JPH0789917B2 - Amylase and method for producing the same - Google Patents

Description

TECHNICAL FIELD The present invention relates to a novel amylase H and a method for producing the same,
More specifically, it relates to a method for culturing a maltohexaose-producing amylase H-producing bacterium belonging to the genus Bacillus, and separating and collecting the amylase H from the culture.

(Background of the Invention and Prior Art) Conventionally, as amylase, glucoamylase, β-amylase, saccharified α-amylase, and these as enzymes for producing starch sugar containing starch, maltose or both sugars from starch Liquefied α-amylase has been developed and used as a pretreatment enzyme for starch to act on the enzyme. However, recently, the usefulness of oligosaccharides such as maltotriose, maltotetraose, maltopentaose, and maltohexaose in which glucopyranose is linearly linked by an α-1,4-glucoside bond has been attracting attention. That is, since maltooligosaccharides have excellent properties such as sweetness, sweetness qualities, viscosity, hygroscopicity, and shapeability, they have been attracting attention in the food industry as well as in other uses as pharmaceuticals and industrial raw materials. . Therefore, it is expected to develop and produce an amylase that specifically produces these oligosaccharides.

Conventionally, regarding maltohexaose-producing amylase,
Aerobacter aerogene
s) produced oxygen (JP-A-48-3968, Biochemica et Biophysica Actor (Biochimica et Biop
hysica Acta), vol.410,333 (1975)), an enzyme produced by Bacillus circulans G-6 (JP-A-57-146590, JP-A-57-146591).
Agricultural Biological Chemistry, vol.46,1539
(1982)) and Bacillus circulance (Bacill
us circulans) Enzyme produced by F-2 (Agricultural Biological Chemistry vol.47,511 (198
3) is known, but maltohexaose-forming amylase, which has an optimum action pH on the high alkaline side (pH 10-11) and has high thermostability like this enzyme, is known. There wasn't. As a result of earnest research on a method for producing amylase having a strong maltooligosaccharide-producing ability, the present inventors have found that a novel microorganism newly belonging to the genus Bacillus isolated from soil is mainly starch or a partial decomposition product of maltohexaose. It was found that amylase, which decomposes into a saccharified product as a component and has its optimum action pH on the high-alkali side and has high thermostability, is produced in a large amount outside the cells.

(Object of the invention) The object of the present invention belongs to the genus Bacillus (pH 7-1).
It is intended to provide a novel maltohexaose-forming amylase having an alkali resistance and a method for producing the same, which is capable of culturing an alkalophilic bacterium capable of growing in the medium 2 and has an extremely stable pH range of pH 5-12, which is extremely wider than that of the culture.

(Structure of the Invention) First, the microorganism used in the present invention is a microorganism isolated from soil collected in Saitama Prefecture, grows well in a medium having the composition shown in Table 1, and the invention described below. Is a microorganism belonging to the genus Bacillus and having the ability to produce the maltohexaose-forming amylase.

If (adjusted with Na ₂ CO ₃₎ (need Table 1 alkaline medium composition sets formed g / soluble starch 20 polypeptone 5 yeast extract _{5 K 2 HPO 4 1 MgSO ·} 7H 2 O 0.2 pH 10 /, the chemically defined medium 20 g / agar may be added to) The present strains have the following mycological properties. In addition, the test of the mycological properties described below is carried out according to the "Bergeys Monual of Determinative Bacteriolo 8th Edition [Bergeys Monual of Determinative Bacteriolo
gy "8th edition (1974)]" and "The Genus Bacillus" (1973)], unless otherwise specified, sodium carbonate %, And the pH of the medium was adjusted to about 10.

[A] Morphology: simple bacillus, size (0.4 to 0.7) x (2.0 to 3.
0) μm, Gram negative, motile. Form spherical to elliptical spores.

[B] Growth state in culture medium a) Meat broth agar plate culture: The shape of the colony is round, the convex ridge of the colony is raised, the peripheral edge is ear-like, viscous, and the color tone is white.

B) Meat broth agar culture; good growth and wart-like growth. The surface is smooth and opaque white.

