JPH0632611B2 - γ-Cyclodextrin synthase and method for producing the same - Google Patents

Description

TECHNICAL FIELD The present invention relates to a novel γ-cyclodextrin synthase and a method for producing the same.

[Conventional technology]

Conventionally, the presence of an enzyme that synthesizes cyclodextrin in Bacillus bacteria is known. That is, Bacillus macerans produces an enzyme that produces a mixture of cyclodextrins whose main component is α-cyclodextrin (amylase symposium Vol.8, (1973), p.21), an alkaliphilic Bacillus It is known that a genus bacterium produces an enzyme that produces a mixture of cyclodextrins containing β-cyclodextrin as a main component (Japanese Patent Publication No. 53-31223).
As described above, microorganisms that produce an enzyme that produces a mixture containing α- and β-cyclodextrin as the main component have been reported in many documents, but produce a cyclodextrin mixture containing γ-cyclodextrin as the main component. No report has been made on a microorganism that produces the enzyme.

If the present inventor can specifically synthesize γ-cyclodextrin and find an enzyme that does not produce α- and β-cyclodextrin, it can be a very useful enzyme for the production of γ-cyclodextrin. From the viewpoint of, when searching for a bacterium capable of producing γ-cyclodextrin synthase, one strain recognized as belonging to the genus Bacillus found in soil specifically identified γ-cyclodextrin. Synthesize and α
It was found that an enzyme that does not synthesize -and β-cyclodextrin is produced, and that this enzyme is a completely new enzyme that is clearly different from the properties of conventional cyclodextrin synthase.

[Structure of Invention]

The present invention has been completed based on the above new findings. The present invention cultivates γ-cyclodextrin synthase A (hereinafter also referred to as “the present enzyme”) having the following physicochemical properties, γ-cyclodextrin synthase A-producing bacteria belonging to the genus Bacillus, and γ-from the culture. The present invention provides a method for collecting cyclodextrin synthase A.

[Physical and chemical properties of γ-cyclodextrin synthase A]

(1) Action and substrate specificity: γ-cyclodextrin is specifically synthesized from starch, amylose, amylopectin, glycogen and the like, and α- and β-cyclodextrin are not produced.

(2) Working pH range: pH 4 to 9 (0.02M buffer solution) [sodium acetate-acetic acid buffer solution (pH 4 to 6), Tris-hydrochloric acid buffer solution (pH 7 to 8), glycine-sodium chloride-sodium hydroxide buffer solution (PH 9-10) 5% in 5% soluble starch
Reaction at 0 ° C. for 60 minutes].

(3) Optimum action pH: around pH 8 (measurement method is the same as (2))
(4) Working temperature: up to about 75 ° C. [reaction in 0.02M Tris-HCl buffer (pH 8), 5% soluble starch for 60 minutes at each temperature].

(5) Optimum working temperature: approx. 65 ° C (measurement method is the same as (4)) (6) Thermal stability: 0.05M Tris-hydrochloric acid buffer solution (pH 7) was heated at each temperature for 30 minutes to remove residual activity. It was measured. As a result, almost no inactivation was observed at temperatures up to 50 ° C, and the enzyme was inactivated by about 19% by heating at 55 ° C for 30 minutes.
Heating at 37 ° C for 30 minutes deactivates about 37%, heating at 65 ° C for 30 minutes deactivates at about 75%, and heating at 70 ° C for 30 minutes almost completely deactivates.

(7) pH stability: stable at pH 6 to 8 (enzyme solution was added to 0.1 ml of 0.1M buffer solution to bring the total volume to 0.2 ml, and then 30 at 50 ° C)
After standing for minutes, the residual activity was measured).

(8) Purification method: Three-fold amount of cold acetone (-2
The resulting precipitate was collected, dialyzed with 0.01 M Tris-HCl buffer (pH 7.5), and CM-Toyopearl (manufactured by Toyo Soda Co., Ltd.) sufficiently equilibrated with the above buffer. Adsorb to the product name). After thoroughly washing with the above buffer, 0.1
Elute with the buffer containing M sodium chloride. After concentration, Sephacryl S-200 (manufactured by Pharmacia Ltd., fully equilibrated with 0.05 M Tris-hydrochloric acid buffer (pH 7) containing 0.2 M sodium chloride)
A single γ-cyclodextrin synthase A was obtained electrophoretically by performing gel filtration with (trade name).

(9) Molecular weight: As a result of measurement by SDS-polyacrylamide gel electrophoresis, a value of 64,000 was obtained.

(10) Isoelectric point: 7.05 as measured by electrophoresis using carrier ampholine (Celvalite manufactured by Selva)
Was obtained.

(11) Titration method: An appropriate amount of enzyme solution was added to 0.5 ml of 0.04 M Tris-hydrochloric acid buffer solution (pH 8) containing 10% soluble starch, and the total amount was adjusted to 1 ml with distilled water, followed by reaction at 50 ° C. for 60 minutes. Then, the produced γ-cyclodextrin was quantified by the brom cresol green method.

