JPH11215996A - Production of l-ribose - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain in an industrially advantageous way L-ribose usable as e.g. an intermediate material for medicines and agrochemical, by subjecting Cellulomonas microorganisms having the ability to produce L-ribose through the isomerization of L-ribulose to L-ribulose. SOLUTION: This L-ribose useful as e.g. an intermediate material for medicines and agrochemicals is obtained in an industrially advantageous way by subjecting microbial cells and/or a treated product thereof of microorganisms belonging to the genus Cellulomonas and having the ability to produce L-ribose through the isomerization of L-ribulose to L-ribulose; wherein the above microorganisms to be used are e.g. Cellulomonas gerida JCM 1490, Cellulomonas biazotea ATCC 486, Cellulomonas flavigena ATCC 482 or the like.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a method for producing L-ribose, and more particularly, to a method for producing L-ribose by isomerizing L-ribulose using a microorganism.

[0002]

2. Description of the Related Art In recent years, non-natural saccharides have attracted attention as intermediate materials for pharmaceuticals and agricultural chemicals. Regarding ribose, the natural D-form is abundantly distributed in all living organisms, not only as a constituent sugar of DNA, but also as a constituent sugar of various vitamins and coenzymes. At present, there is no prospect of supply. As a currently known main production method of L-ribose, there is a synthesis method using L-arabinose as a raw material and using a cobalt catalyst. On the other hand, as a method for producing L-ribose using microorganisms, Acinetobacter calcoaceticus (Acin
It has only been reported that an enzyme produced by etobacter calcoaceticus (DL-28) isomerizes L-ribulose and produces L-ribose (Journal of Fermentatio).
n and Bioengineering Vol.81, No.6, 493-497.1996).

[0003]

When L-ribose is actually produced on an industrial scale using conventional methods, various problems arise. That is, in the synthesis method, L-arabinose as a raw material is expensive, the cobalt catalyst is very expensive, and the reaction yield is never satisfactory.

[0004]

Means for Solving the Problems The present inventors have made intensive studies to solve the above-mentioned problems, and as a result, have found that Cellulomonas
It has been found that a microorganism belonging to the genus ulomonas has the ability to isomerize L-ribulose and produce L-ribose, thereby completing the present invention. That is, the gist of the present invention is to make L-ribulose act on cells of microorganisms belonging to the genus Cellulomonas having the ability to isomerize L-ribulose to produce L-ribose and / or the treated cells. A method for producing L-ribose, further comprising the steps of:
The microorganism belonging to the genus is Cellulomonas gerida
as gerida)), Cellulomon biazotea (Cellulom
onas biazotea) or Cellulomonas flavigena (Ce)
llulomonas flavigena).

[0005] Cellulomonas used in the present invention
The microorganism belonging to onas) is not particularly limited as long as it is a microorganism having an ability to isomerize L-ribulose to produce L-ribose. Or microorganisms belonging to the species Cellulomonas flavigena. The microorganism may be any strain such as a mutant strain or a recombinant strain derived by a genetic technique such as cell fusion or gene recombination. This microorganism may by-produce L-arabinose in addition to L-ribose by isomerization. For example, ultraviolet irradiation, N-methyl-N′-nitro-N-nitrosoguanidine (NTG) treatment, ethyl methanesulfonate ( E
MS) treatment, nitrous acid treatment, acridine treatment and the like, the L-ribose production ratio is good,
-It is effective to obtain a mutant strain with low productivity of arabinose. Further, obtaining a mutant strain which exhibits sufficient activity even without inducing an enzyme with L-ribose or L-ribose at the time of culture by the same mutation treatment is also very effective in improving L-ribose productivity.

[0006]

DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention will be described below in detail.
The microorganism used in the present invention is a microorganism belonging to Cellulomonas gerida, which is JC.
M1490 is Cellulomon biazotea (Cellulomon
ATCC486 as a microorganism belonging to as biazotea)
The Cellulomonas flavigen
Examples of the microorganism belonging to a) include ATCC482. The microorganism may be any strain such as a recombinant strain derived by a genetic technique such as cell fusion or genetic recombination.

