JP2623509B2 - Method for producing branched maltooligosaccharides - Google Patents

Description

Description: TECHNICAL FIELD The present invention relates to a method for producing a branched maltooligosaccharide.

[Prior Art] There are very few reports on cyclodextrinase produced by Bacillus macerans [Biochemistry, Vol. 7, pp. 121-124, 1
968], produced by Bacillus coagulans [Journal of Agricultural Biological Chemistry (Agric. Biol. Chem.), Vol. 47, No. 1441
Pp. 1447, 1983].

Conventionally, as a method for producing a branched maltooligosaccharide, for example, a method for producing from a starch or the like by the action of amylase is known.

On the other hand, using cyclodextrin as a raw material and cyclodextrin glucanotransferase (EC2.4.1.19) produced by Bacillus macerans, etc.,
A method for producing a branched maltooligosaccharide of maltoheptaose or more by catalyzing a coupling reaction between branched cyclodextrin and glucose is known [FEBS Letters, Vol. 6, pp. 182 to 186].
Pp. 1970].

Branched maltooligosaccharides, e.g., 6 ⁴ -0-alpha-glucosyl maltopentaose is [carb Hydrate Research (Carbohydrate Research), 173 pp. The 324-331
^{Page, 1988], 6 3 -0-} α- glucosyl maltotetraose, [Phebus Letters (FEBS Letters), Vol. 6,
Pp ^{182~186, 1970], 6 4 -0} -α- maltosyl maltopentaose is [Journal of Biochemistry (J. Biochem.), Chapter 78, pp. 889-896, 1975] and 6
³ -0-alpha-maltosyl maltotetraose, [Agricultural Biological Chemistry (Agri
c. Biol. Chem.), Vol. 49, pp. 3391-3398, 1985], and the branched maltooligosaccharides are
It is expected that it can be widely applied to medicines and foods as a serum amylase measurement substrate, a nutrient, an excipient, a bulking agent, and the like.

[Problems to be Solved by the Invention] However, the method for producing branched maltooligosaccharides from starch by the action of α-amylase has not been established yet because a method for separating and purifying branched oligosaccharides has not yet been established. The fact is that an efficient method for producing saccharides with high purity and high yield has not been established yet. On the other hand, a method for producing a branched oligosaccharide from a branched cyclodextrin and glucose by utilizing a coupling reaction of cyclodextrin glucanotransferase is based on the decomposition of the product itself or the reaction of the product with the branched cyclodextrin as the reaction proceeds. Since the accumulation of by-products due to the coupling reaction increases, the reaction rate of the product with respect to the branched cyclodextrin must be kept low, and a large amount of unreacted branched cyclodextrin remains in the reaction solution,
There were problems such as difficulty in subsequent purification.

[Means for Solving the Problems] The present inventors have conducted intensive studies for the purpose of rapidly and efficiently obtaining high-purity maltooligosaccharides. As a result, the present inventors have developed a novel cyclodextrinase into cyclodextrin. The inventors obtained a finding that various maltooligosaccharides accumulate from cyclodextrin in high yield when used alone or in combination with a maltooligosaccharide-forming enzyme, and filed a patent application. (Japanese Patent Application No. 1-152257 and Japanese Patent Application
1-221534 specification).

Therefore, the present inventors further conducted various studies to obtain a high-purity branched maltooligosaccharide quickly and efficiently from the branched cyclodextrin as a starting material. As a result, the branched cyclodextrin produced a novel cyclodextrinase and maltooligosaccharide. When the enzymes are contacted simultaneously or sequentially, first, a branched maltooligosaccharide corresponding to the degree of glucose polymerization of the branched cyclodextrin is formed, and then, by the action of the maltooligosaccharide-forming enzyme from the branched maltooligosaccharide, the degree of glucose polymerization is higher than that of the branched maltooligosaccharide. reduced, for example, 6 ⁴ -0-alpha-glucosyl maltopentaose of 6 ³ -0-alpha-glucosyl maltotetraose, 6 ⁴
-0-α- maltosyl maltopentaose, 6 ³ -0-α
-The inventors have obtained knowledge that a branched maltooligosaccharide such as maltosyl maltotetraose can be obtained in a high yield, and completed the present invention.

