JP2559400B2 - Malto-oligosaccharide-forming amylase and method for producing the same - Google Patents

Description

TECHNICAL FIELD The present invention relates to a novel maltooligosaccharide-forming amylase, and more specifically to a maltooligosaccharide-forming amylase that produces maltopentaose and maltohexaose as main components and a method for producing the same. Is.

Malto-oligosaccharide refers to an oligosaccharide produced by hydrolysis of starch, which is a linear sugar having only α-1,4-glucoside bonds and having a degree of polymerization of 2 to 10. The sweetness of malto-oligosaccharide is about 20-25 with a purity of 40-80% assuming sugar as 100. Malto-oligosaccharide syrup has low viscosity, so it is not found in conventional starch sugars with low sweetness and low viscosity. It is expected as a new food improving agent having properties. Further, since it has a high solubility and an excellent moisturizing property, it can be used as a raw material for powdered alcohols and cosmetics, as a tablet raw material, a liquid food for the sick and a calorie supplement sugar and other body reinforcing agents. Also,
High-purity maltooligosaccharides are used as reagents for amylase research and as substrates for measuring α-amylase in human serum and urine in the field of clinical medicine. Malto-oligosaccharides currently used as substrates include maltotetraose (G4), maltopentaose (G5), maltohexaose (G6), maltoheptaose (G7) and the like.

[Conventional technology]

To date, many specific amylases that produce maltooligosaccharides have been reported ([Enzyme Handbook]; supervised by Shiro Akahori, published by Asakura Shoten (Showa 41) and [amylase]; (1986)
Etc.).

As an amylase that produces maltose, β-amylase of higher plant origin has been known since ancient times.
Year Robyt and French et al. Bacillus polymixer (Bacillus) which acts on amylose to produce more than 80% maltose.
I discovered a β-amylase of polymxa origin (J. Robyt
and French. Arch. Biochem. Biophys. 104 338 (1964).
Maltotriose can be easily obtained by degrading pullulan with pullulanase, but in 1975 young people discovered an amylase derived from Streptomyces griseus that acts mainly on starch to produce maltotriose. (Katsuo Wakao, Starch Science 26 175 (19
79)).

In addition to these, Takasaki et al. Recently found a maltotriose-producing amylase originating in the genus Bacillus (Japanese Patent Publication No. 59-17983).

1971 Robyt and A
Pseudomonas pseudom by ckerman
onas stutzeri) (J.Rob)
yt and RJ Ackerman Arch. Biochem. Biophys. 145 105
(1971)).

Also in 1971, Saito was the Bacillus licheniformis (Bacill
It has been discovered that a thermostable α-amylase of us licheniformis) acts on starch to produce mainly maltopentaose (N. Saito Arch. Biochem. Biophys. 155 290 (197).
3)). Furthermore, Kobayashi et al. Yoshiyoshi et al. Recently found an amylase that mainly acts on starch to produce maltopentaose (Shoichi Kobayashi, 1983 Annual Meeting of the Japan Starch Society)
(P301), (Naohiro Yoshigi, 1984 Japan Agricultural Chemistry Conference
P584).

Kainuma et al. 1971 Aerobacter Aerogenes (Aero
It was discovered that partially purified pullulanase derived from bacter aerogenes (Klebsilla pneumoniae) was contaminated with amylase that acts on amylose to produce maltohexaose (K. Kainuma FEBSLett 26 281 (1972)).

An amylase that produces maltohexaose was found by Kennedy et al., Taniguchi et al., And Takasaki et al. (J. Kennedy Starke 31 (1979)), (Taniguchi Hajime, Starch Science 29 107 (1982)), (Takasaki Agric .Biol.Chem.
47 2193 (1983)).

The physicochemical properties of the enzyme of the present invention and the conventionally reported G5 and G6 producing maltooligosaccharide amylase are shown in comparison with Table 1.