[0007] In the production method of the present invention, one or more of the above microorganisms are used. In the production method of the present invention, cells of the above-mentioned microorganisms and / or treated cells are used. Specifically, the cells obtained by culturing the microorganisms as they are, or those obtained by culturing the cells obtained by a known method, that is, those treated with acetone, those subjected to freeze-drying, A treated bacterial cell such as one obtained by physically or enzymatically crushing bacterial cells such as toluene treatment can be used. In addition, an enzyme fraction which acts on L-ribulose and has an ability to produce L-ribose can be extracted from these cells or processed cells and used as a crude product or a purified product. Furthermore, it is also possible to use those obtained by immobilizing the thus obtained cells, cell treated products, enzyme fractions and the like on a carrier such as polyacrylamide gel and carrageenan gel. Therefore, in the present specification, the term "cells and / or treated cells" refers to the above-mentioned cells, treated cells, enzyme fraction,
And a concept containing all the immobilized substances.

Next, the production method of the present invention will be specifically described. In the production method of the present invention, microorganisms are cultured as usual. That is, as the carbon source, carbohydrates such as glucose that can be assimilated by the present microorganism, alcohols such as glycerol, organic acids, and the like, preferably glucose, glycerol, sucrose, and the like, more preferably sucrose, are used. One or more of these carbon sources can be added as needed during the culture.
The medium further contains a nitrogen source that can be assimilated by the microorganism. Nitrogen sources include amino acids, yeast extract, soy peptide, soy flour, corn steep liquor, NZ amine, tryptose, peptone, polypeptone, meat extract, fish extract, other organic nitrogen sources, or sodium nitrate, other inorganic nitrogen sources Preferably, yeast extract, soybean peptide, and polypeptone are used as appropriate. It is effective to add inorganic ions and vitamins as necessary in addition to the above-mentioned carbon source and nitrogen source. As inorganic ions, phosphate ions, magnesium ions, iron ions,
Manganese ions, molybdenum ions and the like are used. Vitamins include thiamine, inositol, pantothenic acid, nicotinamide and the like. Further, the above-mentioned sucrose and other carbon sources, nitrogen sources, inorganic ions, and vitamins may be supplemented and added at an appropriate time during the culture, if necessary.

[0009] The culture is performed under aerobic conditions. In the present invention, it is important to perform aeration sufficiently to express isomerase with high activity. The culturing is performed at a temperature of 20 to 37 ° C, preferably 27 to 32 ° C, for 12 to 48 hours.
The cells obtained by the culture are collected as they are or by a known method such as centrifugation and used for an isomerization reaction. The cells are subjected to an isomerization reaction as it is or by physically or enzymatically disrupting the cells by a known method such as toluene treatment, lysozyme treatment, or ultrasonic disruption. Further, the cells and toluene may be added to the L-ribulose solution. Preferably, the cells are concentrated and separated from the culture termination solution, subjected to a toluene treatment, and then subjected to an isomerization reaction. This isomerization reaction is a reversible reaction, and one unit of the isomerase activity is an amount of an enzyme capable of isomerizing 1 micromol of L-ribose per minute to produce L-ribulose.

