JPH04211369A - Halophilic alkali amylase and production thereof - Google Patents

Abstract

PURPOSE:To obtain the title enzyme efficiently providing maltotriose, from various starch-based substrates by culturing a fungus capable of producing amylase, belonging to the genus Natronococcus, in a salt-containing alkali medium and separating. CONSTITUTION:A fungus capable of producing amylase, belonging to the genus Natronococcus, is cultured in an alkali medium containing >=5wt.% salt. Then, a halophilic alkali amylase is separated from the culture mixture and recovered. The fungus capable of producing a halophilic amylase is preferably Natronococcus sp. Ah-36 strain or Natronococcus Ah-477 strain.

Description

[Detailed description of the invention]

0001

TECHNICAL FIELD The present invention relates to a novel halophilic alkaline amylase and a method for producing the same. To be more specific, Natronococcus
The present invention relates to a method for culturing amylase-producing bacteria belonging to the genus C. ccus and isolating and collecting halophilic amylase whose optimal pH for action is on the alkaline side from the culture.

0002

BACKGROUND OF THE INVENTION Conventionally, various amylase-producing microorganisms have been known that have an optimum pH for action in neutral or acidic conditions. In addition, microorganisms that produce alkaline amylase having an optimum pH on the alkaline side are of the genus Bacillus (e.g.,
(see Japanese Patent Publication No. 50-5272), and Pseudomonas (for example, see Japanese Patent Publication No. 50-5274). Microorganisms that produce so-called halophilic amylases, which exhibit almost no activity in the absence of common salt but exhibit activity in the presence of several percent or more of common salt, also include microorganisms of the genus Micrococcus (e.g., (see Japanese Patent Laid-Open No. 54-49391) and the genus Acinetobacter (for example, see Japanese Patent Application Laid-open No. 54-49391). However, to date, there have been no reports on microorganisms that produce amylase that is halophilic and has an optimum pH on the alkaline side.

0003

[Means for Solving the Problems] As a result of intensive efforts by the present inventors to find a method for producing halophilic amylase using microorganisms, the present inventors have cultivated a new bacterial species of the genus Natronococcus that they have isolated from soil. As a result, we found that halophilic alkaline amylase was produced in significant amounts outside the bacterial cells. This enzyme is different from conventional general amylase in that its optimum pH for action is on the alkaline side, and it is quickly deactivated when dialyzed against water, resulting in sodium chloride,
It has the characteristics of exhibiting remarkable activity when the concentration of salts such as sodium acetate or potassium chloride is 1 molar or more, and we have found that it is a new enzyme that is different from conventional amylases. Completed the invention. The halophilic alkaline amylase (hereinafter referred to as amylase A) of the present invention has the following physical and chemical properties.

1) Action and substrate specificity Amylase A exhibits significant enzymatic activity in the presence of 1 mole or more of sodium chloride, sodium acetate, or potassium chloride, and exhibits no activity in the absence of salt; it is extremely halophilic. It is characterized by being an enzyme, acting specifically on starch, amylose, amylopectin, dextrin, or their partial decomposition products, and producing saccharide decomposition products whose main component is maltotriose.

2) Working pH and optimum pH range (see Figure 1) Working pH
is at pH 5-12, and the optimum working pH is at 8-10.

3) Stable pH range (see Figure 1) under a 20mM buffer containing 2.5M sodium chloride at 4°C.
No deactivation was observed within the pH range of 6 to 12 after standing for 20 hours.

4) Method for measuring titer 75mM containing 2.5 mol of sodium chloride as substrate
Sample 10 containing this enzyme in 200 μl of 1.5% soluble starch solution dissolved in Tris buffer (pH 9.0)
0 μl was added and reacted at 50°C for 15 minutes, and the resulting reducing sugar was quantified by the 3,5-dinitrosalicylic acid reagent method.
The amount of enzyme that generates a reducing power equivalent to 1 μmol of glucose per minute is defined as 1 unit (1 U).

5) Working temperature and optimal working temperature range (see Figure 2) pH
9, it works up to about 80°C, and the optimum working temperature is 55~
It is at 65°C. 6) Thermal stability (see Figure 2) The activity was measured after heating for 20 minutes at each temperature in 50mM Tris buffer (pH 9.0) containing 2.5M sodium chloride. Life is not recognized.

7) Inhibition and activation Amylase A is strongly inhibited by Hg+ and Ni2+. Amylase A is not activated by Ca2+.

