JP3100196B2 - Process for producing starch sugar - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a method for producing starch sugar. More specifically, it is obtained by culturing a novel thermophilic bacterium belonging to Bacillus stearothermophilus , and comprises cyclodextrin (hereinafter referred to as C).
D), especially α-CD (cyclohexamylose)
A novel cyclomaltodextrinase (EC, 3,2,1,54), which mainly acts on malt oligosaccharides derived from the degree of polymerization of the CDs, is produced
The present invention relates to a method for producing starch sugar, wherein D is acted on a saccharide containing D alone or a CD and a branched CD.

[0002]

2. Description of the Related Art There are very few reports on cyclomaltodextrinase. For example, Bacillus macerans , Biochem., 7, 121-124, 19
1986), Bacillus coagula
ns, Agric. Biol. Chem., 47, 1441-1447, 1983
Bacillus sphaericus ,
Japanese Patent Application No. 1-152257 , Xanthomonas campestris (Japanese Patent Application Laid-Open No. 3-83582), and Pseudomonas amiloderamosa ( Pesudomonas am
yloderamosa , Biochim. Biophys. Acta, 391, 96-108
P. 1975).

[0003] All of these enzymes degrade CD well, but since they are intracellular enzymes, not only the preparation of the enzyme is complicated, but also the productivity is limited. Furthermore,
Because of its poor temperature stability and low optimum temperature for the enzymatic reaction, there was a problem in using it for the production of starch sugar as an industrial enzyme. Very few attempts have been made to produce starch sugars using these cyclomaltodextrinases.
No. 2257 and 3-87193.
JP-A-3-83582 describes a method for decomposing a CD inclusion compound using cyclomaltodextrinase.

[0004]

In general, when starch sugar is produced using an enzyme, an enzyme having a high temperature suitable for action and high stability at the temperature is desired. This is because enzymatic reactions at low to medium temperatures cannot prevent microbial contamination in the reaction system,
Not only are unwanted by-products produced by side reactions by contaminating microorganisms, but also a large amount of alkaline agent is required for pH adjustment due to a decrease in the pH during the reaction. Large-scale refining equipment is required to remove methane, which is not economical.

[0005] The optimal pH for the action of the enzyme is preferably a weakly acidic pH of about 4 to 6. This is because, when the enzyme reaction is carried out under neutral to alkaline conditions, the resulting starch sugar isomerized, thereby lowering the required purity of the starch sugar. When the enzyme to be used is produced by a microorganism, the enzyme is preferably produced and accumulated outside the cells (in the medium). This is because, in the case of an intracellular enzyme, the productivity of the enzyme is limited, which is not only economically problematic, but also requires complicated steps for extracting and purifying the enzyme from the cells.

On the other hand, in recent years, maltooligosaccharides and derivatives thereof have been receiving attention as bifidobacterial growth factors in human intestine, research reagents, humectants, and the like, and a method for industrially and efficiently producing maltooligosaccharides has been developed. Is desired. However, among maltooligosaccharides, maltohexaose, maltoheptaose, and maltooctaose were difficult to prepare.

[0007] Further, CDs can incorporate various organic compounds as guest compounds to form inclusion compounds and change various physicochemical or physiochemical effects of the guest compounds. Widely used. Among the CDs, particularly, the branched CD has a property that the CD is hardly soluble in water or an organic solvent, but is easily dissolved in water or an organic solvent, and particularly easily soluble in ethanol. Stabilize by clathrate,
Applications such as solubilization in water and organic solvents are expected. However, it is extremely difficult to fractionate a CD from a branched CD, and it is desired to develop a method for efficiently producing a branched CD.

Accordingly, an object of the present invention is to provide a CDM by using cyclomaltodextrinase which has a high optimal temperature for action, excellent temperature stability, a weakly optimal pH for action and is produced extracellularly. It is an object of the present invention to provide a method for producing a starch sugar such as maltooligosaccharide or branched CD efficiently from a single sugar or a sugar containing CD and branched CD.

[0009]

Means for Solving the Problems The present inventors have found that an enzyme produced at a high temperature suitable for action, excellent in temperature stability, weakly acidic at an optimum action, and produced outside the cells, Malto-oligosaccharides having a degree of polymerization equal to or higher than ose and cyclomaltodextrinases having an action of degrading α-CD (cyclohexaamylose) more strongly than other CDs to produce maltooligosaccharides As a result of broadly searching microorganisms that produce Escherichia coli from the natural world, it was found that one strain (KH-17 strain) belonging to Bacillus stearothermophilus isolated from soil strongly produced the enzyme, and completed the present invention. .

