test negative (4) Indole formation negative (5) Hydrogen sulfide formation negative (6) Starch hydrolysis positive (7) Use of citric acid Not used (8) Use of inorganic nitrogen source Used (9) Dye production Not produced (10) Hydrolysis of gelatin and casein Not decomposed (11) Urease positive (12) Oxidase negative (13) Catalase negative (14) Attitude toward oxygen Aerobic (15) Range of growth Temperature: 30 to 75 ° C (optimal 55 to 60 ° C) pH: 5.5 to 8.0 (optimal pH 6 to 6.5) (16) Lysozyme (17) Resistance to azide (18) Attitude toward sugars D-glucose, L-arabinose, D-xylose,
An acid is produced from D-fructose, D-galactose, D-trehalose, maltose, sucrose, lactose, melibiose, raffinose, cellobiose, glycerin and D-mannite, and no gas is produced.

This strain is a spore-bearing bacterium that is motile,
It is clear that it belongs to the genus Bacillus because it is positive for Gram staining. Furthermore, since it has the ability to hydrolyze starch, cannot grow at pH 5.0, and can grow up to 75 ° C, it is based on the Burjay's Manual of Cysticatic Bacteology, Volume 2, 1984. It was identified as a bacterium belonging to the species R. thermophilus. This Bacillus stearothermophilus KH-17 strain has been deposited at the Institute of Microbial Science and Technology, the Agency of Industrial Science and Technology as Microorganism Research Institute No. 12481.

For the culture of the cyclomaltodextrinase-producing strain, a shaking culture method using a liquid medium, which is adopted for the culture of general aerobic microorganisms, an aeration stirring culture method and the like are used.

As the medium, a medium containing an appropriate nitrogen source, carbon source, vitamins, minerals, etc. may be used. Soluble starch, dextrin, powdered candy, CD
The compounds, pullulan, lactose and the like can be used alone or in combination of two or more kinds. As the nitrogen source, as organic nitrogen compounds, corn steep liquor, polypeptone, various amino acids, soybean meal, meat extract, fish extract, yeast extract, etc., and as inorganic nitrogen compounds, various ammonium salts, nitrates, urea, etc. They can be used alone or in combination of two or more. In addition, vitamins, minerals and the like can be added as appropriate.

The culture temperature is usually 30 to 75 ° C, preferably 40 to 70 ° C, more preferably 50 to 55 ° C. The culture pH is usually 5.5 to 8.0, preferably 6.0 to 7.0. The culture time varies depending on the culture method, but is about 18 to 100 hours. According to the main culturing method, most of cyclomaltodextrinase (about 80
%) Accumulates in the medium (outside the cells), and the rest is localized on the cell surface. After completion of the culture, cyclomaltodextrinase can be obtained by an ordinary method, and an example thereof will be shown below.

First, the culture solution is centrifuged to remove bacterial cells. An equal amount of isopropanol is added to the obtained supernatant to obtain a precipitate. This precipitate was added to 10 mM acetate buffer (p
The crude enzyme obtained by dialysis against H6) was treated with DEAE-
It is subjected to gradient ion exchange chromatography with sodium chloride using Toyopearl 650S, and the eluted active fraction is concentrated with an ultrafiltration membrane having an average molecular weight cut off of 10,000. This concentrated enzyme was subjected to gel filtration using Sephacryl S-300 equilibrated with the same buffer as above to collect an active fraction, and this active fraction was further purified by a chromatofocusing method to obtain a purified enzyme. obtain.

The activity of cyclomaltodextrinase of the present invention was measured as follows. 1% α-CD
Enzyme solution 0.0 appropriately diluted with water in 0.5 mL (ml) of 0.1 M acetate buffer (pH 5.5) containing
After adding 5 mL and reacting at 50 ° C. for 10 minutes, 1.0 mL of Somogyi solution was added to stop the reaction. Then, after boiling for 30 minutes in a boiling water bath,
Quick cooling in running water, Nelson liquid 1.0m
L was added. Water was added to make 10.0 mL, and the reducing sugar produced using 100 μg of glucose treated in the same manner as a standard was measured at 510 nm. Here, 1 unit of this enzyme is
Under the same conditions as above, the amount of an enzyme that produces a reducing sugar corresponding to 1 μmole of glucose from α-CD was used.

