JP2845385B2 - Novel mutant strain and method for producing glycerin using the same - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a novel mutant and a method for producing glycerin using the same, and more particularly, to a novel artificial mutant belonging to the genus Torulopsis sp. The present invention relates to a method for producing glycerin by a fermentation method, which comprises converting a fermentable saccharide to glycerin using the SN-7678 strain and the mutant strain.

[0002]

2. Description of the Related Art Methods for producing polyols by fermentation, especially for producing glycerin using microorganisms belonging to the genus Torulopsis, are known. For example, Japanese Patent Publication No. 47-8589 discloses T. mannitofaciens.
And the method of using
No. 93 discloses Torulopsis anomala (T. anomala)
Are described. Furthermore, GHHajny
Et al., Applied Miclobiology, 8: 5 (1960), describes a method using T. magnoliae.

[0003] However, the microorganisms used in these known methods are not practically used yet because they have low sugar tolerance and are not practical, or the conversion rate to polyols is not always sufficient. Also, Vijaik
ishore et al .; Applied Biochemistry and Biotechnology,
vol 13, 1986, Pichia farino
Although the method using sa) is described, this method requires that the medium be kept alkaline. Therefore, this method has a disadvantage that a large amount of sodium carbonate having a ratio of 10% to sugar must be used, and the burden required for the subsequent treatment is extremely large.

Further, Japanese Patent Application Laid-Open Nos. 62-21509 and 62-96089 disclose Torulopsis sp. SN-18 strain (Micro-organisms No. 7593)
) Is used to produce glycerin from fermentable sugars such as glucose. Although this method makes it possible to produce glycerin using a relatively high concentration of carbohydrate, it has a disadvantage of producing a considerable amount of mannitol as a by-product, and thus has a low glycerin yield and It is still not a satisfactory method in terms of purification complexity.

[0005]

The present inventors have conducted various studies to improve the above-mentioned drawbacks of the conventional method, and as a result, have found that the above-mentioned Torulopsis sp. A mutant strain, Torulopsis sp., Derived from the SN-18 strain (Microtechnological Laboratory No. 7593). The present inventors have found that the strain SN-7678 has a small amount of mannitol by-product and has a high glycerin-producing ability, and has reached the present invention.

That is, in the present invention, the growth pH range is from pH 2.5 to pH 2.5.
7.0, and the amount of mannitol by-produced during the culturing is not more than 1/15 (concentration ratio) of glycerin as a main product. SN-767
8 strain (Microbial Lab. No. 12502) and the Torulopsis sp. The present invention relates to a method for producing glycerin, which comprises inoculating a SN-7678 strain into a medium containing fermentable saccharides, aerobically culturing the medium, and separating and collecting glycerin from the culture solution.

[0007] In the present invention, Torulopsis sp. SN-767
Eight strains are the parent strain Torulopsis sp. A mutant strain produced from an SN-18 strain by artificial mutation means, specifically, Torulopsis sp. It is a novel mutant induced by irradiating the SN-18 strain with ultraviolet light and subsequently treating it with a mutagen such as N-methyl-N'-nitro-N "-nitrosoguanidine.

[0008] The present mutant strain is Torulopsis sp. SN-1
Eight strains were mutated as parent strains and had properties very similar to the parent strains as described later. Therefore, the strains were determined to be novel mutants belonging to the genus Torulopsis, and Torulopsis sp. The strain was named SN-7678 strain.

Next, the bacteriological properties of the present mutant strain will be described. 1. Growth on the culture medium (1) Microscopic findings Vegetative cell size (* 1) 2-6x2-6μ Vegetative cell shape (* 1) Circular vegetative cell growth method (* 1) Multipolar budding mycelium (* 2) Not formed (Note) * 1 Cultured on YM agar at 27 ° C for 5 days * 2 Slide culture on potato glucose agar

(2) Agar slope (* 3) Good growth Glossy white (no gloss) Pigment Orange-yellow colony when cultured for 2 weeks or more (Note) * 3 YM agar medium (3) Liquid culture (* 4) Surface growth film Formation Turbidity Clear Sedimentation Large (Note) * 4 YM agar medium

2. Formation of ascospores Potato glucose agar medium Not formed Cornmeal agar medium Not formed YM agar medium Not formed Carrot extract agar medium Not formed V8 agar medium Not formed

