JP2790320B2 - Thermostable amylase and methods for its production and use - Google Patents

Description

The present invention relates to a novel thermostable amylase capable of acting on raw starch, a method for producing the same, and a method for producing an oligosaccharide using the amylase. Use of this enzyme makes it possible to simplify the saccharification step and save energy.

[Conventional technology]

Maltose is an old starch sugar obtained by hydrolyzing starch, and its use in the food processing field has been expanding in recent years due to its excellent properties as a food material and sweetener.
Since purified maltose is superior to glucose conventionally used for wheel fluid, demand for pharmaceutical use is increasing. In addition, oligosaccharides containing maltotriose, which is a by-product of the production of purified maltose, have mild sweetness, lower viscosity than conventional dextrin-based starch syrup, and high water retention. Therefore, it is recently expected as a new sweetener.

Conventionally, in the process of hydrolyzing starch to produce various sugars, starch granules are gelatinized by heating (85-120 ° C.) with water, liquefied by α-amylase, and then glucoamylase or A method of saccharification by β-amylase has been used. However, gelatinization and liquefaction of high-concentration starch granules require a large amount of energy. In addition, a special device is required because the viscosity of the gelatinized starch is high. Further, when starch is gelatinized at a high temperature and then liquefied, the yield is reduced due to aging of the liquefied product and isomerization of the reducing terminal glucose residue of the liquefied product.

If it becomes possible to saccharify starch by the action of raw starch-degrading enzyme at a temperature below 70 ° C that does not gelatinize, the solution has a low viscosity, which can simplify the process and is also advantageous in terms of energy. Become. Also, isomerization of the reducing terminal glucose residue by high temperature treatment is less economically advantageous. Some studies have been conducted on non-steamed alcohol fermentation using a saccharifying enzyme that has a strong starch-degrading effect, but it has not yet reached practical use. JP-A-60-83595 and JP-A-61-83595 disclose a raw starch-degrading glucoamylase produced by a bacterium belonging to the genus Aspergillus. JP-A-63-59881 and JP-A-63-59887 disclose that a raw starch-degrading glucoamylase produced by a bacterium belonging to the genus Penicillium exhibits higher raw starch-degrading properties in the presence of α-amylase. JP-A-61-47189 discloses a raw starch-degrading glucoamylase produced by a fungus belonging to the genus Humicola. However, no method has been reported for producing maltose or maltotriose directly from raw starch by saccharification.

When raw starch is industrially saccharified to produce maltose and the like, contamination by various bacteria becomes a problem simultaneously with the saccharification rate.
From these points, the reaction temperature is desirably as high as possible within a range where the starch is not gelatinized. Therefore, a thermostable amylase is required.

Conventionally, plant-derived enzymes are used as β-amylase used in the production of maltose, and β-amylase of barley or soybean is used.
Amylases are well known. Β- derived from these plants
Amylase has a problem in terms of reaction temperature, heat resistance or supply. Therefore, a β-amylase derived from a microorganism that can withstand thermal use and can be supplied stably for industrial use is desired. There are many reports on β-amylase derived from microorganisms (amylase; supervised by Michinori Nakamura, Gakkai Shuppan Center, P86 (1986)). However, production of β-amylase by microorganisms has not reached the level of practical use.

Maltose is obtained by using β-amylase in a starch chain and hydrolyzing it to maltose units, whereas conventional β-amylase cannot hydrolyze the α, 1-6 glycosyl bonds in starch and therefore starch. To increase the yield of maltose from starch, ie, α, 1-6
It is a common method to use isoamylase and pullulanase in combination to cleave the glucosyl bond. However, enzymes that cleave these α, 1-6 bonds are expensive.

[Problems to be solved by the invention]

As described above, there are various problems in industrially producing maltose or other oligosaccharides from starch. The present invention seeks to solve these various problems at once from the viewpoint of enzymes. Specifically, (1) it is possible to directly act on raw starch to hydrolyze it, 2)
In that case, starch can be substantially degraded by 100% without using so-called debranching enzymes such as isoamylase,
(3) As a result, the target maltose and maltotriose can be produced predominantly. (4) When used on raw starch, use at a temperature high enough to effectively prevent contamination of various bacteria. Has sufficient heat resistance, and (5)
It provides a novel amylase that can be produced in large quantities by bacteria. Another object of the present invention is to provide a method for producing the novel thermostable amylase and a method for producing maltose and other oligoparticles using the novel method.

