JP3129773B2 - Method for producing D-ribose - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a method for producing D-ribose from D-glucose by fermentation.

[0002] D- ribose is a very important matter with respect to vitamin B ₂ production, thus D- cheaper for ribose production, development of more efficient industrial process has been strongly desired.

[0003] Many fermentation processes for the production of D-ribose are known. However, known fermentation methods are not sufficiently satisfactory as industrial methods for D-ribose production in terms of yield.

According to the present invention, it is possible to produce D-ribose at a higher yield. The inventors of the present invention
It was discovered that a mutant of a bacterium belonging to the genus Bacillus lacking transketolase and having high D-ribose resistance and high D-gluconokinase activity had a remarkably high D-ribose accumulation ability. The present invention has been completed based on this finding.

[0005] The present invention provides a transketolase deficiency,
A microorganism belonging to the genus Bacillus having high D-ribose resistance and high D-gluconokinase activity and capable of producing D-ribose from D-glucose is cultured in a medium in the presence of D-glucose, The present invention relates to a method for producing D-ribose, wherein the obtained D-ribose is recovered from a culture solution.

[0006] In the present specification, transketolase deficiency is defined as a Journal of Biological.
Chemistry 223, 1009 (195
6), Archives of Biochemist
ry and Biophysics 74, 306
Pages (1958) and 74, 315 pages (19
58)). For example,
The amount of oxidation of the reduced form of nicotinamide adenine dinucleotide (NADH) is measured by a spectrophotometer (Kontron KK) at 340 nm by the following method, and the enzymatic activity is measured as described below (see Example 4). The calculation is performed on the cell-free extract prepared in (1), which means that the value does not exceed 0.01 units / mg-protein.

<Method of Analyzing Transketolase Activity> Reaction solution A (0.555 ml): D-ribose-5-phosphate 20 μmol, NAD
H 0.375 μmol, D-ribose-5-phosphate ketol-isomerase and D-ribulose-5-phosphate-3-epimerase 0.5 units each, alpha-glycerin phosphate dehydrogenase (enzyme code 1.1.1.8) .) 0.33 units, sufficient triose phosphate isomerase (enzyme code 5.3.1.
1. ) (Sigma Chemical Co., Lt.)
d. ) And 60 μmol of Tris-HCl buffer (pH 7.
5).

2. Reaction liquid B (0.425 ml): MgCl
₂ 10 μmol, thiamine pyrophosphate 0.215 μmol and Tris-HCl buffer (pH 7.5) 40 μmol.

Each reaction solution was incubated at 30 ° C. for 10 minutes, and the reaction solutions were mixed (total volume: 1 ml). 340n
The change in absorbance at m is measured by a spectrophotometer model UVIKO
N810 (Contron KK) was measured, and after the NADH oxidation rate became linear, 20 μl of the cell-free extract was used.
l was added to the reaction. After the NADH oxidation rate was linear, the change in absorbance at 340 nm was calculated from the plot. One unit of enzyme activity is NA per minute
DH was defined as the amount of enzyme that catalyzes the oxidation of 1 μmol.

[0010] In the present specification, high gluconokinase activity refers to Biochim. Biophys. Act
a. Nicotinamide adenine dinucleotide phosphate (hereinafter referred to as NAD) based on the method described in J. 798, 88-95 (1984).
The amount of reduction of the oxidized form of P) was measured, and the enzyme activity was calculated using the cell-free extract described above, indicating that the value was not less than 0.01 units / mg-protein.

<Method for analyzing gluconokinase activity> The reaction mixture (0.5 ml) was prepared using a Tris-HCl buffer (p
H8.0) 100 μmol, MgCl ₂ 6.6 μmol,
Adenosine triphosphate 3.2μmol, NADP 0.4μ
Mol, sodium gluconate 1.0 μmol, cell-free extract 20 μl and reference 6-phosphogluconate dehydrogenase (enzyme code 1.1.1.44, Sigma Ch
electronic Co. , Led. ) Including 0.005 units. The reaction is started by the addition of the substrate. 340n
change in absorbance at room temperature with a spectrophotometer UVIK
Measured using ON Model 810. One unit of enzyme activity was defined as the amount of enzyme that catalyzed the reduction of 1 μmol of NADP per minute.