3. Physiological properties Oxygen demand Aerobic Growth temperature 36 ° C or less Optimal growth temperature 28-30 ° C Growth pH 2.5-7.0 (initial pH 2.5-8.0) Optimal growth pH 4.0-6.0 KNO ₃ utilization (* 5) Weak (NH ₄ ) ₂ SO ₄ utilization (* 5) Decomposition of fat (* 6) None Decomposition of urea None Liquefaction of gelatin Available

The highest concentration of glucose that can grow (* 7) (70%) The optimum concentration of growth (* 7) (40%) The highest concentration of sodium chloride that can grow (* 8) (13% or more) The optimum concentration of growth (* 8) (7%) Carotenoid formation No Organic acid generation Yes Starch-like substance generation No Vitamin requirement (* 5) Yes (Note) * 5 Judgment by the method of J. Lodder et al. Using Wickerham's synthetic medium * 6 Using tallow * 7 Liquid medium * 8 Growth in 30% (W / V) glucose liquid medium

4. Sugar fermentability (* 5) Glucose + lactose-galactose + melibiose-sucrose + raffinose + maltose-

[0015] 5. Sugar assimilation (* 5) Glucose + D-xylose-galactose + erythritol-D-arabinose + L-arabinose + D-sorbose-L-sorbose + D-ribose-sucrose + L-rhamnose-

Maltose + ethanol-cellobiose-trehalose-adonitol-lactose-dulcitol-melibiose + raffinose + D-sorbitol + melezitose-α-methyl-D-glucoside-inulin + salicin-inositol-soluble starch-lactic acid-co-lactic acid-co-lactic acid-co-lactic acid-co-lactic acid − Citric acid −

This strain has been deposited with the Research Institute of Microbial Industry, National Institute of Advanced Industrial Science and Technology as "Microorganism Accession No. No. 12502". Thus, the mutant Torulopsis sp. The strain SN-7678 is a parent strain (wild strain), Torulopsis sp. It has bacteriological properties very similar to the SN-18 strain. However, this mutant strain is
The H range is slightly narrower than that of the parent strain at pH 2.5 to 7.0, and as shown in Examples described later, high even in a medium mainly composed of corn steep liquor, which is an inexpensive medium suitable for industrial production. The glycerin-producing ability is maintained, the production of by-products such as mannitol is extremely small, and when the strain is cultured under suitable conditions (for example, the culture conditions described herein), the amount of by-products of mannitol is mainly produced. It is different from the parent strain in that it can be suppressed to 1/15 or less (concentration ratio) of glycerin as a product.

Next, a method for producing glycerin using the mutant strain of the present invention will be described. The percentage used in the following description is the capacity (W / V)% unless otherwise specified. The cultivation of the present strain is desirably carried out by stirring culture under aerobic conditions using a liquid medium containing a carbon source, a nitrogen source, inorganic salts and the like.

As the main carbon source of the liquid medium, any one can be used as long as the mutant strain can assimilate and produce the desired glycerin. For example, fermentable sugars such as glucose, fructose and mannose can be used. However, these carbohydrates are used in an amount of 10 to 60%, preferably 20 to 50% of the whole medium.

As the nitrogen source, nitrogen compounds usable by the mutant strain, for example, yeast extract, tryptone, malt extract, casamino acid, corn steep liquor and the like are used. In addition, ammonium salts such as ammonium sulfate, ammonium acetate, and ammonium nitrate can also be used. In addition, as inorganic salts to be added to the medium, for example, salts such as phosphoric acid, magnesium, potassium, calcium, and iron are used. Furthermore, various organic and inorganic substances necessary for the growth of the mutant strain or a commonly used antifoaming agent can be added as necessary.

The cultivation is carried out by inoculating the liquid medium of the above composition directly with the present strain or separately by inoculating a seed culture of the present strain obtained by preculture. This seed culture solution is prepared, for example, by subjecting the strain to a 30% glucose to 1% yeast extract or corn steep liquor to a 30% glucose culture by a conventional method.
Inoculate one platinum loop into a liquid medium of pH 4-6 containing 3%
It is performed by culturing at a temperature of 6 to 36 ° C for 2 to 5 days.

The cultivation temperature is within the range in which the strain can grow, that is, 26 to 36 ° C.
It is in the range of 30 ° C. The pH of the medium is between 2.5 and 7.0, especially 4.5.
A range from 0 to 6.0 is preferred. The culturing period varies depending on the culturing conditions, the type of medium used, the concentration of saccharide as a carbon source, and the like, but is usually about 4 to 8 days. Culture is
The amount of glycerin in the culture was measured by gas chromatography or high-performance liquid chromatography so that the culture could be terminated when the nutrients in the medium were used to the maximum and the amount of glycerin produced in the culture reached the maximum. It is desirable to perform the measurement while measuring by a known method such as lithography.

Glycerin accumulated in the culture solution is separated from the culture solution by a conventional method after completion of the culture. That is,
In such a case, well-known means ordinarily used in the art, such as filtration, centrifugation, ion exchange or adsorption chromatography, solvent extraction, and distillation, are used in combination as necessary. For example, cells are removed from the culture solution by filtration, centrifugation, etc., and then this solution is treated with activated carbon to remove coloring substances, etc., further deionized with an ion exchange resin, and then the solution is concentrated. Syrup. This is further purified by vacuum (vacuum) distillation to obtain glycerin.

[0024]

Next, the present invention will be described in detail with reference to examples. Example 1 (Construction of mutant) Torulopsis sp. Strain SN-18 was converted to glucose 30
% And yeast extract 1% were inoculated into a 100 ml liquid medium and shake-cultured at a temperature of 30 ° C. for 3 days to obtain a culture.
Next, the above culture was diluted 1000-fold with a 30% glucose solution, applied to an agar medium containing 30% glucose, 1% yeast extract, and 1.5% agar in an amount of 100 μl, and irradiated with a 15-w ultraviolet lamp from a distance of 30 cm. (Toshiba germicidal lamp "GL1
5 ") for 45 seconds, followed by stationary culture at 30 ° C. for 4 days. The resulting colony was used as a test medium containing 20% glycerin, 1% yeast extract, and 1.5% agar as a test medium to select a strain having reduced glycerin utilization by the replica method.

Next, the strain selected by the above treatment was inoculated into 100 ml of a liquid medium containing 30% of glucose and 1% of yeast extract, and cultured with shaking at a temperature of 30 ° C. for 3 days to obtain a culture. The cells were separated from the culture obtained above by centrifugation, and the cells were further subjected to 50 mM phosphate buffer (pH =
After washing three times in 7), the cells were suspended again in the same buffer (10).
⁸ cells / ml). The suspension is brought to a final concentration of 250μ.
g / ml of N-methyl-N'-nitro-N "-nitrosoguanidine for 30 minutes.

After the treatment, the cells were separated by a conventional method, washed with a buffer, and then treated with a nystatin antibiotic having a final concentration of 10 μg / ml in a medium containing 20% glycerin and 1% yeast extract for 2 hours. . After washing the cells with buffer,
Apply to the above agar medium containing glucose, and
The culture was allowed to stand for a day. The resulting colonies were selected by a replica method using a medium containing 20% mannitol, 1% yeast extract, and 1.5% agar as a test medium by a replica method.

Next, the strain selected as described above was inoculated into 50 g of a liquid medium containing 30% of glucose and 1% of yeast extract, and cultured with shaking at a temperature of 30 ° C. for 2 days to obtain a culture. This culture was treated to a final concentration of 50 by the same procedure as above.
The cells were treated with 0 μg / ml N-methyl-N′-nitro-N ″ -nitrosoguanidine for 1 hour, and the treated cells were treated with nystatin in a medium containing 10% mannitol and 1% yeast extract as described above. Done.

After the treatment, the bacterial cell solution was applied to the above agar medium containing glucose, and the culture was allowed to stand at 30 ° C. for 4 days, and the grown bacterial cells were selected and the mutant strain Torulopsis sp. SN-76
78 strains (FERM P-12502) were obtained.

Example 2 (a) Preparation of Seed Culture Solution Tolulopsis sp. Was placed on a slant medium consisting of 30% glucose, 1% yeast extract and 1.5% agar. The cells of the SN-7678 strain (FERM P-12502) were applied,
For 2 days. Next, one loopful of the slant-cultured cells of the above strain was inoculated into a 500 ml Erlenmeyer flask containing 50 ml of a liquid medium (pH 5.5) containing 30% of glucose and 1% of yeast extract, and inoculated at 30 ° C. for 2 days. The culture was obtained by shaking culture.

(B) Main culture A liquid medium containing glucose 50%, corn steep liquor 3.27%, ammonium acetate 0.07% and KNO ₃ 0.1% (glucose and corn steep liquor, ammonium acetate, KNO ₃ Use those that have been previously sterilized separately)
3 liters were placed in a 5 liter fermenter, and the Torulopsis sp. 50 ml of a seed culture of the SN-7678 strain (FERM P-12502) was added, the temperature was 30 ° C., the aeration rate was 0.33 vvm, and the rotation speed was 80.
The culture was performed for 6 days while maintaining the pH at 5 with potassium hydroxide at 0 rpm.

After completion of the culture, the culture was analyzed by high performance liquid chromatography (HPLC). As a result, glucose was completely consumed, the glycerin content in the culture was 29% (yield 58%), and mannitol was Content is 0.5
% (Yield 1%).

Next, the cells were removed from the culture by centrifugation, followed by decolorization with activated carbon and desalting with an ion exchange resin (IRA-140: IR-120 = 2: 1). Next, the eluate was concentrated to 30% or more, and glycerin was purified using a column filled with a styrene-divinylbenzene-based cation exchange resin. The purified substance was confirmed to be identical to the standard glycerin by gas chromatography using a TMS derivative, high performance liquid chromatography, and identification by F-kit glycerol.

Example 3 (a) Preparation of Seed Culture Solution Tolulopsis sp. Was placed on a slant medium consisting of 30% glucose, 1% yeast extract and 1.5% agar. The cells of the SN-7678 strain (FERM P-12502) were applied,
For 2 days. Next, a liquid medium containing 30% glucose and 3.3% corn steep liquor (initial pH 5)
One loopful of the slant-cultured cells was inoculated into a 500 ml Erlenmeyer flask containing 50 ml, and at 30 ° C., 220 rpm.
m, shaking culture for 2 days to obtain a culture. Further, the above culture was inoculated into a 5 liter jar fermenter charged with 3 liters of the same medium as above, at 30 ° C., 0.5 vvm,
The cells were cultured at 00 rpm for 2 days to obtain a seed culture solution for main culture.

(B) Main culture A liquid medium (glucose and corn steep) consisting of 40% glucose, 3.4% corn steep liquor, 0.06% ammonium acetate and 0.1% KNO ₃ was placed in a 30-liter jar fermenter. Liquor, ammonium acetate, K
NO ₃ used beforehand was sterilized separately), 15 liters were charged, and the Torulopsis sp. Prepared in (a) above was charged. S
300 ml of seed culture of N-7678 strain (inoculation amount 2
%), And the culture was performed while changing the rotation speed of the stirrer at 230 to 380 rpm. The other culture conditions were as follows: aeration volume of 0.5 vvm, can internal pressure of 0.5 kg / cm ² , temperature of 30
° C, initial and retention pH = 5. After culturing, HPL
C. Glycerin, mannitol and turbidity of the culture solution were measured by a spectrophotometer. Table 1 shows the results.

[0035]

[Table 1]

EXAMPLE 4 Various concentrations of corn steep liquor (CS) were placed in a liquid medium mainly composed of 40% glucose and 0.2% ammonium sulfate.
L) was added to a 30-liter jar fermenter, and 5 liters of the medium was added thereto. SN-7678 strain (FERM P-1
250 ml) of the seed culture solution (300 ml) was inoculated and cultured under the following conditions.

Aeration rate 0.55 vvm Can pressure 0.5 kg / cm ² Culture temperature 30 ° C. Stirring number 280 rpm Inoculation amount 4% Initial pH 5 Holding pH 5

After completion of the culture, glycerin, mannitol and turbidity of the culture solution were measured by HPLC and a spectrophotometer. Table 2 shows the results.

[0039]

[Table 2]

Example 5 Glucose 4 was added to a 30-liter jar fermenter.
0%, corn steep liquor 2.5%, ammonium sulfate
15 liters of a liquid medium consisting of 0.2% was prepared, and the Torulopsis sp. Prepared in Example 3 (a) was prepared. SN-7678
300 ml of seed culture of strain (FERMP-12502)
(2% inoculation amount), and using potassium hydroxide, the first p
After adjusting the H to 5, the culture was carried out by changing the holding pH between 3.0 and 5.0. The results are shown in Table 3.

[0041]

[Table 3]

Example 6 30% glucose, 1.5% yeast extract, potassium tartrate
3 liters of a liquid medium containing 1.5% (added after sterilizing components other than glucose separately) was placed in a 5 liter fermenter, and the Torulopsis sp. Prepared in Example 2 (a) was added.
50 ml of a seed culture of the SN-7678 strain (FERM P-12502) was added, and the temperature was 30 ° C., the aeration rate was 1 vvm,
Culture was performed for 5 days at a rotation speed of 800 tpm.

After completion of the culture, the culture was analyzed by HPLC and spectrophotometer. As a result, glucose was completely consumed, and the glycerin content in the culture was 15.7%.
(Yield 52.4%), mannitol was not formed.

