JPH0269183A - Beta-1,3-glucanase and production thereof - Google Patents

Abstract

PURPOSE:To obtain the subject enzyme having excellent beta-1,3-glucan decomposition capability at a low cost by culturing an alkalophilic microorganism of genus Bacillus capable of producing beta-1,3-glucanase and having a specific property and collecting the produced objective enzyme. CONSTITUTION:An alkalphilic microorganism of genus Bacillus AG430 is cultured in a medium and the beta-1,3-glucanase produced and accumulated in the medium is collected therefrom. The beta-1,3-glucanase is reactive with beta-1,3-glucan having higher polymerization degree than laminaritetraose to decompose the beta-1,3-glucoside bond and produce, glucose laminaribiose and laminaritriose. The microbial strain has an optimum pH of 8.5-10.5, is stable at pH 4-12 under the heating condition at 40 deg.C for 30min and stable under the heating condition of pH7 and 100 deg.C for 20min and has optimum working temperature of about 65 deg.C. It is inactivated within 50min at pH7 and 100 deg.C, has a molecular weight of 32,000-38,000 (by gel-filtration) and and isoelectric point of 3.7-3.9 and is inhibited by mercuric chloride and silver nitrate.

Description

Detailed Description of the Invention [Field of Industrial Application] The present invention relates to a novel β-1,3-glucanase and a method for producing the same. The present invention relates to β-1°3-glucanase which is obtained by culturing a new alkaliphilic microorganism having the enzyme reaction and has an optimum pH for the enzyme reaction on the alkaline side, a method for producing the same, and the above-mentioned novel microorganism.

[Conventional technology]

β-1,3-glucanase optionally hydrolyzes β-L 3-D-glucosidic bonds, which are the main skeletons of laminarin, pachyman, etc., which have β-1,3-D glucosidic bonds in the molecule. It is an enzyme that produces laminario-oligosaccharides such as ribiose and laminalitriose.

First, storage polysaccharides found in brown algae, especially Laminaria genus, are well known as containing β-1,3-glucan.

Other known substances containing β-1,3-glucan include the polysaccharides constituting the cell walls of baker's yeast and molds, and the polysaccharides of the order Salmonales. Conventionally, these polysaccharides have been difficult to utilize industrially and have not been used much.

β-1,3-glucanase, which is known as an enzyme that optionally hydrolyzes β-1,3-glucan, has been the subject of research by many researchers. things have been considered. For example, arthropods [Comp, Bio
chem, Physiol,).

1987, 58.105-110] In particular, filamentous fungi [Enzyme Mic
rob.

Technol,) 1987.9, 89-93),
Actinomycetes [Apple Micropiol Biotychnol (Appl.

Microbiol, Biotechnol, ) 198
4. 20. 207-212], bacteria [Agr, Biol.

Chem, ) 1973.37.1449-1456) and other enzymes have been well studied.

However, many of these enzymes have poor temperature stability or require a long period of time for culturing1, which poses difficulties in using the enzymes industrially at low cost.

[Problem to be solved by the invention]

β-1, which exists in large quantities as a renewable resource in the natural world,
In order to effectively utilize 3-glucan, in particular to efficiently recover and utilize sugars such as glucose 5, laminaribiose, and laminaritriose by enzymatic hydrolysis of the substance, β-1,3-glucan, which has excellent heat resistance and β-1,3- Since extraction operations for glucan from various plants are mainly performed in alkaline conditions, it is necessary to have the optimal pH for enzymatic reactions on the alkaline side in order to simplify the neutralization operation and the decomposition process. is preferred.

Furthermore, by carrying out the enzymatic reaction at a high temperature, it is possible to prevent spoilage, increase the enzyme reaction rate, and increase the utilization and production of products, so it is desirable that the A temperature is also high.

However, as mentioned above, all conventional β1.3-glucanases have drawbacks such as insufficient temperature stability and the need for long culture times to produce the enzyme. Therefore, it has been difficult or unsatisfactory in terms of cost to utilize these enzymes on an industrial scale to obtain hydrolysis products of β-1°3-glucan.

Therefore, the object of the present invention is to develop a new β-1,3-glucan hydrolyzate that satisfies the various requirements in the enzymatic reaction described above and makes it possible to hydrolyze β-1,3-glucan economically and on an industrial scale. An object of the present invention is to provide 1,3-glucanase. Another object of the present invention is to provide a method for producing the above-mentioned novel β-1,3-glucanase. moreover,
Another object of the present invention is to provide a novel microorganism.

