JPS6135792A - Production of d-ribose - Google Patents

Abstract

PURPOSE:To produce a large amount of D-ribose, at a low cost, suppressing the by-production of gluconic acid, by culturing a microbial strain belonging to Bacillus genus and capable of producing D-ribose, in a medium added with a specific compound (or its salt). CONSTITUTION:A microbial strain belonging to Bacillus genus and capable of producing D-ribose, e.g. Bacillus pumilus No.503 (FERM-P No.812) is inoculated in a medium containing sorbitol as a carbon source and corn steep liquor, etc. as a nitrogen source and added with 0.2-2g/l of the compound (or salt) of formula I [R is H, OH, SH, CH3S, or 1-4C alkyl which may have the substituent group of formula II-III (R' is H or NH2); X is group of formula IV-V], and is cultured at 18-45 deg.C and 4.5-9pH for 18-180hr to effect the production and accumulation of D-ribose. The medium is filtered or centrifuged to remove the microbial cells, and the filtrate is decolored, desalted, concentrated, and crystallized with ethanol, etc. to obtain D-ribose.

Description

DETAILED DESCRIPTION OF THE INVENTION Field of Industrial Application The present invention relates to a method for producing D-ribose by an enzymatic method.

According to conventional technology, ribose is contained in all living organisms as a constituent of ribonucleic acid, and its reduced derivative, ribitol, is included as a constituent of vitamin B2 and ribitol tichonic acid, which is a constituent of the cell wall. It is also an extremely important substance physiologically.

-IF, D-ribose has traditionally been used as a raw material for the synthesis of vitamin B2, and in recent years has attracted attention as it is also used as a synthetic raw material for nucleic acid seasonings. This is extremely significant from an industrial perspective.

So far, known methods for producing D-ribose include a single extraction method from natural products, a synthesis method using furan, glucose, etc. as raw materials, and a fermentation method using microorganisms.

Problems to be Solved by the Invention All of the methods described above have drawbacks such as complicated manufacturing processes, expensive raw materials, and low yields. - Repose f is not necessarily a satisfactory manufacturing method.

Means for Solving the rNJ Problem The present inventors have conducted various studies in order to establish a high-yield D-') bose production method using Bacillus bacteria.
- As a result of repeated experiments, Bacillus bacteria capable of producing D-ribose were cultured in a sugar-containing fermentation medium, with the addition of all substances that suppressed the production of gluconic acid but did not affect the growth of the bacteria. It was discovered that a significant amount of D-ribose was produced by this method, and as a result of further research based on this, the present invention was completed.

The present invention uses a microorganism that belongs to the Bacillus species and has D-lipose production ability according to the general formula % formula % () [wherein R is H-iknl to 2 HO-, H8
−.

(In the formula, R' represents ■- or NH2-.) The group f: bt represented by the formula (R' represents ■- or NH2-) is cultured in a medium supplemented with a salt of bamboo shoots, which may be present as a bt m group, and b - A method for producing D-ribose, which is characterized by producing and accumulating ribose and collecting it.

Bacillus used in the method of the present invention
Examples of microorganisms that belong to the genus Bacillus pumilus and have the ability to produce D + ribose include Bacillus pumilus.
Bacillus subtilis (Bacillus pumilus) w Bacillus subtilis (i7) is inhibited, and more specifically, Bacillus bumiμs Asahi 503 (Engineering FO 12600, ATCC 21356
).