As is clear from this table, the enzyme of the present invention acts on starch or starch-based saccharides to produce maltooligosaccharides containing maltopentaose and maltohexaose as the main components. Is
Their physicochemical properties are different.

[Problems to be Solved by the Present Invention]

The object of the present invention is an amylase which acts on starch or starch-based sugars to produce maltooligosaccharides containing maltopentaose and maltohexaose as the main components and a method for producing the same, and a conventional maltooligosaccharide-forming amylase is It is to provide a novel amylase having different physicochemical properties and a method for producing the same.

[Means for solving problems]

As a result of repeated intensive studies to solve the above-mentioned problems, the present inventors newly produced maltopentaose and maltohexaose as main components by culturing microorganisms belonging to the genus Bacillus separated from soil. It was found that a novel maltooligosaccharide-forming amylase can be obtained, and the present invention has been completed.

That is, the physicochemical properties of the maltooligosaccharide-forming amylase of the present invention will be described in detail as follows.

Action and Substrate Specificity It acts on starch or starch-based saccharides to produce mainly maltopentaose (G5) and maltohexaose (G6).

Optimum pH and pH The optimum pH of this enzyme is around 7 (see Fig. 1), and its stable pH is 6 to 10 (see Fig. 3).

Optimum temperature and thermostability The optimum action temperature of this enzyme is 45 ° C to 55 ° C (see Fig. 2). This enzyme is stable up to 50 ° C, but its activity decreases at higher temperatures, and it is almost inactivated at 70 ° C for 30 minutes (see Fig. 4).

Effects of inhibitors, etc. As shown in Table 2, this enzyme is affected by metal ions such as iron, copper and cobalt ions, parachloromercuribenzoate (hereinafter abbreviated as pCMB) and ethylenediamine tetraacetate (hereinafter abbreviated as EDTA). Hindered,
Not inhibited by lithium, magnesium, barium, calcium ions, etc.

Molecular weight The molecular weight of this enzyme was about 58,000 by SDS polyacrylamide gel electrophoresis.

Isoelectric point This enzyme was subjected to electrophoresis in the presence of ampholine and the isoelectric point was measured and found to be around 6.5.

This enzyme mainly produced maltopentaose and maltohexaose when reacted with starch and starch-based sugars. The maltooligosaccharide-forming amylase produced by using maltopentaose and maltohexaose as a main component in the present invention has the above-mentioned properties.

The novel enzyme maltooligosaccharide-forming amylase of the present invention is not limited to the purified enzyme, and may be a crude enzyme solution of a culture of a microorganism, or a standard product during the purification of the enzyme.

It is also possible to support the enzyme or bacterial cell on a water-insoluble carrier and use it in the form of an immobilized enzyme or immobilized bacterial cell.

The enzyme of the present invention acts on starch and starch-based sugars,
Malto-oligosaccharides containing maltopentaose and maltohexaose as the main components are produced, but the reaction conditions when the enzyme acts are preferably a pH of the reaction solution of 6 to 10, particularly 6 to 8, and a reaction temperature of usually 70 ° C or lower. However, 45 to 55 ° C. is preferably selected.

The titer of this enzyme is the unbreakable method (Fuwa.HJBiochem.41
1954).

0.5% dissolved in 0.1M glycine-NaOH buffer (pH8.0)
After adding 1 ml of enzyme solution to 2 ml of soluble starch and reacting at 40 ° C for 5 minutes, 0.2 ml of 0.15N HCL of 2 × 10 ^-4 N iodine
Add 5 ml of the solution to stop the enzymatic reaction, measure the 700 nm absorbance, and calculate from the following formula.

Here, T and To represent the absorbance of the sample and the absorbance of the blank.

Next, a method for producing the novel maltooligosaccharide-forming amylase of the present invention will be described.

