JP2002363279A - METHOD FOR PRODUCING POLY-gamma-GLUTAMIC ACID - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide new microorganisms producing a poly-γ-glutamic acid(PGA) and a method for producing the PGA by utilizing the same microorganisms. SOLUTION: This method for producing the PGA having 200,000-1,200,000 molecular weight in a good efficiency is to use the new microorganisms such as Bacillus pumilus KA strain, Bacillus pumilus E strain or Bacilus pumilus IF014367.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a method for producing poly-γ-glutamic acid, and a novel microorganism used for the production. More specifically, the present invention relates to a method for producing poly-γ-glutamic acid having a relatively small molecular weight using a microorganism belonging to Bacillus pumilus, and a novel microorganism used for the method.

[0002]

2. Description of the Related Art Poly-γ-glutamic acid (hereinafter referred to as PGA)
Is known as a viscous component of natto. In recent years, PGA has attracted attention not only for functional foods and cosmetics, but also as a biodegradable polymer material.
Although PGA is also produced by a chemical method, most of the PGA is produced by a microorganism because the production method by a microorganism is excellent in various points such as production efficiency, purification operation, and purity of the obtained product.

[0003] The microorganisms used for the production of PGA are mainly those belonging to the genus Bacillus, such as Bacillus subtilis, Bacillus licheniformis, and Bacillus licheniformis.
Bacillus anthrasis, Bacillus megaterium and the like have been used.

PGA by these Bacillus microorganisms
Is produced by culturing a microorganism of the genus Bacillus with aeration and agitation in a medium containing L-glutamic acid, and it has been reported that 10 to 50 g / L of PGA was accumulated in the culture solution (biodegradable plastic). Handbook). Also,
Since the molecular weight of PGA produced by Bacillus subtilis (typically, Bacillus natto) is generally 2,000,000 or more, when PGA accumulates in the culture solution, the culture solution becomes a gel, and is aerated and stirred. Inconveniences in production such as difficulties in culturing operations, etc., and difficulty in separating and purifying PGA from a culture solution have occurred. Furthermore, PGA may be hydrolyzed depending on the processing conditions when separating and purifying PGA from the culture solution, and the molecular weight is reduced to about 100,000. Therefore, only a very high molecular weight or a low molecular weight PGA can be obtained, and a PGA having an appropriate molecular weight cannot be obtained.

[0005]

DISCLOSURE OF THE INVENTION The present invention has found a novel microorganism which produces and accumulates poly-γ-glutamic acid having an appropriate molecular weight, and has excellent operability such as culturing, separation and purification using this microorganism. It is intended to provide a method for producing poly-γ-glutamic acid.

[0006]

Means for Solving the Problems The present inventors searched for a microorganism capable of producing and accumulating poly-γ-glutamic acid in a medium containing glutamic acid, and found that Bacillus pumilus (Bacillus) was used.
Us pumilus) have been found to have the ability to produce PGA having an appropriate molecular weight, thereby completing the present invention. By culturing these microorganisms, it has been found that PGA having an appropriate molecular weight is produced, the viscosity of the culture solution is remarkably low, and culturing operations such as aeration and agitation are easily performed. Is completed.

That is, the present invention relates to a method for producing poly-γ-glutamic acid, which comprises a step of culturing a microorganism belonging to Bacillus pumilus capable of producing poly-γ-glutamic acid.

[0008] In a preferred embodiment, the culture is performed in a medium supplemented with glutamic acid.

In a preferred embodiment, the microorganism is Bacillus pumilus strain KA (FERM P-1).
8314), Bacillus pumilus E strain (FERM P
-18315) or Bacillus pumilus IF014
367 strains.

[0010] In another preferred embodiment, the poly-γ-glutamic acid has an average molecular weight of 200,000 to 1.2 million.

[0011] The present invention further relates to a bacterium belonging to Bacillus pumilus capable of producing poly-γ-glutamic acid and having an average molecular weight of 200,000 to 12
It relates to 100,000 poly-γ-glutamic acid.

[0012] The present invention also relates to Bacillus pumilus capable of producing poly-γ-glutamic acid. The present invention
Furthermore, Bacillus pumilus KA strain (FERM P-
18314), and Bacillus pumilus E strain (FE
RM P-18315).

[0013]

BEST MODE FOR CARRYING OUT THE INVENTION
Culturing a microorganism belonging to Bacillus pumilus capable of producing GA. The microorganism belonging to Bacillus pumilus capable of producing PGA is not particularly limited as long as it has the ability to produce PGA. Is preferably used.