In the present specification, glucose dehydrogenase activity in the cell-free extract is determined by Agric B
iol. Chem. 43, 271-278 (197
Measured according to the method described in 9). The amount of reduction of NADP was measured at 340 nm using a spectrophotometer UVIKON model 810.

<Analytical Method of Glucose Dehydrogenase> The analysis mixture (0.5 ml) was prepared by mixing 50 μl of D-glucose.
Mol, 2 μmol of NADP, Tris-HCl buffer (pH
8.0) 0.3 mmol, 5 nmol MnSO ₄ and 20 μl of cell-free extract. The reaction was started by the addition of the substrate. One unit of enzyme activity is NAD per minute
P was defined as the amount of enzyme that catalyzed the reduction of 1 μmol.

In the present specification, high D-ribose resistance is obtained by inoculating a mutant strain of a bacterium belonging to the genus Bacillus deficient in transketolase,
6.5 ° C., 280 rpm. Mean that they show good growth in the presence of more than 70 g / l of D-ribose (absorbance at 590 nm (O
D) is higher than about 5.0). D-glucose 10.0% D-sorbitol 0.2 D-ribose 7.0 or 9.0 Molatein 0.07 Corn steep liquor 0.5 (NH ₄ ) ₂ SO ₄ 0.55 (NH ₄ ) _{2 HPO 4 0.2 KH 2 PO 4} 0.01 K 2 HPO 4 0.03 L- phenylalanine 0.00025 L-tryptophan _{0.00025 FeSO 4 · 7H 2 O 0.00032} MnSO 4 · 6H 2 O 0.00019 CaCO ₃ 4.0

The microorganisms used in the present invention include all strains belonging to the genus Bacillus which lack transketolase and have high D-ribose resistance and high gluconokinase activity. Such strains can be obtained by irradiating the parent strain with ultraviolet light, X-rays, gamma rays, or the like, or treating the parent strain with N-methyl-
By exposure to the action of chemical mutagens such as N'-nitro-N-nitrosoguanidine, nitrogen mustard, ethyl methanesulfonic acid, Bacillus brevis, Bacillus
Bacillus cereus, Bacillus circulus
ans), Bacillus coagulans (Bacillu)
s cougulans), Bacillus licheniformis,
Bacillus megaterium
terium), Bacillus Mesentric (Baci)
Bacillus subtilis, Bacillus pumilus, Bacillus subtilis
Such microorganisms can be derived from microorganisms belonging to the genus Bacillus.

Examples of the mutant strain most preferably used in the present invention include Bacillus pumilus RC15, RE5
And RX13. Each of these microorganisms has been deposited with the Japan Institute of Industrial Science Microorganisms Industrial Technology Research Institute under the following deposit numbers. Bacillus pumilus RC15 FERM-BP No. 2834 (1
March 29, 990) Bacillus pumilus RE5 FERM-BP No. 2833 (1
March 29, 990) Bacillus pumilus RX13 FERM-BP No. 2835 (1
(March 29, 990)

These mutants have high D-ribose resistance. For example, these mutants have the ability to grow in a medium containing D-ribose at a concentration of 70 g / l or more, preferably 120 g / l or more.

In a preferred embodiment of the present invention, D-ribose production is carried out by culturing the microorganism in an aqueous medium containing D-glucose and supplemented with appropriate nutrients under aerobic conditions. . The medium is about 50 g
D-glucose at a concentration of from about 1 g / l to 300 g / l, preferably from about 100 g / l to 250 g / l.

The medium usually needs to contain the following nutrients: D-glucose, D-fructose,
-Mannose, D-sorbitol, D-mannitol,
Assimilable carbon sources such as sucrose, molasses, starch hydrolyzate, starch, acetic acid and ethanol; organic substances such as peptone, yeast extract, soy flour, corn steep liquor, cottonseed meal, dried yeast and meat extract; Digestible nitrogen sources such as inorganics such as ammonium sulfate, ammonium chloride, ammonium phosphate, potassium nitrate and potassium phosphate; vitamins, metals, amino acids and trace elements.