[0028]

EXAMPLES Next, the present invention will be described more specifically by way of examples.

Example 1 Preparation of Crude Enzyme 0.5% Dextrin, 1% Shefton F (US, USA), 0.1% Yeast Extract, 0.1%
Sodium glutamate, 0.1% ammonium nitrate, 0.
_{1% K 2 HPO 4, 0.02} % MgSO 4 · 7H 2 O,
50 mg / L CaCl ₂ .2H ₂ O and 10 mg / L
MnSO ₄ · 7H ₂ O pH 6.3 liquid medium 1
After sterilizing 5 L (liter) at 120 ° C. for 30 minutes,
In advance, using the same medium as above, at 50 ° C. for 20 hours, 18
Bacillus stearothermophilus No. No. cultured with rotary shaking at 0 rpm. KH-17 strain
No.) Inoculate 1 L of the suspension, 250 rpm, 1 v, v, m
Under the above conditions, aeration and agitation culture was carried out at 50 ° C. for 50 hours. After the completion of the culture, the supernatant obtained by centrifuging to remove the bacterial cells was used as a supernatant to obtain an ultrafiltration membrane with an average molecular weight cut off of 5,000 (Romi
(manufactured by Con Co.) and concentrated to about 2 L. The concentrated crude enzyme solution had an enzyme activity of 25.5 units per mL.

Example 2 Purification of enzyme To 1.0 L of the crude enzyme obtained in Example 1 was added an equal amount of isopropanose to obtain a precipitate. This precipitate is
After collecting by centrifuging at 00 × g at 4 ° C. for 10 minutes, 10
It was dialyzed overnight at 4 ° C. against mM acetate buffer (pH 6.0). Then, the supernatant liquid from which insoluble matter was removed by centrifugation was passed through DEAE-Toyopearl 650S equilibrated with the same buffer as above to adsorb the enzyme. The adsorbed enzyme was eluted by a gradient method using the same buffer containing sodium chloride as described above, and then the active fractions were collected to obtain an ultrafiltration membrane (Am having an average molecular weight cut-off of 10,000).
(manufactured by icon) and concentrated. For concentrated enzymes,
Sephacryl (Se) equilibrated with the same buffer as above
Phacryl) S-300 was used for gel filtration chromatography for further purification, followed by concentration using the same ultrafiltration membrane as described above. The concentrated enzyme was further purified by a chromatofocusing method to obtain a purified enzyme. In addition,
The specific activity of this purified enzyme was 71 units / mg protein, which was single on polyacrylamide gel disk electrophoresis.

Example 3 Physicochemical Properties of Enzyme (a) Action 0.2 unit of 10 mM phosphate buffer (pH 6.0) containing 10 mg of various substrates was supplemented with 0.1 unit of the purified enzyme obtained in Example 2. It was added and reacted at 55 ° C. for 3 hours. Then, after heating for 10 minutes in a boiling water bath to inactivate the enzyme, the product was analyzed by HPLC using an MCI gel-CKO4SS column (manufactured by Mitsubishi Kasei Co., Ltd.).
Shown in.

[0032]

[Table 2]

(B) Specificity Table 3 shows the results of the action of the purified enzyme obtained in Example 2 on various substrates. The reaction was measured by a conventional method, and α
-It was expressed as a relative activity with an activity of 100 when CD was used as a substrate. Further, in the case of various maltooligosaccharides, after reacting under the same conditions, the enzyme was inactivated by heating in a boiling water bath for 10 minutes, and then the reducing sugar produced was measured by the Somogy Nelson method.

[0034]

[Table 3] (In Table 3, G6 to G8 represent maltohexaose, maltoheptaose, and maltooctaose, and G1-CD
Is 6-O-α-D-glucosyl-CD, G2-CD
Represents 6-O-α-D-maltosyl-CD. )

(C) Optimum pH 0.1 M acetate buffer (pH 4.2, 5.0, 5.
5, 6.0), 0.1M phosphate buffer (pH 6.0,
6.5, 7.0, 8.0) or glycine-NaOH-N
1% (w) dissolved in aCL buffer (pH 9.0)
/ V) The purified enzyme of the present invention was dialyzed against 1.0 mL of the α-CD solution with water containing 1 mM EDTA overnight, and then dialyzed with water containing no EDTA overnight. Were mixed and the activity was measured by the method described above. The optimum pH is shown as a relative activity with the enzyme activity at pH 5.5 as 100. The result is shown in FIG.