[Failure to solve the problem]

The present inventors conducted a wide search in the natural world to obtain microorganisms capable of producing enzymes having the various properties that β-13-glucanase should possess for industrial use. The present invention was completed based on the discovery that bacteria belonging to the genus Bacillus, which have an optimum pH for growth, produce an enzyme meeting the above-mentioned requirements and can be produced in large quantities.

That is, the present invention provides a novel β-1,3-glucanase, which has the following physicochemical properties.

(a) Action: Decomposes the β-1,3-glucoside bond of β-1,3-glucan to produce glucose, laminaribiose, and laminaritriose.

(b) Substrate specificity: Acts on β-1,3-glucans greater than laminar tetraose.

(c) Whole leopard pH and stable pH range: The optimum pH is 8.5 to 10.5, and it is stable within the pH range of 4 to 12 under heating conditions of 40° C. for 130 minutes.

(d) Stability against temperature: It is stable at pH 7 and heating at 100°C 12O for minutes.

(e) Range of suitable temperature for action: It has an optimum operating temperature around 65°C.

(f) Deactivation conditions: Under the treatment conditions of pH 7 and 100°C, it is completely deactivated in 50 minutes.

(g) Molecular weight by gel filtration method: 32,000-38,000 (h) Isoelectric point: 3.7-3.9 (S) Inhibition and activation: Inhibited by mercuric chloride and silver nitrate.

The present invention also relates to a method for producing the above-mentioned novel β-1,3-glucanase, and according to this method, the β-1,3-glucanase is
lic Bacillus), and the above β-1,3
- A method for producing the β-1,3 glucanase by culturing a glucanase-producing microorganism in a medium, producing and accumulating the enzyme in the medium, and separating and purifying the enzyme.

Furthermore, the present invention also includes the alkaliphilic Bacillus AG430 itself, which grows at a pH of 6.5 to 11.0.

The novel β-1,3 glupotase-producing strain AG 430 used in the method of the present invention was newly searched and isolated from the natural world by the present inventors.
This is a strain that produces 3-glucanase extracellularly. This strain was extracted from Bergey's Manual of Systematic Bacteriology (Bergey's Manual of Systematic Bacteriology).
l of Systematic Bacteriolo
gy) Volume 2 and The Genus Bacillus (The
Genus Bacillus, Department of Agriculture, USA
When identified according to the Cal ture version], it is an aerobic sporobacillus, is motile, has pericenchyma, and has a positive Durham stain and catalase test, so it is clear that it belongs to the genus Bacillus. However, since it grows well on the alkaline side of pH 6.5 to 11.0, it was recognized as a new strain that is taxonomically different from the known Bacillus genus, and it is classified as an alkaliphilic Bacillus genus AG 430.
And so.

Table 1 below shows various mycological properties of the isolated β-1,3-glucanase producing bacterium AG 430.

Table 1 Extracellular β-1° Mycological properties 1, Morphological properties Shape Size Motile Durham staining Sporangia 2, Growth in various media Broth liquid medium Broth agar plate Flesh gelatin Puncture culture 3 - Glucanase producing bacteria Bacillus 0.4-0.5X2. O~3.0μ! Presence (periflagellate) Positive A swollen brittle fungal ring is created, with a white periphery and a convex circular shape, and the color is cloudy 5.0% salt broth liquid medium 7.5% salt broth liquid medium; 3 Biochemical properties * With gelatin Hydrolysis of casein Hydrolysis of starch Utilization of citric acid Reduction of nitrate VP-test Production of indole Hydrogen sulfide Utilization of inorganic nitrogen sources Production of pigment Urease Oxidase Catalase Attitude towards enzymes (anaerobic) * 10: Grows - 2 Does not grow + + ↑ + 10+ or positive or negative 4. Growth pH and temperature Growth pH 6.5-11.0 Growth temperature ~50℃ 5. Sugar assimilation I7-arabinose, L-xylose, D-glucose, D-mannose , D-fructose, D-galactose, maltose, sucrose, lactose, trehalose, raffinose, starch, inositol, D-sorbitol, D
-Assimilates mannitrite and cellulose. Also, L-arabinose, L-xylose, D-mannose, D-galactose, maltose, lactose, trehalose,
Acid is produced from starch, D-sorbitol, and D-mannite, but no gas is produced.

The above-mentioned bacteriophilic Bacillus AG 430 has been deposited with the National Institute of Microbial Technology, Agency of Industrial Science and Technology, with deposit number FERMP-1.
It has been deposited as 0256.

The novel method for producing extracellular β-L 3-glucanase of the present invention will be explained in more detail. β-1,3 as above
- Inoculate glucanase-producing bacteria into an appropriate medium, and from the viewpoint of bacterial growth temperature, at 30-40°C for 48-72 hours.
The culture is carried out aerobically, and the medium contains a carbon source, a nitrogen source, as well as inorganic salts, trace nutrients, etc. as necessary.

