JPH0761264B2 - Novel cyclomaltodextrinase and method for producing the same - Google Patents

Description

TECHNICAL FIELD The present invention relates to a novel enzyme cyclomaltodextrinase, a method for producing the same, and a novel bacterium belonging to the genus Bacillus.

[Conventional technology]

Cyclic maltooligosaccharides alpha cyclodextrin, beta cyclodextrin, gamma cyclodextrin ring-opening, the function of producing a linear maltooligosaccharide, the bacterium Bacillus polymixa, Pseudomonas MSI, amylase produced by mold Aspergillus oryzae, etc.,
It is also known to exist in the cyclodextrin glycosyltransferase (CGTase) produced by the bacteria Bacillus cereus, Bacillus macerans and the like.

On the other hand, cyclomaltodextrinase does not act on high-molecular glucose polymers such as starch, amylose, amylopectin and dextrin at all, and is therefore an enzyme different from the above-mentioned amylases. Also, starch,
Since it does not produce cyclic maltooligosaccharides (alpha cyclodextrin, beta cyclodextrin, etc.) from amylose and amylopectin, it is an enzyme different from the known cyclodextrin glucosyltransferase (CGTase).

Cyclomaltodextrinase is an alpha cyclodextrin (consisting of 6 glucose), beta cyclodextrin (consisting of 7), and gamma cyclodextrin (consisting of 8) which has a cyclic structure in which 6 to 8 glucoses are linked. ), Hydrolyzing the alpha-1,4-linkage, and ring-opening the structure to form a malto-oligosaccharide in which 6 to 8 glucose are linearly linked, that is, malto-oligosaccharide. It has a function of converting into hexaose (6 glucoses are linearly linked), maltoheptaose (7 are linked), and maltooctaose (8 are linked). Furthermore, the action range of the enzyme extends to these three kinds of linear maltooligosaccharides produced, and as a result, the linear maltooligosaccharides produce linear maltooligosaccharides and glucose composed of 2 to 8 low molecular weight glucose. Everywhere

Cyclomaltodextrinase has a limited distribution in microorganisms and animal and plant kingdoms, so there are very few examples of research and application development. So far, only the enzyme produced by Bacillus coagulans is known [S.Ki
tahata, et.al., Agric.Biol.Chem., 47 (7), 1441-144
7, (1983)].

[Problems to be Solved by the Invention]

Therefore, an object of the present invention is to provide a novel cyclomaltodextrinase, a method for producing the same and a microorganism producing the same.

[Means for Solving the Problems]

The present inventors have extensively searched for microorganisms producing a novel cyclomaltodextrinase from the natural world, and Bacillus No. 199 strain belonging to the genus Bacillus isolated from soil produces a novel cyclomaltodextrinase. The present invention has been completed and the present invention has been completed.

That is, the present invention, a new strain No. 199 strain belonging to the genus Bacillus that produces cyclomaltodextrinase, a method for producing a novel cyclomaltodextrinase by culturing this strain in an alkaline medium, and thus obtained It provides a new enzyme.

Next, the mycological properties of the Bacillus genus No. 199 strain of the present invention, the culture method thereof, and the physicochemical properties of the cyclomaltodextrinase of the present invention will be described in detail.

(Microorganism) A microorganism producing cyclomaltodextrinase of the present invention is a bacterial species having the ability to produce cyclomaltodextrinase belonging to the genus Bacillus, and as an example thereof, Bacillus sp. No. 199 (Bacillus sp. No.199) bacteria (hereinafter referred to as “No.199 strain”). In the present invention, of course, natural and artificial mutants of No. 199 strain,
All microorganisms belonging to the genus Bacillus and having the below-mentioned cyclomaltodextrinase-producing ability can be used.

The above No. 199 strain is a soil bacterium isolated from the soil collected in Kofu City, Yamanashi Prefecture, and was deposited at the Institute of Microbial Science and Technology, Institute of Industrial Science on July 21, 1989, and the microorganism deposit number is , Microtechnology Research Institute, No. 10884 "FERM P-10884).

The No. 199 strain has the following mycological properties.

[Mycological properties]

The medium used was one adjusted to pH 10 with the addition of 1% sodium carbonate.

(1) Morphology It is a bacillus having a size of (0.2 to 0.5) × (1.5 to 5) μm. Spores are formed, and the size of spores is (0.5-1) x (1
~ 1.5) μm, ovoid and swelling spores. It is motile and has flagella. Gram stain is positive and acid resistance is negative.