The microorganism used in the production method of the present invention is a microorganism belonging to the genus Bacillus and having a novel maltooligosaccharide-forming amylase-producing ability, and specific examples thereof include Bacillus sp. Fungi No. 879
No. 2), the microorganisms usable in the present invention are not limited to the above.

Bacillus sp. # 707 (Ministry of Industrial Science and Technology No. 8792) is a strain newly isolated from the soil by the present inventor, and its mycological properties are as follows.

In addition, the test and classification method of the mycological properties are described in "Aerobic Sporhoming Bacteria"["Aerobic Sp
oreforming Bacteria "(United State Department of A
griculture, Nov.1952 by NRSmith, REGordon & F.
E.Clark)] and "The Burgers Manual of
Determinative Bacteriology "['Bergey's M
anual of Determinative Bacteriology, 1957)].

(Mycological properties of Bacillus sp. # 707) a. Form 1 15g starch, 10g polypeptone, 5 yeast extract
_{g, K 2 HPO 4 1g,} MgSO 4 · 7H 2 O0.2g, sodium carbonate 10 g,
Table 3 shows the observed morphology on a plate agar medium having a composition (pH 10.0) containing 15 g of agar (however, sodium carbonate is heat-sterilized separately from other components and then added to the medium).

B. Growth state in each medium Table 4 shows the growth state of # 707 bacteria in each medium.

In addition, since there was almost no growth in the medium pH 7.0 and no change was observed, 1% sodium carbonate was added to each medium.
% To pH 10.0.

C. Physiological Properties Regarding the # 707 bacterium, the results obtained by adding 1% of sodium carbonate to the physiological test medium are shown in Tables 5-1 and 5-2.
Shown in

From the mycological properties of the above-mentioned microorganisms, a comparison was made with known bacterial species belonging to the genus Bacillus according to the classification method of the above-mentioned literature, and it was found that Bacillus sp.
No.), the endospores are oval in shape, the endospores are located at the ends, and no spore-induced vegetative cell expansion is observed, and Bacillus stearothermophilus (Bacillus s)
tearothermophilus) is appropriate. However, as shown in Table 6, Bacillus sp. # 707 is positive in the Phosphorus proscalael test (VP test) in physiological properties, whereas Bacillus stearothermophilus (B. stearothermophilus) Is negative, and the growth pH range of the # 707 strain is 9-12, whereas the Bacillus stearothermophilus (B. stearoth
ermophilus) differ in that they are in a weakly acidic neutral range.

From the above results, it is clear that Bacillus sp. # 707 (Microtechnology Research Institute No. 8792) is a microorganism belonging to the genus Bacillus because it is an aerobic spore bacterium,
It was concluded that it is appropriate to identify it as a new bacterial species of aerophilic bacteria, clearly distinguishing it from known bacterial species. The method for culturing a microorganism according to the present invention may be a liquid medium or a solid medium, but a liquid medium is preferable and starch or starch-based sugar is required as a carbon source. Broth extract as a nitrogen source,
Soybean flour, corn steep liquor, corn gluten, etc. can also be used.

An example of a liquid medium is shown. Medium composition per g Starch 15 (pH 10.0) Polypeptone 10 Yeast extract 5 K ₂ HPO ₄ 1 MgSO ₄ / 7H ₂ O 0.2 Sodium carbonate 10 (However, sodium carbonate should be sterilized separately from other ingredients before being added to the medium. Any of static culture, shaking culture and aeration and agitation culture is possible, but shaking culture or aeration and agitation culture is preferable. The medium pH is 9 to 12, preferably pH 10, the culture temperature is 10 to 45 ° C, preferably 37 to 40 ° C, and the microorganism secretes and produces a large amount of amylase in the culture medium in the culture time of 1 to 4 days. To do.

Therefore, after the culture solution is filtered or centrifuged to remove bacteria, amylase is separated and collected from the culture supernatant. This amylase can be collected by a known separation and purification means. Examples of the method for separating and purifying amylase include salting out with a salt such as ammonium sulfate, fractional precipitation with a hydrophilic organic solvent such as alcohol or acetone, adsorption with an ion exchange resin, and molecular sieve chromatography.

