JP2767551B2 - Method for producing bacterial cellulose using non-PQQ-producing strain - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a microorganism belonging to the genus Acetobacter and having the ability to produce cellulosic substances (this microorganism is hereinafter referred to as "cellulose-producing acetic acid bacteria").
And a method for producing a cellulosic substance (bacterial cellulose: BC) using said strain. In particular, the present invention relates to a method for producing a cellulosic substance by culturing the non-PQQ-producing strain in a medium to which PQQ has been added.

[0002]

2. Description of the Related Art Cellulosic substances are edible, are used in the food field, and have excellent water-based dispersibility.
Retention of viscosity of cosmetics or paints, strengthening of food material dough,
It has industrial value in retaining moisture, improving food stability, as a low-calorie additive or as an emulsion stabilizing aid. In addition, the disintegrated product of the cellulosic substance has various industrial uses as a reinforcing agent for polymers, particularly aqueous polymers, based on the structural and physical characteristics of microfibrils. Since such a defibrated material has a high tensile modulus, a material obtained by solidifying the cellulosic deflated product into a paper or solid form is expected to have excellent mechanical properties based on the structural characteristics of microfibrils, and various industrial materials. As an application.

[0003] Conventionally, a method for producing cellulose by culturing a microorganism belonging to the genus Acetobacter has been known. For example, JP-A-62-265990, JP-A-61-221201, and the like disclose such information. As a nutrient medium suitable for culturing cellulose-producing acetic acid bacteria, a Schramm / Hestrin medium comprising a carbon source, peptone, yeast extract, sodium phosphate and citric acid (Schramm et al., J. General Biology, ll. , pp.123-129,
l954) is known. However, when the nutrient medium was shaken or aerated with stirring, the resulting cellulose production was low and the production rate was not always satisfactory. In addition, a medium containing corn steep liquor (CSL), malt extract, or the like in addition to the above nutrient medium is known. Specific components contained in these natural nutrients (peptone, yeast extract, CSL, malt extract, and the like) are known. Is not known to be involved in promoting cellulose production. Inositol, currently known as a factor for promoting the production of cellulose by specific nutrients in the medium,
Phytic acid and pyrroloquinoline quinone (PQQ) (Japanese Patent Publication No. 5-1718); Mitsuo Takai, Journal of Paper Technology Association, No. 42
Vol. 3, No. 3, pp. 237-244), but the amount of cellulose produced is still insufficient, and the effect of these on shaking or aeration and stirring cultures was not clear. On the other hand, the applicant of the present invention has proposed a carboxylic acid or a salt thereof (Japanese Patent Application No. 5-1)
No. 91467), invertase (Japanese Patent Application No. Hei 5-3314).
No. 91) and methionine (Japanese Patent Application No. 5-335564).
Has been found to improve the productivity of cellulosic substances by adding the compound to the medium. Furthermore, it has been found that the addition of an appropriate amount of PQQ to the culture medium promotes the growth of acetic acid bacteria (Agricultural Chemistry Society, Vol. 66, No. 11).
1665-1669, 1992).

[0004]

SUMMARY OF THE INVENTION An object of the present invention is to provide a new method for producing a cellulosic substance economically and at a high yield using a cellulosic acetic acid bacterium.

[0005]

Means for Solving the Problems The present inventors have conducted various studies in order to achieve the above object, and newly created a PQQ non-producing strain belonging to a cellulose-producing acetic acid bacterium. It has been found that by culturing the strain, the productivity of the cellulosic substance is significantly improved. That is, the present invention provides a non-PQQ-producing strain belonging to a cellulose-producing acetic acid bacterium, culturing the non-PQQ-producing strain, and adding cellulose,
Provided are a method for producing a cellulosic substance, which comprises producing and accumulating a cellulosic substance, and collecting the substance, and a method for adding PQQ to the medium at a specific concentration. Further, the present invention provides a cellulosic substance obtained by such a production method.