The cultivation is carried out at a pH of about 4.0 to 9.0, preferably 4.5 to 8.0. The culture time varies depending on the microorganism used and the nutrient medium, but is preferably about 10 to 100 hours. The preferred temperature range for culturing is about 20 to 45 ° C, more preferably 25 to 40 ° C.

The D-ribose thus accumulated can be easily recovered, for example, by the following method. That is, the culture is first filtered or centrifuged, whereby the cells can be removed very easily. Next, the filtrate is desalted and decolorized by treating it with activated carbon and an ion exchange resin, and then concentrated. An organic solvent such as ethanol is added to the concentrate, and D-ribose crystals are separated. Regardless of the method described above or other methods, D-ribose can be easily recovered.

【Example】

The following examples are provided to further illustrate the present invention, but the present invention is not limited thereto.

Preparation of mutant strain RA8 Bacillus pumilus ATCC growing on agar medium
One loop of 31093 was inoculated into 100 ml of a seed culture medium having the following components. D-sorbitol 2% corn steep liquor 2% KH ₂ PO ₄ 0.1% K ₂ HPO ₄ 0.3% L-phenylalanine 0.0025% L-tryptophan 0.0025% (pH 6.7 before sterilization)

The flask was incubated at 30 ° C. for 18 hours. The cells were collected by centrifugation and suspended in 50 ml of a 50 mM phosphate buffer (pH 8.0). A portion (3 ml) of the cell suspension was added with 100 μg / ml (final concentration) of N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) and treated at 30 ° C. for 30 minutes. The treated cells are collected by centrifugation, washed once with sterile water, and
The suspension was resuspended in 1 ml and incubated at 30 ° C. for 2 hours. The culture solution thus prepared was appropriately diluted with sterile water and applied to an agar medium as shown below. D-sorbitol 0.5% Bacto peptone (Difco) 1% Yeast extract (Oriental yeast) 0.2% Nacl 0.2% Agar 1.5% (pH 7.0) Incubate the plate at 36.5 ° C for 2 days did. The colonies that grew on the plates were subcultured on fresh agar plates and incubated at 36.5 ° C. for 24 hours to obtain sufficient cells for tube culture as described below.

Each of the agar cultures thus prepared was used to inoculate 5 ml of the following production medium. D-glucose 14.5% Moratin (dried yeast) 0.07% (Kanebuchi Chemical) D-sorbitol 0.2% Corn steep liquor 0.5% (NH ₄ ) ₂ SO ₄ 0.55% (NH ₄ ) _{2 HPO 4 0.2% KH 2 PO} 4 0.01% K 2 HPO 4 0.03% L- phenylalanine 0.00025% L-tryptophan _{0.00025% FeSO 4 7H 2 O 0.00032} % MnSO 4 6H 2 O 0.00019% CaCO ₃ 4%

The tubes were incubated at 36.5 ° C. for 5 days. Next, the supernatant of the culture solution was obtained by centrifugation, and the level of D-ribose production was analyzed by thin-layer chromatography. 1 μl of the supernatant was applied to a silica gel plate (Kieselg)
el 60 F ₂₅₄ , Merck) and developed with a solvent system consisting of acetone, n-butanol and water (5: 4: 1). Next, a naphthoresorcinol-H ₂ SO ₄ reagent was wiped on a silica gel plate,
For 2-3 minutes to visualize ribose spots. Among the cultured mutant strains, D which is superior to its parent strain
-RA8 was selected as a high ribose producing strain.

Preparation of Mutant Strain RC15 In a manner similar to that described for the preparation of mutant strain RA8,
Strain A8 was transformed with MNNG (100 μg / ml final concentration) and 2-methoxy-6-chloro-9- [3- (ethyl-2-
Chloroethyl) aminopropylamino] -acridine-
Treatment with a mixture of dihydrochloride (10 μg / ml final concentration). Treated cells were treated with D-sorbitol (0.5
%); Bacto peptone (1%) (Difco); Yeast extract (0.2%) (Oriental yeast), NaCl
(0.2%) and 70 g / l D-ribose were inoculated and incubated at 36.5 ° C. for 1 day. A part of the culture solution was transferred to the same fresh medium as described above, supplemented with 90 g / l of D-ribose, and incubated at 36.5 ° C for 2 days. The obtained culture solution was appropriately diluted with sterile water, applied to an agar medium supplemented with 90 g / l of D-ribose,
Incubated at 36.5 ° C for 2 days. As a result, many D-ribose resistant strains were obtained. The D-ribose-producing ability of these mutants was evaluated in the same manner as described for the preparation of mutant RA8.
The ribose was selected as a high producing strain.