That is, the process for producing starch sugar of the present invention allows a novel cyclomaltodextrinase having the following physicochemical properties to act on a saccharide containing cyclodextrin alone or a cyclodextrin and a branched cyclodextrin. It is characterized by the following.

(A) Action The α-1,4-glucopyranoside bond in maltooligosaccharide having a degree of polymerization of maltotriose or higher is hydrolyzed and rearranged to produce a series of maltooligosaccharides. It acts on CDs and produces maltooligosaccharides derived from the degree of polymerization of the CDs as a main component.

(B) Substrate specificity Decomposes maltooligosaccharides and CDs having a degree of polymerization of maltotriose or higher. The rate of hydrolysis of α-CD is greater than other cyclodextrins and linear maltooligosaccharides having the same degree of polymerization. Starch, amylopectin,
Substantially degrades glycogen, panose and isopanose. (C) Optimum pH It works in the range of pH 4 to 8, and the optimum pH is around 5.5. (D) Stable pH range It is stable at pH 6 to 9 when stored at 5 ° C for 24 hours. (E) Appropriate temperature for operation It works in the temperature range of 40 to 75 ° C, and the optimum temperature is around 60 ° C. (F) Inactivation conditions Under storage conditions of 4 ° C. for 24 hours, about 90% is inactivated at pH 4, and completely inactivated at 80 ° C. under conditions of pH 7 and 2 hours. (G) Temperature stability Stable up to 40 ° C. under conditions of pH 7 and 1 hour,
The residual activity at 90 ° C, 60 ° C, 70 ° C and 80 ° C is 90%, 70%, 30% and 13%, respectively. (H) Inhibition and activation Inhibited by divalent cadmium, zinc, mercury, manganese, copper, nickel and pCMB (parachloromercury benzoate). (I) Molecular weight The molecular weight is about 106,000 (gel furnace filtration method). (D) Isoelectric point The isoelectric point is about 4.7 (isoelectric focusing).

[0013] Table 1 shows the differences in physicochemical properties between the enzyme of the present invention and a conventionally known cyclomaltodextrinase.

[0014]

[Table 1]

As is clear from Table 1, the enzyme of the present invention not only acts at a high temperature but also extracellularly (in the medium) as compared with the conventionally known cyclomaltodextrinase.
In that they were generated and accumulated. Since no enzyme having the above properties has been known so far, this cyclomaltodextrinase was determined to be a novel enzyme.

[0016] The cyclomaltodextrinase is obtained from Bacillus stearotherophilus.
rmophilus ), which can be obtained by culturing a microorganism having the ability to produce the enzyme, and collecting the enzyme from the culture.

The microorganism belonging to Bacillus stearothermophilus and producing cyclomaltodextrinase includes Bacillus stearothermophilus No. newly isolated from soil by the present inventors. KH-17
The strain ( Bacillus stearothermophilus No. KH-17) is preferably used. This strain has the following mycological properties.

A. Morphological characteristics (1) Cell shape and size: Bacillus, size (0.6 to
1.0) × (2.5-3.0) μm (micron) (2) Cell polymorphism: None (3) Motility: Motile, with periflagellate (4) Presence of spores: Spores: Swelled slightly Spore shape: elliptical Spore position: slightly terminal (5) Gram stain: positive (6) Acid-fast: negative

B. Growth on various media (1) Gravy agar plate culture Formed white and circular colonies. The center is convex or convex circular. The periphery is all-edge. No pigment production is observed. (2) Gravy agar slant culture Growing in a spread form. (3) Broth liquid culture Growth is weak, slightly turbid and sedimentary, and forms incomplete rings on the surface. (4) Broth gelatin puncture culture Only the surface grows, and no liquefaction of gelatin is observed. (5) Litmus milk No coagulation is observed, and neither acid nor alkali production is observed. (6) No anaerobic growth is observed in the glucose / grass liquid medium. (7) Generation of anaerobic gas from nitrate No gas is generated. (8) Salt broth liquid culture Does not grow in the presence of 3% salt.

C. Physiological properties (1) Nitrate reduction Reduce (2) Denitrification reaction negative (3) VP test negative (4) Indole formation negative (5) Hydrogen sulfide formation negative (6) Starch hydrolysis positive (7) Use of citric acid Not used (8) Use of inorganic nitrogen source Use (9) Pigment not generated (10) Hydrolysis of gelatin and casein Not degraded (11) Urease positive (12) Oxidase negative (13) Catalase negative (14) Attitude to oxygen Aerobic (15) Range of growth Temperature: 30 to 75 ° C (optimal 55 to 60 ° C) pH: 5.5 to 8.0 (optimal pH 6 to 6.5) (16) Lysozyme (17) Resistance to azide Yes (18) Attitude to saccharides D-glucose, L-arabinose, D-xylose,
It produces acid from D-fructose, D-galactose, D-trehalose, maltose, sucrose, lactose, melibiose, raffinose, cellobiose, glycerin, and D-mannitol, and does not produce gas.