[Means for solving the problem]

Properties of thermostable enzyme The present invention firstly provides a novel thermostable amylase capable of acting on raw starch, and an example of this enzyme will be described in detail. The properties of the enzyme shown below are the results obtained using the enzyme obtained in Example 2 described later.

(1) Action: The amylase of the present invention acts on raw starch and mainly produces maltose and maltotriose. Especially 60 ℃
When it acts on raw starch, it degrades raw starch substantially 100%. Easiness of decomposition for various raw starches is in the order of wheat starch> corn starch> sweet potato starch> potato starch (see FIG. 1).

In addition, the heat-resistant amylase having a raw starch-degrading property of the present invention has a strong adsorption power to various raw starch granules, and this adsorption power is based on the degradability of the raw starch.

(2) Optimum pH and pH stability: The optimum pH of this enzyme is around 6.0 (see FIG. 2). Room temperature (22 ° C) in each pH buffer
When treated for 1 hour at pH 5 to 8, it shows a residual activity of 95% or more (see FIG. 2).

(3) Optimum temperature: The optimum temperature measured at pH 7.0 is 70 ° C
(See FIG. 3). When treated at 70 ° C. for 15 minutes, it was not substantially inactivated. When treated for 1 hour, 90% or more at 70 ° C., 70% at 75 ° C., and 50% at 80 ° C. Has residual activity (third
See figure).

(4) Molecular weight: gel filtration method (using FPLS manufactured by Pharmacia,
Column: Sparose TM12, eluent: 5 containing 0.2 M NaCl
52,000 ± 5, 0mM acetic acid-sodium acetate buffer pH5.0)
000.

(5) UV absorption spectrum: μmax in phosphate buffer:
It shows absorption at 275 nm and 190 nm.

(6) Elemental analysis: C; 59.4%, H; 6.4%, N: 15.3%.

Measurement method of oxygen titer The amylolytic activity of this enzyme was measured by adding a certain amount of the enzyme solution to a 1% -soluble starch solution, reacting at a predetermined pH and temperature, and reducing the reducing sugar generated after 5 minutes to 3,3. 5-dinitrosalicylic acid method (DNS method) [Starch and related carbohydrate experiments, Michinori Nakamura et al.,
43 pages, published by Gakkai Shuppan Center (1986)]. The titer for producing a reducing sugar such as glucose 1 μmole phase per minute is defined as one unit. To measure the degradability of raw starch, a solution prepared by dissolving a certain amount of the present enzyme in a buffer solution was added to the solution to give a concentration of raw starch of 1%, and the temperature was 30 to 80 ° C, pH 4.0 to 9.0, and 0 to 24. The sugar generated after time shaking is measured by the above-mentioned DNS method using a calibration curve of glucose. The degree of saccharification is represented by the so-called DE value. The DE value characterizes the degree of starch degradation,
This is a numerical value indicating the reducing power of decomposed starch, assuming that the reducing power of pure dextrose is 100%.

In the present invention, any Bacillus bacterium capable of producing an enzyme having the above properties can be used, and such a bacterium can be isolated from soil, for example, as follows. can do.

Among colonies growing at 60 ° C. on a meat extract-polypeptone agar medium containing 2% raw wheat starch, those forming halos were separated, inoculated into a liquid medium, and cultured for a certain period of time. Choose strong strains.

One of the strains thus isolated was examined for its mycological properties in detail, and was analyzed by the Bergez Manual of Deterministic Bacteriology, 7th edition and 8th edition.
Based on the description in the edition, this strain was identified as a strain belonging to Bacillus stearothermophilus, and furthermore, it was judged to be a new strain because it was different from any of the known strains in terms of raw starch degradability. Therefore, this strain was named Bacillus stearothermophilus B-1 strain,
30th Microorganisms Research Laboratories donated to the Microbiological Research Institute
Deposited as No. 120, and transferred to the International Deposit under the Budapest Treaty on May 26, 1989 as No. 2440 of the JICKEN.