That is, the present invention is a method for producing a branched maltooligosaccharide, comprising simultaneously or sequentially contacting a novel cyclodextrinase and a maltooligosaccharide-forming enzyme having the following physicochemical properties with a branched cyclodextrin.

Action: Cleavage cyclodextrin to produce oligosaccharides derived from the degree of glucose polymerization of cyclodextrin.

Substrate specificity: The hydrolysis rate or affinity for cyclodextrin is greater than that of a polysaccharide or linear oligosaccharide having the same degree of glucose polymerization as cyclodextrin.

Optimum pH and stable pH range: The optimum pH is around 8.0 when β-cyclodextrin is used as a substrate, and the stable pH range is 5.5 to 9.5.

Optimum temperature range for working: Suitable working temperature around 40 ℃.

Deactivation conditions by temperature, etc .: Almost deactivated by treatment at 50 ° C or higher for 15 minutes.

Inhibition and activation: more than 90% inhibited by Hg ²⁺ , Cu ²⁺ , Zn ²⁺ , Ni ²⁺ and Fe ²⁺ , C
10-30% activated by a2 ⁺ and Mg2 ⁺ .

Molecular weight: 144000 by gel filtration, 72000 by SDS PAGE
It is.

 Hereinafter, the present invention will be described in detail.

First, the physicochemical properties of the novel cyclodextrinase will be described.

(1) Action: Acts on cyclodextrin to produce maltooligosaccharides derived from the degree of glucose polymerization of cyclodextrin.

(2) Substrate specificity: The substrate specificity is as shown in Table 1.
The reaction rate parameters for cyclodextrin and maltooligosaccharide are as shown in Table 2.

(3) Optimum pH and stable pH range: The optimum pH is as shown in FIG. 1, and is around pH 8.0 when 1% β-cyclodextrin is used as a substrate. The stable pH range is as shown in FIG.
It was determined by measuring the residual activity after treatment at 24 ° C. for 24 hours. As is clear from FIG. 2, the stable pH range is:
5.5 to 9.5.

(4) Titration method: 500 μl of a 2% β-cyclodextrin solution and 500 μl of 100 mM phosphate buffer (pH 7.5) containing an appropriate amount of the present enzyme
Was reacted at a temperature of 40 ° C. for an appropriate period of time, followed by boiling for 10 minutes to stop the reaction, and maltoheptaose produced was quantified by a high-performance liquid chromatography (hereinafter abbreviated as HPLC) method. When the amount of the enzyme was small, the reducing power was quantified by the Somogyi-Nelson method using glucose as a standard.

The enzyme unit of this enzyme was defined as the amount of enzyme that produces 1 micromol maltoheptaose per minute.

(5) Range of suitable temperature of action As shown in FIG. 3, the present enzyme has a suitable temperature of action around 40 ° C.

(6) Inactivation conditions depending on temperature As shown in FIG. 4, this enzyme was stable in a 100 mM phosphate buffer (pH 7.5) for 15 minutes up to 45 ° C., but it was stable at 50 ° C. Above was deactivated.

(7) Inhibition and activation: Table 3 shows the results of studies on the effect of metal ions on the activity of the present enzyme. As is clear from Table 3, this enzyme was 2
Hg ²⁺ is a valence of the metal ion, Cu ^2+, Zn ^2+, it inhibited more than 90% Ni ²⁺ and Fe ²⁺ and the like, is about 10-30% activated by Ca ²⁺ and Mg ²⁺ .

(8) Purification method: As described in Example 1.

(9) Molecular weight: 72000 by SDS PAGE method, 14400 by gel filtration method
Since it is 0, this enzyme is a dimer composed of subunits having a molecular weight of 72,000.

Table 4 shows the differences in physicochemical properties between this enzyme and a conventionally known cyclodextrinase.