1'h537 (Engineering FO12601, ATCC21357
), Kan 558 (1FO12602, ATCC2135B
), No. 716 [Engineering FO13322 (Consignment date June 3, 1972), FERM-P1491 (Consignment date June 3, 1972)
(June 17, 2016), ATCC21951 (Accession date: 197
(June 4, 3)], Nagi 911 (IFO+3566, FE
RM-P 2260. ATCC31095), Nagi 10
27 (Engineering FO135 [15, FERM-p2466, A
TCC3109B), Description 1083 (Engineering FO13620゜FERM-P 2832.' ATCC31093
), Bacillus zutetilis Nagi 429 (Engineering FO12603
゜ATCC21359), Nagi483 (Engineering FO12604)
゜ATCC21360)', Nagi608 (1FO+332
3゜FERN-P 1490. ATCC21952)
, Nagi957 (Engineering FO13565, rgRiq-p
2259゜ATCC'31096), Nagi 941 (Engineering FO
13573°FERM-P 2360. ATCC31
097), Nagi 1054 (Engineering F0 13586.FE
RM-p2467゜ATCC31091), Na106
7 (Engineering F013588, FERM-P 24681A
TCC31092), Nagi1097 (Engineering Fo 1362)
i.

FERM-P 2833. ATCC31094), etc.

The above Engineering FO number is the accession number of Fermentation Research Institute (Eng. The American Type Cu1t
ure Collection + AI'CC*USA) accession numbers are shown.

Among the above microorganisms, the mycological properties of Bacillus pumilus and Bacillus subtilis are described in the Bacillus Manual p.
・Of Deterministic Bacteriology (Ber
(7ey'o Manual of Determi
nFLtive Bacteriolo(g)ia
These are the same as those described on pages 529 to 534 of the edition. However, regarding the spore-forming ability, it also includes those whose spore-forming ability has been deleted through mutation treatment.

The microorganisms used in the method of the present invention require aromatic amino acids (L-thyronine, L-tryptophan, and phenylalanine) for growth; - A bacterium of the genus Bacillus lacking at least one of the shrimp mefuses.

Furthermore, a pre-spore formation defective strain of the Basinus IIIl bacterium.

Furthermore, the 2-deoxy-D-
This strain has high glucose oxidation activity. When these microorganisms are cultured in a medium containing nutrients necessary for their growth, they produce and accumulate a significant amount of D-lipose in the culture, and at the same time produce gluconic acid as a by-product. During this culture, by adding a compound represented by general formula (I), the production of gluconic acid can be suppressed and the amount of D-lipose produced can be further increased.

Specific examples of alkyl having 1 to 4 prime numbers in compound (I) represented by general formula CI) include methyl, ethyl, n-propyl, isopropyμ, n-butyp
, isobuty7tz, 5ec-butyl.

Examples include tert-buti μ.

Examples include tatoeba 2μ, para-aminobue 2μ.

Specific examples of compound (I) include L-valine, L-, D- or DL-leucine, L-isoleucine, L-methionine, L-synutain, L-cynutin,
Feni p-alanine, DL-α-amino-n-acetic acid, p-
Examples include amino-DI, -phenylalanine, DL-3-7enylserine, glyoxy2+/[, and α-keto-isocaproic acid.

Examples of the salts of compound (I) include IIL-α-aminobutyric acid, glyoxylic acid, α-keto-isocaproic acid, potassium salts, and lysine hydrochloride.

The amount of compound (I) added to the medium is generally about 0.2 f/e), preferably about 0.2 to 2 f/l, and more preferably about 0.2 to 2 f/l. Approximately 0.2
The amount is between 0.6 f/l and 0.6 f/l. , the amount added is
This refers to the total amount added during the culture.

The compound represented by formula (I) may be added to the initial culture medium in an amount of ft, or may be added at an early to middle stage of the culture medium.

As a culture method, a general method of culturing all microorganisms is used, but deep aeration culture is most convenient.

As a culture medium, various nutrient sources, ie, a wide range of sources.

Contains Yasogen, etc. As a fusu source, it is Toba D
-riμcose, D-phyctose, D-mannose, soμpitot/L', D-mannide-μ, Vucrose + fl, 'f honey, 9ml powder of mother's flour hydrolyzate, acetic acid.

Examples include ethanol-μ. In addition, the sources include corn steep liquor, cottonseed meal, yeast extract, dried yeast, buisochumi μ, and meat extract.