C) Broth liquid medium; some turbidity is exhibited as the fungus grows, and a viscous and soft slag is formed.

D) Broth gelatin culture; liquefy gelatin.

E) Litmus milk: Does not coagulate milk.

[C] Physiological properties a) Nitrate reduction: positive b) Denitrification reaction: negative c) VP test: negative d) Indole formation: negative e) Hydrogen sulfide formation: negative f) Starch hydrolysis: positive ) Casein hydrolysis: Positive H) Use of inorganic nitrogen: Negative L) Pigment production: Positive N) Urease: Negative Le) Oxidase: Positive E) Catalase: Positive W) Attitude toward oxygen: aerobic K) Citric acid Utilization: Negative Y) OF test: Aerobically and anaerobically produced acid.

Ta) Salt tolerance: Grows at 12% salt concentration.

(G) Growth range (pH and temperature): pH 7-12; optimum pH 9-10.
Temperature 20-52 ℃; optimum temperature 35-45 ℃.

So) Utilization of carbon source: Aerobically and anaerobically produces an acid from D-glucol, L-arabinose, D-xylose, maltose, sucrose and sorbitol. However, no gas is generated.

T) Nucleic acid (DNA) base composition: GC content [mol%] of DNA is
46.3%.

Based on the above-mentioned mycological properties, a search for the strain in the above literature revealed that it was a microorganism belonging to the genus Bacillus of aerobic spore bacilli. Since it is clearly distinguished from known bacterial species by judging from its mycological properties, in particular, the growth pH range is pH 7-12 and the optimum pH range is pH 9-10 and it is on the alkaline side, this is a new bacterial species. Reached the conclusion that it is reasonable to set
This strain was designated as Bacillus sp. H-167 (Bacillus sp.
-167) (hereinafter referred to as "H-167 strain"). H-
The 167 strain was deposited at the Institute for Microbial Technology, Institute of Industrial Science, on March 5, 1986, and the deposit number is Microindustrial Research Institute No. 8683 (FERM P-8683).

Next, a method for culturing a microorganism for producing the present enzyme will be described.

The culture of the microorganism for producing the enzyme according to the present invention may be either solid or liquid medium. As the nutrient source, known ones conventionally used for culturing Bacillus can be used.
For example, as a carbon source, starch, dextrin, glucose, maltose, sugar raw materials such as sucrose,
As the nitrogen source, it is desirable to use organic substances such as yeast extract, polypeptone, casamino acid, defatted soybean powder. In addition, inorganic salts such as phosphate, magnesium salt and calcium salt may be added. Then, for the growth of this strain and the production of a novel enzyme, maltohexaose-producing amylase H, of the target strain, carbonates such as sodium carbonate and sodium hydrogencarbonate are added to the medium containing the above nutrient sources to adjust the pH to 9-. It is necessary to adjust the pH to 10, and it is most preferable to add 2-3% of sodium hydrogen carbonate to bring the pH of the medium to around 9.5.

The culture is carried out under aerobic conditions, and the suitable temperature for the culture is 35
The temperature is ~ 45 ° C, but in most cases, the culture is performed at around 37 ° C. Thus, the production of maltohexaose-producing amylase from this strain reaches a maximum in 2-3 days in shaking culture.

Since this enzyme is an enzyme produced outside the cells, a crude enzyme solution can be obtained by filtering or centrifuging to remove the bacteria after the completion of culture and collecting the supernatant. This crude enzyme solution may be used as it is, but by applying a suitable combination of known methods such as ammonium sulfate salting-out method, solvent precipitation method, dialysis method, selective adsorption and elution method by ion exchange resin, the crude enzyme solution is used. The enzyme may be obtained.

Next, the method for separating and purifying the maltohexaose-forming amylase H of the present invention and the physicochemical properties of the enzyme will be described.

The crude enzyme obtained from this strain can be purified by appropriately combining column chromatography using an ion exchange resin and column chromatography using a gel filtration method.

For example, the target enzyme is purified by DEAE-Toyopearl (trademark) 650M column chromatography, Toyopearl HW-55 column chromatography, and Sephadex (trademark) G-100 column chromatography, or Ultrogel (trademark) AcA34 column chromatography. be able to. The purified enzyme thus obtained has the following physicochemical properties.