[0014] Such a microorganism is a PG having a moderate molecular weight.
Produce A. An appropriate molecular weight means that the average molecular weight of PGA is between 200,000 and 1.2 million, preferably the average molecular weight is 200,000 to 1,000,000, and more preferably the average molecular weight is 200,000 to 500,000. It is. PGA having such an average molecular weight is preferably used for food applications such as thickeners and mineral absorption promoters, cosmetic applications such as humectants, medical materials, biodegradable absorbent resins and the like.

The average molecular weight is TSK-GEL60.
Gel filtration chromatography using 00PWXL (manufactured by Tosoh Corporation) was performed, PGA was detected with a differential refractometer, and the value was determined from a calibration curve. Gel filtration chromatography is a PGA
Is centrifuged, the supernatant is dialyzed against distilled water, freeze-dried, dissolved in a 50 mM sodium phosphate buffer (pH 7.0), and dissolved in 50 mM phosphate containing 300 mM sodium chloride. Sodium acid buffer (pH 7.
Elution at 0) was performed.

The Bacillus pumilus strain KA and the Bacillus pumilus strain E used in the present invention are novel microorganisms isolated from the soil in Nishi-ku, Kobe by the present inventors. Bacteriological properties and classification of Bacillus pumilus KA strain and Bacillus pumilus E strain are as follows. The test and classification method for mycological properties are described in "Bergey's Manual of Determinative Bacteriology".
teriology) 9th edition (1994) "and"Bergey's Manual of Systematic Bacteriology "
(1986) ".

(1) Bacteriological properties of Bacillus pumilus KA strain Morphological properties (medium composition: broth agar medium) Cell shape: Bacillus Cell size: 0.5-0.8 × 1-2 μm Cell polyplasia: − Mobility: + Spores: − Gram Staining: + B. Cultural properties Standard agar plate culture: (growth) good, (color) yellow-white, (glossy) available Broth agar slant culture: same as plate culture Standard liquid culture: uniformly turbid Standard gelatin stab culture: liquefy gelatin Physiological properties Nitrate reductivity:-VP test: + Indole formation:-Starch hydrolysis:-Casein hydrolysis: + Gelatin hydrolysis: + Citric acid utilization: + Propionic acid utilization:-Catalase activity : + Oxidase activity: + Growth range (pH) 6.8: + 5.7: + Growth range (temperature) 40 ° C.:+50° C.:+w 55 ° C.:- (w: weak) Growth range (NaCl) 2% : + 5%: + 7%: + 10%: + Growth under anaerobic conditions-pH in VP medium: <6 Saccharide utilization and acid production L-arabinose: + D-xylose: + D-glucose: + D-mannose: +

(2) Bacteriological properties of Bacillus pumilus strain E Morphological properties (medium composition: broth agar medium) Cell shape: Bacillus Cell size: 0.5-0.8 × 1-2 μm Cell polyplasia: − Mobility: + Spores: + Gram Staining: + B. Cultural properties Gravy agar plate culture: (growth) good, (color) white, (glossy)
Yes Broth agar slant culture: Same as plate culture Broth liquid culture: Evenly turbid Broth gelatin stab culture: Liquefy gelatin Physiological properties Nitrate reductivity:-VP test: + Indole formation:-Starch hydrolysis:-Casein hydrolysis: + Gelatin hydrolysis: + Citric acid utilization: + Propionic acid utilization:-Catalase activity : + Oxidase activity: + Growth range (pH) 6.8: + 5.7: + Growth range (temperature) 40 ° C.:+50° C.:+w 55 ° C.:- (w: weak) Growth range (NaCl) 2% : + 5%: + 7%: + 10%: + Growth under anaerobic conditions-pH in VP medium: <6 Saccharide utilization and acid production L-arabinose: + D-xylose: + D-glucose: + D-mannose: +

The method for producing PGA of the present invention includes culturing the microorganism belonging to Bacillus pumilus. As a medium used for the culture, either a liquid medium or a solid medium is used. The medium components are not particularly limited, but, in general, a synthetic medium, natural medium, or any other medium containing carbon sources, nitrogen sources, inorganic salts, vitamins, and trace elements at appropriate concentrations used for culturing Bacillus microorganisms can be used. Any medium can be used.

Examples of the carbon source include sugars such as glucose, sucrose, starch, and amylose. As nitrogen sources, organic nitrogen such as peptone and meat extract, ammonia,
Inorganic nitrogen of an ammonium salt (for example, ammonium sulfate or the like) may be mentioned. Examples of the inorganic salts include phosphates (eg, sodium phosphate, sodium hydrogen phosphate, disodium hydrogen phosphate, potassium dihydrogen phosphate, etc.), magnesium salts (eg, magnesium sulfate, etc.), sodium chloride and the like. . Examples of vitamins include biotin. Also, as trace elements, copper,
Metals necessary for the growth of microorganisms, such as iron and manganese, may be mentioned. The above are all examples, and the present invention is not limited to these specific examples.

