JPH0387193A - Production of maltooligosaccharide - Google Patents

Abstract

PURPOSE:To obtain the subject compound of high purity useful as medicines and foodstuffs in a high yield by bringing a new cyclodextrinase having specified physicochemical properties and maltooligosaccharide synthetase into contact with cyclodextrin. CONSTITUTION:With cyolodextrin, a cyclodextrinase obtained from Bacillus sphericus E-244 (FERM EP-2458) and having physicochemical properties such as an activity of production of oligosaccharides by cleavage of cyolodextrin, higher hydrolysis rate and affinity thereto than those to a straight chain oligoeaccharido, about 8.0 optimum pH (stable pH is 5.5-9.5) and about 40 deg.C optimum reaction temperature in case of beta-cyclodextrin substrate, deactivation by heating at 50 deg.C for 15min, >=90% inhibition by Hg<2+>, Cu<2+>, etc., 10-30% activation by Ca<2+> or Mg<2+>, 144000 molecular weight by the gel filtration method and 72000 molecular weight by the SDS PAGE method and maltooligosaccaride synthetase are brought into contact at the same time or in order. The resultant reaction mixture in fractionated using an active carbon column, etc., thus obtaining the objective compound.

Description

[Detailed description of the invention] [Industrial application fields] The present invention relates to a method for producing maltooligosaccharides.

[Prior art] Cyclodextriase [cyclomaltodextrinase]
There are very few reports on Bacillus macerans (also known as E, C, 3゜2.1.54).
produced by Bacillus macerans [
Biochemistry,
7, pp. 121-124 1968], Bacillus coagulans
[Journal of Agricultural Biological Chemistry]
ol, Chem,), Volume 47, Nos. 1441-144
7, 1983] are only known.

Conventionally, a known method for producing malto-oligosaccharides is, for example, a method in which they are produced from starch or the like by the action of amylase, which produces specific oligosaccharides from glucose to maltohexaose. [Arch, Biochem Biophys, 1st
55, pp. 290-298, 1973] On the other hand,
A method for producing malto-oligosaccharides of maltohexaose or higher by using cyclodextrin as a raw material and cleaving cyclodextrin by an acid hydrolysis method (Japanese Patent Application Laid-open No. 1983-1690), or cyclodextrin produced by Bacillus macerans, etc. Glucanotransferase (E, C, 2, 4, 1, 19)
A method is known for producing oligosaccharides greater than maltoheptaose by catalyzing the coupling reaction of cyclodextrin, monosaccharides, and oligosaccharides using [Methods in Enzymology
s in Enzymology), Volume 5, No. 148
-155 pages, 1962] Maltooligo panicles are not only in increasing demand as a substrate for measuring serum amylase, but are also expected to be widely applicable to medicines and foods as nutrients, excipients, bulking agents, etc. has been done.

[Problem to be solved by the invention] However, amylase that specifically produces maltoheptaose salts and malto-oligosaccharides from starch is still unknown, and efficient production using an enzymatic method using starch as a raw material is not yet known. The reality is that the law has not yet been established.

Furthermore, in the case of producing oligosaccharides of maltohexaose or higher using the above-mentioned cyclodextrin as a raw material, the method using acid hydrolysis has disadvantages such as a large amount of by-products and a drastic decrease in the yield of the desired decomposition product. However, in the enzymatic method using cyclodextrin glucanotransferase, as the reaction progresses, the product itself decomposes or the accumulation of by-products due to the coupling reaction between the product and cyclodextrin increases, so the production of cyclodextrin increases. It is necessary to keep the reaction rate of the product low, and there are problems such as a large amount of unreacted cyclodextrin remaining in the reaction solution, making subsequent purification difficult.

[Means for Solving the Problems] The present inventors have conducted extensive studies to solve the above-mentioned difficulties with the aim of rapidly and efficiently obtaining highly pure maltooligosaccharides, and as a result, have developed a novel cyclodextrease. They found that when cyclodextrin is brought into contact with a cyclodextrin, malto-oligosaccharides corresponding to the degree of glucose polymerization of the cyclodextrin are accumulated in high yields, and a patent application has been filed.

− (Specification of Japanese Patent Application No. 1-152257).