First, as a carbon source, any of the conventionally known +i +x materials can be used, and typical examples thereof include Bakiman, Laminaran, and plants containing them.

There are also no particular restrictions on the nitrogen sources, including yeast extract, peptone, meat extract, corn stabilica, amino acid solution,
Examples include organic nitrogen such as soybean meal, and inorganic nitrogen such as ammonium sulfate, ammonium nitrate, and ammonium chloride, which are inexpensive and easily available.

It goes without saying that the organic nitrogen source also serves as a carbon source. Furthermore, in addition to such carbon sources and nitrogen sources, it is also possible to add various commonly used salts, such as inorganic salts such as magnesium salts, potassium salts, phosphates, and iron salts, and vitamins. .

Suitable media for use in the method of the invention include, for example, 1% pachyman, 0.5% polypeptone, 0.5%
yeast extract, 0.1% KH□po, and 0.00
It can be a liquid medium containing 5% VIgSOa.7H20.

Furthermore, since the growth pH of the microorganisms used in the method of the present invention is within the basic range, it is necessary to adjust the pH value of the medium using an appropriate alkali.

For this purpose, 1% sodium hydrogen carbonate can be cited as a typical example, but alkaline reagents such as sodium hydroxide, sodium carbonate, sodium phosphate, and calcium hydroxide can also be used without being limited thereto.

Since the new microorganism used in the method of the present invention, alkalophilic Bacillus AG430, produces β-1,3-glucanase extracellularly, the produced β-13-glucanase is released into the culture medium and stored there. Accumulated. Cultivation of this bacterium can be carried out either by a hatch method or a continuous method, and the produced enzyme can be separated and purified, for example, as follows.

That is, first, the bacterial cells in the culture solution are removed by centrifugation, filtration, etc., and then the resulting supernatant (crude enzyme solution) is directly used as β-1.
, 3-glucan, which would be economically advantageous. It can also be used after further purification. For this purpose, for example, salting out with ammonium sulfate, solvent precipitation with ethanol, acetone, isopropanol, etc., ultrafiltration, gel filtration,
It can be purified by a general enzyme purification method using an ion exchange resin or the like.

Below, one example of a preferred purification method for β-1,3-glucanase of the present invention will be explained.

The AG-430 strain belonging to the alkalophilic Bacillus genus is inoculated into a medium such as the one described above, and the culture solution obtained by culturing it aerobically at 35°C for 72 hours is heated at 8000 rpm, 4
Centrifuge at ℃ for 20 minutes to remove bacterial cells, 1. ! Obtain the supernatant liquid of 1. Then, the supernatant liquid was added with acetone 6 at -20°C.
! Add and leave at -20°C overnight. The supernatant 62 is discarded, and the remaining liquid and the resulting precipitate are centrifuged.

After drying the collected precipitate and removing acetone, it is dissolved in 50 mM phosphate buffer (pH 8,0) and dialyzed twice for 2 hours against the same buffer at 4°C.

The resulting precipitate was centrifuged and the supernatant liquid was equilibrated with the same buffer as DEAE-3 epharose CL-6B.
5 containing 0.1-0.5M NaCl after eluting unnecessary proteins with 50mM phosphate buffer (pH 5, 6).
The enzyme is eluted by a concentration gradient method with 0 mM phosphate buffer (pH 5, 6).

The eluted active fractions were collected and the average fractionated molecular weight was 10.000.
Concentrate using an ultrafiltration membrane. Concentrated enzyme is 5epha
Fill in dex G'-75 and add 10mM phosphate buffer (
Elute using pH 7.0). The eluted active fractions are collected, unnecessary proteins are adsorbed with hydroxyapatite equilibrated with the same buffer solution, and the active fractions are collected and concentrated using an ultrafiltration membrane with an average molecular weight cutoff of 10,000.

Concentrated enzyme was packed into 5 ephadex G-75 and 1
Elute using 0mM phosphate buffer (pH 7,0).

The active fraction thus obtained was concentrated, and 60 mg of a homogeneous enzyme preparation was obtained by polyacrylamide gel electrophoresis (gel concentration 14.0%), with an activity yield of 46%.

The method for measuring β-1,3-glucanase activity and the method for displaying the activity are as follows. That is, 50m1'ITr
1% dissolved in is-HCIll buffer (pH 9,0)
(W/V) Mix 0.2 d of laminaran solution with 0.01 d of appropriately diluted enzyme solution, react for 40°C'' rlo minutes, and then add DNS test solution [J, R, Summer G,
Somers, E. Laboratory Experiments in Biological Chemistry, Academic Press, New York (J.R.Suvwer, G',
E.

