JP2693735B2 - Method for producing D-ribose - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION The present invention is a method for producing D-xylose, xylitol or L-arabinose by fermentation.
-A method of producing ribose.

D-ribose is a very important substance for vitamin B2 production, and therefore there is a strong demand for the development of a cheaper and more efficient industrial method for producing D-ribose.

Many fermentation processes are known for the production of D-ribose. However, the known fermentation method is not sufficiently satisfactory as an industrial method due to its low yield. In particular, accumulation of undesired by-products such as D-gluconic acid often results in low yields of D-ribose from D-glucose. Many of these cases use D-glucose as a starting material. for that reason,
Much effort has been expended through complex fermentation control to prevent the accumulation of D-gluconic acid.

According to the present invention, it is possible to produce D-ribose in a higher yield without accumulation of D-gluconic acid. High mutants of Bacillus bacteria lacking transketolase accumulate D-ribose from D-xylose, xylitol or L-arabinose in a medium containing an assimilable carbon source for growth. It was found to have the ability. Mutants of Bacillus bacteria deficient in transketolase cannot utilize D-xylose, xylitol or L-arabinose as the sole carbon source for their growth. The present invention has been completed based on this finding.

According to the present invention, a microorganism belonging to the genus Bacillus deficient in transketolase is cultured in a medium containing a carbon source capable of assimilation necessary for growth, and the obtained D-ribose is recovered from the culture solution. The present invention relates to a method for producing D-ribose from D-xylose, xylitol or L-arabinose.

[0006] In this specification, transketolase deficiency means Jounal of Biological Chemistry, Vol.
1009 (1956), Archives of Biochemist
ry and Biophysics 74, 306, and 74
Vol. 315 (1958), the amount of oxidized nicotinamide adenine dinucleotide reduced form (NADH) in a cell-free extract obtained from a sample organism was measured at 340 nm. It means that the value does not exceed 0.01 unit / mg protein when measured with a photometer (Contron KK).

"Method for measuring transketolase activity" 1. Reaction solution A (0.555 ml): D-ribose-5-
Phosphoric acid 20 μmol, NADH 0.375 μmol, D
-Ribose-5-phosphate Ketol-isomerase and D-ribulose-5-phosphate 3-epimerase 0.5 units each, sufficient alpha-glycerin containing triose phosphate isomerase (enzyme code 5.3.1.1) Acid dehydrogenase (enzyme code 1.1.1.
8) 0.33 units, and 60 μmol of Tris-HCl buffer (pH 7.5) Reaction liquid B (0.425 ml): magnesium chloride 1
0 μmol, thiamin pyrophosphate 0.215 μmol and Tris-hydrochloric acid buffer 40 μmol (pH 7.5) Each reaction solution was incubated at 30 ° C. for 10 minutes, and the reaction solutions were mixed (total amount 1 ml). The change in absorbance at 340 nm was measured with a spectrophotometer model UVIKON810 (Contron KK), and after the NADH oxidation rate became linear, 20 μl of the cell-free extract was added to the reaction solution. After the NADH oxidation rate became linear, the change in absorbance at 340 nm was calculated from the plot. One unit of enzyme activity was defined as the amount of enzyme that catalyzes the oxidation of 1 μmol NADH per minute.

The microorganisms used in the present invention include all strains belonging to the genus Bacillus deficient in transketolase. Such strains may be obtained by irradiating the parent strain with ultraviolet rays, X-rays, gamma rays or the like, or by irradiating the parent strain with a chemical mutagen such as N-methyl-N′-nitro-N-nitrosoguanidine, nitrogen mustard, ethyl methane sulfonic acid. By exposure to the action of Bacillus brevis (Bacillus
brevis), Bacillus cereus,
Bacillus circulans, Bacillus coagulans, Bacillus licheniformis,
Bacillus megaterium, Bacillus pumilus and Bacillus
It can be derived from a microorganism belonging to the genus Bacillus such as Bacillus subtilis.