Therefore, the present inventors conducted various studies to quickly and efficiently obtain maltooligosaccharides with a lower degree of polymerization using cyclodextrin as a starting material. Or, if the contact is applied sequentially,
First, a maltooligosaccharide corresponding to the degree of glucose polymerization of the cyclodextrin is generated, and then, by the action of a maltooligosaccharide-producing enzyme, a maltooligosaccharide with a lower degree of glucose polymerization than the maltooligosaccharide, such as maltohexaose, maltopentaose, maltotetra, is produced. Obtained the knowledge that malto-oligosaccharides such as ose can be obtained in high yield,
The present invention has been completed.

That is, the present invention is a method for producing maltooligosaccharides, which is characterized by contacting cyclodextrin with a novel cyclodextrinase and a maltooligosaccharide-forming enzyme having the following physical and chemical properties simultaneously or sequentially.

6- ■ Action - Cleavage of cyclodextrin to produce oricosaccharide derived from the degree of glucose polymerization of cyclodextrin.

■Substrate specificity: The rate of water decomposition or affinity for cyclodextrin is greater than that of polysaccharides or linear oligonucleotides with the same degree of glucose polymerization as cyclodextrin.

(2) Optimal pH and stable pH range: When β-cyclodextrin is used as a substrate, the optimum pH is around 80, and the stable pH range is 55-95.

■Suitable temperature range for action: It has an optimal temperature of action around 40°C.

■Conditions for inactivation due to temperature, etc.: It is almost inactivated by treatment at 50°C or higher for 15 minutes.

■Inhibition and activation: 1 g 2 + , (u 2 + , l n 2
+, Ni2+ and Fe are inhibited by more than 90%, and Ca2+ and Mg2+ are activated by 10-30%.

■Molecular weight Nigel filtration method Teha 144. Ooote available, SDS PAG
According to E method, it is 72,000.

The present invention will be explained in detail below.

First, the physicochemical properties of the new cyclodextriase will be described.

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

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

Table (3) Optimum pH and Stable pH Range: The optimal pH is as shown in FIG. 1, and is approximately pH 8.0 when 1% β-cyclodextrin is used as a substrate. The stable pH range is as shown in FIG. 2, and was determined by measuring the residual activity after treatment at each pH for 24 hours at a temperature of 25°C. As is clear from FIG. 2, the stable pH range is 55 to 9.5.

(4) Dodecvalent measurement method: 2% β-cyclodextrin solution at 500μ℃ and 100mM phosphate buffer (pH 7) containing an appropriate amount of this enzyme.
, 5) After mixing 500 μk and reacting at a temperature of 40°C for an appropriate time, the reaction was stopped by boiling for 10 minutes, and the produced maltoheptaose was quantified by high performance liquid chromatography (hereinafter abbreviated as HPLC). did. In addition, when the amount of enzyme was small, the reducing power was determined by the Somogyi-Nelson method using glucose as the standard.

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

(5) Range of suitable temperature for action As shown in Figure 3, this enzyme has a suitable temperature for action near 40°C.

(6) Conditions for temperature-induced inactivation As shown in Figure 4, this enzyme was stable up to 45°C when treated in 100 mM phosphate buffer (pH 7.5) for 15 minutes. It was deactivated at temperatures above 50°C.

(7) Inhibition and activation: The results of the study on the effect of metal ions on the activity of this enzyme were
Shown in the table. As is clear from Table 3, this enzyme supports divalent metal ions such as Hg2+, Cu2+Zn'', and Ni
It was inhibited by more than 90% by 2+ and Fe2+, and activated by about 10-30% by Ca2+ and Mg2+.

No. table (8) Purification method: As described in Example 1.

3 (9) Molecular weight: It is 72,000 according to the SDS PAGE method and 144,000 according to the gel filtration method, so this enzyme is a dimer consisting of subunits with a molecular weight of 72,000.

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

4. As detailed above, this enzyme is a completely new enzyme in that its properties are different from conventionally known cyclodextriases, and in particular it acts best on cyclodextrins.

Next, the method for producing this enzyme will be described.

The microorganism that produces this enzyme belongs to the genus Bacillus,
Any substance that produces this enzyme may be used, for example, Bacillus sphaericus (Bacillus 5
phaericus) E-244 strain. Bacillus sphaericus E-244 strain is a wild strain obtained from soil. The mycological properties of this strain are shown below.