(D) Stable pH Cyclomaltodextrinase of the present invention treated at pH 4 to 10 in the same manner as the enzyme used in (c) at 2 ° C.
FIG. 2 shows the relative activity when the residual enzyme activity when treated for 4 hours at pH 7.0 is 100. The buffers used were acetic acid (pH 4-6), phosphoric acid (pH 6-8).
And glycine-NaOH-NaCL (pH 9 to 10).

(E) Suitable temperature for action The activity of the cyclomaltodextrinase-containing solution of the present invention treated in the same manner as the enzyme used in (c) was measured at each temperature. FIG. 3 shows the relative activity when the enzyme activity measured at 60 ° C. is 100.

(F) Inactivation conditions and (g) Temperature stability The cyclomaltodextrinase-containing solution of the present invention treated in the same manner as the enzyme used in (c) is treated at each temperature of 40 to 80 ° C. The residual activity when treated in the same manner at 4 ° C was 1
The remaining activity when measured as 00 was measured. The results are shown in Fig. 4.

(H) Inhibition and activation Using the cyclomaltodextrinase-containing solution of the present invention treated in the same manner as the enzyme used in (c), the final concentration was 1 mM.
The activity was measured by adding hydrochlorides of various metals to the substrate so that (mercury and pCMB were 0.1 mM), and the relative activity was measured when the activity without addition was taken as 100.
Shown in.

[0040]

[Table 4]

(I) Molecular weight The molecular weight was determined by the gel filtration method using TSK-Gel G3000SW (manufactured by Tosoh Corporation). As a result, as shown in FIG. 5, the molecular weight of cyclomaltodextrinase of the present invention was about 106, It was 000.

(G) Isoelectric point The isoelectric point of the cyclomaltodextrinase of the present invention was determined by isoelectric focusing using Cellvalite precoated gel manufactured by Selva Co., and as shown in FIG. In addition, the isoelectric point was about 4.7.

[0043]

INDUSTRIAL APPLICABILITY As described in detail above, the cyclomaltodextrinase of the present invention has higher temperature stability and can act at high temperature as compared with known cyclomaltodextrinase. It became possible to prevent microbial contamination during the reaction during the production of starch sugar.

Further, since malto-oligosaccharides corresponding to the glucose polymerization degree of CD are accumulated when acting on CD, maltohexaose, maltoheptaose, and maltooctaose, which have been conventionally difficult, can be easily prepared. ..

Furthermore, Bacillus stearothermophilus No. The KH-17 strain produces the above-mentioned cyclomaltodextrinase extracellularly, and is suitable for industrial production of the enzyme.

[Brief description of drawings]

FIG. 1 Optimum pH of novel cyclomaltodextrinase
FIG.

FIG. 2 is a diagram showing a stable pH of the enzyme.

FIG. 3 is a diagram showing a suitable action temperature of the enzyme.

FIG. 4 is a diagram showing conditions for inactivation of the enzyme depending on temperature.

FIG. 5 is a view showing a molecular weight by a gel filtration method using TSK-Gel G3000SW.

FIG. 6 is a diagram showing isoelectric points by isoelectric focusing.

Claims (6)