As described in detail above, the present enzyme is a completely new enzyme which differs in its properties from conventionally known cyclodextrinases, and in particular, it acts best on cyclodextrin.

 Next, a method for producing the present enzyme will be described.

The microorganism that produces the enzyme may be any microorganism that belongs to the genus Bacillus and that produces the enzyme. For example, Bacillus sphaeri
cus) E-244 strain. Bacillus sphaericus E-244
The strain is a wild strain obtained from the soil, and the following shows the mycological properties of the strain.

Bacteriological properties of B. sphaericus strain E-244 (a) Morphology: Bacteria having a cell shape and size of 0.6 to 0.8 × 1.6 to 4.0 microns.

Cell polymorphism Not observed.

Motility Motility with periflagellate.

Spore presence Yes.

Spore swelling Size 0.8-0.9 × 1.1-1.2 microns Oval Position Sub-edge Gram stain Positive Acid-fast Negative (b) Growth in each medium Gravy agar plate culture Form colorless diffusible colonies. The village is smooth and the periphery is smooth. No pigment production is observed.

Gravy agar slope culture Bacterial moss is smooth and the periphery is smooth. No pigment production is observed.

Broth liquid culture Growth is observed throughout the medium, but no precipitate is observed.

Broth gelatin puncture culture Growing only on the upper part of the medium, no liquefaction is observed.

Litmus milk No coagulation is observed, and no acid or alkaline acidification is observed.

(C) Physiological properties Reduction of nitrate Not reduced.

No denitrification reaction.

MR test negative.

VP test negative.

Indole generation No generation.

Generation of hydrogen sulfide Not generated.

Starch hydrolysis Not decomposed.

Use of citric acid Not used.

Use of inorganic nitrogen source Not used.

Dye formation Not generated.

Urease negative.

Oxidase positive.

Catalase positive.

Range of growth Temperature 13-38 ° C pH 6-10.5 Attitude to oxygen Aerobic OF test Negative (no acid production of acid) Attitude to sugar L-arabinose, D-xylose, D-glucose, D-mannose, D- No acid production or gas production from fructose, D-galactose, maltose, sucrose, lactose, trehalose, D-sorbitol, D-mannitol, inosit, glycerin and starch is observed.

(D) Deamination reaction of phenylalanine positive Bacillus sphaericus E-244 strain was identified as a bacterium belonging to the genus Bacillus because it is a gram-positive bacillus that forms spores. Furthermore, since no generation of acid and gas from the sugar was observed, the pH of the VP broth was 7.0 or more, and the deamination of phenylalanine was observed, the Bergez Manual of Cistimatic Bacteriology , Vol. 2, 1984, and identified as a bacterium belonging to the sphaericus species of the genus Bacillus.

In addition, Bacillus sphaericus E-244 was supplied to the Institute of Microbial Industry and Technology by the National Institute of Advanced Industrial Science and Technology
2458).

The cultivation of the strain is basically the same as the method employed for aerobic cultivation of general microorganisms, but usually, a shaking culture method using a liquid medium or an aeration stirring culture method is used. As the medium, a medium containing an appropriate nitrogen source, carbon source, vitamins, minerals, etc. and cyclodextrin, which is an inducing substrate of the present enzyme, is used. The pH is at which the fungus grows
Any range may be used as long as it is in the pH range, but usually a range of 6 to 8 is preferred.

As the culture conditions, for example, shaking culture or aeration-agitation culture is usually performed at 20 to 40 ° C. for 16 hours to 4 days.

Using the culture obtained as described above, a purified product of the present enzyme is obtained, for example, by the following enzyme collecting step. To obtain the enzyme from the above culture, the cells are collected by centrifugation or membrane concentration, and then the cells are disrupted by sonication or treatment with a surfactant, and the bacterial residue is removed by centrifugation or the like. Obtain a liquid. A purified product of the enzyme is obtained by appropriately performing column chromatography such as ion exchange chromatography, hydrophobic chromatography, and gel filtration on the crude enzyme solution.