Preferably, 2 to 10% by weight of glutamic acid or a salt thereof is added to the medium. As the glutamic acid to be added, L-glutamic acid or a salt thereof which is easily used by microorganisms is preferable. D-glutamic acid or a salt thereof is also used as necessary, or according to the use and purpose of PGA. Thus, the resulting PGA can be poly-γ-L-glutamic acid, poly-γ-D, L-glutamic acid, or poly-γ-D-glutamic acid.

The cultivation is preferably performed under aerobic conditions such as shaking culture and aeration and stirring culture. The culture temperature is not particularly limited as long as the microorganism can grow,
50C, preferably about 25-45C, more preferably
About 30-40 ° C. 50 for heat-resistant microorganisms
In some cases, the culture may be carried out at a temperature of at least ℃. The culture time is not limited, but is usually about 3 to 5 days. Of course, a continuous culture method in which a new medium is added while continuously collecting the culture solution can also be employed. Furthermore, the microorganisms may be packed in a column as immobilized cells immobilized on a gel, a porous substance, or the like, and a culture medium may be flowed, continuously cultured, and the effluent may be continuously collected.

In the case of liquid culture, PGA is contained in the culture solution.
If immobilized, it accumulates in the effluent. PGA is collected from the culture, for example, by centrifuging the culture,
The cells are removed and the supernatant is recovered. For separation and purification of PGA from the supernatant, methods commonly used by those skilled in the art, for example, methods such as precipitation with alcohol, salting out, ion exchange, liquid chromatography, gel filtration chromatography, dialysis, and lyophilization are used alone. Alternatively, it may be performed in combination with physical recovery means, for example, centrifugation, filtration and the like.

The average molecular weight of the obtained PGA is about 200,000 to 1.2 million. The average molecular weight is considered to be influenced and changed by factors such as the microorganism used, culture conditions, and the concentration of glutamic acid to be added. Since the molecular weight of the produced PGA is smaller than that of other Bacillus microorganisms such as Bacillus natto, the culture solution or the effluent may be slightly viscous, but has little effect on the culture.

[0025]

EXAMPLES The present invention will be described below in detail with reference to Examples, but the present invention is not limited thereto.

(Example 1) Microorganisms were isolated from soil in Nishi-ku, Kobe, by a conventional method. The isolated microorganism is designated L
-Cultivation in a PGA medium (A) described below containing glutamic acid, centrifugation of the culture to remove cells, dialysis of the supernatant against distilled water, lyophilization, and 50 mM sodium phosphate buffer ( pH 7.0), and dissolved in the above TSK-GEL
PGA-producing strains were screened by column chromatography with 6000PWXL.

By the above screening, two strains of microorganisms, which produce PGA, named KA strain and E strain, were obtained. The KA strain produced PGA with an average molecular weight of 970,000, and the E strain produced PGA with an average molecular weight of 750,000. The mycological properties of these microorganisms are as described above, and based on these mycological properties, they were classified according to the above-mentioned Burgers manual, and all belonged to Bacillus pumilus, but were found to be different strains. Were named Bacillus pumilus KA strain and Bacillus pumilus E strain. These strains have been registered at the National Institute of Advanced Industrial Science and Technology Patent Microorganisms Depositary Center, respectively.
Bacillus pumilus KA strain (FERM P
18314) and Bacillus pumilus E strain (FE
RM P-18315).

(Example 2, Comparative Example 1) The following medium was used as the PGA production medium (A). The percentages are by weight unless otherwise specified. (Component A) Sodium L-glutamate 2% Sodium chloride 0.05% Disodium hydrogen phosphate dodecahydrate 0.42% Potassium dihydrogen phosphate 0.27% Ammonium sulfate 3% Biotin 0.00001% (Component B) Sucrose (sucrose) 5% (component C) magnesium sulfate heptahydrate 0.5% pH 6.4

In preparing the PGA production medium (A),
First, the pH of the mixture of the component A was adjusted to 6.4 with sulfuric acid,
B sterilized under the same conditions after sterilization at 120 ° C. for 20 minutes
The component (sucrose) and the component C (magnesium sulfate heptahydrate) were mixed so that the final concentration was the above concentration.

Bacillus pumilus KA strain (FERM)
P-18314), Bacillus pumilus E strain (FER
MP-18315), and Bacillus pumilus I
The FO14367 strain and, as a comparative example, Bacillus subtilis (hereinafter simply referred to as Bacillus natto) isolated from commercially available natto in a PGA production medium (A) at 37 ° C. for 24 hours with shaking. Obtained. Take 1 ml of each preculture and inoculate into a 500 mL Erlenmeyer flask containing 100 mL of PGA production medium (A), 160 rpm,
The cells were cultured with shaking at 37 ° C. for 120 hours.

