JPS5818074B2 - Production method of α↓-cyclodextrin - Google Patents

Description

[Detailed Description of the Invention] Conventionally, α-cyclodextrin (also known as Schard-
inger α-dextrin or Cyclohex
aamylose) is, for example, U, S, P, 3,640,
No. 837, JP-A No. 51-12,941, No. 51=12
, No. 942, Japanese Patent Publication No. 46-2,380, and Japanese Patent Application Publication No. 1973
-25,743 publications, Journal of the Starch Industry Association, Volume 9, 2
3 pages (1961), Die 5tarke, Volume 8,
280 (1963), Kyoto Prefectural University Press/Agriculture, Vol. 22, p. 106 (1970), Starch Science, Vol. 22, p. 6 (1975), etc. was.

That is, bacteria of the genus Bacillus, such as B and macerans + B, have an optimum pH for growth in the slightly acidic to neutral range.

circulans t B, starother
mophi lus + B.

Cyclodextrin glycosyltransferase (hereinafter referred to as C
It is abbreviated as GTase.

) on starch in the presence of an organic solvent such as tetrachloroethane, trichlorethylene, bromobenzene, fluorobenzene, anthracene, n-decanol, acetone, ethanol, methanol, or the above after acting on starch. By adding an organic solvent, α-2β-2 consisting of 6, 7 or 8 glucose molecules
α-1 derived from the branched structure of γ-cyclodextrin and other higher cyclodextrins or starch
, so-called branched cyclodextrins having 6-glucoside bonds are precipitated outside the reaction system, and α-cyclodextrin is further separated and purified from this precipitate using various organic solvents.

However, this method not only requires very complicated operations, but also has a low yield of α-cyclodextrin as a product per substrate, and also has problems with the fiber layer. Complete removal of cyclodextrin and branched cyclodextrins was difficult.

Moreover, the CGTasj produced by the neutral bacteria used often lacks stability with respect to temperature and pH, and
Since it can only be used under mild conditions of 55° C. or lower and a pH of about 5 to 7, it is susceptible to contamination by various bacteria and easily spoils.

□As a result of intensive study to improve the drawbacks of the conventional method as described above, the present inventors discovered that C produced by neutral bacteria
Unlike conventional methods, we use CGTase produced by alkalophilic bacteria, which differs in substrate specificity and stability from GTase, by directly adding glucose and/or maltose, maltotriose, etc. By acting on a substrate solution containing 20 to 80 parts of chain oligosaccharide at a pH in the range of 4 to 11, we succeeded in accumulating a large amount of α-cyclodextrin in the reaction system.

Furthermore, as a post-treatment, unreacted β-cyclodextrin and trace amounts of γ-cyclodextrin generated as a result of the internal interconversion reaction are
The saccharifying α-amylase produced by bacteria of the genus Bacillus is
- By acting after the cyclodextrin production reaction, it was possible to completely decompose it into glucose, maltose, and maltotriose.

The reaction of producing α-cyclodextrin from β-cyclodextrin in the method of the present invention hardly occurs in CGTase such as Bacillus macerans, which has been conventionally used as an enzyme source.

Furthermore, unlike the starch of the conventional method, the substrate used in the method of the present invention has β
- Since it is a mixture of cyclodextrin and glucose and/or linear oligosaccharides such as maltose and maltotriose, branched cyclodextrins are not generated in the reaction product.

Therefore, according to the method of the present invention, the action of the bacterial saccharification type α-amylase used in the post-treatment is completely carried out, and β-cyclodextrin and γ-cyclodextrin are completely decomposed into glucose, maltose, and maltotriose. It was possible to create a simple system consisting only of α-cyclodextrin and these non-cyclic carbohydrates.

When bacterial saccharifying α-amylase is used as a post-treatment for the conventional process of producing α-cyclodextrin from starch, the branched α-cyclodextrin cannot be degraded and the reaction is incomplete. Become.

This branched α-cyclodextrin is mostly α-1
, 6-glucosyl-α-cyclodextrin or α-1,6-maltosyl-α-cyclodextrin, and it has already been reported that it cannot be degraded by glucoamylase or pullulanase (a starch cutting enzyme). (Analytical Biochemist
ry, 39.521 (1971); cereal
Chemistry, 43°111 (1966)
;Archives of Bioche-mister
y and Biophysics, 1
37, 483 (1970), etc.).

On the other hand, unlike the conventional method, the carbohydrate after the reaction according to the present invention has a simple composition consisting of only α-cyclodextrin, glucose, maltose, and maltotriose, so the use of a problematic organic solvent is avoided. It only needs to be done once, which not only makes it possible to significantly reduce costs, but also
Problems with pollution control measures such as wastewater treatment will also be almost eliminated.

