JP3027449B2 - Novel cyclomaltodextrinase, method for producing the same, and microorganism producing the enzyme - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

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

[0002]

2. Description of the Related Art There are very few reports on cyclomaltodextrinase. For example, Bacillus macerans , Biochem., 7, 121-124, 19
Bacillus coagula (68)
ns, Agric. Biol. Chem., 47, 1441-1447, 1983
Bacillus sphaericus ,
Japanese Patent Application No. 1-152257 , Xanthomonas campestris (Japanese Patent Application Laid-Open No. 3-83582), and Pseudomonas amiloderamosa ( Pesudomonas am
yloderamosa , Biochim. Biophys. Acta, 391, 96-108
P. 1975).

[0003] All of these enzymes degrade CD well, but since they are intracellular enzymes, not only the preparation of the enzyme is complicated, but also the productivity is limited. Furthermore, it is inferior in temperature stability and low in the optimum temperature of the enzymatic reaction, so that there is a problem when used for production of starch sugar as an industrial enzyme.

[0004]

In general, when starch sugar is produced using an enzyme, an enzyme having a high temperature suitable for action and high stability at the temperature is desired. This is because enzymatic reactions at low to medium temperatures cannot prevent microbial contamination in the reaction system,
Not only are unwanted by-products produced by side reactions by contaminating microorganisms, but also a large amount of alkaline agent is required for pH adjustment due to a decrease in the pH during the reaction. Large-scale refining equipment is required to remove methane, which is not economical.

[0005] The optimal pH for the action of the enzyme is preferably a weakly acidic pH of about 4 to 6. This is because, when the enzyme reaction is carried out under neutral to alkaline conditions, the resulting starch sugar isomerized, thereby lowering the required purity of the starch sugar.

Further, when the enzyme to be 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 an intracellular enzyme, the productivity of the enzyme is limited, which is not only economically problematic, but also requires complicated steps for extracting and purifying the enzyme from the cells.

[0007] Accordingly, an object of the present invention is to provide a novel cyclomaltodextrinase which is an enzyme produced outside of the cells, which has a high optimal temperature for action, excellent temperature stability, a weakly optimal pH for action and an extracellularly produced enzyme. It is to provide a production method and a microorganism producing the enzyme.

[0008]

Means for Solving the Problems The present inventors have found that an enzyme produced at a high temperature suitable for action, excellent in temperature stability, weakly acidic at an optimum action, and produced outside the cells, Cyclomaltodextrinase having an action of decomposing maltooligosaccharides and CDs, particularly α-CD (cyclohexamylose), having a degree of polymerization equal to or higher than that of other CDs more strongly than other CDs to produce maltooligosaccharides As a result of broadly searching for microorganisms that produce Escherichia coli from the natural world, it was found that one strain (KH-17 strain) belonging to Bacillus stearothermophilus isolated from soil strongly produced the enzyme, and completed the present invention. did.

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, comprising culturing the strain and collecting the novel cyclomaltodextrinase from the culture. It is.

Hereinafter, the physicochemical properties of the cyclomaltodextrinase of the present invention will be described.

(A) Action The α-1,4-glucopyranoside bond in maltooligosaccharide having a degree of polymerization of maltotriose or higher is hydrolyzed and rearranged to produce a series of maltooligosaccharides. It acts on CDs and produces maltooligosaccharides derived from the degree of polymerization of the CDs as a main component. (B) Substrate specificity Decomposes maltooligosaccharides and CDs having a degree of polymerization of maltotriose or higher. The rate of hydrolysis of α-CD is greater than other CDs and linear maltooligosaccharides having the same degree of polymerization. Starch, amylopectin, glycogen,
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 It is stable at pH 6 to 9 when stored at 5 ° C for 24 hours. (E) Appropriate temperature for operation It works in the temperature range of 40 to 75 ° C, and the optimum temperature is around 60 ° C. (F) Inactivation conditions Under storage conditions of 4 ° C. for 24 hours, about 90% is inactivated at pH 4, and completely inactivated at 80 ° C. under conditions of pH 7 and 2 hours.

(G) Temperature stability Stable up to 40 ° C. under conditions of pH 7 and 1 hour,
The residual activity at 90 ° C, 60 ° C, 70 ° C and 80 ° C is 90%, 70%, 30% and 13%, respectively. (H) Inhibition and activation 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). (D) Isoelectric point The isoelectric point is about 4.7 (isoelectric focusing).

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

[0015]

[Table 1]

As is clear from Table 1, the enzyme of the present invention not only acts at a high temperature but also extracellularly (in the medium) as compared with the conventionally known cyclomaltodextrinase.
In that they were generated and accumulated. To date, no enzyme having the above properties has been known, and thus the cyclomaltodextrinase of the present invention was determined to be a novel enzyme.