The enzyme is referred to as "amylase H".

[Physicochemical properties of amylase H]

(1) Action and Substrate Specificity It specifically acts on starch, amylose, amylopectin, dextrin or a partial degradation product thereof to produce a sugar degradation product containing maltohexaose as a main component.

(2) Working pH and optimum pH range The working pH is pH 5-12, and the optimum working pH is pH 10-11.

(3) Stable pH range The stable pH is pH 5-12. (50 mM, in buffer, 50 ° C, 30
(4) Treatment method (4) Method for measuring titer 50μm sample containing this enzyme in 300μm of 1% soluble starch solution dissolved in 50mM glycine buffer (pH 9.0) as a substrate
Was added and reacted at 40 ° C for 10 minutes, and the resulting reducing sugar was added to 3,5
-Quantified by dinitrosalicylic acid reagent method, 1 per minute
One unit is the amount of enzyme that produces a reducing power equivalent to μmol glucose.

(5) Working temperature and optimum working temperature range It works up to about 80 ℃ at pH 9.0, and the optimum working temperature is 55-6.
It is at 0 ° C.

(6) Thermal stability After heating in 50 mM glycine buffer (pH 9.0) or 50 mM Tris buffer (pH 9.0) at each temperature for 30 minutes, the residual activity was measured. As a result, it is stable up to 55 ° C and deactivates by about 30% by heating at 60 ° C for 30 minutes.

(7) Inhibition Inhibited by mercury and zinc ions.

(8) Stabilization No increase in thermal stability is observed due to calcium ions.

(9) Purification method and molecular weight Crude enzyme powder was dissolved in 20 mM Tris-HCl buffer at pH 7.0, and D
Apply to an EAE-Toyopearl 650M column, elute with an aqueous sodium chloride solution, and further purify the two fractions obtained by gel filtration using a Toyopearl HW-55 column and then gel filtration using a Sephadex G-100 column or Ultrogel AcA34 column. Purify.

This enzyme is separated into three components by the above purification method. The molecular weights of each component are about 60,000, 73,000 and 80,000 respectively, and the isoelectric points are pl3.5, 4.1 and 4.3, respectively.
No difference was observed in other physicochemical properties.

The enzyme according to the present invention produces a saccharified product containing maltohexaose as a main component from a starchy substrate, and maltose other than maltohexaose from the initial reaction, maltotetraose,
In terms of producing maltopentaose, Bacillus amylase G6 {JP-A-57-146590, JP-A-57-146591
No. and Agricultural Biological Chemistry, 46, 1539 (1982).
Year)}, but the enzyme of the present invention is more stable on the alkaline side than Bacillus amylase G6 and
The pH range is characteristically different from that of pH 5-12 and optimum action pH 10-11. The thermostability of Bacillus amylase G6 is about 50% inactivated by heating at 55 ° C for 10 minutes, whereas the enzyme is stable and about 70% by heating at 60 ° C for 30 minutes.
A significant difference is also seen in the fact that the activity of 100% remains, which is different from the conventional enzyme.

Table 2 shows the results of comparison of the above physicochemical properties.

The method of the present invention will be described below with reference to examples.

Example 1 Soluble starch powder 1%, polypeptone 0.5%, 0.5% yeast extract, K ₂ HPO ₄ 0.1% aqueous solution of sodium and sodium hydrogen carbonate prepared in another sterilized medium containing MgSO ₄ · 7H ₂ O0.02% The aqueous solution was aseptically added to a final concentration of 0-3.0%, and the pH was adjusted.

50 ml of the above medium was placed in a 300 ml Erlenmeyer flask that had been sterilized in advance, and 0.5 ml of a preculture liquid of the H-167 strain (Microtech Lab. No. 8683) was inoculated, and the mixture was subjected to rotary shaking culture at 37 ° C. for 3 days. . After culturing, the culture broth was centrifuged to remove the bacteria, and the supernatant was collected and used as a crude enzyme solution. The results of comparing the activities are shown in Table 3.