8) Purification method and molecular weight One example of a purification method for amylase A is to add 2.
Add 5-3 volumes of cold saturated ammonium sulfate solution, dissolve the precipitated protein in 20mM Tris buffer (pH 8.0) containing 2.5M sodium chloride, and gel filtrate with Sephacryl™ S-200 column. , and can be further carried out by high performance liquid chromatography using a Synchropack (trademark) GPC100 column. The molecular weight of the purified enzyme thus obtained was calculated from SDS-acrylamide gel electrophoresis, and was 70,000 to 80,000.
It was 00.

[0011] The present invention also provides a method for cultivating Natronococcus (N
amylase-producing bacteria belonging to the genus Atronococcus are cultured in an alkaline medium containing 5% by weight or more of salt, and halophilic alkaline amylase is isolated from the culture,
This is a method for producing halophilic alkaline amylase, which comprises recovering the halophilic alkaline amylase.

[0012] The present invention is directed to the use of Natronococcus
There is a method characterized by culturing amylase-producing bacteria belonging to the genus P. nococcus and separating and collecting halophilic alkaline amylase from the culture.

The present invention will be explained in detail below.

First, the microorganism used in the present invention is an amylase-producing bacterium belonging to the genus Natronococcus, and a specific example of this microorganism is that the present inventors isolated and identified it from soil on the shores of Lake Magadi, Kenya. Examples include Natronococcus Ah-36 bacteria and Ah-471 bacteria. This bacterium grows in a bacterial growth medium containing salt, particularly in a medium with the composition shown in Table 1, and belongs to the genus Natronococcus, which efficiently produces the halophilic alkaline amylase of the present invention. It is a microorganism.

[0015]

[Table 1]

[0016] The mycological properties of the above-mentioned bacterial strains used in the examples of the present invention are shown below. The mycological tests described below are based on Bergey's Manual of Determinative Bacteriology.
nual of Determinative Bac
treriology) 8th edition (1974), "Experimental Methods for Chemical Classification of Microorganisms" (1982) edited by Kazuo Komagata, and Ross (Ross) described in Journal of Genetic Microbiology, Vol. 123, p. 75. method (Ross et al.,
Journal of Genetic Micro
biology, 123, p75 (1981)
Unless otherwise specified, a medium containing 15% by weight of sodium chloride was used in all cases, with the pH adjusted to approximately 9.5 by adding 1.5% of sodium carbonate. .

Ah-36 strain (1) Morphology a) Cell shape and size Cocci (1 to 2 μm in diameter) form irregular groups. b) Motility None c) Spores None (2) Growth status in medium a) Juicy agar plate medium The shape of the colony is circular, and the surface of the colony is circularly raised. The color is pink with a reddish tinge. b) Meat juice liquid medium becomes reddish as the bacteria grow. There is no viscosity. c) Broth gelatin medium Does not liquefy gelatin. (3) Physiological properties a) Nitrate reduction Positive b) Indole production Positive c) Pigment production Positive d) Starch hydrolysis Positive e) Inorganic nitrogen utilization Negative f) Oxidase Positive g) Catalase Positive h) Oxygen attitude
Aerobic i) Utilization of citric acid Negative j) Salt tolerance Grows well in the presence of 8% by weight or more of salt, particularly in the presence of 15 to 20% by weight, and also grows in saturated salt. k) Growth range pH 8-10;
Optimum pH 9-9.5 Temperature 20-50°C; Optimum temperature 35-45°C 1) Utilization of carbon sources: Utilizes glucose, maltose, sucrose, lactose, fructose, starch, no gas generation. m) Nucleic acid (DNA) Base sequence DNA GC content (mol%) is 63.5% n) Cell membrane lipid Glyceryl diether skeleton C20, C20 diether core lipid o) Antibiotic susceptibility test Anisomycin, bacitracin, erythromycin, rifampicin , sensitive to tetracycline. Not susceptible to ampicillin, chloramphenicol, polymyxin B, and streptomycin.