Examples of the alkaliphilic Bacillus bacterium that supplies CGTase used in the present invention include, for example, Patent No. 886
, 583 specification and Japanese Patent Publication No. 53-31,223, Bacillus genus 4382 (Feikoken Bacteria No. 614) and Bacillus 4135 (Feikoken Bacteria No. 617)
, A169 bacterium (Feikoken Bibori, No. 618), Attendance 13
Bacillus obensis (Feikoken Bacterial Report No. 611), Bacillus 17-1 (Feikokuken Bacterial Report No. 612), and Bacillus obensis described in Japanese Patent Publication No. 52-31,949. 1.990), etc.

The enzymatic activity of the above CGTase was measured by the following method: 0. ．． 0 containing 0.5% (w/v) amylose
．． Add 0.2 ml of the test enzyme solution diluted with distilled water to 0.31 nl of IN phosphate buffer (pH 7.0) and incubate at 40°C.
4 m of 0.2 N hydrochloric acid was added to the reaction solution for 10 minutes.
1 of distilled water to stop the reaction, add 4 rf of distilled water to this.
0.02% iodine and 0.2% iodine were added to the prepared solution to which Ll had been added, and to a blank prepared in the same manner as above using the test enzyme solution that had been previously heated and inactivated.
An iodine solution containing 0.5% iodopotassium was added, and the absorbance at 700 mμ was measured in 1 crrL of the liquid layer, and the amount of enzyme that reduced this absorbance by a factor of 10 was defined as 1 unit.

In addition, CGTase produced by Bacillus macerans (IFO3490) is prepared by adjusting the pH of the substrate to pH 6 with a phosphate buffer.
It was adjusted to 0 and measured in the same manner.

The raw materials (substrates) of the present invention are β-cyclodextrin and linear oligosaccharides such as glucose and/or maltose and maltotriose (hereinafter referred to as auxiliary substrates).

) with respect to the mixing ratio of β-cyclodextrin/
The co-substrate ratio is 5. If the reaction rate is above, the internal interconversion reaction rate from β-cyclodextrin to α-cyclodextrin becomes extremely low, and almost no α-cyclodextrin is produced.

When the above ratio is 1.25 or less, the internal interconversion reaction rate is very slow, but due to the coupling action of CGTase, almost no α-cyclodextrin is produced, and a large amount of acyclic carbohydrates are produced. It is industrially disadvantageous because it is produced in

As a result of intensive studies on the conditions for producing α-cyclodextrin from β-cyclodextrin through internal interconversion reactions, the present inventors discovered that glucose and/or maltose, Linear oligosaccharides such as ose are suitable;
1 when the β-cyclodextrin/cosubstrate ratio is less than or equal to 5.
．． It has been discovered that a mixture of 25 or more is suitable as a substrate for the α-cyclodextrin production reaction.

In addition, the concentration of the substrate when carrying out the method of the present invention is 10%.
(W/V) or more, preferably 20-30% (W/V)
It is better to do it.

In the method of the present invention, the amount of enzyme added to the substrate is 1 g of total (solid content) of β-cyclodextrin and auxiliary substrate.
Appropriately, 50 to 300 units of CGTase and 5 to 30 units of post-treatment saccharified α-amylase are appropriate.

Furthermore, by using glucoamylase in combination with the bacterial saccharification type α-amylase reaction, which is the second stage of the reaction of the present invention, it is possible to simplify the system to a mixed system of α-cyclodextrin and glucose. Therefore, glucose adsorption resins or cycloaxtri
1-136,889 as described in each publication.

), it is also possible to produce α-cyclodextrin without using any organic solvent.

The present invention will be explained in more detail below with reference to Examples.

Example 1 15% (W/V) β-cyclodextrin (manufactured by Nihon Shokuhin Kako (a), trade name Seldex-N) and 6% (W/V)
CGTas produced by Bacillus A38-2, an alkalophilic bacterium, is added to 1 g of a mixed solution containing glucose /V).
Powder of e (40,000 units/g) was added at a solid content of 200 units/g, and the mixture was reacted at pH 7, Q and temperature of 60° C. for 50 hours.

This reaction solution was heated at 120°C for 5 minutes to denature and deactivate CGTase, and then the bacterial saccharification type α-amylase (
Daiwa Kasei Co., Ltd., 10,000 units/7) was added at a solid content of 20 units/g and reacted at pH 5.5 and temperature 50°C for 24 hours to remove unreacted β-cyclodextrin and internal interconversion reaction. A trace amount of by-produced γ-cyclodextrin was decomposed into linear oligosaccharides.

The content of α-cyclodextyl-IJ contained in the reaction completed solution was determined from Starch Science, Vol. 25, No. 1, p. 19 (1).
It was measured according to the high performance liquid chromatography method described in 1997).

That is, using a DuPont-Shimadzu model 830 high-performance liquid chromatograph, the column was Nippon Waters' Microband Pack CH (4 x 3 oom); solvent: a mixture of water/acetonitrile nibs 25/75 (V/V); column temperature. The analysis was performed under the conditions of 50° C. and a flow rate of 1 rILl/min.