In the isomerization reaction, the cells are added to an aqueous L-ribulose solution adjusted to pH 4 to 9.5, preferably pH 8.5 to 9.0, at least 2 units per gram of ribulose.
Preferably, it is added so that it becomes 6 units or more, and the temperature 2
It is carried out at 0 ° C to 40 ° C, preferably at 27 ° C to 32 ° C. Since the pH of the reaction solution decreases as the reaction proceeds, it is preferable to control the pH to around 9.0 with an alkaline solution such as sodium hydroxide. Depending on the amount of cells used, the reaction is completed within a few seconds to several days when L-ribose and L-ribulose reach equilibrium. The produced L-ribose can be separated and purified from the reaction completed solution by a known method. That is, after completion of the reaction, the microbial cells are first removed by an existing method such as centrifugation. Prior to the eradication, heat may be applied to the culture-finished solution to deactivate the enzyme and reduce the content. Further, carbonation before or after eradication is very effective for subsequent purification. The bacteria-removed solution contains a few carbohydrates such as L-ribose and L-arabinose in addition to L-ribose, and also substances derived from bacterial cells and culture media. These can be removed by a usual method, that is, using a chromatography or crystallization technique. Industrially, it is also possible to perform separation by pseudo mobile phase chromatography using an ion exchange resin. In the step of separation and purification, if necessary, ordinary sugar purification operations such as desalting and decoloring can be added.

[0011]

The present invention will be described in more detail with reference to the following examples, but ordinary modifications in the technical field of the present invention can be made without departing from the gist of the present invention. Example 1 a) Microorganism used

[0012]

TABLE 1 Strain A Cellulomonas gerida JCM1490 Strain B Cellulomonas biazotea ATCC486 Strain C Cellulomonas flavigena ATCC482

B) Culture method Sucrose 2.0 g / L, yeast extract 5.0 g /
L, soy peptide 5.0 g / L, NaCl 5 g /
L, K ₂ HPO ₄ 3 g / L, KH ₂ PO ₄ 1 g / L, L
A medium containing 15 g / L ribose (pH 7.0)
Each 0 mL was dispensed into a 200 mL baffle flask, and each of the strains A to C described in a) above was inoculated. These baffled flasks were set on a shaking incubator rotating at 160 rpm, and cultured at 30 ° C. for 18 hours.

C) Method for Isomerization Reaction Each of the culture-end solutions obtained by the culture described in b) above was centrifuged and collected. To each cell pellet, glycine-hydrochloride buffer (50 mM, pH 9.0) was added in 2 mL portions to suspend uniformly, and then toluene was added in 60 μL portions and mixed vigorously for 15 minutes. The toluene-treated cells thus prepared were added to 5 mL of a 10 g / L L-ribulose solution, and the mixture was reacted at 30 ° C. for 15 hours with gentle stirring so as to be uniform. On the way, the pH was controlled at 9.0 with 1N NaOH.

D) Confirmation of L-ribose production The reaction-terminated liquids described in the above c) were each centrifuged to remove microbial cells. The L-ribose content contained in the obtained supernatant was measured by high performance liquid chromatography under the following conditions. The retention time of L-ribose is 24.0 minutes under the following analysis conditions.

[0016]

[Table 2] High-performance liquid chromatography analysis conditions Column: MCI GEL CK08EC 8 mm ID × 300 mm (manufactured by Mitsubishi Chemical Corporation) Eluent: Water Flow rate: 0.6 ml / min Column temperature: 75 ° C Detection Instrument: RI d) Result The production results of each strain are as shown in the following table.

[0017]

TABLE 3 Strain L-Ribulose L-ribose g / L g / L A 2.5 5.9 B 1.8 4.1 C 1.8 4.3

[0018]

According to the production method of the present invention, L-ribose can be isomerized using a microorganism to produce L-ribose.

Claims (3)

[Claims] 1. Cellulomonas having the ability to isomerize L-ribulose to produce L-ribose.
s) L-characterizing that a cell of a microorganism belonging to the genus and / or a processed product of the cell is allowed to act on L-ribulose.
Method for producing ribose. 2. The microorganism belonging to the genus Cellulomonas is a cellulomonas gerid.
a), Cellulomonas biazotea
tea) or Cellulomonas flavigena (Cellulomonas)
2. The method according to claim 1, which is a microorganism belonging to the species flavigena). 3. Cellulomonas g
erida) is JCM1490, microorganisms belonging to Cellulomonas biazotea are ATCC486, and microorganisms belonging to species of Cellulomonas flavigena are A.
The method of claim 1, wherein the method is TCC482.