[Claims] 1. A novel cyclomaltodextrinase having the following physicochemical properties. (A) Action The α-1,4-glucopyranoside bond in the maltooligosaccharide having a degree of polymerization of maltotriose or higher is hydrolyzed and rearranged to produce a series of maltooligosaccharides. Further, it acts on cyclodextrins and produces maltooligosaccharides derived from the degree of polymerization of the cyclodextrins as the main component. (B) Substrate specificity It decomposes maltooligosaccharides and cyclodextrins having a degree of polymerization of maltotriose or higher. The hydrolysis rate of α-cyclodextrin is higher than that of other cyclodextrins and linear maltooligosaccharides having the same degree of polymerization. It hardly decomposes starch, amylopectin, glycogen, panose and isopanose. (C) Optimum pH It works in the range of pH 4 to 8, and the optimum pH is around 5.5. (D) Stable pH range Stable at pH 6-9 when stored at 5 ° C for 24 hours. (E) Optimum temperature of action It works in the temperature range of 40 to 75 ° C, and the optimum temperature is around 60 ° C. (F) Inactivation condition About 90% of pH 4 is inactivated under storage conditions of 4 ° C and 24 hours, and completely inactivated at 80 ° C under conditions of pH 7 and 2 hours. (G) Temperature stability pH 7 is stable up to 40 ° C. under the condition of 1 hour, 50
The residual activities at 90 ° C, 60 ° C, 70 ° C and 80 ° C are 90%, 70%, 30% and 13%, respectively. (H) Inhibition and activation It is inhibited by divalent cadmium, zinc, mercury, manganese, copper, nickel and pCMB (parachloromercury benzoate). (I) Molecular weight The molecular weight is about 106,000 (gel filtration method). (G) Isoelectric point The isoelectric point is about 4.7 (isoelectric focusing method). 2. Bacillus stearothermophilus ( Ba
cillus stearothermophilus ), which has a capacity to produce the novel cyclomaltodextrinase according to claim 1 and is cultured, and the novel cyclomaltodextrinase according to claim 1 is collected from the culture. A method for producing a novel cyclomaltodextrinase. 3. A novel cyclomaltodextrinase-producing bacterium belonging to Bacillus stearothermophilus No. KH-17 strain ( Ba
cillus stearothermophilus No.KH-17, Microbiology Research Institute
No. 2481), the process for producing the novel cyclomaltodextrinase according to claim 2. 4. The method for producing a novel cyclomaltodextrinase according to claim 2 or 3, wherein the culture is aerobically carried out at 40 to 70 ° C. 5. The method for producing a novel cyclomaltodextrinase according to claim 2, wherein the pH of the culture solution is in the range of 5.5 to 8.0. 6. Bacillus stearothermophilus ( Ba
cillus stearothermophilus ), which has the ability to extracellularly produce the novel cyclomaltodextrinase of claim 1 and has the following mycological properties: Bacillus stearothermophilus No. KH-17 strain ( Bacillus s
tearothermophilus No.KH-17, Microtech Lab.
issue). A. Morphological features (1) Cell shape and size: bacillus, size (0.6 ~
1.0) × (2.5 to 3.0) μm (microns) (2) Cell polymorphism: None (3) Motility: Motile, with periflagellates (4) Presence or absence of spores: Ascospore: Swelling slightly Spore shape: oval Position of spores: slightly terminal side (5) Gram stainability: positive (6) acid resistance: negative B. Growth on various media (1) Meat broth agar plate culture It is white and forms circular colonies. The center is convex or convex. The rim is full-edged. No pigment production is observed. (2) Slop culture of broth agar Grows in a spread pattern. (3) Broth liquid culture The growth is weak, and a slight turbidity and sediment are formed, and an imperfect ring is formed on the surface. (4) Meat broth gelatin stab culture Only the surface was grown and no liquefaction of gelatin was observed. (5) Litmus milk No coagulation and no acid or alkali production. (6) No anaerobic growth with glucose / broth liquid medium is observed. (7) Generation of anaerobic gas from nitrate No gas is generated. C. Physiological properties (1) Reduction of nitrate Reduction (2) Denitrification reaction Negative (3) VP test negative (4) Indole formation negative (5) Hydrogen sulfide formation negative (6) Starch hydrolysis positive (7) Use of citric acid Not used (8) Use of inorganic nitrogen source Used (9) Dye production Not produced (10) Hydrolysis of gelatin and casein Not decomposed (11) Urease positive (12) Oxidase negative (13) Catalase negative (14) Attitude toward oxygen Aerobic (15) Range of growth Temperature: 30 to 75 ° C (optimal 55 to 60 ° C) pH: 5.5 to 8.0 (optimal pH 6 to 6.5) (16) Lysozyme (17) Resistance to azide (18) Attitude toward sugars D-glucose, L-arabinose, D-xylose,
An acid is produced from D-fructose, D-galactose, D-trehalose, maltose, sucrose, lactose, melibiose, raffinose, cellobiose, glycerin and D-mannite, and no gas is produced.