After completion of the culture, each culture was centrifuged to obtain a supernatant. PGA was purified by dialysis and freeze-drying as described above, and the amount and molecular weight of the produced PGA were measured. Table 1 shows the results.

[0032]

[Table 1]

The amount of PGA was determined from the area ratio of a purified PGA sample on a gel filtration chromatograph. The molecular weight of PGA was measured by the above method.

The results show that Bacillus subtilis produces PGA with an average molecular weight of 2.6 million, whereas microorganisms belonging to Bacillus pumilus have about 92%.
It shows that PGA having a molecular weight of about 10,000 to 1.1 million is produced. Also, the viscosity of the medium was greatly reduced. The viscosity was measured by a method using a B-type viscometer.

(Example 3, Comparative Example 2) PG having the following composition
A production medium (B) was prepared. % Represents% by weight.

The medium was sterilized at 120 ° C. for 20 minutes, and glucose was sterilized and added separately as in Example 2. In the case of Bacillus natto of Comparative Example, the glucose concentration was 8%. Bacillus pumilus E strain and Bacillus natto were shake-cultured in this PGA production medium (B) at 37 ° C. for 24 hours to obtain a preculture. 1m of each preculture
50 mL containing 100 mL of PGA production medium (B)
Inoculate 0 mL Erlenmeyer flask, 160 rpm, 37 ° C,
Shaking culture was performed for 120 hours. After completion of the culture, P
The amount of GA and the average molecular weight were measured in the same manner as in Example 2.
Table 2 shows the results.

[0037]

[Table 2]

The results show that the Bacillus pumilus E strain produces almost the same amount of PGA as Bacillus natto, its estimated average molecular weight is about 240,000, and the average of PGA produced by Bacillus natto in Comparative Example This indicates that the molecular weight is about 1/10 of 2.2 million. In addition, Bacillus pumilus E
The viscosity of the culture of the strain was found to be about 110 times lower than that of Bacillus natto.

Example 4 A 3 L jar fermenter containing 2 L of PGA production medium (A) was inoculated with 100 ml of a preculture of Bacillus pumilus E strain that had been precultured overnight in the same medium. 2 L / 500 rpm, 37 ° C.
The cells were cultured for 120 hours with aeration at a rate of 1 minute (aeration and stirring culture). Take a portion of the culture and add Bacillus pumilus
E determined the molecular weight and the amount of PGA produced in the culture solution. The yield is 9.6 g / L and the average molecular weight is about 55
0000. The viscosity of the culture solution is 170 mPa · s
Met. The pH of the culture was adjusted to 3, the cells were removed by centrifugation, and the resulting culture supernatant was concentrated with an ultrafiltration membrane. A 4-fold amount of methanol was added to this concentrated solution, and the resulting white precipitate was collected by filtration, washed with methanol, and dried to obtain 13.7 g of poly-γ-glutamic acid (recovery rate: about 71%).

[0040]

By culturing a microorganism belonging to Bacillus pumilus, a polysaccharide having a molecular weight of 200,000 to 1.2 million can be obtained.
γ-glutamic acid (PGA) is produced. This PGA
Since the solution has a lower viscosity than conventional PGA having a molecular weight of 2,000,000 or more, it is excellent in operability such as culture, separation and purification, and can be produced extremely efficiently, easily and inexpensively.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI theme coat ゛ (reference) (C12P 13/14 C12R 1:07) F-term (reference) 4B064 AE02 CA02 CD13 CE03 DA10 4B065 AA15X AC14 BA22 BB12 BD14 CA16 CA41 CA50 4J001 EA36 GA20

Claims (8)

[Claims] 1. A method for producing poly-γ-glutamic acid, comprising a step of culturing a microorganism belonging to Bacillus pumilus capable of producing poly-γ-glutamic acid. 2. The method according to claim 1, wherein the culturing is performed in a medium supplemented with glutamic acid. 3. The microorganism according to claim 1, wherein the microorganism is Bacillus pumilus K.
The method according to claim 1 or 2, wherein the strain is A strain (FERM P-18314), Bacillus pumilus E strain (FERM P-18315) or Bacillus pumilus IF014367 strain. 4. The production method according to claim 1, wherein the poly-γ-glutamic acid has an average molecular weight of 200,000 to 1.2 million. 5. A poly-γ-polysaccharide having an average molecular weight of 200,000 to 1.2 million obtained by culturing a microorganism belonging to Bacillus pumilus capable of producing poly-γ-glutamic acid.
Glutamic acid. 6. A Bacillus pumilus capable of producing poly-γ-glutamic acid. 7. A Bacillus pumilus KA strain (FERM)
P-18314). 8. A Bacillus pumilus E strain (FERM)
P-18315).