 Next, a method for producing a branched maltooligosaccharide will be described.

Branched cyclodextrin to cyclo dextrinase and maltooligosaccharides forming enzyme described above simultaneously or by sequentially contacting act, depending on maltooligosaccharides produced enzyme from the reaction mixture, for example, 6 ⁴ -0-alpha-glucosyl maltopentaose, 6 ³ -0-α-glucosyl maltotetraose,
6 ⁴ -0-alpha-maltosyl maltopentaose, 6 ³ -O-
A branched maltooligosaccharide such as α-maltosyl maltotetraose is obtained.

The branched cyclodextrin may be any, for example, 6-0-α-maltosyl-β-cyclodextrin, 6-0-α-glucosyl-β-cyclodextrin, 6-0-α-glucosyl-α-cyclodextrin or 6-0-α-maltosyl-α-cyclodextrin and the like are preferred.

As for the substrate concentration of the branched cyclodextrin, the higher the concentration, the better, but for example, a concentration range of 0.1 to 30% is preferable.

The maltooligosaccharide-forming enzyme varies depending on the target oligosaccharide, and examples thereof include known enzymes such as β-amylase and glucoamylase.

The reaction conditions for bringing the cyclodextrinase and the maltooligosaccharide-forming enzyme into contact with the branched cyclodextrin simultaneously or sequentially may be any condition as long as the action pH and temperature of the cyclodextrinase and the maltooligosaccharide-forming enzyme are in the range. For example, pH 5.0-8.0
And a temperature of 35 to 45 ° C. is preferred. Further, if necessary, an organic solvent or the like can be added to the reaction product. The reaction time is
Depends on the stability of the reaction product, but preferably from 30 minutes to
It is about 48 hours. The amount of the enzyme is not particularly limited, but may be adjusted so that the product is maximized within the reaction time, and the necessary amount may be appropriately added. It is desirable to stop the reaction by, for example, acid or heat treatment when the product reaches a maximum.

Next, the desired branched maltooligosaccharide is obtained from the branched maltooligosaccharide-containing reaction solution obtained as described above, and any method may be used as long as it is a conventional oligosaccharide separation method, such as a distribution absorption column, a gel filtration column, or the like. By fractionating using an activated carbon column or the like, a highly pure branched maltooligosaccharide solution can be easily obtained.

[Effects of the Invention] According to the present invention, a branched maltooligosaccharide having a clear structure can be efficiently and industrially produced from a branched cyclodextrin, which is industrially extremely significant.

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

Example 1 1% β-cyclodextrin, 1% peptone, 0.5% N
100 ml of a liquid medium (using tap water, pH 7.0) composed of aC1 and 0.1% yeast extract is placed in a 500 ml Sakaguchi flask, and 1
Sterilization treatment was performed at 20 ° C. for 20 minutes. One loopful of this was inoculated from a stock slant of Bacillus sphaericus strain E-244 (FERM BP-2458) and cultured with shaking at 30 ° C. for 1 day. 50 ml of the main culture was inoculated into a 3 liter mini jar containing 2 liters of medium prepared under the same medium composition and sterilization conditions as above,
Aeration and agitation culture was performed at 30 ° C., 1 vvm and 350 rpm for 2 days. After completion of the culture, cells were separated from the culture solution by centrifugation at 8000 rpm for 20 minutes, and 2% Triton X-10 was added.
The cells were suspended in 500 ml of 10 mM phosphate buffer (pH 7.0) containing 0 and stirred at 25 ° C. for 1 day. 12000 r. From the suspension.
After removing cell debris by centrifugation at pm for 20 minutes, the supernatant was added to 10 mM phosphate buffer (pH 7.0).
Dialyzed for hours. The dialysate was centrifuged at 12000 rpm for 20 minutes to remove insolubles, and the supernatant was used as a crude enzyme solution (1).

