JP2623507B2 - Method for producing maltooligosaccharides - Google Patents

Description

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

[Prior Art] There are very few reports on cyclodextrinase (also called cyclomalodextrinase EC3.2.1.54), and it is produced by Bacillus macerans [Biochemistry] (Biochemistry), Volume 7, Volume 12
1-124, 1968], Bacillus coagulans (Bacillu
s coagulans) [Journal of Agricultural Biological Chemistry (Ag
ric. Biol. Chem.), Vol. 47, pp. 1441-147, 1983] and the like.

BACKGROUND ART Conventionally, as a method for producing maltooligosaccharides, for example, a method of producing maltooligosaccharides from starch or the like by utilizing an amylase that produces specific oligosaccharides from glucose to maltohexaose is known. [Arc Biochem. Biophys, Vol. 155, 290-298
On the other hand, cyclodextrin is used as a raw material, and a cyclodextrin is cleaved by an acid hydrolysis method to produce a maltooligosaccharide of maltohexaose or higher (JP-A-61-191690), or
Using cyclodextrin glucanotransferase (EC2.4.1.19) produced by Bacillus macerans, etc. to catalyze the coupling reaction of cyclodextrins, monosaccharides and oligosaccharides to produce oligosaccharides of maltoheptaose or higher There are known ways to do this. [Methods in Enzymol
ogy), Vol. 5, pp. 148-155, 1962] Maltooligosaccharides are not only increasing in demand as a substrate for measuring serum amylase, but also widely used as nutrients, excipients, bulking agents and the like. It is expected to be applicable to medicines and foods.

[Problems to be Solved by the Invention] However, an amylase that specifically produces maltooligosaccharides of maltoheptaose or more from starch has not yet been known, and an efficient production method using an enzyme method using starch as a raw material has not been known. The fact is that it has not been established yet.

Furthermore, in the case of the production of oligosaccharides of maltohexaose or more using the above cyclodextrin as a raw material, the method using acid hydrolysis has disadvantages such as a large amount of by-products and a remarkable decrease in the yield of a target decomposed product. There is also an enzymatic method using cyclodextrin glucanotransferase, as the reaction proceeds, the decomposition of the product itself, or
Since the accumulation of by-products due to the coupling reaction between the product and the cyclodextrin increases, the reaction rate of the product with respect to the cyclodextrin must be kept low, and a large amount of unreacted cyclodextrin remains in the reaction solution,
There were problems such as difficulty in subsequent purification.

[Means for Solving the Problems] The present inventors have made intensive studies to solve the above-mentioned difficulties for the purpose of obtaining high-purity maltooligosaccharides quickly and efficiently, and as a result, a novel cyclodextrinase was obtained. Was contacted with cyclodextrin, and it was found that maltooligosaccharides accumulating in a high yield depending on the degree of glucose polymerization of cyclodextrin, etc., and applied for a patent. (Japanese Patent Application No. 1-152257).

Therefore, the present inventors further conducted various studies to obtain a maltooligosaccharide having a lower degree of polymerization quickly and efficiently using cydextrin as a starting material, and as a result, a novel cyclodextrinase and maltooligosaccharide-forming enzyme were added to cyclodextrin. Simultaneously or sequentially, a malto-olidose sugar corresponding to the degree of glucose polymerization of cyclodextrin is produced, and then the degree of glucose polymerization is reduced from the maltooligosaccharide by the action of the maltooligosaccharide-forming enzyme from the maltooligosaccharide. For example, the present inventors have found that maltooligosaccharides such as maltohexaose, maltopentaose, and maltotetraose can be obtained in high yield, and have completed the present invention.

That is, the present invention is a method for producing maltooligosaccharides, which comprises simultaneously or sequentially contacting 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: The optimum pH is around 8.0 when β cyclodextrin and a substrate are used, 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: 144,000 by gel filtration, 72,00 by SDS PAGE
It is 0.

 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, and produces maltooligosaccharides derived from the degree of glucose polymerization of the cyclodextrin.