SOMERS, “Laboratory Expert
iments in Biological Chemi
stry,' Academic Press, Ne
York), 3435 pages, 1944) 1.0
d to stop the enzymatic reaction. Heat in a boiling water bath for 5 minutes, then rapidly cool, add 4 ml of water, and stir thoroughly. Using a similarly treated 0.2 mg/m glucose solution as a standard, the degree of coloration is measured using a spectrophotometer (wavelength: 510 nm). The unit of enzyme activity is expressed as one unit, which is the amount of enzyme required to produce reducing sugar equivalent to 1 mg of glucose per minute under the above-mentioned conditions.

Table 2 shows a comparison of the physicochemical and enzymatic properties of β-1,3-glucanase obtained by the method of the present invention and β-1,3-glucanase derived from conventionally known microorganisms.

(Margins below this page) [Action] β-1, which exists in large quantities as a renewable resource in nature,
3-glucan is not very effectively utilized.

In order to effectively utilize this β-1,3-glucan, it is necessary to develop an enzyme (β-1,3-glucanase) that can efficiently hydrolyze it. In other words, it is important to obtain an enzyme that can hydrolyze β-1°3-glucan with high efficiency. It is important for purposes such as recovering and utilizing this, or decomposing and removing it after using it as β-1,3-glucan itself.

In such uses, it is extremely desirable from the viewpoint of industrial application that the β-1,3-glucanase be stable at high temperatures and have an optimum pH for the enzymatic reaction on the alkaline side.

For this purpose, β-1゜3-
Although glucan hydrolases have been discovered, none of them have been fully satisfactory from an industrial standpoint. That is,
All of the enzymes that have been studied so far have had poor high temperature stability, or the culture time of enzyme-producing microorganisms has been extremely long.

Therefore, the present inventors conducted various searches and found that bacteria belonging to the genus Alkaliphilic Bacillus produce effective β-1,3-glucanase at a high production rate.

This enzyme satisfies all of the requirements for the hydrolysis reaction of β-1,3-glucan, and has solved all of the problems of conventionally known enzymes.

That is, first of all, since the β-1,3-glucanase enzyme of the present invention is produced extracellularly by the above-mentioned microorganism, isolation and purification are extremely simple, and significant improvements are expected in terms of labor and production costs. can.

Furthermore, it has excellent high-temperature stability and has an optimal pH for enzymatic reactions on the alkaline side, so it can be easily neutralized after extracting β-1,3-glucan from various plants under alkaline conditions. Since it is possible to subject the enzyme to the enzymatic decomposition reaction as it is without further treatment, the work is significantly simplified, and since the use of extra reagents becomes unnecessary, the production cost of the decomposition product can also be reduced.

Thus, according to the novel enzyme of the present invention, it becomes possible to degrade and utilize β-1,3-glucan on an industrial scale. It is also extremely advantageous in terms of cost performance.

〔Example〕

Hereinafter, the present invention will be explained in more detail with reference to Examples.

Example 1 Alkaliphilic Bacillus AG-430 strain (FERM P
10256) in a 500 d Erlenmeyer flask, Bakiman 2% polypeptone 0.5%, yeast extract 0.5%, K
zHPOao, 1%Mg5O, 7H200,005
%, and culture solution 100 containing 1% sodium bicarbonate.
m1pH9) and incubated at 33°C for 48 hours at 20Orp.
Shaking culture was carried out at m. Then, β-1 in the culture supernatant,
As a result of measuring 3-glucanase activity as described above, 1
It was 6 units/d.

Example 2 Alkaliphilic Bacillus AG-430 strain (FERM P
10256) wo2 Bakiman 1%, polypeptone 0.5%, yeast extract 0.5%, K in an A-volume Erlenmeyer flask
lLPo, 0.1%, Mg5O, 7H200,00
5%, and 600% culture medium containing 1% sodium carbonate.
rnl (pH 10) and cultured aerobically at 37°C for 72 hours, the resulting culture solution was incubated at 8000 rpm.
4 Centrifuge for 20 minutes at 500℃ to remove bacterial cells.
Obtain the supernatant liquid of d.

The β-1,3-glucanase activity in the culture supernatant was measured as described above and was found to be 6 units/ml.

Next, acetone 21 at -20°C was added to the supernatant, and -
Leave at 20°C overnight and centrifuge the precipitate.