Then, about 500 ml of the crude enzyme solution (1) (total activity 200 units, specific activity 0.1, pH 7.0) was added to 10 mM phosphate buffer (pH 7.0).
Column packed with DEAE Sepharose (φ34x170m)
m), the enzyme was adsorbed, and then eluted with a gradient of 0 to 0.5 M NaCl. The elution pattern of this DEAE Sepharose column chromatography is shown in FIG. Collect the active fraction and crude enzyme solution (2)
105 ml (total activity 145 units, specific activity 0.58, yield 72.5%) were obtained.

Then, the crude enzyme solution (2) was mixed with 1M sodium sulfate.
Ether 5PW equilibrated with 100mM phosphate buffer (pH 7.0)
After applying to a packed column (φ21.5 × 150 mm) to adsorb cyclodextrinase, elution was performed with a gradient gradient of 1M to 0M sodium sulfate. This elution pattern is shown in FIG. The active fractions were collected to obtain 50 ml of a crude enzyme solution (3) (total activity 72 units, specific activity 2.93, yield 36%).

Next, the crude enzyme solution (3) was ultra-concentrated to 1.5 ml with a collodion bag, and 0.2 ml was added to TSK gel G300.
The mixture was subjected to gel filtration using 0SW, and eluted with 100 mM phosphate buffer (pH 7.0) containing 0.2 M NaC1. This elution pattern is shown in FIG. The active fractions were collected, and purified cyclodextrinase solution (1.4 ml, total activity 2.2 units, protein amount 0.1 ml).
24 mg, specific activity 9.17, yield 1%). The cyclodextrinase was single in SDS PAGE. (FIG. 8) Example 2 6-0-α-glucosyl-β-cyclodextrin
To a solution of 200 mg in 10 ml of 10 mM phosphate buffer (pH 7.5), 0.43 unit of the crude enzyme solution of cyclodextrinase obtained in Example 1 was added, and the reaction was carried out at 40 ° C. for 3 hours. The reaction was performed. The reaction solution was boiled for 10 minutes to stop the reaction, and the boiled solution was concentrated to dryness by a rotary evaporator and then redissolved in 1 ml of deionized water. This solution was separated and purified by an HPLC operation using a PA-43 HPLC column for carbohydrate fractionation (manufactured by YMC, packed column for distribution and adsorption chromatography). The result of analyzing the purified product by HPLC using a TSKgel Amide80 column (manufactured by Tosoh Corporation, a packed column for distribution and adsorption chromatography) is shown in FIG. 9. The purity of the obtained 6n-0-α-glucosyl maltoheptaose was about 95%. About 3 mg of the present 6n-0-α-glucosyl maltoheptaose was dissolved in 290 μl of 10 mM acetate buffer (pH 5.8), and 10 μl of β-amylase (manufactured by Sigma, 5600 U / ml in Sigma amylase units) was added. Add 40 ℃
, And the reaction was stopped by boiling for 10 minutes. FIG. 10 shows the results of HPLC analysis of this boiling solution using a TSKgel Amide 80 column (manufactured by Tosoh Corporation, a packed column for distribution and adsorption chromatography). From 6n-0-α-glucosyl maltoheptaose,
6 ⁴ -0-alpha-glucosyl maltopentaose and maltose are mainly generated, 6 ⁴ -0-α- glucosyl maltopentaose of 1.69mg was obtained.

Example 3 6-0-α-maltosyl-β-cyclodextrin
To a solution of 200 mg in 10 ml of 10 mM phosphate buffer (pH 7.5) was added 0.43 unit of the crude enzyme solution of cyclodextrinase obtained in Example 1, and the reaction was carried out at 40 ° C. for 3 hours. The reaction was performed. The reaction was stopped by boiling the reaction solution for 10 minutes, and the boiling solution was concentrated to dryness using a rotary evaporator and then redissolved in 1 ml of deionized water. The solution was separated and purified by HPLC using a PA-43 HPLC column for carbohydrate fractionation (packed column for distribution and adsorption chromatography, manufactured by YMC). The results of analyzing the purified product by HPLC using a TSKgel Amide80 column (manufactured by Tosoh Corporation, packed column for distribution and adsorption chromatography) are shown in FIG.
The purity of the obtained 6n-0-α-maltosyl maltoheptaose was about 95%.