The cyclomaltodextrinase of the present invention is obtained from Bacillus stearothermophilus
arothermophilus ), which can be obtained by culturing a microorganism capable of producing the enzyme and collecting the enzyme from the culture.

The microorganism belonging to the Bacillus stearothermophilus used in the present invention and producing cyclomaltodextrinase includes Bacillus stearothermophilus No. 1 having the following mycological properties. KH-17 strain ( Ba
cillus stearothermophilus No. KH-17) is preferably used.

A. Morphological characteristics (1) Cell shape and size: Bacillus, size (0.6 to
1.0) × (2.5-3.0) μm (microns) (2) Cell polymorphism: None (3) Motility: Motile, with periflagellate (4) Presence of spores: Spores: Swelled slightly Spore shape: elliptical Spore position: slightly terminal (5) Gram stain: positive (6) Acid-fast: negative

B. Growth on various media (1) Gravy agar plate culture Formed white and circular colonies. The center is convex or convex circular. The periphery is all-edge. No pigment production is observed. (2) Gravy agar slant culture Growing in a spread form. (3) Broth liquid culture Growth is weak, slightly turbid and sedimentary, and forms incomplete rings on the surface. (4) Broth gelatin puncture culture Only the surface is grown, and no liquefaction of gelatin is observed. (5) Litmus milk No coagulation is observed, and neither acid nor alkali production is observed. (6) No anaerobic growth is observed in the glucose / grass liquid medium. (7) Generation of anaerobic gas from nitrate No gas is generated.

C. Physiological properties (1) Nitrate reduction Reduce (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 Use (9) Pigment not generated (10) Hydrolysis of gelatin and casein Not degraded (11) Urease positive (12) Oxidase negative (13) Catalase negative (14) Attitude to 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 Yes (18) Attitude to saccharides D-glucose, L-arabinose, D-xylose,
It produces acid from D-fructose, D-galactose, D-trehalose, maltose, sucrose, lactose, melibiose, raffinose, cellobiose, glycerin, and D-mannitol, and does not produce gas.

This strain is a spore-forming bacillus which is motile,
It is clear that the bacterium belongs to the genus Bacillus because of positive Gram staining. Furthermore, it has the ability to hydrolyze starch, cannot grow at pH 5.0, and can grow up to 75 ° C., and thus can be grown according to the Burges Manual of Sistimatic Bacteriology, Vol. 2, 1984. It was identified as a bacterium belonging to the species Loserophilus. This Bacillus stearothermophilus KH-17 strain has been deposited with the National Institute of Microbial Industry and Technology of the National Institute of Advanced Industrial Science and Technology as Microorganisms Bacteria No. 12481.

For culturing the above-mentioned cyclomaltodextrinase-producing strain, a shaking culturing method using a liquid medium, an aerated stirring culturing method, or the like, which is employed for culturing general aerobic microorganisms, is used.

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

The culturing temperature is usually 30 to 75 ° C, preferably 40 to 70 ° C, more preferably 50 to 55 ° C. Culture pH is usually 5.5 to 8.0, preferably 6.0 to 7.0. The culturing time varies depending on the culturing method, but is about 18 to 100 hours. By this culture method, most of the cyclomaltodextrinase (about 80%) was obtained.
%) Accumulates in the medium (extracellularly) and the rest is localized on the cell surface. After completion of the culture, cyclomaltodextrinase can be obtained by a usual method. One example is shown below.

First, the culture is centrifuged to remove cells. An equal amount of isopanol is added to the obtained supernatant to obtain a precipitate. This precipitate is washed with a 10 mM acetate buffer (p
The crude enzyme obtained by dialysis against H6) was subjected to DEAE-
The resulting fraction is subjected to gradient ion exchange chromatography on 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. The concentrated enzyme was subjected to gel filtration using Sephacryl S-300 equilibrated with the same buffer as above to collect an active fraction, and the active fraction was further purified by a chromatofocusing method to obtain a purified enzyme. obtain.

The activity of the cyclomaltodextrinase of the present invention was measured as follows. 1% α-CD
The enzyme solution 0.0 appropriately diluted with water in 0.5 mL (milliliter) 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 in a boiling water bath for 30 minutes,
Quench in running water, Nelson liquid 1.0m
L was added. Water was added to make 10.0 mL, and the reducing sugar generated using 100 μg of glucose treated similarly as a standard was measured at 510 nm. Here, one unit of the enzyme is
Under the same conditions as above, the amount of the enzyme that produced reducing sugar corresponding to 1 μmole of glucose from α-CD was defined as the amount of enzyme.