Ah-471 strain (1) Morphology a) Cell shape and size Cocci (3 to 6 μm in diameter) form irregular groups. b) Motility None c) Spores None (2) Growth status in medium a) Juicy agar plate medium The shape of the colony is circular, and the surface of the colony is circularly raised. Pink color. b) Meat juice liquid medium becomes reddish as the bacteria grow. There is no viscosity. c) Broth gelatin medium Does not liquefy gelatin. (3) Physiological properties a) Nitrate reduction Positive b) Indole production Positive c) Pigment production Positive d) Starch hydrolysis Positive e) Inorganic nitrogen utilization Negative f) Oxidase Positive g) Catalase Positive h) Oxygen attitude
Aerobic i) Utilization of citric acid Negative j) Salt tolerance Grows well in the presence of 5% by weight or more of salt, particularly in the presence of 10 to 15% by weight, and also grows in saturated salt. k) Growth range pH 7.5-1
1; Optimum pH 9-9.5 Temperature 20-50°C; Optimum temperature 35-45°C 1) Utilization of carbon sources Glucose, maltose, sucrose, lactose, fructose, starch are used, no gas is generated. m) Nucleic acid (DNA) Base sequence DNA GC content (mol%) is 63.9% n) Cell membrane lipid Glyceryl diether skeleton C20, C20 diether core lipid o) Antibiotic susceptibility test Anisomycin, erythromycin, rifampicin,
Sensitive to tetracycline. ampicillin, bacitracin, chloramphenicol,
Not sensitive to polymyxin B and streptomycin.

Based on the above mycological properties, we searched for this strain in the literature and found that it is a microorganism belonging to the genus Halococcus, a highly halophilic bacterium, which requires a high concentration of sodium chloride for growth. However, this strain differs from the genus Halococcus in the various characteristic properties shown above, particularly in that the growth pH is on the alkaline side.

[0020] In recent years, Tindall et al. have investigated a group of bacteria that are highly saline and grow at alkaline pH, and as a result of their investigation, they have found a group of cocci that is highly hypersaline and grows at alkaline pH. As a group of bacteria clearly different from the genus Halococcus, these bacteria have been newly named the genus Natronococcus [Systematic Applied Microbiology (Tindall e.g.
tal. , Systematic Applied
Microbiology) Volume 5, Page 41 (19
1984)]. Since this strain is a halophilic and alkalophilic bacterium, it is identified as a microorganism belonging to the genus Natronococcus, which was named by Tyndall et al.; Since it is clearly distinguishable from known bacterial species, judging from the fact that it is a lipid, we came to the conclusion that it is appropriate to designate this strain as a new bacterial species.
Ah-36 (Natronococcus sp.
Ah-36) (hereinafter referred to as Ah-36 strain) and Natronococcus sp. Ah-471 (Natronococcus sp.
coccus sp. Ah-471) (hereinafter referred to as Ah
-471 strain). Ah-36 strain and A
The h-471 strain was deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology on September 25, 1989, and its accession numbers are FAIKENBIBIKI NO.
It is No. 25.

[0021] The present invention is not limited to the above-mentioned bacterial cells or their variants and mutants, but is effective against all bacteria that belong to the genus Natronococcus and have the ability to produce the halophilic alkaline amylase. It is possible to use.

Next, enzyme production according to the present invention will be described. The above-mentioned microorganisms for enzyme production may be cultured in either solid or liquid media. It is desirable to use carbohydrate raw materials such as starch, amylopectin, dextran, dextrin, and glucose as a carbon source, and organic substances such as yeast extract, casamino acids, and peptone as a nitrogen source. In addition, inorganic salts such as potassium salts, magnesium salts, iron salts, and calcium salts may be added. In order to grow this strain and produce the target amylase, 10% or more of sodium chloride is added to the medium containing the above nutrients, and carbonates such as sodium carbonate and sodium hydrogen carbonate are added to adjust the pH. It is necessary to adjust the amount of sodium chloride to 9 to 10, and it is particularly desirable to add 10 to 20% of sodium chloride.

[0023] Cultivation is carried out under aerobic conditions, and the appropriate temperature for culturing is 35 to 45°C, but in most cases it is cultured at around 37°C. The amylase of the present invention is secreted outside the bacterial cell.

[0024] In order to collect and purify the enzyme of the present invention from the above-mentioned culture solution, known purification methods can be used alone or in combination. For example, after completion of the culture, the culture solution is filtered or centrifuged to remove bacterial cells, and then known methods such as ammonium sulfate salting out, solvent precipitation, and dialysis are applied in combination as appropriate. Further, column chromatography using a gel filtration method, hydroxyapatite chromatography, column chromatography using an ion exchange resin, etc. can be carried out in an appropriate combination.

The properties of the halophilic alkaline amylase (hereinafter referred to as amylase A) obtained by the present invention are as described above.