(2) Growth condition Meat broth agar plate culture Good growth, round and flat shape, all edges are opaque, lustrous, and light yellowish brown.

Meat juice agar slope culture It grows in the form of filaments or strips, has the same shape as that of the meat juice agar plate culture, and does not secrete pigment into the medium.

Liquid culture of broth Good growth, slight precipitation, and pellicle formation on the surface.

Broth gelatin culture It grows well and hydrolyzes gelatin.

 Litmus milk Due to alkaline medium, no color change is apparent.

(3) Physiological properties Nitrate reduction: Positive Denitrification reaction: Negative MR test: Methyl red discoloration is not observed because the medium is alkaline VP test: Negative indole formation: Negative hydrogen sulfide formation: Negative starch hydrolysis Decomposition: Positive Use of citric acid: Not used (Coser and Christensen's medium) Use of inorganic nitrogen source: Not used (Nitrate and ammonium salts) Pigment formation: Negative urease: Negative oxidase: Positive catalase: Positive Growth range: pH7 to 11 Temperature 20-45 ° C Oxygen attitude: Aerobic OF test: Fermentation (glucose) Presence or absence of acid and gas production from sugars: L-arabinose, D-xylose, D-glucose, D-mannose, D-fructose, Maltose, sucrose,
Utilizes lactose, trehalose, D-mannitol and starch to produce acid but no gas.

D-galactose, D-sorbitol, inositol,
Do not use glycerin.

(4) Other properties Sodium chloride tolerance: Grow in 10% sodium chloride. GC content of DNA: 35.8 mol% or more. The mycological properties are classified into Vergy's Manual of Systematic Bacteriology Volume 2 (1986). According to the method, it was compared with known bacterial species of the genus Bacillus.

As a result, the strain of the present invention is similar to Bacillus circulans, but it is clearly different in the point of positive oxidase, the point of growth under alkaline condition, and the point of growth in the presence of 10% NaCl. There is.

Therefore, since this strain is distinguished from known strains, it was determined to be a new strain belonging to the genus Bacillus and named Bacillus No. 199 strain.

(Culture method and purification method) In obtaining the cyclomaltodextrinase of the present invention, the enzyme-producing bacterium belonging to the genus Bacillus may be cultured by a usual method for producing an enzyme, except that an alkaline medium is used. Good. The form of culture may be liquid culture or solid culture, and for industrially advantageous culture, inoculation of a spore suspension or culture solution of the above-mentioned production bacterium into a medium, shaking culture, or aeration stirring culture is performed. Just go.

The nutrient source of the medium is not particularly limited, and a carbon source, a nitrogen source and the like usually used for culturing microorganisms can be contained in the medium. As a carbon source, starch,
Yeast extract, dextrin, glycerin, glucose,
Sucrose, galactose, inositol, mannitol and the like are used, and as the nitrogen source, peptone, soybean powder, meat extract, rice bran, malt, urea, corn steep liquor and other organic nitrogen compounds are used. In addition, inorganic salts such as sodium chloride, phosphates, sulfates, potassium, calcium, magnesium, zinc, manganese,
Metal salts such as iron may be appropriately added, and as necessary, anti-foaming agents such as animal, plant, and mineral oils may be added.
The culturing conditions such as culturing temperature and culturing time are selected so as to be suitable for the growth of the bacterium to be used and to maximize the production of this enzyme. For example, the pH of the medium is alkaline, preferably 8
As described above, more preferably 9 to 11, most preferably 10, and the temperature is 30 to 45 ° C, preferably 37 to 40 ° C. However, the culture composition, the hydrogen ion concentration of the medium, the culture temperature, the culture conditions such as stirring conditions should be appropriately adjusted so that preferable results can be obtained depending on the type of strain to be used and external conditions. Needless to say.
In order to obtain the present enzyme from the culture thus obtained, it is possible to appropriately collect the metabolites by appropriately utilizing the means usually used for collecting the metabolites. For example, any of the means utilizing the difference in solubility between the present enzyme and impurities, the means utilizing the difference in ionic binding force, the means utilizing the difference in adsorption affinity, the means utilizing the difference in molecular weight, respectively, or, They are used in appropriate combination or repeatedly. Specifically, since most of the enzyme is present in cells, the culture solution was filtered or centrifuged, and the collected cells were crushed by ultrasonic waves, ultrahigh pressure, a homogenizer, etc. The cell extract may be purified by combining various ion exchange chromatography, gel filtration chromatography, adsorption chromatography, liquid chromatography, cellulose partition chromatography and the like.