Next, the present invention will be described with reference to examples, but the present invention is not limited to these examples.

〔Example〕

Example 1 Bacillus sp. # 707 bacterium (Microtechnology Research Institute No. 8792) was used as starch 1.5%, polypeptone 1.0%, yeast extract 0.5%, dipotassium phosphate 0.1%, magnesium sulfate 0.02%, carbonic acid. Sodium 1.0% (However, sodium carbonate is sterilized separately from other ingredients and then added to the medium)
The medium 5 containing was put in a 10-volume jar fermenter, sterilized at 120 ° C. for 20 minutes, inoculated, and cultured at 37 ° C. for 2 days. Jar fermenter culture is 15 rpm
It was carried out at 0 rpm and an aeration rate of 5 / min. After the completion of the culture, the cells are removed by centrifugation, ammonium sulfate is added to the crude enzyme solution obtained as a supernatant to a saturation of 40%, and the resulting precipitate is removed by centrifugation. Next, ammonium sulfate is added to the centrifugation supernatant at 65% saturation, and the resulting precipitates are collected by centrifugation and dissolved in 10 mM Tris-HCl buffer (pH 8.0). This solution was used as a dialysis membrane with cellophane tube and 10 mM Tris-HCl buffer (pH
Dialyze overnight at 8.0).

The dialyzed enzyme solution was previously adjusted to 10 mM Tris-HCl buffer (pH
Pour the solution through a column (1.5 × 45 cm) of DEAE-Toyopearl 650M (Toyo Soda Co., Ltd. Japan) equilibrated with 8.0). By this operation, the enzyme is adsorbed on the ion-exchange resin, and the unadsorbed protein which is not adsorbed is washed with the same buffer solution.
The maltooligosaccharide-forming amylase is eluted with the above buffer containing 0.4 M potassium chloride using the ion gradient method.
Next, this active fraction was mixed with 10 mM Tris-HCl buffer (pH 8.0).
After dialysis with DEAE-TOYOPEARL 650M (1.5 × 30c
m) through which it is adsorbed, washed thoroughly with the same buffer, and then
The maltooligosaccharide-forming amylase was eluted with the same buffer containing ~ 0.2 M potassium chloride as in the first run.

This active fraction was then ultrafiltered using an ultrafiltration membrane (PM-10 Amicon) to about 1
Sephacryl S-200 concentrated 0 times and buffered with 10 mM Tris-HCl buffer (pH 8.0) containing 0.1 M potassium chloride
(Falasia Sweden) column (2.2 x 90c
m) to obtain the purified enzyme.

Example 2 Using the enzyme preparation obtained in Example 1, starch was saccharified. 0.1 ml of the enzyme solution was added to 2 ml of 2% potato starch gelatinization solution dissolved in 10 mM Tris-hydrochloric acid buffer (pH 8.0), and the mixture was reacted at 45 ° C. for 24 hours. After reaction, heat (100 ℃
After deactivating (× 10 minutes), the produced sugar was analyzed by the high performance liquid chromatograph method.

FIG. 5 is a drawing showing a high-performance liquid chromatogram. The saccharified products obtained from the enzyme preparation obtained in Example 1 contained maltopentaose (G5) 26% and maltohexaose (G6) 28.
%Met.

〔The invention's effect〕

TECHNICAL FIELD The present invention relates to a novel maltooligosaccharide-forming amylase that produces maltopentaose and maltohexaose as main components and a method for producing the same.

According to the present invention, it becomes possible to efficiently produce maltooligosaccharides containing maltopentaose and maltohexaose as a main component, and conventionally, since mass production is difficult,
Many applications can be expected as a new food improving agent in the food field that could not be used. Further, if used as a source of high-purity maltopentaose or maltohexaose currently used as a reagent for amylase research or as a reagent for measuring α-amylase in human serum and urine in the field of clinical medicine, it is relatively High-purity maltopentaose and maltohexaose can be mass-produced at low cost by a simple refining process, which is of great industrial significance.