Comparative Example 1 50 ml of a medium containing 1% of yeast extract and 30% of glucose
Into a 500 ml Erlenmeyer flask at 120 ° C.
Sterilize for 15 minutes in a cooler
N-18 strain) or a mutant strain (SN-7678 strain FER)
The cells of MP-12502) were inoculated one loop each.
After culturing at 30 ° C. for 2 days, the cells were separated by a centrifuge and washed three times with fresh glucose solution. 20 cells after washing
% And 30% of the glucose solution, and dispensed in 4 ml portions into test tubes. Potassium tartrate was added to a final concentration of 0.5%, and shaking culture was performed for 6 days.

After the completion of the culture, the culture solution was analyzed by HPLC. As a result, the yield of glycerin per glucose consumed and the yield of mannitol as a by-product were as shown in Table 4.

[0046]

[Table 4]

Comparative Example 2 The mutant strain of the present invention (SN-7678 FERM) was prepared by changing the concentration of carbon (carbohydrate) in the medium and the type of nitrogen source in various ways.
P-12502) and the parent strain (SN-18 strain) were cultured according to the following method, and the glycerin production (yield) and mannitol production (yield) of both strains were compared.

1) Test A 2.6% Corn steep liquor, 0.05% ammonium sulfate
%, 30%, 40% or 50% of glucose were put into 500 ml volumetric Erlenmeyer flasks and sterilized at 120 ° C. for 15 minutes. After cooling, the pH of the medium was adjusted to 5.0, the seed culture was inoculated with 1%, and cultured at 30 ° C. and 220 rpm for 4 to 7 days while adjusting the pH of the culture to 5 or more. . After completion of the culture, the amounts of glycerin, mannitol and glucose in the culture solution were measured by HPLC. The results are shown in Table 5.

[0049]

[Table 5]

2) Test B The medium was Difco Nitrogen Base W / O Amino Acid & Ammoniu
m Sulfate and ammonium sulfate (A
S), ammonium acetate (AAc), casamino acid, yeast extract, corn steep liquor (CSL), and other nitrogen sources were used. The addition amount of the nitrogen source was 0.5% (in terms of standard final concentration), and 1% of potassium tartrate was additionally added. After culturing, each medium was inoculated with 1% of a seed culture solution, and cultured with shaking at 30 ° C. for 7 days. After completion, the contents of the test tube were appropriately diluted to determine the amounts of glycerin (GL) and mannitol (MN). Was measured. The results are shown in Table 6.

[0051]

[Table 6]

From the results shown in Tables 5 and 6, the characteristics of the mutant of the present invention such as high productivity of glycerin and low productivity of by-product mannitol are almost influenced by differences in the type and concentration of the substrate in the medium. It was confirmed that they were always maintained without any problem.

[0053]

Industrial Applicability The novel mutant strain SN-7678 of the present invention has an extremely high conversion rate from fermentable saccharides to glycerin (yield to sugar) and extremely low production of by-products such as mannitol. have. Therefore, commercial fermentation production of glycerin is possible by using this strain.

Continued on the front page (72) Inventor Keiji Kainuma 2-29-4 Higashi, Tsukuba, Ibaraki Pref. (72) Inventor Kiyotaka Nishida 1-1-28-201, Jonan, Iwatsuki, Saitama 5-10-2 Mochida (72) Inventor Toshiro Ochiai 2-1-18 Asahi, Kawaguchi City, Saitama Prefecture LG Kawaguchi 309 (72) Inventor Takeshi Kawaguchi 21-6 Ichiricho, Gyoda City, Saitama Prefecture (72) Inventor Tsuneo Oda 128 Akigawa-shi, Tokyo 128 (58) Fields studied (Int. Cl. ⁶ , DB name) C12P 7/20 C12N 1/16 CA (STN)

Claims (2)

(57) [Claims] 1. The growth pH range is from pH 2.5 to 7.0,
And the amount of mannitol by-produced during the culturing is not more than 1/15 (concentration ratio) of glycerin which is a main product, and the artificial mutant strain Torulopsis sp. SN-7678
Bacterial strain (Microtechnical Laboratory No. 12502). 2. A method according to claim 1, wherein said torropsis sp. A method for producing glycerin, comprising inoculating a SN-7678 strain (Microtechnical Laboratories No. 12502) into a medium containing fermentable saccharides and cultivating aerobically, separating and collecting glycerin from the culture solution. Method.