The novel cyclomaltodextrinase of the present invention thus obtained has the following physicochemical properties.

[Physical and chemical properties]

(1) Action and substrate specificity The α-1,4-glucoside bond of cyclic maltooligosaccharide is cleaved to form a linear maltooligosaccharide. This enzyme is an alpha-former of the linear maltooligosaccharides produced.
It cleaves 1,4-glucoside bonds to produce low-order linear maltooligosaccharides and glucose.

Table 1 shows an example of the ratio of each maltooligosaccharide after the enzymatic reaction when the cyclic maltooligosaccharides, alpha cyclodextrin, beta cyclodextrin and gamma cyclodextrin, were used as substrates.

From Table 1, a linear maltooligosaccharide containing maltohexaose is produced from alpha cyclodextrin, a linear maltooligosaccharide containing maltoheptaose is produced from beta cyclodextrin, and a maltooctaose is contained from gamma cyclodextrin. It is clear that linear maltooligosaccharides are produced.

That is, the present enzyme is a linear oligosaccharide containing a linear maltooligosaccharide in which one position of the alpha-1,4-glucoside bond is cleaved, regardless of the number of glucose groups of the cyclic maltooligosaccharide as a substrate. It is characterized by generating.

(2) Optimum pH and stable pH range The effect of the substrate on pH was examined based on the following activity measurement method. Table 2 shows the relative activity at each pH with the maximum activity being 100%. From this, it can be seen that the optimum pH is 5.5 to 6.5.

The stability of pH is shown in Table 3. This is a variety of
A buffer solution of pH was added to the enzyme solution and heated at 40 ° C for 30 minutes, and the remaining activity was measured. The unheated enzyme activity at each pH was defined as 100% and expressed as relative activity. is there.

From Table 3, it can be seen that the stable pH range is about pH 6 to 10, which is relatively stable on the alkaline side.

(3) Range of suitable temperature of action The enzyme reaction was carried out at each temperature according to the following activity measuring method. The results are shown in Table 4.
The activity at ° C was defined as 100%, and the activity was expressed as relative activity.

As is clear from Table 4, the optimum temperature for the action of this enzyme is 40-50.
℃.

(4) Temperature stability After heat treatment at pH 6 for 10 minutes at each temperature, the residual activity was examined. The untreated enzyme activity was defined as 100%, and its residual activity is shown in Table 5.

As shown in Table 5, this enzyme is stable at 30 to 50 ° C.

(5) Inactivation conditions As shown in Table 2, when heat-treated at pH 4 for 30 minutes at 40 ° C, the enzyme is almost inactivated.

(6) Isoelectric point This enzyme has an isoelectric point around pH5.

(7) Molecular weight The molecular weight determined by gel filtration using Toyopearl HW55F was about 120,000.

The molecular weight obtained by SDS-polyacrylamide electrophoresis was about 68,000.

(8) Purification The present enzyme can be purified by centrifugation after the completion of culture, and then using a separation and purification means known in the art, for example, an ion exchanger gel filtration agent.

The method for purifying this enzyme will be specifically described in Examples below.

[Activity measurement method]

Of the cyclic maltooligosaccharides, beta cyclodextrin is used as the reaction substrate.

100m containing 0.2% (W / V) beta cyclodextrin
To 1 ml of M phosphate buffer (pH 6.0), add 0.1 ml of enzyme solution, mix, and incubate at 40 ° C for 30 minutes.

After the reaction, add 0.81 ml of 100 mM phosphate buffer (pH 6.0) to the test tube in advance, add 0.11 ml of the reaction solution to make the total volume 1 ml, and immediately add 3.5 ml of 30 mM sodium hydroxide solution. Then 0.5 ml of 5 mM sodium carbonate solution containing 0.02% (W / V) phenolphthalein is added. After leaving it at room temperature for exactly 30 minutes, measure the absorbance at 550 nm.

100 mM with several known amounts of beta-cyclodextrin
For the phosphate buffer (pH 6.0), replace the enzyme solution with distilled water and perform the same operation to create a calibration curve.

Using the calibration curve, the residual amount of beta cyclodextrin after the reaction is determined, and the amount of decomposed beta cyclodextrin is calculated.

One unit of enzyme was defined as a value converted based on the value calculated under the above reaction conditions.