(2) Substrate specificity: The substrate specificity is as summarized 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) Titer measurement method: 500 μ of a 2% β-cyclodextrin solution and 500 μ of a 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 the enzyme that produces 1 micromol of 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⁺ , Cu ²⁺ , Zn ²⁺ , Ni ²⁺ and Fe ²⁺
It was inhibited by 0% or more, and activated by 10 to 30% by Ca ²⁺ and Mg ²⁺ .

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

(9) Molecular weight: 72,000 by SDS PAGE method, 144, by gel filtration method
000, the enzyme is a dimer composed of 72,000 molecular weight subunits.

Table 4 shows the differences in physicochemical properties between the present 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 for purifying the present enzyme may be any microorganism that belongs to the genus Bacillus and that produces the present enzyme. For example, Bacillus sphaeris (Bacillus sphaeri)
cus) E-244 strain. Bacillus sphaericus E-
The 244 strain is a wild strain obtained from soil. The bacteriological properties of this strain are shown below.

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

 Presence or absence of cell polymorphism Not observed Presence or absence of motility Has periflagellate and motility.

 Spore presence Yes.

 Spore swelling Size 0.8-0.9 × 1.1-1.2 micron Oval position Subvertebral Gram stain Positive Acid-fast Negative (b) Growth condition 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.

Growth of liquid broth liquid culture medium is observed, but no precipitate is observed.

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

 No litmus milk coagulation and no acid or alkali production.

(C) Physiological properties Reduction of nitrate Not reduced.

 No denitrification reaction.

 MR test negative VP test negative Indole generation Not generated.

 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 Growth Range Temperature 13-38 ° C PH 6-10.5 Attitude to Oxygen Aerobic OF Test Negative (no acid production) Attitude to Sugars L-arabinose, D-xylose, D-glucose , D-mannose, D-fructose, D-galactose, malt tongue, sucrose, lactose, trehalose, D-sorbitol, D-mannitol, inosit, glycerin and starch. .

(D) Deamination reaction of phenylalanine positive The 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.
BP-2458).

The cultivation of the strain is basically the same as the method employed in 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, and the like, and cyclodextrin, which is a substrate for induction of the present enzyme, is used. As for pH, this bacterium 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 for the culture conditions, for example, shaking culture or aeration and stirring 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 combining the crude enzyme solution with column chromatography such as ion exchange chromatography, hydrophobic chromatography, and gel filtration.

 Next, a method for producing maltooligosaccharide will be described.

The cyclodextrin is contacted with the cyclodextrinase and the maltooligosaccharide-forming enzyme simultaneously or sequentially, depending on the maltooligosaccharide-forming enzyme from the reaction solution.For example, maltohexaose, maltopentaose, maltotetraose, etc. You get maltooligosaccharides.

Any cyclodextrin may be used, and for example, β cyclodextrin or α cyclodextrin is preferable.

The concentration of the cyclodextrin substrate is preferably, for example, a concentration equal to or higher than the Km value for the cyclodextrinase substrate.

The maltooligosaccharide-forming enzyme varies depending on the target oligosaccharide, and examples thereof include known enzymes such as maltotetraose-forming enzyme, maltopentaose-forming enzyme, and maltohexaose-forming enzyme.

The reaction conditions for simultaneously or sequentially contacting cyclodextrin with cyclodextrinase and maltooligosaccharide-forming enzyme may be any conditions as long as they are within the action pH and temperature range of cyclodextrinase and maltooligosaccharide-forming enzyme. For example, pH 7.0-8.0
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
Depending on the stability of the reaction product, it is preferably 30-48
About an hour. The amount of the enzyme may be any amount, and 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 target maltooligosaccharide is obtained from the maltooligosaccharide-containing reaction solution obtained as described above, and any method may be used as long as it is a normal oligosaccharide separation method.
In the case of this production method, high-purity maltooligosaccharide liquid can be easily obtained by removing unreacted cyclodextrin from the reaction solution and, if necessary, fractionating it using an activated carbon column or the like. As a method for removing the unreacted cyclodextrin from the reaction solution, separation and removal can be performed by a known method such as a cooling treatment, an organic solvent addition treatment, or a cyclodextrin adsorption column treatment.