Example 2 1% potato starch, 2% polypeptone, yeast extract
_{0.1%, K 2 HPO 4 0.1} %, MgSO 4 · 7H 2 O0.02%, CaCl 2 · 2H 2 O
Amylase H-producing bacterium (Microtechnology Research Institute No. 8683) was inoculated into Medium 6 containing 0.02% and 12.5% NaHCO ₃ 2.5%, and after aerobically culturing at 37 ° C for 50 hours, the culture solution was centrifuged to inoculate the bacteria. The body was removed, and about 18 cold acetone was added to the obtained supernatant of about 6 to precipitate a protein, which was dehydrated and dried to obtain a crude enzyme powder. Dissolve this in 20 mM Tris-HCl buffer at pH 7.0 and add DEA.
Purification was carried out using an E-Toyopearl 650M column (1.6φ × 40 cm), and two active fractions were obtained. That is, the fraction (H-1) eluted at a sodium chloride concentration of 280 to 300 mM
Fractions (H-2) eluted at 300-320 mM were obtained, these two fractions were each collected and concentrated. Furthermore, H-1, H-
The two fractions were each purified by gel filtration using a Toyopearl HW-55 column (pH 7.0) of 2.6φ x 70 cm, and the H-1 fraction was purified using a Sephadex G-100 column (pH 7.0) of 1.6φ x 70 cm. ), Gel filtration, for H-2 fraction 1.6φ
When gel filtration was performed using a × 70 cm Ultrogel AcA34 column (pH 7.0), two more active fractions (H-1-1, H-1-2) from the H-1 fraction and from the H-2 fraction were obtained. A single active fraction was obtained. Purified preparations were obtained by collecting and concentrating each active fraction. The purified sample shows a single band on disk electrophoresis, and its molecular weight and isoelectric point are
H-1-1 is about 73,000, pl4.1, H-1-2 is about 6
, P13.5, H-2 was about 80,000, pl4.3.

Test example 50 mM glycine buffer solution (pH 10.4) containing 1.0 g of soluble starch
4 units of this enzyme solution was added to 10 ml, reacted at 50 ° C for 1 hour, and then subjected to high performance liquid chromatography (column: Nucleosil 5
When analyzed using NH ₂ and eluent acetonitrile 60%: water 40%), a sugar degradation product mainly containing maltohexaose was obtained as shown in FIG. 4 after the reaction under the above reaction conditions
The yield of maltohexaose at the hour was 0.25 g.

[Brief description of drawings]

FIG. 1 is a diagram showing a chromatogram obtained by high-performance liquid chromatography of a sugar degradation product when the enzyme was allowed to act on starch, and FIG. 2 is a diagram showing optimum pH and stable pH of the enzyme. FIG. 3 is a diagram showing the optimum temperature of action and thermostability of the present enzyme.

Claims (3)

[Claims] 1. An amylase H having the following physicochemical properties. 1. Action and Substrate Specificity It acts specifically on starch, amylose, amylopectin, dextrin or partial degradation products thereof, and produces a sugar degradation product containing maltohexaose as a main component. 2. Working pH and optimum pH range Working pH is 5-12 and optimum pH is 10-11. 3. Stable pH range Stable pH is at pH 5-12. 4. Working temperature and optimum working temperature range It works up to about 80 ℃ at pH 9.0, and the optimum working temperature is 55-6.
It is at 0 ° C. 5. Thermal stability 50 mM glycine buffer (pH 9.0) or 50 mM Tris buffer (p
It is stable up to 55 ℃ under H9.0) and deactivates by 30% at 60 ℃ for 30 minutes. 6. Inhibition Inhibited by mercury and zinc ions. 7. Molecular weight The molecular weight is 60,000 to 80,000. 2. A method for producing amylase H, which comprises culturing an amylase H-producing bacterium belonging to the genus Bacillus and separating and collecting amylase H from the culture. 3. An amylase H-producing bacterium belonging to the genus Bacillus is Bacillus sp. H-167 (Bacillus sp. H-16).
The manufacturing method according to claim (2), which is 7).