[Brief description of drawings]

Fig. 1 shows the optimum pH of this enzyme, and shows the relative activity when the enzyme solution was reacted at each pH, and Fig. 2 shows the optimum temperature of this enzyme. And showing the relative activity when the enzyme solution was reacted at each temperature,
Fig. 3 shows the pH stability of this enzyme, and shows the relative activity after the enzyme solution was left at 40 ° C for 1 hour at each pH. Fig. 4 shows the thermostability of this enzyme. FIG. 5 is a diagram showing the activity, showing the residual activity as a relative activity after the enzyme solution was maintained at each temperature for 30 minutes, and FIG. 5 is a diagram showing the sugar composition of saccharified products separated by high performance liquid chromatograph. is there. 1 ... Glucose, 2 ... Maltose, 3 ... Maltotriose, 4 ... Maltotetraose, 5 ... Maltopentaose, 6 ... Maltohexaose.

Claims (3)

(57) [Claims] 1. A maltooligosaccharide-forming amylase having the following physicochemical properties. Action and Substrate Specificity It acts on starch or starch-based saccharides to produce mainly maltopentaose (G5) and maltohexaose (G6). Optimum pH and pH stability The optimum pH of this enzyme is around 7, and its stable pH is 6-10.
Is. Optimum temperature and thermostability The optimum action temperature of this enzyme is 45 ℃ ~ 55 ℃, and is stable up to 50 ℃, the activity decreases at higher temperature,
Almost deactivated at 70 ° C for 30 minutes. Effect of inhibitors etc. This enzyme is a metal ion such as iron, copper and cobalt ion, parachloromercuribenzoate (hereinafter abbreviated as pCMB),
Inhibited by ethylenediaminetetraacetate (hereinafter abbreviated as EDTA), lithium, magnesium,
Not inhibited by barium, calcium ions, etc. Molecular weight The molecular weight of this enzyme was about 58,000 by SDS polyacrylamide gel electrophoresis. Isoelectric point This enzyme was subjected to electrophoresis in the presence of ampholine and the isoelectric point was measured and found to be around 6.5. 2. A microorganism belonging to the genus Bacillus and having the following physicochemical properties and capable of producing a maltooligosaccharide-forming amylase is cultured in a medium, and the maltooligosaccharide-forming amylase is collected from the culture. A method for producing a maltooligosaccharide-forming amylase. Action and Substrate Specificity It acts on starch or starch-based saccharides to produce mainly maltopentaose (G5) and maltohexaose (G6). Optimum pH and pH stability The optimum pH of this enzyme is around 7, and its stable pH is 6-10.
Is. Optimum temperature and thermostability The optimum action temperature of this enzyme is 45 ℃ ~ 55 ℃, and is stable up to 50 ℃, the activity decreases at higher temperature,
Almost deactivated at 70 ° C for 30 minutes. Effect of inhibitors etc. This enzyme is a metal ion such as iron, copper and cobalt ion, parachloromercuribenzoate (hereinafter abbreviated as pCMB),
Inhibited by ethylenediaminetetraacetate (hereinafter abbreviated as EDTA), lithium, magnesium,
Not inhibited by barium, calcium ions, etc. Molecular weight The molecular weight of this enzyme was about 58,000 by SDS polyacrylamide gel electrophoresis. Isoelectric point This enzyme was subjected to electrophoresis in the presence of ampholine and the isoelectric point was measured and found to be around 6.5. 3. The method according to claim 2, wherein the microorganism belonging to the genus Bacillus is the alkaline bacterium Bacillus sp. # 707 bacterium (Microtechnology Research Institute No. 8792). A method for producing a malto-oligosaccharide-forming amylase.