Preparation of Mutant RE5 In a manner similar to that described for the preparation of mutant RC15,
The C15 strain was treated with 100 μg / ml (final concentration) of MNNG, and the treated cells were spread on an agar medium supplemented with 70 g / l of D-ribose. Among the D-ribose resistant mutants, mutant RE5 was selected as a D-ribose-high producing strain superior to its parent strain.

Preparation of Mutant RX13 In a manner similar to that described for the preparation of mutant RC15, D-ribose-
A resistant mutant was obtained. Among the D-ribose-resistant mutants, mutant RX13 was selected as a D-ribose-high producing strain superior to its parent strain.

Example 1 One loop of agar culture of the mutant strain RA8, RC15, RE5 or RX13 was inoculated into 7 ml of a seed medium in a test tube. The components of the medium are as shown below. D-sorbitol 2.0% corn steep liquor 2.0% KH ₂ PO ₄ 0.1% K ₂ HPO ₄ 0.3% L-phenylalanine 0.0025% L-tryptophan 0.0025% (pH 6.7)

The inoculated test tube is placed on a test tube shaker,
Incubated at 6.5 ° C. for 6 hours. The seed culture (4 ml) thus prepared was inoculated into a production medium in a 500 ml Erlenmeyer flask to make 40 ml.
The components of the production medium are as follows. Moratin _{0.07% (NH 4) 2 SO} 4 0.55% (NH 4) 2 HPO 4 0.2% FeSO 4 · 7H 2 O 0.00032% MnSO 4 · 6H 2 O 0.00019% CaCO 3 4 % D-glucose (shown in Table 1) Corn steep liquor (shown in Table 1) (pH about 7.0)

The flask was heated at 36.5 ° C. and 220 rpm.
And incubated for 4 days. Mutants RA8, RC1
Table 1 shows the D-ribose productivity of 5, RE5 and RX13. Parent strain, Bacillus pumilus ATCC 31093
Was cultured in the same manner as described above, and the results are shown in the same table. Mutants RE5 and RX13 were each 1.
It showed 5- and 1.7-fold higher D-ribose productivity.

[0033]

Table 1 D-ribose-producing D-ribose-producing amount of mutant strains RA8, RC15, RE5 and RX13 Strain D-glucose Corn steep liquor (g / l) (G / l) (g / l) 3 days 4 days RA8 145 3 70.9 73.8 RC15 145 3 68.9 77.8 RE5 145 3 83.7 85.1 RE5 172 6 94.5 100.3 RX13 200 3 77.5 118.8 Parent strain 145 3 68.5 66.6 Parent strain 172 6 67.9 68.9

Example 2 In the same manner as described in Example 1, a seed culture of the mutant strain RE5 was prepared, and the concentrations of D-glucose and corn steep liquor (Lot. No. 630315B Nippon Shokuhin Kako Co., Ltd.) were prepared. Were inoculated into the same production medium, except that was 145 and 4.5 g / l, respectively. The free amino acid concentration in corn steep liquor measured by Hitachi Automatic Amino Acid Analyzer, Model 835 is as follows. Amino acid concentration (mmol / 100 g corn steep liquor) L-aspartic acid 0.17 L-threonine 1.75 L-serine 2.79 L-glutamic acid 2.68 L-proline 7.41 L-glycine 1.93 L -Alanine 10.87 L-cystine not determined L-valine 3.60 L-methionine 1.55 L-isoleucine 1.57 L-leucine 6.16 L-tyrosine 2.11 L-phenylalanine 2.38 L-lysine 2.82 L-histidine 1.05 L-tryptophan 0.18 L-arginine 3.56 Incubate the flask for 3 days and add D-ribose 8
7.0 g / l were obtained.