In addition to inorganic nitrogen compounds such as peptone, casamino acids, other astringent mineral resources, ammonia water, ammonia gas, ammonium sulfate, ammonium nitrate, ammonium chloride, ammonium carbonate, ammonium phosphate, and sodium nitrate, organic compounds such as urea and amino acids Nitrogen compounds are used. Various metals, vitamins, amino acids, and the like necessary for the growth of the microorganisms used as nitrogen source #1 are added to the medium as appropriate.

Culture conditions, ie, culture temperature, medium pli, culture time, etc., are not particularly limited, but the culture temperature is generally about 18 to 45°C, more preferably about 25 to 45°C.
The pH of the medium is generally about 4.5 to 9.
The culture time is generally about 18 to 180 hours, more preferably about 36 to 120 hours.

In order to collect this D-lipose from the culture solution, a conventionally known method for separating and collecting Nori-lipose is employed. For example, after removing the bacterial cells by filtering or centrifuging the culture solution, the bacterial cell-removed liquid is treated with activated carbon, decolorized and desalted by ion exchange resin treatment, condensed, and concentrated with ethyl alcohol, etc. It is preferable to add an organic solvent for crystallization. In this case, if the culture results contain carbohydrates other than the target D-lipose, they should be treated with gμconuoxidase or yeast or a strain of bacteria that assimilates the carbohydrate tl- without assimilating D-lipose. This can also be removed from the cultured body by treatment with.

Below, DIJ when the additive of the present invention is added to the medium
The amount of porcelain and gluconic acid produced is shown.

The microorganisms and culture medium used were the same as those used in Example 1 described below, and the culture conditions and other operations were the same as those in Example 1 described later.

Table 1 shows the amounts of D-lipose and gluconic acid produced in the culture medium.

As described above, by adding the additive of the present invention to the culture medium and culturing, it is possible to increase the number of 30 to 501 Da/
The amount of gluconic acid produced can be suppressed to 4 to 20 ul/r:l, and as a result, the by-product of gluconic acid can be suppressed and the total amount of D-lipose produced can be increased.

EXAMPLES The present invention will be explained in detail below using Examples, but the content of the present invention is not limited thereto.

Example 1 Bacillus pumi/V7-f Mutant strain Nagi 716 (TECH FO13322, FERM-1) obtained by several times of ultraviolet irradiation
1491. ATcc 21951 +1/2%, cornstarch liquor 2%, dipotassium phosphate 0.396, potassium phosphate 0.1t
I6 to fxle medium + (1?) 411LL, 36℃2
After culturing for 4 hours and using this as a seed, D-gluco-71896,
Cornstap Liquor 296, Ammorum Sulfate D
, 596, was inoculated into medium 1001 consisting of 1.6% acid-selective μsum, 0.005% L-tryptophan, and 0.02596 L-valine.

When this was cultured with aeration at 38°C for 72 hours, D-lipose was accumulated at a ratio of 80 and 17417me. ]]-
The amount of repose produced was measured by the Oμ Shino μ method. This document is about Methods in Carbohydrate Chemistry.
e Chem: Letry) Volume 1, page 484 (19
62).

After removing bacterial cells by filtration from the D-Lipose fermentation solution, it was concentrated to about half its volume, and about % of ethanol was added to remove all sediments. After desalting with cation and anion exchange # fat, activated charcoal was added. The entire column was passed through to decolorize. This decolorizing solution was condensed with t-S, and about 4 borrowed amounts of ethanol/l' were added thereto to obtain D-lipose crystals 7.4#.

Example 2 Bacillus spumi/I/nu strain hermitage 71 used in Example 1
6 (ENGFO+3322.FERM-P Nagi1491,
ATCC21951), Tulpitou A/296, corn staple liquor 296, dipotassium phosphate 0.3%,
It was inoculated into medium 104 consisting of 0.1% potassium phosphate and cultured at 37°C for 24 hours.
1 ammonium 0.5y6, 1 takasum 1
．． 6%, Manganese C acid 0.05%, Leucine 0.05
96, L-) It was inoculated into 10 liters of a medium containing 0.00596 L-lyptophan, and cultured with aeration at 3[gamma][deg.]C for 72 hours. D-lipose was accumulated in the culture solution at a rate of 90.6 W/yτ. This culture solution was treated in the same manner as in Example 1 to obtain D-lipose crystal 8.3#.

