Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Process involving micro-organisms of different genera in the...
Reexamination Certificate
2000-11-27
2002-02-19
Lilling, Herbert J. (Department: 1651)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Process involving micro-organisms of different genera in the...
C435S094000, C435S105000
Reexamination Certificate
active
06348326
ABSTRACT:
TECHNICAL FIELD
The present invention relates to a method for producing L-ribose by multiple steps of biological reactions starting from an inexpensive saccharide.
RELATED ART
In recent years, non-naturally occurring saccharides attracts attention as intermediate materials of pharmaceutical drugs and agricultural chemicals. As for ribose, almost no prospect can be currently seen for supply of the non-naturally occurring L-isomer, in spite of the fact that naturally occurring D-isomer is abundantly distributed all over the living world not only as the constitutional saccharide of DNA as a matter of course, but also as the constitutional saccharide of various vitamins or coenzymes. As the major method for producing L-ribose known at present, the synthetic method starting from L-arabinose as the raw material and utilizing a cobalt catalyst can be mentioned. On the other hand, as for a method for producing L-ribose using a microorganism, there is only one report that an enzyme produced by
Acinetobacter calcoaceticus
DL-28 strain isomerizes L-ribulose to produce L-ribose (Journal of Fermentation and Bioengineering Vol. 81, No. 6, 493-497, 1996).
As methods for producing L-ribulose, which is the raw material of the aforementioned enzymatic reaction, there are known a method utilizing isomerization of L-arabinose by L-arabinose isomerase (Methods Enzym. Anal. (3rd Ed.) vol. 6, 442-449, 1984) and a method utilizing a microbial reaction starting from purified ribitol as the starting material (Biochem. J., vol. 64, 385, 1956). However, it is hard to say for the both methods that the raw materials of L-arabinose and ribitol are inexpensive, and there has not been reported any process for stably supplying L-ribulose in an industrial scale.
As microbiological production methods of ribitol, which is the raw material used in one of the aforementioned two kinds of methods for producing L-ribulose, there have been reported production methods by an enzymatic reaction using D-ribose as a raw material (Japanese Patent Publication Nos. 45-2071/1972 and 49-12718/1974), production methods by fermentation of glucose etc. (Japanese Patent Publication Nos. 6-30591/1988, 6-30592/1988, 6-30593/1988, 6-30594/1988) and a production method by carbon dioxide fixation of an algae (Japanese Patent Publication No. 44-16350/1969).
However, any combination of the aforementioned techniques could not produce L-ribose in an industrial scale, and therefore no technique is known at all for producing L-ribose in an industrial scale as well as isolating and purifying L-ribose with high purity and high yield from the produced solution containing L-ribose.
In fact, when it is attempted to biologically produce L-ribose in an industrial scale using a conventional method, various problems arise. That is, for the first step, i.e., the production of ribitol, a method by fermentation of an inexpensive raw material such as the glucose is desirable among the conventional methods. However, such a conventional method cannot necessarily provide satisfactory productivity of ribitol even though it can efficiently produce sugar alcohols such as erythritol.
As for the technique for the second step, i.e., the conversion of ribitol into L-ribulose, in conventional methods, ribitol as the raw material must be purified. However, crystallized ribitol is very expensive as the raw material. Moreover, when the aforementioned fermented ribitol is used, it contains a large amount of polyalcohols such as erythritol and glycerol, and it is very difficult to separate and purify ribitol alone from it with good yield. Therefore, there has been desired a method of directly converting a fermentation broth containing ribitol or roughly purified ribitol into L-ribulose without purifying the ribitol in order to industrially produce L-ribulose at a low cost.
Further, even if L-arabinose is used as the raw material instead of ribitol, L-arabinose cannot be said to be inexpensive, and it also provides low yield of L-ribulose.
Also in the third stage, i.e., the reaction of converting L-ribulose into L-ribose, L-ribulose as the raw material must be purified in the conventional techniques. Purified L-ribulose is a very expensive raw material, and it is very difficult to isolate and purify it from the aforementioned liquid containing L-ribulose. Therefore, there has been desired a method of directly converting a fermentation broth containing ribitol or roughly purified ribitol into L-ribulose without purifying the ribitol and further directly converting the L-ribulose contained in the broth containing L-ribulose into L-ribose without purifying the L-ribulose, in order to industrially produce the objective product, L-ribose, at a low cost.
Further, since any method for isolating and purifying L-ribose from an L-ribose containing liquid including a lot of impurities by multiple-step biological reactions is not known at all, it has been necessary to develop an effective method.
DISCLOSURE OF THE INVENTION
The inventors of the present invention assiduously studied in order to solve the aforementioned problems. As a result, they found a method that can efficiently produce non-naturally occurring type L-ribose, which is an important intermediate of drugs and agricultural chemicals, at a low cost by using an inexpensive starting material such as glucose and combining several steps of microbial reactions and purification procedures, and thus accomplished the present invention.
That is, the present invention provides a method for producing L-ribose wherein L-ribose is produced from a saccharide raw material such as glucose as a starting material by using several steps of biological procedures. More specifically, the present invention provides a method for producing L-ribose, which comprises a step of producing ribitol from glucose by using a microorganism having an ability to produce ribitol from a saccharide raw material such as glucose (also referred to as the “first step” hereafter), a step of producing L-ribulose from the ribitol by using a microorganism having an ability to produce L-ribulose from ribitol (also referred to as the “second step” hereafter), and a step of producing L-ribose from the L-ribulose by using a microorganism having an ability to produce L-ribose from the L-ribulose (also referred to as “third step” hereafter).
Furthermore, as preferred embodiments of the present invention, there are provided the aforementioned method that further comprises contacting an L-ribose containing liquid produced by the aforementioned biological procedures with a gel type filtration medium, or adding an organic solvent to the L-ribose containing liquid so that L-ribulose alone should be deposited without depositing unreacted L-ribulose remaining in the reaction mixture.
Hereafter, the present invention will be explained in detail.
The method of the present invention is characterized in that L-ribose is produced starting from a saccharide raw material such as glucose by using several steps of biological procedures, and an example thereof is the method comprising the aforementioned first step, second step and third step. Although the following explanation will be made by exemplifying this method, the present invention is not limited to that method.
The first step of the production method of the present invention is a step of producing ribitol from a saccharide material such as glucose. The microorganism used in the first step is not particularly limited so long as it is a microorganism having an ability to produce ribitol from a saccharide material such as glucose. Preferred examples thereof include microorganisms belonging to the genus Trichosporonoides. More preferred examples include microorganisms belonging to the species
Trichosoporonoides madida, Trichosoporonoides nigrescens, Trichosporonoides oedocephalis, Trichosoporonoides megachillensis
and
Trichosporonoides spathulata
. Further preferred examples include microorganisms belonging to the species
Trichosoporonoides oedocephalis
and
Trichosporonoides megachillensis
. Examples of the microorganisms belon
Hara Mari
Kawaguchi Tomoko
Ueda Makoto
Lilling Herbert J.
Mitsubishi Chemical Corporation
Wenderoth , Lind & Ponack, L.L.P.
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