The amount of enzyme that produces 1 mg of γ-cyclodextrin per hour under the above reaction conditions was defined as 1 unit.

The physicochemical properties of γ-cyclodextrin synthase A of the present invention and conventionally reported cyclodextrin synthase are shown in the following table for comparison.

As is clear from this table, this enzyme is recognized as a novel γ-cyclodextrin synthase which is not classified into any of the conventionally known cyclodextrin synthases.

This enzyme can be collected from a culture by culturing a microorganism belonging to the genus Bacillus and having the ability to produce γ-cyclodextrin synthase A. As an example of such a microorganism, Bacillus sp. NO. 313 (Bacillus sp NO.
313) can be mentioned. Bacillus SP N
O.313 was deposited at the Institute of Microbial Science and Technology of the Agency of Industrial Science on July 22, 1960, and the deposit number is Micromachine Research Institute of Microbiology No. 8366 (FERM P-8366). The mycological properties of Bacillus sp. No. 313 are as follows.

(1) Morphology: Bacillus (0.6-0.8μ × 2-3μ) with motility and forming spores, the size of which is 0.8-1.0μ × 0.8-1.0
It is a circle of μ. No clear swelling of spores is observed. There is neither cell polymorphism nor acid resistance. Gram stain is positive.

(2) Meat broth agar plate culture: The shape of the community is circular, the surface of the community is flat, and the periphery of the community is wavy. In addition, the color tone of the village is white and has no gloss.

(3) Slop culture of broth agar: Good growth, filamentous growth.

(4) Liquid culture of broth: good growth and pellicle formation.

(5) Litmus milk: Peptone.

(6) Gelatin: Does not liquefy.

(7) Reduction of nitrate: No reduction.

(8) Denitrification reaction: None (9) MR test: Positive (10) VP test: Positive (11) Indole formation: None (12) Hydrogen sulfide formation: None (13) Starch hydrolysis: Positive (14) ) Casein hydrolysis: Positive (15) Utilization of citric acid: Positive (16) Utilization of inorganic nitrogen source: Positive (17) Formation of pigment: Not produced.

(18) Urease: Positive (19) Oxidase: Positive (20) Catalase: Positive (21) Salt broth: Does not grow with 10% or more of salt.

(22) Growth range: pH 4-9 Temperature 23-48 ° C (23) Attitude toward oxygen: aerobic (24) Availability of sugars: L-arabinose, D-xylose, D-glucose, D-mannose, D- Fructose, maltose, sucrose, lactose, trehalose, D-sorbit, D-mannitol, inosit, and glycerin are used to generate an acid. No gas is generated.

It also utilizes D-galactose and starch, but produces neither acid nor gas.

About the above mycological properties
It was confirmed that the microorganism should be identified as Bacillus subtilis with reference to the 8th Edition of Determinating Bacteriology.

In culturing the γ-cyclodextrin synthase A-producing bacterium, a commonly used medium can be widely used. Generally, it is cultured in a medium containing a carbon source such as starch and partially decomposed products of starch such as dextrin. In addition, as a nitrogen source, peptone, casein, meat extract, yeast extract, corn steep liquor or extracts such as soybean or soybean meal, and to supplement this, inorganic nitrogen sources, phosphates, magnesium salts, calcium salts, A culture medium containing the same is used. Culture at pH 4-9, temperature 30-40 ° C, 3
It is performed with aeration for 5 days.

In the obtained culture, γ-cyclodextrin synthase A is out of the cells and is present in the culture solution. The culture is filtered to remove the bacterial cells, and the filtrate is concentrated and then precipitated by adding an organic solvent such as acetone, ethanol, methanol, or isopropanol, or by further precipitating with a salting out material such as ammonium sulfate. be able to.

Γ-Cyclodextrin synthase A thus obtained
In order to synthesize γ-cyclodextrin using, the present enzyme is added to at least one of starch, amylose, amylopectin, and glycogen to adjust pH 4 to 9, preferably 6
-8, the temperature is 40 to 60 ° C, preferably about 55 ° C.

Preferably, after the completion of the reaction, after deactivating the enzyme, the reaction solution is treated with glycoamylase or bacterial liquefaction type α-amylase, preferably both in combination to decompose sugars other than γ-cyclodextrin into glucose. Later, γ-
Collect cyclodextrin. For collecting γ-cyclodextrin, a conventional method, for example, a column method,
A solvent precipitation method or the like is appropriately used.

〔Example〕

 Examples of the present invention will be shown below.