The non-PQQ-producing strain used in the present invention is a cellulose-producing acetic acid bacterium, for example, Acetobacter sp. BPR2001.
Strain, Acetobacter xylinu
m ) ATCC 23768, Acetobacter xylinum ATCC 23768, Acetobacter pasturianus ( A. pasteurianus ) ATCC 10245, Acetobacter xylinum ATCC 14851, Acetobacter xylinum ATCC 11142, and Acetobacter xylinum ATCC 10821, etc. using NTG (nitrosoguanidine). Can be created by chemical mutation treatment according to the method of (1). Among these, Acetobacter species BPR2001 ( Acetobacter s
(p.BPR2001) The taxonomic properties of the strain named strain were bacillus in morphology, negative in Gram stain, negative in sporulation, aerobic in oxygen, positive in catalase, negative in oxidase, and ethanol. Is positive for acetic acid production, positive for acetate oxidation, and positive for lactate oxidation, and is suitable for creating a non-PQQ-producing strain of the present invention. Therefore, a PQQ non-producing strain belonging to Acetobacter species is one of the preferred embodiments of the present invention. The Acetobacter sp. BPR2001 strain was deposited on February 24, 1993 at the Patent Microorganisms Depositary Center, National Institute of Bioscience and Human-Technology, Ministry of International Trade and Industry (accession number: FERM P-1).
3466) and subsequently transferred to a deposit under the Budapest Treaty on the International Recognition of Patent Deposits on February 7, 1994 (Accession No. FERM BP-4545).

[0007] Methods for chemical mutation treatment using a mutagen such as NTG include, for example, Bio Factors, Vol. 1, p. 297-302.
(1988) and J. Gen. Microbiol, Vol. 135, p. 2917-2.
929 (1989). Therefore, those skilled in the art can use the PQQ of the present invention based on these known methods.
Non-producing strains can be obtained. The non-PQQ-producing strain of the present invention can also be obtained by other mutation methods, for example, irradiation. BP which is an example of the non-PQQ non-producing strain
The R3001C strain was deposited on May 2, 1994 at the Patent Microorganisms Depositary of the Institute of Biotechnology and Industrial Technology, Ministry of International Trade and Industry, and has been assigned the accession number FERM P-14297. It goes without saying that the scope of the present invention is not limited to this deposited strain. BPR3001C
The strain is only one specific example of the present invention. If the strain belongs to a cellulose-producing acetic acid bacterium and does not substantially produce PQQ, the “PQQ of a cellulose-producing acetic acid bacterium” of the present invention is used.
Non-producing strains ".

[0008] In the composition of the medium used in the production method of the present invention, sucrose, glucose, fructose, mannitol, sorbitol, galactose,
Maltose, erythrit, gadnit, glycerin,
Ethylene glycol, ethanol and the like can be used alone or in combination. Furthermore, starch hydrolyzate, citrus molasses, beet molasses, beet juice, sugarcane juice, fruit juices including citrus fruits, etc. containing these can also be used in addition to shoe cloth. Also,
As a nitrogen source, ammonium sulfate, ammonium chloride,
An organic or inorganic nitrogen source such as ammonium salt such as ammonium phosphate, nitrate, and urea can be used, or Bact-Peptone, Bact-Soyton
e, Yeast-Extract, and soybeans may be used. Amino acids, vitamins, fatty acids, nucleic acids, 2,7,9-tricarboxy-1H pyrrolo [2,3-5] -quinoline-4,5 as organic micronutrients
-A dione may be added. When using an auxotrophic mutant that requires amino acids or the like for growth, it is necessary to supplement the required nutrients. As the inorganic salts, phosphates, magnesium salts, calcium salts, iron salts, manganese salts, cobalt salts, molybdates, red blood salts, chelate metals and the like are used.

The pH of the culture is between 3 and 7, preferably 5
Control near. The cultivation temperature is in the range of 10 to 40 ° C, preferably 25 to 35 ° C. The concentration of oxygen supplied to the culture tank may be 1 to 100%, preferably 21 to 80%. Those skilled in the art can appropriately select the composition ratio of each component in the medium, the inoculation of the cells into the medium, and the like, depending on the culture method. It has been found that the use of a non-PQQ-producing strain in the production method of the present invention improves the yield of cellulosic substance several to several tens of times as compared with the conventional method. Furthermore, in the production method of the present invention, a specific concentration of PQQ in the medium, that is, about 0.001 ng / ml to about 0.1 ng / ml, particularly about 0.001 ng / ml to about 0.01 ng.
An unexpected and surprising finding was that the addition of PQQ at a concentration of / ml further increased the productivity of the cellulosic material. In the method of the present invention, the culture method is not limited, and may be any of stationary, shaking, and aeration / agitation culture. One of the features of the method of the present invention is that the cellulosic productivity is not affected even by culturing under shaking or aeration and stirring. In addition, any of so-called batch fermentation, feed batch fermentation, repeated batch fermentation, and continuous fermentation can be used. Further, the stirring means can be arbitrarily selected from conventionally known means such as an impeller, an air-lift fermenter, a pump-driven circulation of fermentation broth, and a combination of these means.

The cellulosic substance produced by the method of the present invention may be recovered as it is, or may be subjected to a treatment for removing substances other than cellulosic substances such as bacterial cells contained in the substance. To remove impurities, washing with water, dehydration under pressure, washing with diluted acid, washing with alkali, treatment with a polar organic solvent such as toluene and ethyl acetate, treatment with a bleach such as sodium hypochlorite and hydrogen peroxide, and bacteria such as lysozyme Removal of impurities from cellulosic substances by treatment with solubilizing enzymes, treatment with surfactants such as sodium lauryl sulfate and deoxycholic acid, and heating and washing in the range from room temperature to 200 ° C, alone or in combination Can be. The cellulosic material thus obtained in the present invention includes cellulose, a substance containing a heteropolysaccharide having cellulose as a main chain, and a substance containing glucan such as β-1,3, β-1,2. Components other than cellulose in the case of heteropolysaccharide are mannose, fructose, galactose, xylose,
Hexoses such as arabinose, rhamnose, glucuronic acid,
Pentose and organic acids. These polysaccharides may be a single substance, or two or more polysaccharides may be mixed by hydrogen bonding or the like.

[0011]

The present invention will be described in more detail with reference to the following examples. Example 1 A BPR3001C strain, which is a non-PQQ-producing strain of the present invention, was created by the method described below. (1) Mutation treatment with NTG BPR2001 was converted from Cglyc-F
The cells were inoculated into a 750 ml roux flask containing 100 ml of ru medium, and cultured at 28 ° C. for 3 days. After the culture, the roux flask was shaken well to remove the cells from the cellulose membrane, and then the culture solution was aseptically filtered with gauze to obtain a bacterial solution as a filtrate.
2 ml of the obtained bacterial solution was inoculated into a 20 ml CSL-Fru medium (100 ml flask) containing 0.2% cellulase (Meisserase P1), and cultured at 28 ° C. for 24 hours with shaking at 180 rpm. After the culture, 10 ml of the culture solution was inoculated into 10 ml (100 ml flask) of a CSL-Fru medium containing 0.2% cellulase, and further cultured under shaking under the same conditions for 3 hours. The cells were collected from the culture by centrifugation (5000 rpm, 5 min), and further washed with 50 mM phosphate buffer (pH 6.5). After washing, the cells were resuspended in 20 ml phosphate buffer. An NTG solution (100 μg / m 2) was added to 4.5 ml of the cell suspension.
l) Add 0.5 ml and add 3 ml in a 20 ml assist tube.
Shake at 0 ° C. for 30 minutes. At this time, a control to which 0.5 ml of sterilized water was added was also prepared as a control. After completion of the shaking, the cells were collected by centrifugation, and further washed three times with a CSL-Fru medium. 0.2% after washing
The cells were suspended in 5 ml of CSL-Fru medium containing cellulase, and 0.1 ml of the suspension was used for measurement of viable cell count.
The resultant was spread on a u-plate, and the rest was cultured with shaking at 30 ° C. overnight to fix the mutation. Add an equal volume of 30% glycerol to the culture solution and dispense 1 ml at a time into the assist tube.
Stored frozen at -80 ° C. The survival rate under the above conditions was about 1.4%.

(2) Screening for non-PQQ-producing strain NTG-treated BPR2001 was plated on a YPG plate containing 100 mg / l bromocresol green, and cultured at 28 ° C for 3 to 5 days. After the culture, a colony that did not produce a yellow halo was selected. Further, by selecting a strain having no ethanol oxidase activity from the selected colonies, the BPR3001C strain and other P
A QQ non-producing strain was obtained.

[0013] Next, the bacteria are separated from the glycerol stock by CSL.
-Inoculated in a 750 ml roux flask charged with 100 ml of Fru medium, and incubated at 28 ° C. for 3 days. After the culture, shake the roux flask well to remove the cells from the cellulose membrane, and then add 12.5 ml of the bacterial solution to CS containing 0.2% mecerase.
Inoculating a flask containing 112.5 ml of L-Fru medium,
Shaking culture was performed at 28 ° C. and 180 rpm for 24 hours. About these fungi, M. Ameyama, M. Nonobe, E. Shinagawa,
K. Matsushita, and O. Adachi, Analytical Biochem.,
151, 263-267 (1985). The PQQ concentration was measured. Table 1 shows the results.

[0014]

[Table 1] From Table 1, it can be seen that the BPR3001C strain does not substantially produce PQQ in the medium and the cells.

Example 2 Using the BPR3001C strain created in Example 1, a cellulosic substance was produced by the production method of the present invention.
The culture conditions by flask culture are as follows. -Flask culture 75 containing 100 ml of medium from glycerol stock
The cells were inoculated in a 0-ml roux flask and cultured at 28 ° C. for 3 days. After the culture, the roux flask was shaken well to remove the cells from the cellulose membrane, and 12.5 ml of the bacterial solution was inoculated into a 500 ml vertical baffle flask containing 112.5 ml of a medium.
The cells were cultured at 180 ° C. for 4 days. The medium is CSL-
Glc was used. The culture conditions for the jar culture are as follows.・ 1l jar culture 100ml of CSL-Fru medium from glycerol stock
And inoculated in a 750 ml roux flask,
The culture was allowed to stand for a day. After the culture, shake the well flask to remove the cells from the cellulose membrane.
Inoculate a 500 ml flask containing 12.5 ml of medium,
The cells were cultured at 8 ° C. and 180 rpm for 3 days. The culture solution is aseptically disintegrated with a blender, and 60 ml of the mixture is mixed with 540 ml of C
Inoculate a 1-liter jar charged with SL-Glc medium,
Main culture is performed while controlling the rotation speed automatically so that the dissolved oxygen amount (DO) becomes 3.0% or more while controlling the pH to 4.9 to 5.1 with NH ₃ gas and 1NH ₂ SO _4. Was. The above results are shown in Tables 2 and 3.

[0016]

[Table 2] Carbon source glucose

[0017]

[Table 3] Carbon source Glucose Incubation time 45 hours

Example 3 Next, the non-PQQ-producing strain BPR3001C of the present invention was
L-Suc medium (however, sucrose 70 g / l, CSL
10 ml / l), and PQQ was further reduced to 0.001 to 10
After adding ng / ml, the cells were cultured in a 1-liter jar in the same manner as in Example 2. The results obtained are shown in Table 4 below.