Examples of the mutant strain most preferably used in the present invention include Bacillus pumilus ATCC 31093 and its mutant strains RC15, RE5 and RX13. The mutant strains RC15, RE5 and RX13 were obtained as a D-ribose overproducing strain from D-glucose as described in Japanese Patent Application No. 187,208. Each of these microorganisms has been deposited at the Institute of Biotechnology, Japan Institute of Technology, with the following deposit numbers.

Bacillus pumilus RC15 FERM-BP No. 283
4 (March 29, 1990) Bacillus pumilus RE5 FERM-BP No. 2833 (1990
March 29, 2013) Bacillus pumilus RX13 FERM-BP No. 2835 (1990)
March 29, 2013) These mutants lack transketolase activity.

In a preferred embodiment of the present invention, an aqueous medium containing an assimilable carbon source and D-xylose, xylitol or L-arabinose necessary for growth together with appropriate nutrients under aerobic conditions. Then, D-ribose is produced by culturing the microorganism. The medium may contain D-xylose, xylitol or L-arabinose at a concentration of about 20 g / l to 200 g / l, preferably about 50 g / l to 150 g / l.

The medium is usually an assimilable carbon source such as D-glucose, D-fructose, D-mannose, D-sorbitol, D-mannitol, glycerol, sucrose, molasses, starch hydrolyzate, acetic acid and ethanol; peptone. , Organic sources such as yeast extract, soybean flour, corn steep liquor, cottonseed meal, dry yeast and meat extract and digestible nitrogen sources such as ammonium sulfate, ammonium chloride, ammonium phosphate, inorganics such as potassium nitrate and potassium phosphate It is required to include nutrients consisting of: vitamins; metals; amino acids; and trace elements.

Culturing is carried out at a pH of about 4.0 to 9.0, preferably 5.0 to 8.0. The culture time varies depending on the microorganism used and the nutrient medium, but it is preferably about 10 to 100 hours. A preferable temperature range for carrying out the culture is about 20 to 45 ° C, more preferably about 25 to 40 ° C.

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

[0015]

The following examples serve to illustrate the invention in more detail, without restricting it in any way. Percentage values are weight / volume.

Preparation of Mutant Strain RA8 One platinum loop of Bacillus pumilus ATCC 31093 growing on an agar medium was inoculated into 100 ml of a seed culture medium containing the components shown below.

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 50 mM phosphate buffer (pH 8.
0) Suspended in 50 ml. Part of cell suspension (3 ml)
N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) 100 μg / ml (final concentration) was added to
It was treated at 30 ° C for 30 minutes. The treated cells are collected by centrifugation,
The cells were washed once with sterile water, resuspended in 5 ml of seed medium, and further incubated at 30 ° C for 2 hours. The culture solution thus prepared was appropriately diluted with sterilized water and applied to the agar medium shown below.

D-sorbitol 0.5% Bactopeptone (Difco) 1% Yeast extract (Oriental yeast) 0.2% NaCl 0.2% Agar 1.5% (pH 7.0) Plates at 36.5 ° C. And incubated for 2 days. Colonies grown on the plates were subcultured on fresh agar plates and further incubated at 36.5 ° C. for 24 hours to obtain sufficient amounts of cells for test tube culture as shown below. Each of the agar cultures thus prepared was used to inoculate 5 ml of the production medium shown below.

D-Glucose 14.5% Moratein (dry yeast, Kanebuchi Kagaku) 0.07% 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 the _{4 · 4~6H 2 O 0.00019% CaCO} 3 4% tubes were incubated for 5 days at 36.5 ° C.. Next, the supernatant of the culture broth was obtained by centrifugation and analyzed for D-ribose production level by thin layer chromatography. Supernatant 1
Add μl to a silica gel plate (Kieselgel 60F ₂₅₄ , Merc
k) spotted on and developed with a solvent system consisting of acetone, n-butanol and water (5: 4: 1). Next, the silica gel plate was sprayed with the naphthresorcinol-H ₂ SO ₄ reagent and heated at 100 ° C. for 2 to 3 minutes to visualize the spots of D-ribose. RA among the cultured mutants as a D-ribose high-producing strain superior to its parent strain
Eight strains were selected.