After drying the collected precipitate and removing acetone, it is dissolved in 50 mM phosphate buffer (pH 8,0) and dialyzed twice for 2 hours against the same buffer at 4°C. The resulting precipitate was centrifuged and the supernatant liquid was equilibrated with the same buffer as DEAE-
Adsorbed to Sepharose GL-5B, 50mM
After eluating unnecessary proteins with phosphate buffer (pH 5, 6),
The enzyme is eluted by a concentration gradient method of 50 mM phosphate buffer (pH 5,6) containing 1-0.5 M NaC+. The eluted active fractions were collected and the average fractional molecular weight was 10,00.
Concentrate using a 0.0 ultrafiltration membrane. Concentrated enzyme is 5ep
hdex G-75 and 10mM phosphate buffer (
Elute using pH 7.0). The eluted active fractions are collected, unnecessary proteins are adsorbed with hydroxyapatite equilibrated with the above buffer solution, and the active fractions that have passed through are collected.

The active fraction thus obtained was concentrated and 20 mg of the enzyme preparation was obtained.
I got it. The activity yield was 52%, and the β-1,3-glucanase activity was 7O units/d.

[Effect of the invention] As described in detail above, the novel β-1゜3-
Glucanases have an aph of the enzyme reaction on the alkaline side and also have excellent high temperature stability. Therefore, β-1,3
- The enzymatic decomposition reaction of glucan can be carried out on the alkaline side, which makes it possible to carry out the enzymatic decomposition reaction immediately after the β-1,3-glucan extraction step.

Furthermore, since the enzymatic reaction can be carried out at high temperatures, the reaction rate can be significantly increased.

Thus, with the β-1,3-glucanase of the present invention, it is possible to industrially advantageously produce a decomposition product of β-1,3-glucan, with high decomposition efficiency and production of decomposition products. This makes it possible to achieve high performance and reduce manufacturing costs.

Furthermore, since the β-1,3-glucanase of the present invention can be obtained from a microorganism belonging to the alkalophilic Bacillus genus that is produced extracellularly, it can be easily isolated and purified, and therefore can be mass-produced at low cost. .

Claims (1)

[Scope of Claims] (1) Action of a novel β-1,3-glucanase (a) having the following physicochemical properties: Decomposes the β-1,3-glucoside bond of β-1,3-glucan, , laminaribiose, and laminalitriose. (b) Substrate specificity: Acts on β-1,3-glucans greater than laminar tetraose. (c) Optimal pH and stable pH range: The optimal pH is 8.5 to 10.5, and is stable within the pH range of 4 to 12 under heating conditions at 40° C. for 30 minutes. (d) Stability against temperature: Stable at pH 7, 100° C., and heating for 20 minutes. (E) Range of optimum temperature for action: The optimum temperature for action is around 65°C. (f) Inactivation conditions: Under the treatment conditions of pH 7 and 100°C, the activity is completely inactivated in 50 minutes. (g) Molecular weight by gel filtration method: 32,000-38,000 (h) Isoelectric point: 3.7-3.9 (li) Inhibition and activation: Inhibited by mercuric chloride and silver nitrate. (2) The following physical and chemical properties: (a) Action: Decomposes the β-1,3-glucoside bond of β-1,3-glucan to produce glucose, laminaribiose, and laminaritriose. (b) Substrate specificity: Acts on β-1,3-glucans greater than laminar tetraose. (c) Optimal pH and stable pH range: The optimal pH is 8.5 to 10.5, and is stable within the pH range of 4 to 12 under heating conditions at 40° C. for 30 minutes. (d) Stability against temperature: Stable at pH 7, 100° C., and heating for 20 minutes. (E) Range of optimum temperature for action: The optimum temperature for action is around 65°C. (f) Inactivation conditions: Under the treatment conditions of pH 7 and 100°C, the activity is completely inactivated in 50 minutes. (g) Molecular weight by gel filtration method: 32,000-38,000 (h) Isoelectric point: 3.7-3.9 (li) Inhibition and activation: Inhibited by mercuric chloride and silver nitrate. A microorganism belonging to the alkalophilic Bacillus genus, which has the ability to produce β-1,3-glucanase and whose growth pH is on the alkaline side, is cultured in a medium, and the β-1,3-glucanase is produced and accumulated in the medium. The above-mentioned method for producing β-1,3-glucanase, which is characterized in that the β-1,3-glucanase is collected. (3) The β-1,3-
Method for producing glucanase. (4) The method for producing β-1,3-glucanase according to claim 2 or 3, wherein the pH of the culture solution is within the range of 5 to 10. (5) Alkaliphilic Bacillus AG430 with a growth pH of 6.5 to 11.0