6n-0-α-maltosyl maltoheptaose about 3mg
Was dissolved in 290 μl of 10 mM acetate buffer (pH 5.8), and 10 μl of β-amylase (manufactured by Sigma, 5600 U / ml in Sigma amylase unit) was added thereto, followed by reaction at 40 ° C. for 1.5 hours. Thereafter, the reaction was stopped by boiling for 10 minutes. This boiling solution is applied to a TSKgel Amide80 column (manufactured by Tosoh Corporation,
Using a packed column for distribution and adsorption chromatography)
The results of analysis by HPLC are shown in FIG. 6n-0-α-
From maltosyl maltoheptaose, 6 ⁴ -0-α- maltosyl maltopentaose and maltose are mainly generated, 6 ⁴ -0-α- maltosyl maltopentaose of 2.51mg was obtained.

Example 4 6-0-α-glucosyl-α-cyclodextrin
To a solution of 200 mg in 10 ml of 10 mM phosphate buffer (pH 7.5) was added 0.43 unit of the crude cyclodextrinase enzyme solution obtained in Example 1, and the reaction was carried out at 40 ° C. for 20 hours. The reaction was performed. The reaction was stopped by boiling the reaction solution for 10 minutes, and the boiling solution was concentrated to dryness using a rotary evaporator and then redissolved in 1 ml of deionized water. This solution was separated and purified by an HPLC operation using a PA-43 HPLC column for carbohydrate fractionation (manufactured by YMC, packed column for distribution and adsorption chromatography). Purify the product with TSKgel Amide80 column (manufactured by Tosoh Corporation, packed column for distribution and adsorption chromatography)
FIG. 13 shows the results of the analysis by HPLC using. The purity of the obtained 6n-0-α-glucosylmaltohexaose was about 95%.

About 6 mg of this 6n-0-α-glucosyl maltohexaose
Was dissolved in 290 μl of 10 mM acetate buffer (pH 5.8), and 10 μl of β-amylase (manufactured by Sigma, 5600 U / ml in Sigma amylase unit) was added thereto, followed by reaction at 40 ° C. for 1.5 hours. Thereafter, the reaction was stopped by boiling for 10 minutes. This boiling solution is applied to a TSKgel Amide80 column (manufactured by Tosoh Corporation,
Using a packed column for distribution and adsorption chromatography)
The results of analysis by HPLC are shown in FIG. 6n-0-α-
From glucosyl maltohexaose, 6 ³ -0-α- glucosyl maltotetraose and maltose are mainly generated, 6 ³ -0-α- glucosyl maltotetraose 2.34mg was obtained.

Example 5 6-0-α-maltosyl-α-cyclodextrin
To a solution of 200 mg in 10 ml of 10 mM phosphate buffer (pH 7.5) was added 0.43 unit of the crude cyclodextrinase enzyme solution obtained in Example 1, and the reaction was carried out at 40 ° C. for 20 hours. The reaction was performed. The reaction solution was boiled for 10 minutes to stop the reaction. The boiled solution was concentrated to dryness using a rotary evaporator and then redissolved in 1 ml of deionized water. This solution was separated and purified by an HPLC operation using a PA-43 HPLC column for carbohydrate fractionation (manufactured by YMC, packed column for distribution and adsorption chromatography). The results of analyzing the purified product by HPLC using a TSKgel Amide80 column (manufactured by Tosoh Corporation, a packed column for distribution and adsorption chromatography) are shown in FIG.
The purity of the obtained 6n-0-α-maltosyl maltohexaose was about 95%.

About 6 mg of this 6n-0-α-maltosyl maltohexaose
Was dissolved in 290 μl of 10 mM acetate buffer (pH 5.8), and 10 μl of β-amylase (manufactured by Sigma, 5600 U / ml in Sigma amylase unit) was added thereto, followed by reaction at 40 ° C. for 1.5 hours. Thereafter, the reaction was stopped by boiling for 10 minutes. This boiling solution is applied to a TSKgel Amide80 column (manufactured by Tosoh Corporation,
Using a packed column for distribution and adsorption chromatography)
The results of analysis by HPLC are shown in FIG. 6n-0-α-
From maltosyl maltohexaose, 6 ³ -0-α- maltosyl maltotetraose and maltose are mainly generated, 6 ³ -0-α- maltosyl maltotetraose 2.35mg was obtained.

[Brief description of the drawings]

FIG. 1 is a diagram showing the optimum pH of the novel cyclodextrinase, FIG. 2 is a diagram showing the stable pH of the enzyme, and FIG. 3 is a diagram showing the action temperature of the enzyme. And the fourth
The figure is a diagram showing the conditions of inactivation by the temperature of the enzyme,
FIG. 5 is a diagram showing the results of column chromatography of the enzyme using DEAE Sepharose, FIG. 6 is a diagram showing the results of HPLC of the enzyme using TSKgel ether 5PW, and FIG. FIG. 8 is a diagram showing a result of HPLC using TSKgel G3000SW, FIG. 8 is a diagram showing a result of SDS PAGE of the enzyme, and FIG. 9 is a diagram showing purified 6n-0-α-glucosyl maltoheptaose and TSKgel Amide80. The chart pattern analyzed by HPLC used, FIG. 10 is a chart pattern analyzed by HPLC using TSKgel Amide80, and the reaction solution after the β-amylase was allowed to act on 6n-0-α-glucosylmaltoheptaose. FIG. 11 shows that purified 6n-0-α-maltosyl maltoheptaose was purified by HP using TSKgel Amide80.
The chart pattern analyzed by LC, FIG. 12 shows 6n-0-α
-A reaction pattern obtained after allowing β-amylase to act on maltosyl maltoheptaose was analyzed by HPLC using TSKgel Amide 80, and a chart pattern, FIG. 13 shows purified 6n-0-α-
A chart pattern of glucosylmaltohexaose analyzed by HPLC using TSKgel Amide80, FIG. 14 shows 6n-0
-The reaction solution after the action of β-amylase on α-glucosylmaltohexaose was subjected to HPLC using TSKgel Amide80.
The chart pattern analyzed in Fig. 15 is purified 6n-0-
Chart pattern of α-maltosyl maltohexaose analyzed by HPLC using TSKgel Amide 80, FIG. 16 shows 6n
The reaction solution obtained by reacting β-amylase with 0-α-maltosyl maltohexaose was used with TSKgel Amide80.
It is a figure showing the chart pattern analyzed by HPLC.

Claims (1)

(57) [Claims] 1. A process for producing maltooligosaccharides, comprising simultaneously or sequentially contacting a branched cyclodextrin with a novel cyclodextrinase having the following physicochemical properties and a maltooligosaccharide-forming enzyme. Action: Cleavage cyclodextrin to produce oligosaccharides derived from the degree of glucose polymerization of cyclodextrin. Substrate specificity: The hydrolysis rate or affinity for cyclodextrin is greater than that of a polysaccharide or linear oligosaccharide having the same degree of glucose polymerization as cyclodextrin. Optimum pH and stable pH range: Optimum pH is determined by using β-cyclodextrin as a substrate.
It is around 8.0 and the stable pH range is 5.5-9.5. Optimum temperature range for working: Suitable working temperature around 40 ℃. Deactivation conditions by temperature, etc .: Almost deactivated by treatment at 50 ° C or higher for 15 minutes. Inhibition and activation: 90% or more inhibited by Hg ²⁺ , Cu ²⁺ , Zn ²⁺ , Ni ²⁺ and Fe ²⁺ , Ca
10% to 30% activated by ²⁺ and Mg ²⁺ . Molecular weight: 144,000 by gel filtration, 72000 by SDS PAGE.