The mycological properties of Bacillus stearothermophilus B-1 are as follows.

1. Microscopic (including electron microscopic) findings Gram staining: Positive Form: Bacillus Size: 0.6 × 1.0 × 2-3.5 μm Polymorphism: None Mobility: Yes Flagella: None Spore: Yes, camellia round And exists at the tip of the cell at a size of 1 to 1.8 μm.

Acid resistance: None 2. Culture findings Gravy agar plate: Uneven colonies with a diameter of about 5 mm after culturing for 24 hours. White with irregular edges.

Gravy agar slope: white. Not flowing.

Juice Liquid: cloudy. White sediment Juice gelatin puncture: Liquefied gelatin.

Litmus milk: acid / coagulation 3. Physiological properties Nitrate reduction: + MR test:-VP test:-Indole formation:-Hydrogen sulfide formation:-Starch hydrolyzability: + Citric acid availability:-Nitric acid Use of (NO ₃ ⁻ ): − Use of ammonia (NH ₄ ⁺ ): + Dye formation: +. Produces a slightly water-soluble brown pigment.

Catalase: + Oxidase: ± Growth range (pH): 5.5 to 8.0 (Temperature): 45 ° C to 75 ° C Attitude to oxygen: Aerobic OF test: O 4. Oxidation of sugars, gas generation and utilization Saccharide Acid gas Qualification L-arabinose +-+ D-xylose +-+ D-glucose +-+ D-mannose +-+ D-fructose +-+ D-galactose ±-± maltose ±-+ sucrose +-+ Lactose − − − Trehalose ± − + D-sorbit − − − D-mannitol + − + Inosit − − − Glycerin ± − ± Starch + − + 5. Other properties Raw starch degradability: on agar medium containing raw starch The bacterium forms a halo, but Bacillus stearothermophilus IFO 12
550 does not form a halo.

The present invention further relates to a method for producing a thermostable amylase having the above-mentioned properties, wherein the method comprises culturing a Bacillus bacterium capable of producing the thermostable amylase in a medium, and culturing the bacterium. And collecting the enzyme from the enzyme.

In the cultivation as described above, various starches, starch hydrolysates, corn meal, wheat flour, molasses and the like can be used alone or in combination as the carbon source of the medium. The amount of use varies depending on the type, but is preferably 0.1% to 30%.
As the nitrogen source, soybean flour, cottonseed oil residue, peptone, casein, meat extract, yeast extract, malt extract, inorganic ammonium salt, inorganic nitrate and the like can be used alone or in combination. The amount used depends on the type, but is 0.
05% to 20% is preferred. In addition, inorganic salts such as phosphates, magnesium salts, FeSO ₄ , KCl, and CaCl ₂ , and if necessary, organic trace nutrients can be added to the medium.

The production of the amylase of the present invention is carried out by using a microorganism having the amylase-producing ability in a medium usually used for amylase production.
Solid or liquid culture may be performed at a temperature of 45 to 75 ° C, preferably 50 to 65 ° C, and a pH of 5.5 to 8.0, preferably 6.0 to 7.5.
In 3 days, a significant amount of thermostable amylase having raw starch degradability can be obtained.

Purification of the enzyme of the present invention can be performed according to a method commonly used for purifying an enzyme. For example, the culture solution obtained as described above is first treated by a method such as centrifugation or filtration to remove bacterial cells. The culture filtrate or supernatant thus obtained can be subjected to a sulfur fractionation, and further subjected to gel filtration, ion-exchange column separation, and the like to obtain a purified enzyme.

The present invention further relates to a method for producing an oligosaccharide. By reacting the enzyme of the present invention on raw starch, an oligosaccharide mixture mainly composed of maltose and maltotriose is produced. These can be collected together to obtain an oligosaccharide product, or a mixture thereof can be separated according to a conventional method to obtain maltose and / or maltotriose alone. All of these methods fall within the scope of the present invention.