Halophilic alkaline amylase A, which is a specific example of the method of the present invention, has an optimum pH on the alkaline side and exhibits activity under high salt concentrations. , had the characteristic of acting on dextrin or its decomposition products to produce glycolysates containing maltotriose as the main component.

For example, to give one example, a 50ml solution containing 2.5% soluble starch and 2.5M sodium chloride
Add 2 units of purified amylase A to 10 ml of M. Tris buffer (pH 9.0), perform an enzyme reaction at 60°C for 5 hours, keep this reaction solution at 100°C for 10 minutes to inactivate the enzyme, and remove the ion exchange resin. After desalting using paper chromatography (developing solvent: n-butanol/pyridine/
water = 6:4:3 (v/v)) and high performance liquid chromatography (column: Shinpack CLC NH2,
The eluate was analyzed using acetonitrile/water = 65:35 using a detection differential refractometer). As a result, a glycolysate containing maltotriose as a main component was obtained as shown in FIG. When the starch decomposition product was confirmed by liquid chromatography, the maltotriose content was approximately 70%.

Conventional enzymes that hydrolyze starch-type substrates into maltotriose include Streptomyces griseus.
s) Amylase produced by strain N-A468 (Special Publication No. 57
-6915 Publication), Bacillus YT
-Amylase produced by strain 1004
315) and amylase produced by Bacillus KP1071 strain (Japanese Patent Publication No. 1-16157). However, the present enzyme A clearly differs in properties from any of the known reported enzymes in various respects. For example, this enzyme A is stable on the alkaline side, with an effective pH range of 5 to 12, and an optimal operating pH of 8 to 1.
It differs greatly from other enzymes in that it is on the alkaline side. Furthermore, the present enzyme A has completely different properties from other enzymes in that it is a halophilic amylase. That is, this enzyme A is rapidly inactivated when dialyzed against water, and exhibits significant activity when the concentration of salt such as sodium chloride, sodium acetate, or potassium chloride is 1 molar or more.

On the other hand, examples of halophilic amylases are disclosed in, for example, Japanese Patent Publication No. 50-5273 (method for producing halophilic α-amylase), Japanese Patent Publication No. 49391-1983 (method for producing halophilic α-amylase),
Although there have been reports on methods for producing amylase 1 and/or 2), there have been no known amylases that specifically produce maltotriose.

Alkaline amylase has been reported in, for example, Japanese Patent Publication No. 50-5272 (Production method of amylase) and Japanese Patent Publication No. 50-5274 (Production method of alkaline amylase using microorganisms). No activity is expressed in the inside.

[0031] As described above, the halophilic alkaline amylase of the present invention was found to be a novel amylase not found in the past, judging from its various properties.

By using the bacteria Ah-36 and Ah-471 belonging to the genus Natronococcus, which are specific examples of the present invention, a novel halophilic alkaline amylase can be obtained, and by using this enzyme, various starches can be produced. Maltotriose can be effectively produced from the system substrate. Maltotriose is used in various fields such as low-sweetness food ingredients, food modifiers, low-cariogenic food bases, intravenous fluid components, and parenteral nutritional supplements, and the effective use of this enzyme is desired. It is an oligosaccharide.

[0033]

[Examples] The present invention will be explained in more detail and concretely below with reference to Examples, but the present invention is not limited to these Examples.

Example 1 Soluble starch 1%, casamino acids 0.75%, yeast extract 1%, potassium chloride 0.2%, magnesium sulfate 0.
2%, ferrous sulfate 0.005% and sodium carbonate 1
．． A separately sterilized sodium chloride solution was aseptically added to a pH 9.5 medium containing 5% to give a final concentration of 0 to 25% (w/v). Pour 50 ml of the above medium into a 300 ml Erlenmeyer flask that had been sterilized in advance.
24) or Ah-471 strain (Feikoken Bibori No. 11)
025) was inoculated with 2.5 ml of preculture solution and incubated at 37°C for 4 hours.
The cells were cultured with rotational shaking for days. After culturing, the culture solution was centrifuged to remove bacteria, and the supernatant was collected and used as a crude enzyme solution to measure amylase activity. The results are shown in Table 2.