As a result, 6.8% (W/V) α-cyclodextrin was produced in the liquid, and all unreacted β-cyclodextrin and by-produced γ-cyclodextrin were decomposed. It was recognized that

This means that about 45 molecules of the β-cyclodextrin used were changed to α-cyclodextrin.

Next, 6]9 tetrachloroethane was added to the above-mentioned reaction completed liquid, and after leaving it for 3 days with stirring at room temperature, the precipitate of α-cyclodextrin produced was separated by P,
The solvent was removed by steam distillation.

Then, the α-cyclodextrin solution obtained by decolorizing with activated carbon and filtration was freeze-dried to obtain 61 g of α-cyclodextrin.

? Example 2 10% (W/V) β-cyclodextrin and 3
% (W/V) of maltose,
CG produced by alkalophilic bacterium Bacillus 417-1
200 units/g of Tase powder (30,000 units/!9)
- Solid content was added and reacted at pH 10.0 and temperature 55°C for 50 hours.

Thereafter, the sample was treated with bacterial saccharification type α-amylase in the same manner as in Example 1, and approximately 41 molecules of the β-cyclodextrin used were converted to α-cyclodextrin.

5 (L) of cyclohexane was added to the reaction solution after completion of the reaction, and the mixture was allowed to stand for 3 days with stirring at room temperature to precipitate α-cyclodextrin. The precipitate was separated and subjected to steam distillation to remove the solvent.

Then, the α-cyclodextrin solution obtained by decolorizing with activated carbon was freeze-dried to obtain 75 g of α-cyclodextrin.

Example 3 Instead of glucose as an auxiliary substrate in Example 1, bimaltose syrup obtained by hydrolyzing starch using pullulanase (starch cutting enzyme) and β-amylase (glucose 3 in solid content) was used. %, maltose 86%1
Using a mixed substrate solution with the addition of 11% of other sugars,
A treatment reaction was carried out in the same manner as in Example 1 except that the enzyme reaction was carried out at pH 5.0 to obtain α-cyclodextrin of 57.9.

The above-mentioned bimaltose syrup was prepared by the following method: A 5% (W/V) potato starch suspension adjusted to pH 6.0 was heated at 130°C for 30 minutes to homogeneously gelatinize it. ℃, and add 30 units/g of starch pullulanase powder (manufactured by ABM, trade name: Pullzyme) and 2
After adding β-amylase powder (manufactured by Nagase Sangyo Co., Ltd.) containing 0 units/g of starch and saccharifying the mixture for 60 hours, decolorization and filtration with activated carbon were performed in a conventional manner.

Example 4 Glucose was added to 15% (W/V) of β-cyclodextrin at 0% (W/V) and 1.5% (W/V), respectively.
/V), 3% (W/V), 8% (W/V), 12% (
W/V), 15% (W/V) and 20% (W/V)
1 g of each of the added mixed solutions was prepared, and treated and reacted under the same operating conditions as in Example 1.

As a result, the conversion rate of β-cyclodextrin to α-cyclodextrin was 2% and 8%, respectively.

The percentages were 31, 48%, 23%, 3%, and 0.2.

That is, 3% (W/V) to 12% (W/V) (
7) When glucose was used, the conversion rate to α-cyclodextrin was dog.

Example 5 The reaction was carried out in the same manner as in Example 4 except that maltose was used instead of glucose.

As a result, the conversion rates of β-cyclodextrin to α-cyclodextrin were 1.2%, 6, 28, 43%, 20, 1.7, and 0.1, respectively.

That is, in this case as well, from 3% (W/V) to 12% (W/V) to 15% (W/V) β-cyclodextrin.
) when maltose was used, the conversion rate to α-cyclodextrin was dog.

Comparative Example 1 In place of the CGTase used in Example 1, 200 units/g solid content of CGTase powder (manufactured by Nagase Sangyo Co., Ltd., approximately 10,000 years/9) produced by Bacillus macerans (IFO3490) was used. The treatment and reaction were carried out in the same manner as in Example 1, except that the pH of the solution was changed to 565.

As a result, only 6 g of α-cyclodextrin was obtained.

Claims (1)

[Claims] 1 Cyclodextrin glycosyltransferase (Cyclodextrin glycosyltransferase) obtained by culturing alkaliphilic bacteria that belong to the genus Bacillus and have an optimum pH for growth on the alkaline side.
yc-osyl transferase) to 100 parts of β-cyclodextrin, glucose and/or
Alternatively, saccharification obtained by reacting a mixed substrate solution containing 20 to 80 parts of linear oligosaccharides such as maltose and maltotriose at a pH range of 4 to 11, and then culturing Bacillus bacteria in the reaction solution. Decomposing unreacted β-cyclodextrin and by-produced γ-cyclodextrin into glucose, maltose and/or maltotriose by using α-amylase, and separating and collecting α-cyclodextrin from this decomposition liquid. A method for producing α-cyclodextrin, characterized by: