JPH08280A - Production of maltooligosaccharide - Google Patents

Abstract

PURPOSE:To produce a maltooligosaccharide useful for adhesive, dispersion agent, film agent, etc., with a simple operation in high efficiency and yield by treating a specific cyclodextrin with cyclodextrinase in the presence of glucose. CONSTITUTION:This maltooligosaccharide of formula II is produced by culturing Bacillus sphaericus E-244 (FERM BP-2458), etc., to produce a cyclodextrinase and treating a cyclodextrin of formula I, ((n) is 6 or 7) with the cyclodextrinase in the presence of glucose.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a novel method for producing maltooligosaccharides, and more particularly to a method for efficiently producing maltooctaose or maltononaose. Is.

[0002]

Maltooctaose or maltononaose is used as, for example, an adhesive, a dispersant, a film agent, a substrate for measuring α-amylase activity, etc., and has recently attracted attention as a moisturizer, a reagent for research and the like. ing. Examples of the method for producing this maltooctaose include, for example, a method of cleaving γ-cyclodextrin with cyclomaltodextrinase (JP-A-5-68579), a method of acid-decomposing γ-cyclodextrin [“carbohydrate. Research (Carbohydrate Research
rch) ", vol. 242, pp. 1-9, 1993.
Year] is known. Further, as a method for producing maltononaose, for example, a method of acid-decomposing δ-cyclodecyltrin is known (Japanese Patent Laid-Open No. 61-19169).
No. 0). However, both of these methods
The yield is extremely low, about 10%, and development of a production method with high yield is desired.

[0003]

The present invention has been made for the purpose of providing a method for producing a large amount of maltooctaose or maltononaose in high yield.

[0004]

The inventors of the present invention have conducted various studies to achieve the above object, and as a result, cyclodextrinase, which is a hydrolase, also has a strong glucose transfer activity. And by reacting β-cyclodextrin or γ-cyclodextrin with the cyclodextrinase in the presence of glucose,
1 each of malto octaose and maltononaose
The present inventors have found that it can be efficiently and easily produced by an enzymatic reaction in the process, and have completed the present invention based on these findings.

That is, the present invention has the general formula

Embedded image A cyclodextrin represented by the formula (wherein n is 6 or 7) is reacted with cyclodextrinase in the presence of glucose.

[Chemical 4] The present invention provides a method for producing a maltooligosaccharide represented by the formula (n has the same meaning as described above).

Hereinafter, the present invention will be described in detail. The cyclodextrin represented by the general formula (I) which is the starting material in the present invention may be produced by any method, for example, commercially available β-cyclodextrin (n
6) or γ-cyclodextrin (n is 7) (the degree of glucose polymerization is 7 and 8 respectively) and the like are used.

Next, cyclodextrinase (hereinafter referred to as CDase) acting on cyclodextrin represented by the general formula (I) is a hydrolase, and cyclomaltodextrinase (cyclomaltodextrinase).
It is also called e) and belongs to EC 3.2.154. The CDase may be of any type and is not particularly limited and its origin is also not particularly limited. For example, Bacillus magellanes (Ba
Examples thereof include well-known cyclodextrinases having the following physicochemical properties.

Physicochemical properties (a) Action: It has the action of cleaving cyclodextrin to produce maltooligosaccharide derived from the degree of glucose polymerization of cyclodextrin. (B) Substrate specificity: Hydrolyzation rate or affinity for cyclodextrin is higher than that of linear oligosaccharides having the same degree of glucose polymerization as polysaccharides or cyclodextrins. (C) Optimum pH and stable pH range: When β-cyclodextrin is used as a substrate, it has an optimum pH in the vicinity of pH 8.0 and has a stable pH range of 5.5 to 9.5. (D) Optimum temperature of action: Has a proper temperature of action around 40 ° C. (E) Deactivation: It has a property of being almost deactivated by treatment at a temperature of 50 ° C. or higher for 15 minutes.

(F) Inhibition and activation: Hg ²⁺ , Cu ²⁺ ,
90% or more inhibition by Zn ²⁺ , Ni ²⁺ and Fe ²⁺ ,
It has the property of being activated by Ca ²⁺ and Mg ²⁺ by 10 to 30%. (G) Molecular weight: Molecular weight determined by gel filtration is 144,00
0, the molecular weight by SDS PAGE method was 72,000.
Is. That is, this enzyme is a dimer composed of subunits having a molecular weight of 72,000.

The titer of this enzyme is 2% (w / v).
Concentration of β-cyclodextrin solution 500 μl and 100 mM concentration of phosphate buffer (p
H7.5) (500 μl) was mixed and allowed to react at a temperature of 40 ° C. for an appropriate time, the reaction was stopped by boiling for 10 minutes, and the generated maltoheptaose was quantified by high performance liquid chromatography. Also,
When the amount of enzyme was small, it was determined by measuring the reducing power by the Somogi-Nelson method using glucose as a standard. Regarding the enzyme unit of this enzyme, the amount of enzyme that produces 1 micromol of maltoheptaose per minute was 1 unit.

The enzyme having such a property belongs to, for example, the genus Bacillus, and a microorganism producing the enzyme, for example, Bacillus sphericas.
ricus) E-244 strain [deposited at the Institute of Microbial Science and Technology, Institute of Industrial Science and Technology (currently Institute of Biotechnology, Institute of Biotechnology, Institute of Industrial Science and Technology) as Microtechnology Research Institute No. 2458 (FERM BP-2458)] and the like It is obtained by culturing in a medium and collecting from this culture. The physicochemical properties of the enzyme and its producing bacterium Bacillus sphaericus E-
The mycological properties of the 244 strain (FERM BP-2458) and the method for producing an enzyme using the same are known [Japanese Patent Laid-Open No. 15384/1993, 86701/1993].
Publication, "Appl Microbiol Biote
chnol ", Vol. 39, pp. 714-719 (1
993) etc.].

When the CDase is allowed to act on the cyclodextrin represented by the general formula (I) in the presence of glucose, the transfer activity of the enzyme causes the transfer of glucose only once at an unspecified position of the cyclic sugar chain. A reaction solution of the maltooligosaccharide represented by the general formula (II) is obtained. In addition, OH in this general formula (II) is OH
It indicates that the group is an α-anomer, a β-anomer or a mixture of both. The concentration of the cyclodextrin (starting material) in the enzyme reaction is not particularly limited, but it may be C
It is preferable to adjust the concentration so that the concentration is equal to or higher than the Km value for the substrate of Dase. The amount of glucose to coexist is not particularly limited, and is usually 10% of that of cyclodextrin.
To 100 times the molar amount. Further, the amount of enzyme is not particularly limited, so that the necessary amount may be appropriately added so that the amount of the product is maximized within the reaction time.
It is selected in the range of 0.1 to 10 units per 1 g of cyclodextrin.

The enzyme reaction conditions are not particularly limited as long as they are in the range of action pH and action temperature of CDase, but the reaction is usually carried out under conditions of pH 7.5 to 9.0 and temperature 35 to 45 ° C. A reaction time of 30 minutes to 48 hours is suitable. Furthermore, in this reaction, if necessary, 1,
A water-soluble organic solvent such as 4-dioxane, acetone, N, N-dimethylformamide (DMF), ethanol or dimethylsulfoxide (DMSO) may be added.
In addition, after completion of the enzymatic reaction with CDase, for example, acid treatment using hydrochloric acid, acetic acid, or the like is performed at 75 to 100 ° C. for 30 minutes.
It is preferable to deactivate the enzyme by heat treatment for about 180 minutes.

Next, the desired maltooligosaccharide represented by the general formula (II), that is, maltooctaose or maltononaose, is separated and purified from the obtained reaction solution containing maltooligosaccharide. Is not particularly limited, and a conventional method for separating and purifying maltooligosaccharides can be used. For example,
Precipitation removal method of cyclodextrin of starting material with toluene, activated carbon column chromatography, ODS column chromatography, silica gel column chromatography,
A method of collecting fractions by using thin layer chromatography or the like can be appropriately combined and employed.

The above-mentioned general formula (I
As one of its uses, the maltooligosaccharide represented by I) is itself used as a substrate for measuring α-amylase activity as described above. Furthermore, the maltooligosaccharide has an aromatic group at the 1-position of the reducing terminal glucose of the maltooligosaccharide by a conventional method. A maltooligoside (α-, obtained by introducing a color-forming group such as a 2-chloro-4nitrophenyl group or a p-nitrophenyl group).
Anomer or β-anomer) is particularly suitable as a substrate for measuring α-amylase activity.

[0016]

According to the method of the present invention, maltooctaose or maltononaose is used as a starting material with inexpensive cyclodextrin and glucose as a starting material, and the enzyme reaction is carried out in one step, and the operation is simple, and the yield is unprecedented. It can be mass-produced efficiently.

[0017]

Examples are shown below. The specific optical rotation in each example is a value measured by the D line of sodium at 25 ° C.

Example 1 Production of maltooctaose (1) Preparation of CDase 1% (w / v) β-cyclodextrin, 1% (w / v)
v) peptone, 0.5% (w / v) NaCl and 0.1
100 ml of a liquid medium (using tap water, pH 7.0) consisting of% (w / v) yeast extract was placed in a 500 ml Sakaguchi flask and sterilized at 120 ° C. for 20 minutes.
In addition, Bacillus sphaericus E-244 (FERM
One platinum loop was inoculated from the stored slant of BP-2458), and cultured at 30 ° C. for 1 day with shaking. 50m of this culture
1 ml was inoculated into a 3000 ml mini jar containing 2000 ml of medium prepared by the same medium composition and sterilization conditions as above, and aerated and agitated for 2 days at 30 ° C., 1 vvm and 350 rpm. The cells were separated from the culture solution by centrifugation at 8,000 rpm for 20 minutes, and 500 ml of a 10 mM phosphate buffer solution (pH 7.0) containing 2% (w / v) Triton X-100.
The microbial cells were suspended and stirred at 25 ° C for 1 day. After the bacterial cell residue was removed from the suspension by centrifugation at 12000 rpm for 20 minutes, the supernatant was dialyzed against 10 mM phosphate buffer (pH 7.0) for 16 hours. The dialysate obtained was centrifuged at 12000 rpm for 20 minutes to remove insoluble materials, and the supernatant was used as a crude enzyme solution (1).

Then, about 500 ml of this crude enzyme solution (1)
(Total activity 200 units, protein amount 2083 mg, specific activity 0.1, pH 7.0) in 10 mM phosphate buffer (pH
It was applied to a DEAE sepharose packed column (φ34 × 170 mm) equilibrated with 7.0) to adsorb the enzyme, and then elution was performed with a gradient gradient of 0 to 0.5 M NaCl. The active fractions thus obtained were collected to obtain 105 ml of crude enzyme solution (2) (total activity 145 units, specific activity 0.58, yield 72.5%). Then, 20 ml of this crude enzyme solution (2) (total activity 31 units, protein amount 29 mg) was added to 100 m containing 1M sodium sulfate.
Ether 5 equilibrated with M phosphate buffer (pH 7.0)
After applying to a PW packed column (φ21.5 × 150 mm) to adsorb the enzyme, elution was performed with a gradient of 1M to 0 sodium sulfate. 50 ml of the crude enzyme solution (3) was collected by collecting the active fractions thus obtained.
(Total activity 72 units, specific activity 2.93, yield 36%) was obtained.

(2) Production of maltooctaose Commercial β-cyclodextrin [Wako Pure Chemical Industries, Ltd.]
Manufactured] 100 g (88.1 mmol) and commercially available glucose [manufactured by Wako Pure Chemical Industries, Ltd.] 666 g (3.7 mol)
Was dissolved in 1 l of 10 mM phosphate buffer (pH 7.5). 125 ml (total activity 180 units) of the crude enzyme solution (3) of CDase obtained by repeating the above (1) was added thereto,
After reacting at 40 ° C. for 6 hours while stirring, the enzyme was inactivated by boiling for 30 minutes in a boiling water bath. Subsequently, the reaction solution was cooled to room temperature, 20 ml of toluene was added, and the mixture was stirred at room temperature for 5 hours, and then the precipitate was radiolyzed (# 10).
0) and a membrane filter (0.45)
(μm), and concentrated under reduced pressure to 300 ml. The obtained concentrated liquid is subjected to activated carbon column chromatography for purification, and an ethanol-water mixed liquid (volume ratio 0% → 3
(0% gradient), and the 25-30% ethanol-eluted fraction was concentrated to dryness and maltooctaose 45.0 g
(34.2 mmol, yield 38.8%) was obtained.

Melting point (° C): 223 to 225 Infrared absorption spectrum (cm ^-1 ): 3280, 2930,
1650, 1420, 1360, 1150, 1080,
1020 Nuclear magnetic resonance spectrum (200 MHz) ppm (D
₂ O): 3.28 to 4.01 (48H, m), 4.65.
(Ca.0.5H, d, J = 7.8Hz), 5.23
(Ca.0.5H, d, J = 3.4Hz), 5.35
(7H, d, J = 3.9Hz) High performance liquid chromatography [TSKge manufactured by Tosoh Corporation]
l Amide-80 column (4.6 mm ID x 250
mm), RI detection, eluent: acetonitrile / water = 3:
2 (v / v), flow rate: 1.0 ml / min]: t _R = 1
8.0 min Specific rotation [α]: (c = 1.00, H ₂ O); +180
° Elemental analysis value: As C ₄₈ H ₈₂ O ₄₁

Example 2 Production of Maltononaose A commercially available γ-cyclodextrin [Wako Pure Chemical Industries, Ltd.]
20 g (15.4 mmol) of commercially available glucose and 111 g (616 mol) of commercially available glucose [manufactured by Wako Pure Chemical Industries, Ltd.] were dissolved in 0.2 l of 10 mM phosphate buffer (pH 7.5). CDa obtained therein in the same manner as in Example 1 (1) above
After adding 25 ml of the crude enzyme solution (3) of se (total activity 36 units) and stirring the mixture for 6 hours at 40 ° C., the mixture was boiled in a boiling water bath for 30 minutes to inactivate the enzyme. Then, the reaction solution was cooled to room temperature, filtered with radiolite (# 100), and further filtered with a membrane filter (0.45 μm).
After filtration by, it was concentrated under reduced pressure to 50 ml. The obtained concentrated liquid was subjected to activated carbon column chromatography for purification, and an ethanol-water mixed liquid (volume ratio 0% → 45
% Gradient) and the elution fraction of 40-45% ethanol was concentrated to dryness to obtain about 10 g of a mixture of maltononaose and γ-cyclodextrin. Then, the mixture is subjected to ODS column chromatography for purification,
It was eluted with a water-acetonitrile mixture (volume ratio 2%), concentrated to dryness, and maltononaose 5.8 g (3.92 mmo).
1, yield 25.4%) was obtained.

Melting point (° C): 231-235 Infrared absorption spectrum (cm ^-1 ): 3300, 2930,
1650, 1420, 1360, 1150, 1080,
1020 Nuclear magnetic resonance spectrum (200 MHz) ppm (D
₂ O): 3.28 to 4.01 (54H, m), 4.65
(Ca.0.5H, d, J = 8.1Hz), 5.23
(Ca.0.5H, d, J = 3.9Hz), 5.35
(7H, d, J = 3.9Hz) High performance liquid chromatography [TSKge manufactured by Tosoh Corporation]
l Amide-80 column (4.6 mm ID x 250
mm), RI detection, eluent: acetonitrile / water = 3:
2 (v / v), flow rate: 1.0 ml / min]: t _R = 2
1.0 min Specific rotation [α]: (c = 1.00, H ₂ O); +181
° Elemental analysis value: As C ₅₄ H ₉₂ O ₄₆

Claims (1)

[Claims] 1. A compound of the general formula The cyclodextrin represented by the formula (n is 6 or 7) is reacted with cyclodextrinase in the presence of glucose. A method for producing a maltooligosaccharide represented by the formula (n has the same meaning as described above).