In the present invention, the thermostable amylase used in the method for producing an oligosaccharide means not only an isolated or purified thermostable amylase, but also any enzyme source containing such an amylase. That is, a culture solution obtained by culturing an enzyme-producing bacterium can be used as it is as an enzyme source. Advantageously, cells are separated from the culture solution, and a culture filtrate or supernatant is used as an enzyme solution. Further, if necessary, it can be used as a partially purified product or a completely purified high-purity enzyme by a method usually used for purification of an enzyme such as an ammonium sulfate fractionation method, a membrane separation method, and a chromatographic separation method.

In saccharification, raw starch is suspended in water (usually 10 to 50%) and directly decomposed by a heat-resistant amylase having raw starch-degrading activity without gelatinization by heating. In this case, unlike the conventional saccharification of starch, liquefaction of starch by so-called liquefied α-amylase is not required. Saccharification of raw starch is from room temperature to 70
C., pH 4.5-9.0. The gelatinization temperature of the starch varies depending on the type of the starch, and in this case, it is desirable to set the reaction temperature to a high temperature within a range where the starch does not gelatinize and remains granular. More preferably, 50 to 65 ° C. and pH 5 to 8 are desirable. Although the decomposition reaction can be carried out while standing, it is desirable to carry out the stirring so that the starch milk is uniformly suspended.

The amount of enzyme used per 1 g of starch is suitably 1 to 300 units, but it is desirable to adjust the amount of enzyme depending on the reaction temperature and reaction time. The starch to be decomposed can be used regardless of underground starch or above-ground starch. The ease of decomposition of various starches is in the order of wheat starch> corn starch> potato starch> potato starch.

 Next, an example will be described.

Example 1 Culture for Enzyme Production Bacillus stearothermophilus in a medium containing 0.5% meat extract, 1% polypeptone, 0.5% NaCl and 1% soluble starch, adjusted to pH 7.0 with NaOH, and then sterilized. The B-1 strain was inoculated and shake-cultured at 55 ° C. for 24 hours. The supernatant obtained by removing the cells by centrifugation has an amylase activity of 1.06 units / m.
l showed.

Example 2. Purification method of enzyme The culture solution was centrifuged (10,000 rpm, 20 minutes), and ammonium sulfate was added to the supernatant from which the cells had been removed so as to be 70% saturated, and the mixture was allowed to stand for 24 hours. A crude enzyme precipitate was obtained by centrifugation (10,000 rpm, 20 minutes). Crude enzyme was added to 10 mM phosphate buffer (pH 7.2)
After centrifugation (15,000 rpm, 20 minutes) of the insoluble material, the supernatant was subjected to gel filtration using Sephadex G-100 to obtain a fraction showing amylase activity.

Then, the amylase activity fraction was adsorbed on DEAE cellulose 32 and eluted with 10 mM phosphate buffer (pH 8.5) to collect the amylase activity fraction. The amylase activity fraction was again adsorbed on DEAE cellulose 32 and eluted with 10 mM phosphate buffer (pH 7.2) to collect the amylase activity fraction. It was dialyzed against pure water and freeze-dried to obtain an enzyme preparation. This enzyme preparation showed a single band on disk electrophoresis. Table 1 shows the progress of this purification.

Example 3 Measurement of Degradation Rate of Raw Starch In a 50 ml Erlenmeyer flask, 40 mg of various raw starches were put, and 4 ml (5.925 units, 60 ° C.) of an enzyme solution prepared with a phosphate buffer (pH 7.2) was added to 50 ° C. And shaken at 60 ° C. Samples were taken every 6 hours, centrifuged (1,000 rpm, 5 minutes), diluted appropriately, and the resulting reducing sugar was measured by the DNS method. The amount of generated reducing sugar was shown by DE value (FIG. 1). The composition of reducing sugars produced by raw starch decomposition was determined by HPLC (apparatus: Hitachi 665A-12).
LC, detector: Water refractometer Elecxtronics Uni
t, column: Hanoi Cosmosil Packed column 4.6 × 150 mm, after elution: acetonitrile / water = 75/25).
Maltose was the main component in the resulting reducing sugar, followed by maltotriose and glucose (maltose>maltotriose> glucose). Considering the DE value and the reducing sugar composition, the raw starch decomposition rate at 60 ° C was almost 100%.