Example 3 Mutant strain Nagi 716 (
IFO13322, FERM-p 1491, AT
cc 21951) in Tulpitou/I/296, Corn Stamp Liquor 296, Dipotassium Phosphate 0.3%
, inoculated into medium 104 consisting of 0.1% potassium phosphate and cultured at 37°C for 24 hours. Carbonic power 1.696,
L-)butophane 0.00596, α-aminobutyric acid 0
．． Plate on medium 1001 consisting of 02596 and heat at 37°C.
Culture was carried out with aeration and stirring for 72 hours. D-ribose was accumulated in the culture solution at a rate of 78.2 W/lxl. This culture solution was treated in the same manner as in Example 1 to obtain D-ribose crystals.
Got 4 for 2.

Effects of the Invention By culturing a microorganism that belongs to Bacillus deer and has the ability to produce D-ribose in a medium supplemented with the compound (I) of the present invention or its salt, the by-product of gluconic acid can be suppressed and D-
Ribose production can be increased. Furthermore, since the amount of by-product gluconic acid is small, the separation and removal operation of gluconic acid is easy.

Procedural Amendment (Target) July 1, 1985 (July 1, 1985) Case Description 1982 Patent Application No. 145854 2 Title of the invention D-Production method of ribose 3 Relationship to the sound case to be amended Patent applicant address: 2-27 Doshomachi, Higashi-ku, Osaka Name (2)
93) Takeda Pharmaceutical Co., Ltd. Representative: Kanetai Iku 4th Department 4, Agent Address: 2-17-85-5, Jusohonmachi, Yodoyo-ku, Osaka City, Detailed Description of the Invention in the Specification Subject to Amendment, Column 6, Amendment Contents (1) r FERM-P on page 4, lines 14-15 of the specification
Correct 1491J to r FERM BP-812J.

(2) Same book, page 12, lines 11-12, page 13, lines 17-
[FERM-P on line 18 and page 14, lines 12-13
No, 1491j is corrected to [FERMBP-8+2j.

1. Notification of Change in Accession Number February 30, 1985 1. Indication of Case Patent Application No. 145854 of 1988 2. Title of Invention Method for Manufacturing D-ribose 3. Person who filed the procedure Relationship with the case Patent Applicant Address 2-27 Doshomachi, Higashi-ku, Osaka City Name
(293) Takeda Pharmaceutical Co., Ltd. Representative: Kim Taiiku 4, Agent address: 2-17-85-5, Jusohonmachi, Yodogawa-ku, Osaka, Japan
Name of the old depositary institution: Institute of Microbial Technology, Agency of Industrial Science and Technology 6 Old accession number: FERM-PNo. FERM, BP-812 9, List of attached documents (1) One or more documents certifying the new accession number

Claims (1)

[Claims] A microorganism belonging to the genus Bacillus and having the ability to produce D-ribose is defined by the general formula R-X-COOH [wherein R is H- or 1 or 2 HO-, HS-
, CH_3S-, ▲ There are mathematical formulas, chemical formulas, tables, etc. ▼ or formula ▲ There are mathematical formulas, chemical formulas, tables, etc. ▼ (In the formula, R' represents H- or NH_2-) as a substituent. X represents a group ▲There are numerical formulas, chemical formulas, tables, etc.▼ or ▲There are numerical formulas, chemical formulas, tables, etc.▼, respectively. A method for producing D-ribose, which comprises culturing in a medium to which the compound represented by the formula or a salt thereof is added, producing and accumulating D-ribose in the culture solution, and collecting the D-ribose.