Preparation of mutant strain RC15 (FERM-BP No. 2834) In the same manner as described in the method for preparing mutant strain RA8,
The RA8 strain was treated with MNNG (100 μg / ml final concentration) and 2-methoxy-6-chloro-9- [3- (ethyl-2-
Treated with a mixture of chloroethyl) aminopropylamino] acridine-dihydrochloride (10 μg / ml final concentration). Treated cells with D-sorbitol (0.5
%); Bactopeptone (1%; Difco); Yeast extract (0.2%; Oriental yeast); NaCl (0.2
%) And 70 g / l of D-ribose.
Incubated at 36.5 ° C for 1 day. A part of the culture was inoculated into the same fresh medium 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 sterilized water, spread on an agar medium supplemented with 90 g / l of D-ribose, and incubated at 36.5 ° C for 2 days. As a result, many D
A ribose resistant strain was obtained. The mutant strain RC15 (FERM-BP No. 2834) was selected as a D-ribose high-producing strain by evaluating the D-ribose-producing ability of these mutant strains by the same method as described in the preparation of the mutant strain RA8.

Preparation of mutant strain RE5 (FERM-BP No.2833) RC15 (FERM-BP No.2834) strain was prepared in the same manner as described in the preparation of mutant strain RC15 (FERM-BP No.2834). MN
NG was treated with 100 μg / ml (final concentration), and the treated cells were plated on agar medium supplemented with 70 g / l of D-ribose. Among the D-ribose resistant mutants, mutant RE5 (F
ERM-BP No. 2833) was selected as a D-ribose high-producing strain superior to its parent strain.

Preparation of Mutant Strain RX13 (FERM-BP No.2835) The strain RE5 (FERM-BP No.2833) was prepared in the same manner as described in the preparation of mutant strain RC15 (FERM-BP No.2834). A D-ribose resistant mutant was obtained by iterative mutagenesis. Among the D-ribose resistant mutants, mutant RX13 (F
ERM-BP No. 2835) was selected as the D-ribose high-producing strain superior to its parent strain.

Example 1 Bacillus pumilus wild type strain, ATCC 31093, RC15 (FERM
-BP No. 2834), RE5 (FERM-BP No. 2833) and RX13 (FER
M-BP No. 2835) agar culture with a platinum loop of 500 ml Erlenmeyer flask 100 ml of seed medium shown below
Was inoculated.

[0024] D- sorbitol 2% peptone (Oriental Yeast) 0.25% dry yeast (Oriental _{Yeast) 0.2% K 2 HPO 4 0.3} % KH 2 PO 4 0.1% MgSO 4 · 7H 2 O 0 0.05% L-phenylalanine 0.0025% L-tryptophan 0.0025% L-tyrosine 0.0025% L-valine 0.0025% Flasks were incubated for 9 hours at 37 ° C and 240 rpm, and 0.1 ml of each culture was added. 100 ml of a seed medium having the following medium composition in a 500 ml Erlenmeyer flask was inoculated.

D-sorbitol 4% corn steep liquor 4% K ₂ HPO ₄ 0.6% KH ₂ PO ₄ 0.2% L-phenylalanine 0.005% L-tryptophan 0.005% Flask at 37 ° C. and 240 rpm. Incubated for 16 hours. 5 seed cultures (4 ml) prepared in this way
The production medium in a 00 ml Erlenmeyer flask was inoculated to make 40 ml. The components of the production medium are as follows.