[0028]

Next, the present invention will be described more specifically with reference to examples.

Example 1 Preparation of Crude Enzyme 0.5% dextrin, 1% Shefton F (USA, manufactured by Shefton, USA), 0.1% yeast extract, 0.1%
Sodium glutamate, 0.1% ammonium nitrate, 0.1%
1% K ₂ HPO ₄ , 0.02% MgSO ₄ .7H ₂ O,
50 mg / L CaCl ₂ .2H ₂ O and 10 mg / L
Of MnSO ₄ · 7H ₂ Liquid Medium 1 pH6.3 containing O
After sterilizing 5 L (liter) at 120 ° C. for 30 minutes,
In advance, use the same medium as above at 50 ° C. for 20 hours,
Bacillus stearothermophilus no. KH-17 strain (Microbial Research Laboratories No. 12481)
No.) 1 L of the suspension was inoculated, and 250 rpm, 1 v, v, m
Under the conditions described above, aeration and stirring culture was performed at 50 ° C. for 50 hours. After completion of the culture, the supernatant obtained by removing the cells by centrifugation was subjected to ultrafiltration with an average molecular weight cutoff of 5,000 (Romi).
con.) and concentrated to about 2 L. This concentrated crude enzyme solution had an enzyme activity of 25.5 units per mL.

Example 2 Purification of Enzyme An equal amount of isopropanose was added to 1.0 L of the crude enzyme obtained in Example 1 to obtain a precipitate. This precipitate is added to 10.0
After collection by centrifugation at 00 × g and 4 ° C. for 10 minutes,
Dialysis was performed overnight at 4 ° C. against a mM acetate buffer (pH 6.0). Subsequently, the supernatant liquid from which insolubles were 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 the active fractions were collected to obtain an ultrafiltration membrane (Am) having an average molecular weight cut off of 10,000.
(conicon). For concentrated enzymes,
Sephacryl (Se) equilibrated with the same buffer as above
After further purification by gel filtration chromatography using S. phacrylyl S-300, the resultant was concentrated 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 the purified enzyme was 71 units / mg protein, which was single on polyacrylamide gel disk electrophoresis.

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

[0032]

[Table 2]

(B) Specificity Table 3 shows the results obtained by allowing the purified enzyme obtained in Example 2 to act on various substrates. The reaction was measured by a conventional method, and α
-The relative activity was expressed as a relative activity where the activity when CD was used as a substrate was defined as 100. In addition, 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 the generated reducing sugar was measured by the Somogyi 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, 5.0)
5, 6.0), 0.1 M 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 previously dialyzed against 1.0 mL of the α-CD solution against water containing 1 mM EDTA overnight, and further dialyzed overnight against water containing no EDTA, 0.05 mL of a cyclomaltodextrinase-containing solution. 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 being 100. The result is shown in FIG.

(D) Stable pH The cyclomaltodextrinase of the present invention, which has been treated in the same manner as the enzyme used in (c) in the pH range of 4 to 10, at 2 ° C for 2 hours.
FIG. 2 shows the relative activities, where the residual enzyme activity when treated at pH 7.0 after treatment for 4 hours is 100. The buffers used were acetic acid (pH 4 to 6) and phosphoric acid (pH 6 to 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. The relative activity when the enzyme activity measured at 60 ° C. is taken as 100 is shown in FIG.

(F) Deactivation 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 appropriately treated at each temperature of 40 to 80 ° C. The residual activity when treated similarly at 4 ° C was 1
The activity remaining when the value was set to 00 was measured. FIG. 4 shows the results.

(H) Inhibition and activation Using the cyclomaltodextrinase-containing solution of the present invention treated in the same manner as in (c), a final concentration of 1 mM
(Hercury and pCMB are 0.1 mM). The results obtained by adding the hydrochloride of various metals to the substrate and measuring the activity are shown in Table 4.
Shown in

[0040]

[Table 4]

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

(N) Isoelectric point The isoelectric point of the cyclomaltodextrinase of the present invention was determined by isoelectric point electrophoresis using Selvalite precoated gel manufactured by Selva, as shown in FIG. The isoelectric point was about 4.7.

[0043]

As described above in detail, according to the cyclomaltodextrinase of the present invention, it has higher temperature stability and can operate at a higher temperature than known cyclomaltodextrinase. It has become possible to prevent microbial contamination during the reaction during the production of starch sugar.