[0035]

[Table 2]

Example 2 Potato starch 1%, casamino acids 0.75%, yeast extract 1%, potassium chloride 0.2%, magnesium sulfate 0.
2%, ferrous sulfate 0.005%, sodium carbonate 1.5
Amylase A-producing bacteria (Feikoken Bacteria No. 11024) were inoculated into 4 liters of a medium containing 15% sodium chloride and 15% sodium chloride, and after culturing aerobically at 37°C for 72 hours, the culture solution was centrifuged to remove the bacterial cells. was removed, and about 12 liters of cold saturated ammonium sulfate solution (pH 8) was added to about 4 liters of the resulting supernatant to precipitate proteins. The obtained precipitate was dissolved in 20 mM Tris buffer (pH 8) containing 2.5 M sodium chloride, and dialyzed in the above buffer at 4° C. for 24 hours to obtain about 350 ml of a crude enzyme solution. The amount of enzyme obtained was 1800 units (recovery rate 70%). The crude enzyme solution was concentrated with polyethylene glycol 6000 and then treated with Sephacryl S-200.
Gel filtration was performed using a column (2.6 x 83 cm),
The obtained activity classification was transferred to Synchropack GPC100 (7.
By performing high-performance liquid chromatography using a 8 x 300 mm sample and separating the active fraction, we obtained 136 units of a purified sample (yield approximately 8% from the crude enzyme solution) that showed a single band electrophoretically. Obtained.

[0037]

As described above, according to the present invention, novel amylase-producing bacteria belonging to the genus Natronococcus, such as Natronococcus sp. Ah-36 strain and Natronococcus sp. Ah-471 strain, can be produced. Halophilic alkaline amylase is obtained. This enzyme can effectively produce maltotriose from various starch-based substrates. This maltotriose is useful as a low-sweet food component, a food modifier, a low-cariogenic food base, an infusion component, an intravenous nutritional supplement, and the like.

[Brief explanation of the drawing]

Figure 1 shows the optimal action pH and stable p of the enzyme amylase A.
FIG. 2 is a graph showing the optimum temperature for action and thermal stability of this enzyme, and FIG. 3 is a graph showing paper chromatography and liquid chromatography showing the saccharide decomposition products when this enzyme is applied to starch. It is a graph.

Claims (4)

[Claims] Claim 1: A halophilic alkaline amylase having the following physical and chemical properties. 1) Action and substrate specificity It is a highly halophilic enzyme that exhibits significant enzymatic activity in the presence of 1 mol or more of sodium chloride, sodium acetate, or potassium chloride, and exhibits no activity in the absence of salt. , starch, amylose, amylopectin,
Acts specifically on dextrin or its partial decomposition products,
Generates saccharide decomposition products whose main component is maltotriose. 2) Working pH and optimum pH range Working pH is between pH 5 and 12, and optimum working pH is between 8 and 1.
It is at 0. 3) stable pH range under 20mM buffer containing 2.5M sodium chloride;
No deactivation was observed in the pH range of 6 to 12 when left at 4°C for 20 hours. 4) Working temperature and optimum working temperature range It works up to about 80°C at pH 9, and the optimum working temperature is 5.
It is between 5 and 65 degrees Celsius. 5) Thermal stability After heating in 50mM Tris buffer (pH 9.0) containing 2.5 mol of sodium chloride at each temperature for 20 minutes, the activity was measured, and almost no inactivation was observed until heating to 60°C. do not have. 6) Inhibition, activation Strongly inhibited by Hg+ and Ni2+, not activated by Ca2+. 7) Molecular weight The protein precipitated by adding a saturated ammonium sulfate solution was dissolved in 20mM Tris buffer containing 2.5M sodium chloride, permeated through the gel, and further purified by high performance liquid chromatography.The molecular weight of the protein was determined by SDS-acrylamide gel electrophoresis. 70,000 to 80, calculated according to the law.
It is 000. Claim 2: Natronococcus
5% by weight of amylase-producing bacteria belonging to the genus
A method for producing halophilic alkaline amylase, which comprises culturing in an alkaline medium containing salt as described above, and separating and recovering halophilic alkaline amylase from the culture. [Claim 3] The halophilic alkaline amylase-producing bacterium is Natronococcus sp. Ah-36 (Natron
ococcus sp. 3. The method for producing halophilic alkaline amylase according to claim 2, wherein the strain is Ah-36). 4. The halophilic alkaline amylase producing bacterium is Natronococcus sp. Ah-471 (Natro
nococcus sp. 3. The method for producing halophilic alkaline amylase according to claim 2, wherein the halophilic alkaline amylase is a strain of A.Ah-471).