Moratein 0.07% (NH ₄ ) ₂ SO ₄ 0.55% (NH ₄ ) ₂ HPO ₄ 0.2% MgSO ₄ .7H ₂ 0 0.00038% MnSO ₄ .4-6H ₂ O 0. the 00038% CaCO ₃ 4% corn steep liquor 1.5% d-xylose 6% d-sorbitol 4% flasks were incubated for 2 days at 37 ° C. and 240 rpm. Bacillus pumilus wild type strain, ATCC 31093,
RC15 (FERM-BP No. 2834), RE5 (FERM-BP No. 2833) and
Table 1 shows the D-ribose productivity of the RX13 (FERM-BP No. 2835) strain.

Table 1 D-ribose production of Bacillus pumilus wild type strain, ATCC 31093, RC15 (FERM-BP No. 2834), RE5 (FE RM-BP No. 2833) and RX13 (FERM-BP No. 2835). Sex (unit: g / l) strain D-ribose D-xylose D-sorbitol wild-type strain 0 0 0 ATCC 31093 43.5 12.3 0 RC15 (FERM-BP No.2834) 44.5 11.00 RE5 ( FERM-BP No.2833) 45.0 10.0 0 RX13 (FERM-BP No.2835) 60.0 0 0 Bacillus pumilus ATCC 31093 strain produced D-ribose, but the wild-type strain produced it. I didn't. Mutant RX13
(FERM-BP No. 2835) showed 1.5 times higher productivity of D-ribose than the ATCC 31093 strain.

Example 2 RX13 was prepared in the same manner as described in the preparation of the seed medium above.
The (FERM-BP No. 2835) strain was inoculated into a production medium of an Erlenmeyer flask of 500 ml, and the culture was performed at 40 ml. The composition of the production medium is as follows.

[0029] Moratein _{0.07% (NH 4) 2 SO} 4 0.55% (NH 4) 2 HPO 4 0.2% MgSO 4 · 7H 2 O 0.00038% MnSO 4 · 4~6H 2 O 0. 0380% corn steep liquor 1.5% D-sorbitol 4% pentose or pentitol (in Table 2) 6% Flasks were incubated for 2 days at 37 ° C and 240 rpm. Table 2 shows D-ribose production from various pentoses or pentitols.

Table 2 D-ribose production from various pentoses or pentitols Pentose or pentitol ribose production (g / l) D-xylose 49.6 xylitol 62.8 L-arabinose 39.3 L-arabitol 1 .8 D-arabinose 2.0 D-arabitol 1.3 L-lyxose 3.0 D-lyxose 1.8 L-xylose 5.1 ribitol 0.6 no addition 2.0 D-ribose production was D-xylose. Shown only when using xylitol and L-arabinose.

Example 3 RX13 (FERM-BP No. 2835) was mixed with 6% xylitol and 4%.
The culture was performed in the same manner as described above, except that 10% xylitol and 4% carbon source as shown in Table 3 were used instead of D-sorbitol of. The results are summarized in Table 3.

Table 3 Effect of carbon source on D-ribose production (unit: g / l) Carbon source D-ribose xylitol Carbon source D-sorbitol 84.8 2.00 Glycerol 58.5 26.4 0 D-mannitol 47 .4 47.4 0 D-fructose 43.3 50.8 0 D-glucose 41.9 51.00 sucrose 64.4 39.9 0 no addition 2.0 98.9 0 D-ribose production is D -Sorbitol, glycerol,
It was observed when D-mannitol, D-fructose, D-glucose, and sucrose were present, whereas D-ribose accumulation did not occur when only xylitol was present.