Example 3 One platinum loop of agar culture of the mutant strain RE5 was inoculated into 100 ml of the following seed medium in a 500 ml Erlenmeyer flask. 0.2% D-sorbitol 2% Bacto peptone 0.25% yeast extract _{K 2 HPO 4 0.3% KH 2} PO 4 0.1% MgSO 4 · 7H 2 O 0.05% L- phenylalanine 0.0025 % L-tryptophan 0.0025% L-tyrosine 0.0025% L-valine 0.0025%

The flasks were incubated at 36.5 ° C. and 220 rpm for 9 hours, and 0.1 ml of each culture was transferred to two 500 ml Erlenmeyer flasks each containing 150 ml of a seed medium having the following medium components. 4% D-Sorbitol 4% corn steep liquor _{KH 2 PO 4 0.2% K 2} HPO 4 0.6% L- phenylalanine 0.005% L-tryptophan 0.005%

The flask was incubated at 36.5 ° C. and 220 rpm for 19 hours, and the resulting culture was used for inoculating a 3 liter jar fermentation. After inoculation, the seed culture (170 ml) was inoculated into a production medium prepared to be 1.7 liters. The components of the production medium are shown below. D- Glucose 17.2% Moratein 0.07% corn steep liquor _{0.3% (NH 4) 2 SO} 4 0.55% (NH 4) 2 HPO 4 0.2% FeSO 4 · 7H 2 O 0.00032 _{% MnSO 4 · 6H 2 O 0.00019} %

Cultivation was carried out at 36.5 ° C. and 500 rpm with aeration at 0.85 l / min. During the culture,
The pH of the medium was adjusted between 5.3 and 7.0 using 6N sodium hydroxide. After 71 hours of culture, D-
The amount of ribose production was 94.0 g / l. From this culture, the cells were removed by filtration and the filtrate was concentrated to half its original volume. Next, about 1/4 of the volume of ethanol was added to remove the precipitate. The residue was desalted with a cation and anion exchange resin, and then decolorized with an activated carbon column. The decolorized solution is concentrated and about 4 times its volume of ethanol is added, whereby D-ribose crystals 15
9.8 g were obtained.

Example 4 Bacillus pumilus ATCC 31093, mutant R
A8, RC15, RE5, or RX13
A test tube containing 7 ml of seed medium of the components as described in the preparation for mutant RA8 was inoculated and incubated at 36.5 ° C. and 280 rpm for 5 hours. Next, D-
The glucose concentration is 100 g / l and in some cases D-
Except that ribose was added to a final concentration of 70, 90 or 120 g / l, 0.6 ml of each seed culture was added to 6 ml of the production medium described in the preparation of mutant strain RA8.
Was inoculated.

The test tube was heated at 36.5 ° C. and 280 rpm for 2 hours.
Incubated for days. The growth of the tested microorganisms was measured optically at 590 nm on days 1 and 2. Table 2 shows the results. Mutants RC15, RE5 and RX13 were derived from the parent strain Bacillus pumilus ATC.
It showed significantly higher resistance to D-ribose 70 and 90 g / l than C 31093 and mutant RA8.

[0041]

[Table 2]

Enzyme activity In a manner similar to that described in Example 3, Bacillus pumilus ATCC 31093 and mutants RA8, RC
15, RE5 and RX13 were cultured in a 3 liter jar fermenter. When D-glucose in the medium completely disappeared, 50 ml of the culture was withdrawn from the fermenter. The culture was centrifuged at 6,000 xg for 10 minutes, and the precipitated cells were washed with 10 mM Tris-HCl buffer (pH
7.5) Suspended in 10 ml. 6,000x cell suspension
After centrifugation at g for 10 minutes, the precipitated cells were washed again in the same manner as described above. The obtained cells were frozen at −20 ° C. until use.

The frozen cells were thawed, suspended in 10 ml of 10 mM Tris-HCl buffer (pH 7.5), and diluted with 6,000 × g at 1 ml.
Centrifuged for 0 minutes. 2 ml of the precipitated cells in the same buffer
And resuspended in lysozyme (Sigma Chemical
l Co. ) Was added to a final concentration of 200 μg / ml. Then, the mixture was stirred (240 rpm) while the temperature was 37 ° C.
For 1 hour to lyse the cells. The lysate thus obtained was centrifuged at 6,000 × g for 10 minutes. The obtained supernatant was used as a cell-free extract.