◎Mycological properties of Bacillus sphaericus E-244 strain (a) Morphology ■ Cell shape and size ■ Presence or absence of cell pleomorphism ■ Presence or absence of motility 06-0.8 X 1.6-4.0 microns It is a bacillus.

unacceptable Has periflagella and is motile can be.

■Presence or absence of spores Yes.

Sporangia Swelling size 0.8-0.9 x 1.1
~12 micron shape, oval position, sub-end ■Gram staining positive ■acid-fast negative (b) Growth status in each medium■Meat juice agar plate culture Forms colorless, diffusive colonies. The settlement is smooth with smooth edges. No pigment production is observed.

■ Flesh agar slant culture The fungal moss is smooth with smooth edges.

Pigment production is unacceptable.

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

■Meat juice gelatin puncture culture ■ Ridmus milk physiological properties Nitrate reduction Denitrification reaction MR test ■P test Generation of indole Production of hydrogen sulfide Hydrolysis of starch Use of citric acid Utilization of inorganic nitrogen sources production of pigments Grows only on the top of the medium, No liquefaction is observed.

No coagulation was observed, acid, Alkali production was also observed. I can't.

No refunds.

none.

negative negative Not generated.

Not generated.

Do not disassemble.

Not used.

Not used.

Not generated.

8 ■ Urease negative ■ Oxidase positive ■ Catalase positive ■ Growth range Temperature 13-38°C pH 6-105 [Phase] Attitude toward oxygen Aerobic [phase] O-F test Negative (no acid production recognized) ■ Attitude toward sugars L-arabinose, D-xylose, D-glucose,
Neither acid production nor gas production from D-mannose, D-fructose, D-galactose, maltose, sucrose, lactose, trehalose, D-sorbitol, D-mannite, inosit, glycerin, and starch is observed.

(d) Deamination reaction of phenylalanine The 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 acid or gas generation from sugar was observed, the pH of the VP broth was 7.0 or higher, and deamination of phenylalanine was observed, Purge Aids Manual of Systematic Bacteriology, Vol. 2, 1984, it was identified as a bacterium belonging to the species Sphaericus of the genus Bacillus.

In addition, Bacillus sphaericus E-244 was submitted to the Institute of Microbial Technology, Agency of Industrial Science and Technology, as part of the Microbiological Research Institute No. 2458 (F
ERM BP-2458).

Cultivation of the bacterial strain is in principle the same as the method used for aerobic cultivation of general microorganisms, but usually a shaking culture method using a liquid medium or an aerated agitation culture method is used. The medium used includes a suitable nitrogen source, carbon source, vitamins, minerals, etc., and cyclodextrin, which is an inducing substrate for this enzyme. The pH is the pH at which this bacterium grows.
Any value in the H range may be used, but a range of 6 to 8 is usually preferred.

As for the culture conditions, for example, shaking culture or aerated 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 enzyme collection process shown below. To obtain the enzyme from the above culture, the bacteria are collected by centrifugation or membrane concentration, and then the cells are crushed by ultrasonication or surfactant treatment, and the bacterial residue is removed by centrifugation to obtain the crude enzyme. Get the liquid. A purified product of the present enzyme is obtained by subjecting the crude enzyme solution to appropriate combinations of column chromatography such as ion exchange chromatography, hydrophobic chromatography, and gel filtration.

Next, a method for producing maltooligosaccharide will be described.

Cyclodextrin is contacted with the above-mentioned cyclodextriase and maltooligosaccharide-forming enzyme simultaneously or sequentially, and malto-oligos such as maltohexaose, maltopentaose, maltotetraose, etc. are extracted from the reaction solution according to the maltooligosaccharide-forming enzyme. We get sugar.

Any cyclodextrin may be used, and preferred examples include β-cyclodextrin and α-cyclodextrin.

Regarding the substrate concentration of cyclodextrin, e.g.
The concentration is preferably equal to or higher than the Km value for the substrate of the above-mentioned cyclodextriase.

Examples of maltooligosaccharide-producing enzymes include known enzymes such as maltotetraose-producing enzymes, maltopentaose-producing enzymes, and maltohexaose-producing enzymes, although they vary depending on the target oligosaccharide.