Example 4 RX13 (FERM-BP No. 2835) strain was mixed with 8% of the medium composition instead of 6% D-xylose in the same manner as described in the preparation of seed culture (300 ml) above. 5 containing the same production medium as described in Example 1 except using xylitol
A liter jar was inoculated, and the amount of the liquid after inoculation was set to 3 liters and cultured. The fermentation was carried out at 37 ° C. and 500 rpm and aerated at 1.5 liter / min. The production yield of D-ribose was 79.9 g / l when 8% xylitol was used in 40 hours fermentation. Cells were removed from the fermentation broth by centrifugation (3000 g for 10 minutes) and filtration, the filtrate was desalted with cation and anion exchange resins and then decolorized through a column of activated carbon. The decolorization solution was concentrated and then about twice its volume of ethanol was added, thereby obtaining 210 g of D-ribose crystals.

Example 5 RX13 (FERM-BP No. 2835) was prepared in the same manner as the above culture.
The strains were cultured in production medium containing radiolabeled D- sorbitol radiolabeled xylitol or ¹⁴ C in 500ml Erlenmeyer flask ¹⁴ C (40ml). The fermentation was carried out at 37 ° C and 240 rpm. At the start of fermentation and after 42 hours, 2 ml of culture solution was obtained. Then, after removing the cells by centrifugation (3000 g, 10 minutes) and filtration, 5 μl of the filtrate was applied to a silica gel plate.
(TLC; Kieselgel 60F ₂₅₄ , Merck) was spotted and developed with three different solvent systems with the compositions shown below.

1) Acetone / n-butanol / water (5:
4: 1) 2) 2% phenylboric acid in ethyl acetate / 2-propanol / water (6: 3: 1) 3) n-propanol / ethanol / 50 mM potassium phosphate buffer (pH 7.0) (55) : 25:20) The radioactivity on thin layer chromatography was measured by a TLC radio scanner (Aloka, JTC601). Radioactivity and D
-The yields of ribose, xylitol and D-sorbitol were as shown in Table 4.

Table 4 Parasitic radioactivity of xylitol and D-sorbitol emitted during ribose production Time D-ribose xylitol D-sorbitol (hours) (g / l) (cpm) (g / l) (cpm) ( g / l) (cpm) ¹⁴ C-xylitol 0 0 0 98.0 646 39.0 0 42 83.9 539 7.1 31 4.0 0 ¹⁴ CD-sorbitol 0 0 0 96.8 0 38.5 858 42 91.2 0 7.2 0 0 0 As shown, the origin of D-ribose was identified as xylitol rather than D-sorbitol.

Claims (7)

(57) [Claims] 1. A method of culturing a microorganism belonging to the genus Bacillus deficient in transketolase in a medium containing an assimilable carbon source for growth and recovering the obtained D-ribose from the culture solution. A method for producing D-ribose from D-xylose, xylitol or L-arabinose, which is characterized. 2. The microorganism belonging to the genus Bacillus is 0.01
The production method according to claim 1, which has a transketolase activity of not more than a unit / mg protein. 3. The microorganism is a Bacillus brevis sp.
us brevis), Bacillus cereu
s), Bacillus circulan
s), Bacillus coagulan
s), Bacillus licheniformis species (Bacillus lich
eniformis), Bacillus megaterium species (Bacillus me)
gaterium), Bacillus pumilu
s) and Bacillus subtilis species
The manufacturing method according to claim 1 or 2, which belongs to. 4. The microorganism is Bacillus pumilus ATCC 310.
93, Bacillus pumilus RC15 (FERM-BP No. 2834), Bacillus pumilus RE5 (FERM-BP No. 2833), or Bacillus pumilus RX13 (FERM-BP No. 2835).
Production method described in 1. 5. The initial concentration of D-xylose, xylitol or L-arabinose in the culture medium is about 20 g / l.
To 200 g / l, preferably about 50 g / l to 1
It is 50 g / l, The manufacturing method in any one of Claims 1-4. 6. The method according to claim 1, wherein the culturing is carried out at a pH of about 4.0 to 9.0, preferably 5.0 to 8.0. 7. The production method according to claim 1, wherein the culturing is carried out at a temperature of 20 to 45 ° C., preferably about 25 to 40 ° C.