This strain is a spore-forming bacillus and has a motility,
It is clear that the bacterium belongs to the genus Bacillus because of positive Gram staining. Furthermore, it has the ability to hydrolyze starch, cannot grow at pH 5.0, and can grow up to 75 ° C., and thus can be grown according to the Burges Manual of Sistimatic Bacteriology, Vol. 2, 1984. It was identified as a bacterium belonging to the species Loserophilus. This Bacillus stearothermophilus KH-17 strain has been deposited with the National Institute of Microbial Industry and Technology of the National Institute of Advanced Industrial Science and Technology as Microorganisms Bacteria No. 12481.

The above-mentioned cyclomaltodextrinase-producing strain is cultured by a shaking culture method using a liquid medium or an aeration stirring culture method, which is generally used for culturing aerobic microorganisms.

The medium may be a medium containing an appropriate nitrogen source, carbon source, vitamin, mineral and the like. Carbon sources include soluble starch, dextrin, powdered candy, CD
, Pullulan, lactose and the like can be used alone or in combination of two or more. Nitrogen sources include organic nitrogen compounds such as corn steep liquor, polypeptone, various amino acids, soybean meal, meat extract, fish extract, yeast extract, etc., and inorganic nitrogen compounds such as various ammonium salts, nitrates, urea They can be used alone or in combination of two or more. In addition, vitamins, minerals, and the like can be appropriately added.

The culturing temperature is usually 30 to 75 ° C, preferably 40 to 70 ° C, more preferably 50 to 55 ° C. Culture pH is usually 5.5 to 8.0, preferably 6.0 to 7.0. The culturing time varies depending on the culturing method, but is about 18 to 100 hours. By this culture method, most of the cyclomaltodextrinase (about 80%) was obtained.
%) Accumulates in the medium (extracellularly) and the rest is localized on the cell surface. After completion of the culture, cyclomaltodextrinase can be obtained by a usual method. One example is shown below.

First, the culture is centrifuged to remove cells. An equal amount of isopanol is added to the obtained supernatant to obtain a precipitate. This precipitate 10 mM acetate buffer (pH
The crude enzyme obtained by dialysis against 6) is subjected to gradient ion exchange chromatography of sodium chloride using DEAE-Toyopearl 650S, and the eluted active fraction is collected and desalted. Next, the active fraction is concentrated with an ultrafiltration membrane having an average molecular weight cut off of 10,000. The concentrated enzyme is subjected to gel filtration using Sephacryl S-300 equilibrated with the same buffer as described above to collect an active fraction, and the active fraction is further purified by a chromatofocusing method to obtain a purified enzyme. . The cyclomaltodextrinase thus obtained has the physicochemical properties as described above.

In the present invention, the activity of cyclomaltodextrinase was measured as follows. 0.5 mL (milliliter) 0.1 M containing 1% α-CD
0.05 mL of an enzyme solution appropriately diluted with water was added to an acetate buffer (pH 5.5), and the mixture was reacted at 50 ° C. for 10 minutes. Then, 1.0 mL of a Somogyi solution was added to stop the reaction. Then, the mixture was boiled in a boiling water bath for 30 minutes, quenched in running water, and 1.0 mL of Nelson solution was added. Water was added to make 10.0 mL, and the reducing sugar generated using 100 μg of glucose treated similarly as a standard was measured at 510 nm. Here, one of the enzymes
The unit was the amount of enzyme that generates reducing sugar corresponding to 1 μmole of glucose from α-CD under the same conditions as above.

In the present invention, the purified enzyme can be used, but it may be used as a crude enzyme such as a culture supernatant or a concentrate thereof, which is more economical.

Next, a method for producing starch sugar using the above enzyme will be described.
A carbohydrate containing D and a branched CD is used. CDs are of the genus Bacillus or Klebsiella
CD-generating enzyme (Cyclomaltodextrin g
lucanotransferase (hereinafter abbreviated as "CGTase")
After acting on starch, it is produced through purification operations such as crystallization, organic solvent precipitation, and various types of chromatography. In addition, branched CD is produced as a by-product when CD is produced by allowing CGTase to act on starch. In addition, a branched mixture of starch such as pullulanase, isoamylase, etc., using a mixture of high concentration maltooligosaccharide and CD as a substrate is used. It is also produced by the reverse synthesis of enzymes.