The reaction conditions for contacting cyclodextrin with cyclodextrinase and maltooligosaccharide-forming enzyme simultaneously or sequentially may be any conditions as long as they are within the action pH and temperature range of cyclodextrinase and maltooligosaccharide-forming enzyme, for example, pH7.0～
8.0 and a temperature of 35-45°C is preferred. Furthermore, if necessary, it is also possible to add an organic solvent or the like to the reaction product. The reaction time varies depending on the stability of the reaction product, but is preferably about 30 minutes to 48 hours. The amount of enzyme may be any amount, and it is sufficient to adjust the amount of enzyme so that the maximum amount of the product within the reaction time is 2-, and add the required amount as appropriate. It is desirable to stop the reaction by, for example, acid or heat treatment when the product reaches its maximum level.

Next, the desired malto-oligosaccharide is obtained from the malto-oligosaccharide-containing reaction solution obtained in duplicate, but any conventional oligosaccharide separation method may be used.
In the case of this production method, a highly purified maltooligosaccharide solution can be easily obtained by removing unreacted cyclodextrin from the reaction solution and further fractionating it using an activated carbon column or the like if necessary. As a method for removing unreacted cyclodextrin from the reaction solution, it can be separated and removed by, for example, known methods such as cooling treatment, organic solvent addition treatment, cyclodextrin adsorption column treatment, and the like.

[Effects of the Invention] According to the present invention, maltooligosaccharide having a chain length derived from a maltooligosaccharide-forming enzyme can be efficiently produced from cyclodextrin by contacting cyclodextrinase and a maltooligosaccharide-forming enzyme simultaneously or sequentially. This is extremely meaningful industrially.

[Example] The present invention will be explained in more detail below by giving examples.

Example 1 1% β-cyclodextrin, 1% peptone, 0.5%
Liquid medium consisting of NaCl and 0.1% yeast extract (tap water used, pH 7.0) 100 mR to 500 m
The mixture was placed in a 1-volume Slope flask and sterilized at 120°C for 20 minutes. In addition, Bacillus sphaericus E-2
One platinum loop was inoculated from 44 strain (FERM BP-2458) preserved slant and cultured with shaking at 30°C for 1 day. 50 ml of the main culture solution was inoculated into a 3°C mini-jar containing 22 media prepared with the same medium composition and sterilization conditions as above, and aerated for 2 days at 30°C, 1 vvm, 350 r, p, m. Perform agitation culture, and after completion of culture, 8.00 Or, p, m from the culture solution.

The bacterial cells were separated by centrifugation for 20 minutes, and 2%
Suspend the bacterial cells in 500 rnu of 10mM phosphate buffer (pH 7.0) containing Triton x-ioo.
The mixture was stirred at 5°C for 1 day. From the suspension 12.00O
After removing bacterial cell residue by centrifugation at R, P, and M for 20 minutes, the supernatant was diluted with 10 mM phosphate buffer (pH
70) for 16 hours. 12.00 for dialysate
The mixture was centrifuged at Or, p, m for 20 minutes to remove insoluble matter, and the supernatant was used as a crude enzyme solution (1).

Next, approximately 500 mj of this crude enzyme solution (1) (200 units in total, specific activity 0.1, pH 7.0) was placed in a DEAE Sepharose-packed column (φ34 170 mm) to adsorb this enzyme, then 0 to 0.5 M NaCl
Elution was performed using a gradient gradient of This DEA
The elution pattern of E-Sepharose column chromatography is shown in FIG. Collect the active fraction and make crude enzyme solution (2) 105 mf! (145 total units, specific activity 0.58, yield 725%) was obtained.

Next, this crude enzyme solution (2) was equilibrated with 1M sodium sulfate-containing 100 mM phosphate buffer (pH 7.0) in an ether 5 PW packed column (φ215 x 150
After adsorption of cyclodextlyase, elution was performed using a gradient gradient from IM to OM sodium sulfate. This elution pattern is shown in FIG. Collect active fractions and add crude enzyme solution (3) 50 m
fl (total linkage 72 units, specific activity 2.93, yield 36
%) was obtained.

Next, this crude enzyme solution (3) was ultraconcentrated to a concentration of 1.5 using a collodion bag, and then 0.2 ml was added to TS.
Subjected to gel filtration using K gel G3000SW,
100 mM phosphate buffer containing 0.2 M NaCl (
pH 7.0). This elution pattern was
As shown in the figure. The active fractions were collected to obtain 1.4 mR of purified cyclodextlyase solution (22 units in total, protein amount 0.24 mg, specific activity 9.17, yield 1%).