[0019]

[Table 4] From these results, it can be seen that the production of cellulosic substances is further improved by adding an appropriate amount of PQQ during the cultivation of the non-PQQ-producing strain of the present invention. At this time, the growth itself of the strain was not affected by the addition of PQQ. The composition of the medium described in the present specification is as follows.

CSL-Fru medium

Table 5 Component final concentration (mM) (NH ₄ ) ₂ SO ₄ 25 KH ₂ PO ₄ 7.3 MgSO ₄ 1.0 FeSO ₄ 0.013 CaCl ₂ 0.10 Na ₂ MoO ₄ 0.001 ZnSO ₄₀ 0.006 MnSO ₄ 0.006 CuSO ₄ 0.0002 Vitamin mixture (described below) 10 ml / 1 carbon source qs CSL qs Defoamer 0.01 v / v% Final pH = 5.0 ± 0.2 (Unless otherwise specified, Fructose 40g / l, C
SL 20 ml / l) CSL-Glc medium The fructose of the CSL-Fru medium was replaced with glucose (unless otherwise specified, glucose 40 g / l).
l, CSL 20 ml / l) CSL-Suc medium The fructose of the CSL-Fru medium was changed to sucrose (unless otherwise specified, sucrose 40 g / l, CSL
20ml / l)

[0021]

[Table 6]

YPF medium Yeast extract (Difco) 5 g / l Bacto peptone 5 g / l Fructose 40 g / l YPG medium Yeast extract (Difco) 5 g / l Bacto peptone 5 g / l Glucose 40 g / l Agar medium is added with 2% agar. .

In the above table, the amount of cellulose (g / l)
After the completion of the culture, the solid matter in the jar fermenter was accumulated, and the components were removed by washing with water. After that, the cells were treated in a 1% NaOH aqueous solution at 110 ° C. for 20 minutes to remove the cells. Further, the resulting cellulose was washed with water until the washing liquid became nearly neutral, and then vacuum-dried at 80 ° C. for 12 hours, and the dry weight was measured. The yield (%) was determined as follows. Calculation of yield to consumed sugar (%) The yield to consumed sugar was calculated as the yield to consumed sugar as follows.

[Number 1] _{Y BC = BC / (RC MF} -RC BF) * 100 Y BC: vs. consumption sugar yield (%) BC: BC accumulation amount (g / l) RC _MF: medium sugar concentration (g / l ) RC _BF : Sugar concentration (g / l) of medium after culturing

[0024]

According to the present invention, in the production of a cellulosic substance by the method of the present invention, by using a non-PQQ-producing strain of cellulosic acetic acid bacteria, the productivity of the cellulosic substance is remarkably improved as compared with the conventional method. Furthermore, by adding an appropriate amount of PQQ to the medium, the production of cellulosic substances is further improved. This indicates that the method of the present invention can produce cellulose efficiently and at low cost.

──────────────────────────────────────────────────続 き Continued on the front page (72) Inventor Fumihiro Yoshinaga 3-2-1 Sakado, Takatsu-ku, Kawasaki City, Kanagawa Prefecture Within Biopolymer Research Inc. (72) Inventor Masanobu Matsuoka 3-chome Sakado, Takatsu-ku, Kawasaki City, Kanagawa Prefecture No. 2 No. 1 Biopolymer Research Inc. (58) Field surveyed (Int. Cl. ⁶ , DB name) C12P 19/04 CA (STN)

Claims (3)

(57) [Claims] 1. A PQQ belonging to a cellulose-producing acetic acid bacterium.
Non-producing strain. 2. A method for producing a cellulosic substance, comprising culturing the non-PQQ-producing strain according to claim 1, producing and accumulating a cellulosic substance in a medium, and collecting the substance. 3. The method according to claim 1, wherein PQQ is contained in the medium in an amount of about 0.001 to about 0.
The method according to claim 2, wherein 1 ng / ml is added.