According to the method of the present invention, for example, a saccharide containing CD or a saccharide containing CD and a branched CD obtained by the above method is used as a substrate, and the above-mentioned cyclomaltodextrin is used as a substrate. The desired starch sugar can be obtained by allowing the enzyme to act. The amount of the enzyme used is changed according to the amount, type, substrate concentration, and reaction time of CD in the reaction solution. The reaction conditions are pH 5
6. The temperature is preferably 55-60 ° C, and the reaction time is 1-50.
Time is preferred.

In the present invention, when the above-mentioned cyclomaltodextrinase is allowed to act on a saccharide containing CD, maltooligosaccharide derived from the degree of polymerization of the CD is produced as a main component. That is, maltohexaose when acting on α-CD, maltoheptaose when acting on β-CD, and maltooctaose as a main component when acting on γ-CD. Generate. Therefore, maltohexaose, maltoheptaose, and maltooctaose, which have conventionally been considered difficult, can be easily prepared.

When the above-mentioned cyclomaltodextrinase is allowed to act on a saccharide containing CD and branched CD, CD is more selectively decomposed, and most of the branched CD remains without being decomposed. For this reason, it becomes easy to fractionate the branched CD, and it can be sufficiently fractionated by a simple and economical method, for example, ultrafiltration membrane treatment, a method using a strongly acidic cation exchange resin, and the like. Can be obtained at

In the present invention, after performing the enzyme reaction as described above to produce the target starch sugar, various treatments can be performed as required. For example, cyclomaltodextrinase is allowed to act on a saccharide containing CD,
When maltooligosaccharides are produced, they can be used as syrups as they are by decolorization with activated carbon and deionization with an ion exchange resin, or as sugar alcohols after reduction treatment. In addition, if necessary, gel filtration chromatography, carbon column chromatography, cation exchange resin column chromatography, or, by using a membrane fractionation method or crystallization method, the target high purity maltooligosaccharides It can be easily prepared.

When cyclomaltodextrinase is allowed to act on a saccharide containing CD and branched CD to selectively decompose CD, for example, ultrafiltration membrane treatment, strong acid cation exchange resin Fractionated CD can be obtained in high yield by fractionation by a method, a method using a resin carrier for gel filtration, or the like. The ultrafiltration membrane has an average molecular weight of 70
It is preferable to use about 0 to 1500, and more preferably about 1000.

[0034]

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

Example 1 Preparation of Crude Enzyme 0.5% dextrin, 1% Shefton F (USA, manufactured by Shefton, USA), 0.1% yeast extract, 0.1%
Sodium glutamate, 0.1% ammonium nitrate, 0.1%
1% K ₂ HPO ₄ , 0.02% MgSO ₄ .7H ₂ O,
50 mg / L CaCl ₂ .2H ₂ O and 10 mg / L
Of MnSO ₄ · 7H ₂ Liquid Medium 1 pH6.3 containing O
After sterilizing 5 L (liter) at 120 ° C. for 30 minutes, the same medium was used in advance at 50 ° C. for 20 hours for 180 hours.
Bacillus stearothermophilus no. KH-17 strain (Microbial Research Laboratories No. 12481)
No.) 1 L of the suspension was inoculated, and 250 rpm, 1 v, v, m
Under the conditions described above, aeration and stirring culture was performed at 50 ° C. for 50 hours.
After completion of the culture, the supernatant liquid from which the cells were removed by centrifugation was passed through an ultrafiltration membrane (Romicon) having an average molecular weight cut-off of 5,000 and concentrated to about 2 L. This concentrated crude enzyme solution had an enzyme activity of 25.5 units per mL.

Example 2 Action on CD 0.5 unit of the concentrated crude enzyme obtained in Example 1 was added to 0.2 mL of 10 mM phosphate buffer (pH 6.0) containing 10 mg of various CDs, After reacting for 24 hours, the enzyme was inactivated by boiling in a boiling water bath for 10 minutes.
Next, the solution was passed through an ODS (octadecylated silica gel) column to adsorb unreacted CD, and a flow-through fraction was obtained. Table 2 shows the results obtained by analyzing the flow-through fraction by HPLC using an MCI-gel CKO4SS column (manufactured by Mitsubishi Kasei Kogyo).

[0037]

[Table 2] (In the table, G1 to G8 represent glucose, maltose, maltotriose, maltotetraose, maltopentaose, maltohexaose, maltoheptaose and maltooctaose, respectively.)

As is evident from Table 2, three CDs were obtained from each CD under the above conditions by the action of cyclomaltodextrinase.
Maltooligosaccharides corresponding to the degree of glucose polymerization of CD were produced in a yield of 0% or more.

