Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound containing saccharide radical
Reexamination Certificate
1998-05-13
2004-01-06
Saidha, Tekchand (Department: 1652)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing compound containing saccharide radical
C435S196000, C435S252300, C435S252320, C435S252330, C435S320100, C435S069100, C536S023200
Reexamination Certificate
active
06673576
ABSTRACT:
TECHNICAL FIELD
The present invention relates to a process for producing 5′-inosinic acid or 5′-guanylic acid for use in seasonings or the like from inosine or guanosine or a precursor thereof using adenosine triphosphate (ATP)-producing microorganisms containing a DNA encoding a protein that has the activity of forming 5′-inosinic acid or 5′-guanylic acid from inosine or guanosine.
Further, the present invention relates to a novel protein having the inosine-guanosine kinase activity, a gene encoding said protein, a recombinant DNA containing said gene, and a microorganism which is transformed with said recombinant DNA.
BACKGROUND ART
In order to produce 5′-inosinic acid by phosphorylating inosine using microorganisms, a method using p-nitrophenyl phosphate (Japanese Patent Publication No. 29,858/1964), a method using inorganic phosphoric acids (Japanese Patent Publication Nos. 1,186/1967 and 44,350/1974), a method using acetyl phosphate (Japanese Patent Application Laid-Open No. 82,098/1981), and a method using ATP (Japanese Patent Application Laid-Open No. 230,094/1988) have been developed so far. However, the accumulation of 5′-inosinic acid which is produced by these methods has not necessarily been satisfactory. As an improved method of phosphorylating inosine with ATP, a method which comprises obtaining a gene encoding inosine-guanosine kinase of
Escherichia coli
, preparing an
E. coli
strain having the increased inosine-guanosine kinase activity by recombinant DNA technique, and phosphorylating inosine or guanosine using this strain to produce 5′-inosinic acid or 5′-guanylic acid, has been also developed (WO 91/08286). However, this method requires that a microorganism for regenerating ATP to be consumed in the reaction is separately cultured and that its cells are added to the reaction solution. Accordingly, a method of obtaining 5′-inosinic acid or 5′-guanylic acid more efficiently has been in demand.
Moreover, it is only known that the inosine-guanosine kinase gene is presents in some microorganisms such as
E. coli
[J. Gen. Microbiol., 135, 1263-1273 (1989)].
The present inventors have developed a process for producing 5′-inosinic acid and/or 5′-guanylic acid more efficiently. Consequently, they have found that 5′-inosinic acid and/or 5′-guanylic acid can be produced easily in a high yield by introducing a gene encoding an inosine-guanosine kinase into a microorganism having sufficient ability to regenerate ATP to be consumed in the reaction. They have also found a novel inosine-guanosine kinase having an amino-acid sequence which is different from that of an inosine-guanosine kinase derived from
E. coli.
DISCLOSURE OF THE INVENTION
The present invention relates to a process for producing 5′-inosinic acid or 5′-guanylic acid for use in seasonings or the like from inosine or guanosine or a precursor thereof easily in a high yield. More specifically, the present invention is to provide a process in which a gene encoding a protein that has the activity of converting inosine and/or guanosine into 5′-inosinic acid and/or 5′-guanylic acid is introduced into a microorganism having a sufficient ability to regenerate ATP to be consumed in the reaction, whereby 5′-inosinic acid and/or 5′-guanylic acid is easily obtained efficiently in a high yield in the presence of only the microorganism having the gene introduced therein without separately culturing another microorganism for regenerating ATP to be consumed in the reaction and adding it to the reaction solution.
Further, the present invention is to provide a novel protein which can be obtained from microorganisms belonging to
Exiguobacterium acetylicum
and which has the activity of converting inosine and guanosine into 5′-inosinic acid and 5′-guanylic acid, respectively, a gene encoding said protein, a recombinant DNA containing said gene, a microorganism which is transformed with said recombinant DNA, and a process for producing 5′-inosinic acid and/or 5′-guanylic acid using this microorganism.
The protein of the present invention is novel in that the amino-acid sequence of the protein of the present invention is vastly different from that of a known protein having inosine-guanosine kinase activity. An inosine-guanosine kinase derived from
E.coli
has been already known. The present inventors have found that a protein having the inosine-guanosine kinase activity is also produced in microorganisms belonging to the genus Exiguobacterium which were not known before to have the inosine-guanosine kinase activity, and they have succeeded in isolating this protein and cloning the gene encoding the protein. This protein is much different from the known protein with respect to the amino-acid sequence.
It has been found for the first time by the present inventors that the protein having the amino-acid sequence quite different from that of the known protein having the inosine-guanosine activity has the same activity as the known protein.
That is, the present invention relates to the following:
(1) a process for producing 5′-inosinic acid or 5′-guanylic acid, which comprises contacting a transformant obtained by introducing a gene encoding a protein having inosine-guanosine kinase activity into a microorganism capable of reproducing ATP, with inosine or guanosine or a precursor thereof, an energy source and a phosphate donor, accumulating 5′-inosinic acid or 5′-guanylic arid in the reaction solution, and collecting the same therefrom,
(2) a process for producing 5′-inosinic acid or 5′-guanylic acid according to (1), wherein the microorganism capable of reproducing ATP belongs to a genus selected from the group consisting of
Corynebacterium, Escherichia, Saccharomyces, Staphylococcus
and
Candida,
(3) a process for producing 5′-inosinic acid or 5′-guanylic acid according to (1), wherein the microorganism capable of reproducing ATP belongs to
Corynebacterium ammoniagenes,
(4) a process for producing 5′-inosinic acid or 5′-guanylic acid according to any one of (1) to (3), wherein the gene encoding the protein having inosine-guanosine kinase activity is a gene derived from
Exiguobacterium acetylicum
or a gene capable of hybridizing said gene,
(5) a process for producing 5′-inosinic acid or 5′-guanylic acid according to any one of (1) to (3), wherein the gene encoding the protein having inosine-guanosine kinase activity is a gene derived from Escherichia coli or a gene capable of hybridizing said gene,
(6) a transformant obtained by introducing a gene encoding a protein having inosine-guanosine kinase activity into a microorganism capable of reproducing ATP,
(7) a transformant according (6), wherein the microorganism capable of reproducing ATP belongs to a genus selected from the group consisting of
Corynebacterium, Escherichia, Saccharomyces, Staphylococcus
and
Candida,
(8) a transformant according to (6), wherein the microorganism, capable of reproducing ATP belongs to
Corynebacterium ammoniagenes,
(9) a transformant according to any of claims 6 to 8, wherein the gene encoding a protein having inosine-guanosine kinase activity is a gene derived from
Exiguobacterium acetylicum
or a gene capable of hybridizing said gene,
(10) a transformant according to any of (6) to (8), wherein the gene encoding a protein having inosine-guanosine kinase activity is a gene derived from
Escherichia coli
or a gene capable of hybridizing said gene,
(11) a recombinant DNA being capable of replicating in
Corynebacterium ammoniagenes
and containing a gene encoding a protein having inosine-guanosine kinase activity,
(12) a recombinant DNA according to (11), wherein the gene encoding a protein having inosine-guanosine kinase activity is a gene derived from
Exiguobacterium acetylicum
or a gene capable of hybridizing said gene,
(13) a recombinant DNA according to (11), wherein the gene encoding a protein having in
Kawasaki Hisashi
Shimaoka Megumi
Usuda Yoshihiro
Utagawa Takashi
Ajinomoto Co. Inc.
Oblon & Spivak, McClelland, Maier & Neustadt P.C.
Saidha Tekchand
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