Example 1 21% potato starch, 1% peptone, KH ₂ PO ₄
0.3%, yeast extract 0.1%, NaCl 0.1%, MgSO ₄ .
500 ml of a medium containing 1% and CaCl ₂ 0.02% was placed in a 2-volume Erlenmeyer flask and sterilized by a conventional method, and then Bacillus sp. NO. 313 (FERM P-8366) was inoculated,
The cells were cultured at 37 ° C for 5 days with shaking. After the culture was completed, the cells were removed by a centrifuge, and the amount of enzyme produced was measured. As a result, it was 0.33 units per 1 ml of the medium.

Example 2 The same composition as in Example 1 was added to the above Bacillus sp. N.
Inoculated with O.313 (FERM P-8366), 37 ℃
After culturing with shaking for 5 days, the cells were removed by a centrifuge, and the obtained crude enzyme solution was made into acetone dry powder by a usual method. It was possible to obtain 6.9 g of γ-cyclodextrin synthase A (specific activity 43 units / g) per culture solution.

Reference example 5% potato starch gelatinization liquid 20 (with 1N NaOH
240 units of γ-cyclodextrin synthase A was added to (adjusted to pH 8.0) and reacted at 55 ° C. for 20 hours to produce γ-cyclodextrin. After the reaction, inactivate the enzyme by heating (100 ° C, 30 minutes), then add 1N HCl to pH
It was adjusted to 5.5, 2,000 units of glucoamylase and 4,000 units of bacterial liquefied α-amylase were added, and the mixture was reacted at 50 ° C for 5 hours to decompose sugars other than γ-cyclodextrin into glucose. After the reaction solution was treated with activated carbon, γ-cyclodextrin was precipitated with trichloroethylene. The precipitate was separated by filtration and trichloroethylene was removed by steam distillation. The obtained γ-cyclodextrin solution was freeze-dried to obtain 10 g of γ-cyclodextrin.

The γ-cyclodextrin obtained in the reference example was analyzed by high performance liquid chromatography (filler nucleosyl 5NH ₂ , 4.6
× 25 cm, acetonitrile / water-65 / 35, flow rate 1 m
(l / min) and the bromcresol-green method.

FIG. 1 is a drawing showing a high performance liquid chromatogram,
(A) is a sample containing preparations of glucose (G), α-CD (cyclodextrin), β-CD and γ-CD, (B) is a sample containing oligosaccharides having 1 to 7 glucose units, (C) ) Is a sample containing γ-CD of the present invention,
(D) is a chromatogram of the sample which mixed the sample (A) and the sample (C). From the above chromatogram, it can be seen that the retention time of γ-CD of the present invention completely matches the retention time of the γ-CD standard.

Figure 2 shows γ-C by the Brom-cresol-green method.
It is a figure which shows the absorption spectrum of D, and (gamma) -C of this invention.
It can be seen that the spectrum of D completely matches the spectrum of the γ-CD standard.

[Brief description of drawings]

FIG. 1 is a drawing showing a high performance liquid chromatogram, and FIG. 2 is a drawing showing an absorption spectrum by the brom cresol green method.

Claims (3)

[Claims] 1. A γ-cyclodextrin synthase A having the following physicochemical properties. Action and substrate specificity: γ-cyclodextrin is specifically synthesized from starch, amylose, amylopectin, glycogen and the like. Formation of α- and β-cyclodextrin is not observed. Working pH range: pH 4 to 9 (in 0.02M buffer) Optimal working pH: around pH 8 Working temperature range: approx. 75 ° C or less Optimal working temperature: approx. 65 ° C Thermal stability: in 0.05M Tris-HCl buffer By heating for 30 minutes, it hardly deactivates below 50 ° C, but 70 ° C
With the above, it is almost completely deactivated. pH stability: Stable at pH 6 to 8 Molecular weight: 64,000 (measured by SDS-polyacrylamide electrophoresis method) Isoelectric point: 7.05 (measured by electrophoresis method using carrier ampholine) 2. A γ-cyclodextrin synthase A, which belongs to the genus Bacillus and has the following physicochemical properties, is cultured, and γ-cyclodextrin synthase A is collected from the culture. Process for producing cyclodextrin synthase A. Action and substrate specificity: γ-cyclodextrin is specifically synthesized from starch, amylose, amylopectin, glycogen and the like. Formation of α- and β-cyclodextrin is not observed. Working pH range: pH 4 to 9 (in 0.02M buffer) Optimal working pH: around pH 8 Working temperature range: approx. 75 ° C or less Optimal working temperature: approx. 65 ° C Thermal stability: in 0.05M Tris-HCl buffer By heating for 30 minutes, it hardly deactivates below 50 ° C, but 70 ° C
With the above, it is almost completely deactivated. pH stability: Stable at pH 6 to 8 Molecular weight: 64,000 (measured by SDS-polyacrylamide electrophoresis method) Isoelectric point: 7.05 (measured by electrophoresis method using carrier ampholine) 3. A γ-cyclodextrin synthase A-producing bacterium belonging to the genus Bacillus is Bacillus sp. NO.31.
No. 3 (Bacillus sp NO.313)
The method described in (2).