Example 3 Action on CD and Branched CD 1.9 g of α-CD, 5.6 g of maltose and 3 mM
-10 mL of a carbohydrate mixture containing calcium chloride (pH
4.5) to 0.05 mL of pullulanase (manufactured by Amano Pharmaceutical Co., Ltd.)
(Trade name DB-250), and reacted at 65 ° C. for 72 hours to produce a branched α-CD.
D19.8%, maltosyl α-CD 10.3%, dimaltosyl α-CD 2.7% and other oligosaccharides 67.2
% Of the carbohydrate mixture was obtained. Then 30%
(W / v) 0.5 g per 1 g of solid content of the saccharide mixture
Table 3 shows the saccharide composition of the saccharide mixture obtained by adding the units of cyclomaltodextrinase prepared in Example 1 and reacting the mixture at pH 5.5 and 60 ° C. for 70 hours.

The sugar composition was Asahi Pack NH ₂ P-5.
The mobile phase was 65% acetonitrile using 0.1 column.
It was used at a flow rate of 8 mL / min, and carbohydrate was measured by high performance liquid chromatography using an RI detector.

[0041]

[Table 3]

As is apparent from Table 3, this cyclomaltodextrinase is obtained from α-C present in the carbohydrate mixture.
Despite hydrolysis of all (99.5%) of D, 78.8% of maltosyl α-CD and 96.3% of dimaltosyl α-CD remained undegraded.

[0043]

As described above, according to the present invention,
By reacting a novel cyclomaltodextrinase on a carbohydrate containing CD, maltooligosaccharides corresponding to the degree of polymerization of CD can be produced. Therefore, maltohexaose, maltoheptaose and maltooctaose can be produced. It can be easily prepared. In addition, by acting a novel cyclomaltodextrinase on a saccharide containing CD and branched CD, CD can be selectively degraded, so that fractionation of branched CD becomes easy,
The branched CD can be industrially efficiently produced. In addition, since this cyclomaltodextrinase has high temperature stability and can act at a high temperature, it is possible to prevent microbial contamination during the reaction during starch sugar production. Further, since this cyclomaltodextrinase is an enzyme produced outside the cells, the preparation of the enzyme is easy and mass production is possible.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification FI (C12N 9/24 C12R 1:07) (C12P 19/14 C12R 1:07) (58) Investigated field (Int.Cl. ⁷ , DB name) C12P 19/00-19/64 C12N 1/00-1/38 C12N 9/00-9/99 BIOSIS (DIALOG) MEDLINE (STN) WPI (DIALOG)

Claims (1)

(57) [Claims] 1. A method for producing a starch sugar, which comprises reacting a novel cyclomaltodextrinase having the following physicochemical properties on a saccharide containing cyclodextrin alone or a cyclodextrin and a branched cyclodextrin. . (A) Action The α-1,4-glucopyranoside bond in maltooligosaccharides having a degree of polymerization of maltotriose or higher is hydrolyzed and rearranged to produce a series of maltooligosaccharides. In addition, it acts on cyclodextrins, and produces maltooligosaccharides derived from the degree of polymerization of the cyclodextrin as a main component. (B) Substrate specificity Decomposes maltooligosaccharides and cyclodextrins having a degree of polymerization of maltotriose or higher. The rate of hydrolysis of α-cyclodextrin is greater than other cyclodextrins and linear maltooligosaccharides having the same degree of polymerization. Substantially degrades starch, amylopectin, glycogen, panose and isopanose. (C) Optimum pH It works in the range of pH 4 to 8, and the optimum pH is around 5.5. (D) Stable pH range When stored at 5 ° C. for 24 hours, it is stable at pH 6 to 9. (E) Appropriate temperature for operation It works in the temperature range of 40 to 75 ° C, and the optimum temperature is around 60 ° C. (F) Inactivation conditions Under storage conditions of 4 ° C. for 24 hours, about 90% is inactivated at pH 4, and completely inactivated at 80 ° C. under conditions of pH 7 and 2 hours. (G) Temperature stability Stable up to 40 ° C. under conditions of pH 7 and 1 hour,
The residual activity at 90 ° C, 60 ° C, 70 ° C and 80 ° C is 90%, 70%, 30% and 13%, respectively. (H) Inhibition and activation Inhibited by divalent cadmium, zinc, mercury, manganese, copper, nickel and pCMB (parachloromercury benzoate). (I) Molecular weight The molecular weight is about 106,000 (gel furnace filtration method). (D) Isoelectric point The isoelectric point is about 4.7 (isoelectric focusing).