Example 4. 5 g of corn starch was weighed, and 0.1 M acetate buffer (pH 6.
0) Suspended in 15 ml, 5 ml of an enzyme solution prepared to contain 50 units of thermostable amylase was added, and the mixture was shaken at 55 ° C for 12 hours. The reaction solution was analyzed by HPLC (apparatus: Hitachi 665-12LC, detector:
Water Refractometer Electoronics Unit, columns:
Hanoi Cosmosil Packed column 4.6 × 150 mm, eluent: acetonitrile / water = 75/25). 2.6% glucose, 56.5% maltose,
32.8% of maltotriose was produced.

Example 5 The substrate in Example 2 was changed to wheat starch, and the reaction temperature was changed to 60 ° C.
The raw starch was decomposed by the same method as described in Example 2 except that
An oligosaccharide solution containing 2.1%, maltose 51.3%, and maltotriose 39.4% was obtained.

Example 6 Corn starch, potato starch, potato starch, and wheat starch were each weighed 5 g each, and heat-resistant amylase 10
Add 0.1M acetate buffer (pH 6.0) containing 0 units, and add 24 units at 60 ° C.
Shake time. The sugar in the reaction solution was analyzed by HPLC, and the composition of each reaction solution was as shown in Table 2.

[Effect of the Invention] In producing maltose and maltotriose,
When this method of directly decomposing raw starch using a heat-resistant amylase having raw starch-degrading activity is employed, compared with the conventional method using β-amylase, it does not require a so-called debranching enzyme, and also requires equipment and energy. This makes it possible to construct a starch saccharification process that is more advantageous.

[Brief description of the drawings]

FIG. 1 is a graph showing the time course of the decomposition of various raw starches by the thermostable amylase having a raw starch degrading activity of the present invention. In FIG. 2, the left side is a graph showing the optimum pH of the enzyme, and the right side is a graph showing the remaining enzyme activity when the enzyme was treated at each pH at room temperature (22 ° C.) for 1 hour. In FIG. 3, the left side is a graph showing the optimum temperature of the enzyme, and the right side is a graph showing the temperature stability, that is, the residual enzyme activity of the enzyme after treatment at each temperature for a certain time.

Claims (6)

(57) [Claims] 1. The following properties: Action: Acts on raw starch to produce mainly maltose and maltotriose, and at 60 ° C. substantially converts the raw starch
Decomposes 100%; Optimum pH: around 6.0; pH stability: pH when treated for 1 hour at room temperature in buffer
5 to 8 show 95% or more residual activity; optimum temperature: 70 ° C; temperature stability: substantially inactive when treated at 70 ° C for 15 minutes at pH 7.0, 1 hour at 70 ° C A thermostable amylase having a residual activity of at least 90% when treated; and having a molecular weight of 52,000 ± 5,000 as determined by gel filtration. 2. The method for producing a thermostable amylase according to claim 1, wherein a bacterium belonging to the genus Bacillus having the ability to produce the amylase is cultured in a medium, and the amylase is collected from the culture. And how. 3. The bacterium of the genus Bacillus is Bacillus stearothermophilus B-.
3. The method according to claim 2, which is one strain. 4. A method for producing maltooligosaccharides containing maltose and maltotriose as main components, wherein the heat-resistant amylase according to claim 1 is allowed to act on raw starch. 5. A method for producing maltose, wherein the method comprises the steps of:
A maltose and maltotriose-based maltooligosaccharide are produced by allowing the heat-resistant amylase described in 1 to act on raw starch, and then maltose is isolated. 6. A method for producing maltotriose, wherein the heat-resistant amylase according to claim 1 is allowed to act on raw starch to cause maltose and maltooligosaccharides containing maltotriose as main components to act. A method comprising isolating maltotriose.