That is, the amount of enzyme that decomposes 1 micromolar of beta cyclodextrin in 60 minutes was defined as 1 unit.

[Operation and effect of the invention]

As described above, with the discovery of the novel cyclomaltodextrinase of the present invention and the Bacillus genus No. 199 strain producing the same, the research and application of cyclomaltodextrinase, which has been known only to one type so far, , Can provide a new place.

Among cyclodextrins, alpha cyclodextrin composed of 6 glucoses and beta cyclodextrin composed of 7 glucoses can be easily produced from a starch raw material, and are currently produced in large quantities at low prices in Japan. If linear maltooligosaccharides can be produced from these alpha cyclodextrin and beta cyclodextrin as raw materials, conventional starch can be used as raw material to produce linear maltooligosaccharides having a chain length of 1 to 7 glucose. Compared with the case, it can be greatly simplified and speeded up. This is because, when starch is used as a raw material, branched dextrin is produced, so that the use of an enzyme (for example, pullulanase) for cutting the branched structure is unavoidable. Alternatively, undegraded starch or branched dextrin remains in the final product and contains impurities.

By reacting the enzyme according to the present invention with cyclodextrin, which is supplied inexpensively and in large quantities, as a raw material, it becomes possible to produce a highly pure product containing only a linear maltooligosaccharide by a one-step reaction. That is, it provides a new production method for the production of maltooligosaccharide, which is widely used as a sweetener replacing sugar in the food industry.

Heretofore, no example has been known in which the present enzyme is used for the purpose of producing a linear maltooligosaccharide. In addition, there is no known example in which the present enzyme was found to be produced by Bacillus bacteria growing in an alkaline medium.

〔Example〕

Example 1 Soluble starch 2.5 g, polypeptone 1.25 g, yeast extract
225 g of water is added to 1.25 g, phosphoric acid-potassium hydrogen 0.25 g, and magnesium sulfate (heptahydrate) 0.05 g, sterilized at 120 ° C. for 15 minutes, and 45 ml is dispensed into five 300 ml Erlenmeyer flasks.

Sodium carbonate 0.1 g, 0.25 g, 0.5 g, 0.75 g and 1 g are each dissolved in 5 ml of water, sterilized at 120 ° C. for 15 minutes, and added to each of the above 5 Erlenmeyer flasks. This gives a medium containing 0.2%, 0.5%, 1%, 1.5% and 2% of sodium carbonate. The pH at this time is respectively pH8.6, pH9.8, pH10.3,
The pH was 10.6 and pH 10.8.

These mediums were inoculated with the No. 199 bacterium pre-cultured for 18 to 20 hours, and shake culture was carried out at 37 ° C for 24 hours.

The cells were collected by centrifugation, washed with distilled water, and then suspended in 25 ml of 50 mM phosphate buffer (pH 7.5). The suspension was ultrasonically disrupted, and the supernatant was collected by centrifugation to obtain a cell-free extract.

When the activity of these crude enzyme solutions was measured, they were 0.5 U / ml, 2.1 U / ml, 10.0 U / ml under the culture conditions of sodium carbonate content 0.2%, 0.5%, 1%, 1.5%, 2%, respectively. , 1
The activity values of 8.0 U / ml and 5.6 U / ml were obtained.

Example 2 10 g of soluble starch, 5 g of polypeptone, 5 g of yeast extract,
Phosphoric acid-potassium hydrogen 1g, magnesium sulfate (heptahydrate)
Add 900 ml of water to 0.2 g and sterilize at 120 ° C for 15 minutes. Dissolve 15 g of sodium carbonate in 100 ml of water, sterilize at 120 ° C for 15 minutes, and add. Dispense 100 ml each into a 300 ml Erlenmeyer flask,
Inoculate 1 ml each of No. 199 bacteria pre-cultured for 18 to 20 hours, and
Shaking culture was performed at 7 ° C. for 24 hours.

Collect the cells by centrifugation and wash with distilled water, then add 500 ml of 5
The cells were suspended in 0 mM phosphate buffer (pH 7.5). The suspension was sonicated and the supernatant was collected by centrifugation to obtain a cell-free extract having a specific activity of 15.7 U / ml protein.

5% of the nucleic acid removing agent C-9 (manufactured by Kurita Water Industries) was added to this crude enzyme solution.
5 ml of the solution was added and left overnight in the refrigerator, and the precipitate was removed by centrifugation.