[Effects of the Invention] According to the present invention, a cyclodextrinase and a maltooligosaccharide-forming enzyme are simultaneously or sequentially brought into contact with each other to efficiently produce a maltooligosaccharide having a chain length derived from the maltooligosaccharide-forming enzyme from cyclodextrin. Can be industrially very 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 consisting of aCl and 0.1% yeast extract (using tap water, pH 7.0) is placed in a 500 ml dissolution flask, and
Sterilization treatment was performed at 20 ° C. for 20 minutes. In addition, Bacillus
One loopful of loops was inoculated from a stock of Sphericus strain E-244 (FERM BP-2458) and cultured with shaking at 30 ° C for 1 day. 50 ml of the main culture solution was inoculated into a 3-volume mini jar containing the medium 2 prepared under the same medium composition and sterilization conditions as above, and aerated and agitated at 30 ° C., 1 vvm, 350 rpm for 2 days. After the culture is completed, remove the culture solution at 8,000 rpm for 20 minutes.
The cells were separated by centrifugation for 5 minutes, suspended in 500 ml of 10 mM phosphate buffer (pH 7.0) containing 2% Triton X-100, and stirred at 25 ° C. for 1 day. 1 from the suspension
After removing bacterial cell residues by centrifugation at 2,000 rpm for 20 minutes, the supernatant was dialyzed against 10 mM phosphate buffer (pH 7.0) for 16 hours. The dialysate is centrifuged at 12,000 rpm for 20 minutes to remove insolubles, and the supernatant is used as a crude enzyme solution (1).
And

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 (φ34 × 170m)
m), the enzyme was adsorbed, and then eluted with a gradient 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 and the crude enzyme solution (3) 50 ml (total activity 72 units, specific activity 2.93, yield 36%)
I got

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 NaCl. 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 A solution of 10 mg of β-cyclodextrin in 1 ml of 100 mM phosphate buffer (pH 7.0) was mixed with 0.043 units of the crude enzyme solution of cyclodextrinase obtained in Example 1 and Add about 1,000 units of maltopentaose-forming enzyme prepared by the method described in Example 1 of JP-B-50-25552,
The reaction was carried out at 0 ° C. for 16 hours to obtain a reaction solution. The reaction was stopped by adding hydrochloric acid to the reaction solution to adjust the pH to about 2.0, and further neutralized with sodium hydroxide solution.
The solution was passed through an S (octadecylated silica gel) column to adsorb unreacted β-cyclodextrin, and a flow-through fraction was obtained. The flow-through fraction was applied to a TSK gel Amide 80 column (Tosoh Corporation,
Partition / adsorption chromatography solution packed column)
The results of analysis by HPLC are shown in FIG. The proportion of maltopentaose in the crude maltooligosaccharide was about 45%.

Example 3 To 100 ml of β cyclodextrin dissolved in 10 ml of 10 mM phosphate buffer (pH 7.0) was added 0.43 unit of the crude enzyme solution of cyclodextrinase obtained in Example 1, and the mixture was added at 40 ° C. After 2 hours, the reaction was stopped by boiling for 10 minutes. Further, this reaction solution was
After adding about 1,000 units of maltopentaose-forming enzyme prepared by the method described in Example 1 of Japanese Patent No. 25552, the reaction was carried out at 40 ° C. for 30 minutes, and the reaction was stopped by boiling for 10 minutes. The reaction solution was passed through an ODS (octadecylated silica gel) column to adsorb unreacted β-cyclodextrin, and a flow-through fraction was obtained. Pass the flow-through fraction to TSK gel Amid
The results of HPLC analysis using an e80 column (Tosoh Corporation, packed column for distribution and adsorption chromatography)
Figure 10 shows. The proportion of maltopentaose in the crude maltooligosaccharide was about 45%.

[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 working temperature of the enzyme. Yes, 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 TSK gel ether 5PW, and FIG. FIG. 8 is a view showing a result of HPLC of the enzyme using TSK gel G3000SW, FIG. 8 is a view showing a result of SDS PAGE of the enzyme, and FIGS. It is a chart pattern analyzed by HPLC using 80.

Claims (1)

(57) [Claims] 1. A method for producing maltooligosaccharides, comprising simultaneously or sequentially contacting 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, 72,000 by SDS PAGE
It is.