Bacillus pumilus ATCC 3109
3 and mutants RA8, RC15, RE5 and RX1
D-glucose dehydrogenase (GDH), D-glucose-6-phosphate dehydrogenase (G6PDH), gluconokinase (GAK) and 6-phosphogluconate dehydrogenase (6PGD)
Table 3 shows the results obtained by measuring the enzyme activity of H). Mutants RE5 and RX13 were found to have 10-fold higher gluconokinase activity than the parent strain.

[0045]

Table 3 Bacillus pumilus ATCC 31093 and mutants RA8, RC15, RE5 and RX13 Enzyme-active enzyme activity in cell-free extracts Bacillus enzyme pumilus Mutant Mutant Mutant Mutant ATCC 31093 RA8 RC15 RE5 RX13 GDH 0.067 * 0.073 0.074 0.091 0.088 (0.019) ** (0.023) (0.025) (0.036) (0.035) GAK 0.009 0.021 0.051 0.075 0.083 (0.003) (0.007) (0.017) (0.030) (0.041) ) * Unit / ml-culture solution ** unit / mg-protein

Embodiment Bacillus pumilus ATCC 31093 and mutant RE5 were cultured in a test tube in a seed culture medium in the same manner as described in Example 1. After 5.5 hours of culture, a sample of the culture was drawn out, and the morphological observation of the mutant strain was performed under a microscope. Their micrographs are shown in FIG.
Bacillus pumilus ATCC 31093, compared to the formation of short chains of two or three cells,
In No. 5, the formation of a long filament composed of 10 or more cells was observed.

[Brief description of the drawings]

FIG. 1 is a photomicrograph (1,100 ×, 2,800 ×) showing the morphology of an organism and showing the morphology of Bacillus pumilus ATCC 31093 and mutant RE5.

──────────────────────────────────────────────────続 き Continuation of front page (72) Inventor Yutaka Setoguchi 1-12-13-805 Katase Kaigan, Fujisawa-shi, Kanagawa (58) Field surveyed (Int. Cl. ⁷ , DB name) C12P 19/00-19 / 02 BIOSIS (DIALOG)

Claims (10)

(57) [Claims] Claims 1. A transketolase-deficient, high D-
A microorganism belonging to the genus Bacillus having ribose resistance and high D-gluconokinase activity and capable of producing D-ribose from D-glucose is cultured in a medium in the presence of D-glucose. And recovering the obtained D-ribose from a culture solution. 2. The microorganism belonging to the genus Bacillus is 70 g / l.
The production method according to claim 1, which has the ability to grow in a culture solution containing D-ribose at a concentration of preferably 120 g / l or more. 3. The microorganism belonging to the genus Bacillus is 0.01 to 3.
2. The production method according to claim 1, which has a D-gluconokinase activity of unit / mg protein or more. 4. The method according to claim 1, wherein the microorganism is Bacillus pumilus sp.
2. The method according to claim 1, wherein the method belongs to the group (illus pumilus). 5. The method according to claim 5, wherein the microorganism is Bacillus pumilus RC15.
(FERM-BP No. 2834), Bacillus pumilus RE5 (FERM-BP No. 2833) or RX13
(FERM-BP No. 2835). 6. The initial concentration of D-glucose in the culture solution is about 50 g / l to 300 g / l, preferably about 1 g / l.
The production method according to any one of claims 1 to 5, wherein the amount is from 00 g / l to 250 g / l. 7. The method according to any one of claims 1 to 6, wherein the culturing is carried out within a pH range of about 4.0 to 9.0, preferably 5.0 to 8.0. 8. About 20 to 45 ° C., preferably about 25 ° C.
The culturing is performed at a temperature in the range of 1 to 40 ° C.
The production method according to any one of the above items. 9. A transketolase-deficient, high D-
Bacillus pumilus species having ribose resistance and high D-gluconokinase activity and having the ability to produce D-ribose from D-glucose.
milus). 10. A transketolase deficient and high D
-Bacillus pumilus RC15 (FERM-BP) having ribose resistance and high D-gluconokinase activity and having an ability to produce D-ribose from D-glucose.
No. 2834), Bacillus pumilus RE5 (FER
M-BP No. 2833) and Bacillus pumilus RX
13 (FERM-BP No. 2835).