The salting-out fraction with ammonium sulfate was carried out under the condition of 40 to 70% saturation. After dialyzing this with 50 mM phosphate buffer (pH 7.5) and passing it through DEAE Toyopearl 650M (manufactured by Tosoh) equilibrated with the same buffer, the enzyme is adsorbed, and this is adsorbed on sodium chloride from 0 to 0.5M concentration. Gradient elution was performed with the same buffer containing The active fraction was dialyzed against 50 mM phosphate buffer (pH 7.5) containing 0.1 M sodium chloride, and then passed through DEAE Toyopearl 650 M equilibrated with the same buffer. This was subjected to gradient elution with the same buffer containing 0.1 to 0.5 M sodium chloride. Toyopearl HW55F (manufactured by Tosoh) equilibrated with 50 mM phosphate buffer (pH 7.5) containing 0.1 M NaCl after removing sodium chloride by dialysis to remove the active fraction and concentrating
Was used for gel filtration. The active fraction was dialyzed against 50 mM phosphate buffer (pH 6.2) and then passed through DEAE Toyopearl 650M equilibrated with the same buffer. This was subjected to gradient elution with the same buffer containing sodium chloride at a concentration of 0 to 0.3M.
When the active fractions were collected and subjected to polyacrylamide gel electrophoresis, a single band was detected. Specific activity is 860 U / mg
It was protein.

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Claims (4)

[Claims] 1. A cyclomaltodextrinase having the following physicochemical properties. (A) Action and substrate specificity The α-1,4-glucoside bond of cyclic maltooligosaccharide is cleaved to produce a linear maltooligosaccharide. (B) Optimum pH and stable pH range The optimum pH is 5.5 to 6.5. The stable pH range is about pH 6-10. (C) Optimum temperature range for action The optimum temperature range for action is 40 to 50 ° C. (D) Temperature stability It is stable at 30 to 50 ℃ at pH6. (E) Deactivation condition Heat treatment at 40 ° C for 30 minutes at pH 4 deactivates. (F) Isoelectric point The isoelectric point is around 5. (G) Molecular weight The molecular weight obtained by gel filtration using Toyopearl HW55F is approximately 12
The molecular weight by SDS-polyacrylamide electrophoresis is about 68,000. 2. A cyclomaltodextrinase-producing bacterium belonging to the genus Bacillus is cultivated, and cyclomaltodextrinase is collected from the culture.
A method for producing the described cyclomaltodextrinase. 3. The method according to claim 2, wherein the cyclomaltodextrinase-producing bacterium is Bacillus sp. No. 199. 4. Bacillus sp. No. 199 characterized by having the following mycological properties. (A) Morphology It is a bacillus having a size of (0.2 to 0.5) × (1.5 to 5) μm. Spores are formed, and the size of spores is (0.5-1) x (1
~ 1.5) μm, ovoid and swelling spores. It is motile and has flagella. Gram stain is positive and acid resistance is negative. (B) Growth state Meat broth agar plate culture Good growth, round and flat shape, all edges are opaque, lustrous, and light yellowish brown. Meat juice agar slope culture It grows in the form of filaments or strips, has the same shape as that of the meat juice agar plate culture, and does not secrete pigment into the medium. Liquid culture of broth Good growth, slight precipitation, and pellicle formation on the surface. Broth gelatin culture It grows well and hydrolyzes gelatin. Litmus milk Due to alkaline medium, no color change is apparent. (C) Physiological properties Nitrate reduction: Positive Denitrification reaction: Negative MR test: Methyl red discoloration is not observed because the medium is alkaline VP test: Negative Indole formation: Negative hydrogen sulfide formation: Negative starch hydrolysis Decomposition: Positive Use of citric acid: Not used (Coser and Christensen's medium) Use of inorganic nitrogen source: Not used (nitrate and ammonium salts) Pigment formation: Negative urease: Negative oxidase: Positive catalase: Positive Growth range: pH7 to 11 Temperature 20-45 ℃ Attitude toward oxygen: Aerobic OF test: Fermentation (glucose) Presence of acid and gas generation from sugars: L-arabinose, D-xylose, D-glucose, D-mannose, D-fructose, Maltose, sucrose, lactose, trehalose, D-mannitol, de It uses acid to produce acid, but no gas. D-galactose, D
-Do not use sorbitol, inositol or glycerin. (D) Other properties Sodium chloride tolerance: Growth of 10% sodium chloride GC content of DNA: 35.8 mol%