Further, maltohexaose, maltoheptaose, and maltooctaose, which have conventionally been considered difficult, can be easily prepared since maltooligosaccharides accumulate according to the degree of glucose polymerization of CD when acted on CD. .

Further, the Bacillus stearothermophilus no. The KH-17 strain is suitable for industrial production of the cyclomaltodextrinase because it produces the cyclomaltodextrinase outside the cells.

[Brief description of the drawings]

FIG. 1. Optimal pH of a novel cyclomaltodextrinase
FIG.

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

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

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

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

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

Continuation of the front page (51) Int. Cl. ⁷ Identification code FI C12R 1:07) (56) References Journal of fermentation and Bioengineering, Vol. 71, No. 4, (1991) p. 226-229 (58) Fields investigated (Int. Cl. ⁷ , DB name) C12N 9/00-9/99 C12N 1/00-1/38 BIOSIS (DIALOG) WPI (DIALOG)

Claims (6)

(57) [Claims] 1. A novel cyclomaltodextrinase having the following physicochemical properties: (A) Action The α-1,4-glucopyranoside bond in maltooligosaccharides having a degree of polymerization of maltotriose or higher is hydrolyzed and rearranged to produce a series of maltooligosaccharides. In addition, it acts on cyclodextrins, and produces maltooligosaccharides derived from the degree of polymerization of the cyclodextrin as a main component. (B) Substrate specificity Decomposes maltooligosaccharides and cyclodextrins having a degree of polymerization of maltotriose or higher. The rate of hydrolysis of α-cyclodextrin is greater than other cyclodextrins and linear maltooligosaccharides having the same degree of polymerization. Substantially degrades 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 When stored at 5 ° C. for 24 hours, it is stable at pH 6 to 9. (E) Appropriate temperature for operation It works in the temperature range of 40 to 75 ° C, and the optimum temperature is around 60 ° C. (F) Inactivation conditions Under storage conditions of 4 ° C. for 24 hours, about 90% is inactivated at pH 4, and completely inactivated at 80 ° C. under conditions of pH 7 and 2 hours. (G) Temperature stability Stable up to 40 ° C. under conditions of pH 7 and 1 hour,
The residual activity at 90 ° C, 60 ° C, 70 ° C and 80 ° C is 90%, 70%, 30% and 13%, respectively. (H) Inhibition and activation 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). (D) Isoelectric point The isoelectric point is about 4.7 (isoelectric focusing). 2. Bacillus stearothermophilus ( Ba)
cillus stearothermophilus ), wherein a microorganism capable of producing the novel cyclomaltodextrinase according to claim 1 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 Bacillus stearothermophilus no. KH-17 strain ( Ba
cillus stearothermophilus No.KH-17
2481). The method for producing a novel cyclomaltodextrinase according to claim 2, wherein 4. The method for producing a novel cyclomaltodextrinase according to claim 2, wherein the culturing is performed aerobically 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 ), having the ability to produce the novel cyclomaltodextrinase according to claim 1 outside the cells, and having the following mycological properties: Bacillus stearothermophilus no. KH-17 strain ( Bacillus s
tearothermophilus No.KH-17, No. 12481
issue). A. Morphological characteristics (1) Cell shape and size: Bacillus, size (0.6 to
1.0) × (2.5-3.0) μm (microns) (2) Cell polymorphism: None (3) Motility: Motile, with periflagellate (4) Presence of spores: Spores: Slightly swollen Spore shape: elliptical Spore position: slightly distal (5) Gram staining: positive (6) Acid-fast: negative Growth on various media (1) Gravy agar plate culture Formed white and circular colonies. The center is convex or convex circular. The periphery is all-edge. No pigment production is observed. (2) Gravy agar slant culture Growing in a spread form. (3) Broth liquid culture Growth is weak, slightly turbid and sedimentary, and forms incomplete rings on the surface. (4) Broth gelatin puncture culture Only the surface is grown, and no liquefaction of gelatin is observed. (5) Litmus milk No coagulation is observed, and neither acid nor alkali production is observed. (6) No anaerobic growth is observed in the glucose / grass liquid medium. (7) Generation of anaerobic gas from nitrate No gas is generated. C. Physiological properties (1) Nitrate reduction Reduce (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 Use (9) Pigment not generated (10) Hydrolysis of gelatin and casein Not degraded (11) Urease positive (12) Oxidase negative (13) Catalase negative (14) Attitude to 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 Yes (18) Attitude to saccharides D-glucose, L-arabinose, D-xylose,
It produces acid from D-fructose, D-galactose, D-trehalose, maltose, sucrose, lactose, melibiose, raffinose, cellobiose, glycerin, and D-mannitol, and does not produce gas.