Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Patent
1997-01-21
2000-01-04
Houtteman, Scott W.
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
536 266, C12Q 168
Patent
active
060108517
DESCRIPTION:
BRIEF SUMMARY
TECHNICAL FIELD
The present invention relates to a method for producing nucleoside-5'-phosphate ester. The present invention also relates to a novel acid phosphatase, a gene coding for the acid phosphatase, a recombinant DNA containing the gene, and a microorganism harboring the recombinant DNA which are useful to produce nucleoside-5'-phosphate ester. Nucleoside-5'-phosphate ester is useful as a seasoning, a pharmaceutical, and a row material for producing such substances.
BACKGROUND ART
Methods for biochemically phosphorylating nucleoside to produce nucleoside-5'-phosphate ester by using the following phosphate group donors are known, including a method which uses p-nitrophenyphosphoric acid (Japanese Patent Publication No. 39-29858), a method which uses inorganic phosphoric acid (Japanese Patent Publication No. 42-1186), a method which uses polyphosphoric acid (Japanese Patent Laid-open No. 53-56390), a method which uses acetylphosphoric acid (Japanese Patent Laid-open No. 56-82098), and a method which uses adenosine triphosphate (ATP) (Japanese Patent Laid-open No. 63-230094). However, these methods have not been satisfactory to produce nucleoside-5'-phosphate ester efficiently and inexpensively because the substrates to be used are expensive, or because by-products are produced in the reaction.
Thus the present inventors have developed a method for efficiently producing nucleoside-5'-phosphate ester without by-producing 2'-, 3'-nucleotide isomers by allowing cells of a specified microorganism to act under an acidic condition on a nucleoside and a phosphate group donor selected from the group consisting of polyphosphoric acid or a salt thereof, phenylphosphoric acid or a salt thereof, and carbamyl phosphate or a salt thereof (Japanese Patent Laid-open No. 7-231793).
However, even this method has had the following drawbacks. Namely, for example, a part of the substrate is degraded during the reaction due to a nucleoside-degrading activity which unfortunately exists in a slight amount in the cells of the microorganism to be used. Moreover, if the reaction is continued, produced and accumulated nucleoside-5'-phosphate ester is degraded. Therefore, by-products are produced in a reaction solution, and it has been impossible to obtain a sufficient yield. In addition, the reaction cannot be performed if the substrate is added at a high concentration because of a low transphosphorylation activity per microbial cell.
DISCLOSURE OF THE INVENTION
An object of the present invention is to provide a method for inexpensively and efficiently producing nucleoside-5'-phosphate ester. Another object of the present invention is to provide an enzyme, a gene coding for the enzyme, a recombinant DNA containing the gene, and a microorganism harboring the recombinant DNA which are useful for the method for producing nucleoside-5'-phosphate ester.
As a result of various investigations made by the present inventors in order to develop a method for producing nucleoside-5'-phosphate ester which is more efficient than the conventional methods, it has been found out that nucleoside-5'-phosphate ester can be efficiently produced at a high yield by allowing an acid phosphatase purified from a cell-free extract of a microorganism to act under a condition of pH 3.0 to 5.5 on a nucleoside and a phosphate group donor selected from the group consisting of polyphosphoric acid or a salt thereof, phenylphosphoric acid or a salt thereof, and carbamyl phosphate or a salt thereof. Further, the present inventors have succeeded in obtaining wild type genes coding for acid phosphatases from various bacteria and genes coding for acid phosphatases having lowered phosphomonoesterase activities from bacterium belonging to the genus Morganella and bacterium belonging to the genus Escherichia. Moreover, the present inventors have found out that productivity of nucleoside-5'-phosphate ester is remarkably improved by expressing the gene in a large amount in accordance with genetic engineering techniques. Thus the present invention has been completed
REFERENCES:
Abstract for Congress of the Society for Fermentation and Bioengineering, p. 356, Oct. 10, 1994, Y. Asano, et al., "Phosphorylation of Nucleosides With Pyrophosphate As A Phosphate Donor" (with English Translation).
Nippon Nogeikagaku Kaishi, vol. 69, p. 270 (20a10), Jul. 5, 1995, Y. Mihara, et al., "Enzymatic Phosphorylation Reaction Of Nucleoside Using Pyrophosphate As A Phosphate Donor" (With English Translation).
Efstratiadis et al., Nucleic Acids Research, 4(12):4165-74, Abstract, Dec. 1977.
Asano Yasuhisa
Mihara Yasuhiro
Utagawa Takashi
Yamada Hideaki
Ajinomoto Co. Inc.
Houtteman Scott W.
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