Cyclodextrinase was single on SDS PAGE. (Figure 8) Example 2 10 mg of β-cyclodextrin was added to 1 ml of 100 mM phosphate buffer (pH 7.0)! 0.043 unit of the crude enzyme solution of cyclodextrease obtained in Example 1 and Japanese Patent Publication No. 50-25552,
Approximately 1,000 units of the maltopentaose-generating enzyme prepared by the method described in Example 1 was added, and the reaction was carried out at 40° C. for 16 hours to obtain a reaction solution. The reaction was stopped by adding hydrochloric acid to the reaction solution to bring the pH to about 20, and after neutralization with a sodium hydroxide solution, the solution was passed through an 0DS (octadecylated silica gel) column to remove unreacted β-cyclodextrin. was adsorbed to obtain a permeable fraction. The flow-through fraction was analyzed by HPLC using a TSK gel Am1de 80 column (manufactured by Tosoh Corporation, packed column for partition/adsorption chromatography), and the results are shown in FIG. The proportion of maltopentaose in crude malto-oligosaccharide is approximately 45%
Met.

Example 3 100mg of β-cyclodextrin was added to 10mM phosphate buffer (pH 7.0) in 10mf! To the solution was added 0.43 units of the crude enzyme solution of cyclodextrease obtained in the example process, the reaction was carried out at 40°C for 2 hours, and then the reaction was stopped by boiling for 10 minutes. . Furthermore, about 1,000 units of maltopentaose-generating enzyme prepared by the method described in Example 1 of Japanese Patent Publication No. 5025552 was added to this reaction solution, and the reaction was carried out at 40° C. for 30 minutes, followed by boiling for 10 minutes. The reaction was stopped. This reaction solution was passed through an ODS (octadecylated silica gel) column to adsorb unreacted β-cyclodextrin, and a passed-through fraction was obtained. The flow-through fraction was transferred to TSK gelA.
The results of HPLC analysis using a mide 80 column (manufactured by Tosoh Corporation, packed column for partition/adsorption chromatography) are shown in FIG. The proportion of maltopentaose in the crude malto-oligosaccharide was approximately 45%.

[Brief explanation of drawings]

FIG. 1 is a diagram showing the optimum pH of the novel cyclodextriase, FIG. 2 is a diagram showing the stable pH of the enzyme, and FIG. 3 is a diagram showing the operating temperature of the enzyme. , FIG. 4 is a diagram showing the conditions for temperature-dependent inactivation of the enzyme, FIG. 5 is a diagram showing the results of column chromatography of the enzyme using DEAE Sepharose, and FIG. Enzyme TSK get HP by ether 5PW
FIG. 7 shows the results of LC, and FIG. 7 shows the TSK of the enzyme.
FIG. 8 is a diagram showing the results of HPLC using gelG3000SW, FIG. 8 is a diagram showing the results of SDS PAGE of the enzyme, and FIGS. 9 and 10 are diagrams showing the results of SDS PAGE of the enzyme. FIGS. HP
This is a chart pattern analyzed by LC.

Claims (1)

[Scope of Claims] A method for producing maltooligosaccharides, which comprises contacting cyclodextrin with a novel cyclodextriase and a maltooligosaccharide-forming enzyme having the following physical and chemical properties simultaneously or sequentially. [1] Action: Cleaves cyclodextrin to generate oligosaccharides derived from the degree of glucose polymerization of cyclodextrin. [2] Substrate specificity: The water dissolution rate or affinity for cyclodextrin is greater than that of a polysaccharide or a linear oligosaccharide with the same degree of glucose polymerization as cyclodextrin. [3] Optimal pH and stable pH range: When β-cyclodextrin is used as a substrate, the optimal pH is around 8.0, and the stable pH range is 5.5 to 9.5.
It is. [4] Range of suitable temperature for action: The suitable temperature for action is around 40°C. [5] Conditions for deactivation due to temperature, etc.: Almost all deactivation occurs by treatment at 50° C. or higher for 15 minutes. [6] Inhibition and activation: Hg^2^+, Cu^2^+, Zn^2^+, Ni^2
It was inhibited by more than 90% by ^+ and Fe^2^+, and Ca^2
It is activated by 10-30% by ^+ and Mg^2^+. [7] Molecular weight: 144,000 by gel filtration method, SDSPAGE
According to the law, it is 72,000.