Method for producing nucleoside-5′-phosphate ester

Details Method for producing nucleoside-5′-phosphate ester Method for producing nucleoside-5′-phosphate ester

1999-10-13

2001-03-27

Nashed, Nashaat T. (Department: 1652)

Chemistry: molecular biology and microbiology

Enzyme , proenzyme; compositions thereof; process for...

Hydrolase

C435S194000, C435S195000

Reexamination Certificate

active

06207435

ABSTRACT:

BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to a method for producing nucleoside-5′-phosphate esters. 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 for producing nucleoside-5′-phosphate esters. Nucleoside-5′-phosphate esters are useful as, for example, seasoning and pharmaceuticals.
2. Description of the Background
Methods for biochemically phosphorylating nucleosides to produce nucleoside-5′-phosphate esters by using the following phosphate group donors are known, such as 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 esters efficiently and inexpensively because the substrates are expensive, or because undesirable 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, phenylphosphonic 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 portion 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, the produced and accumulated nucleoside-5′-phosphate ester is degraded. Therefore, by-products are produced in the 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.
OBJECTS OF THE INVENTION
It is an object of the present invention is to provide a method for inexpensively and efficiently producing nucleoside-5′-phosphate esters via biochemical synthesis using an acid phosphatase. It is another object of the present invention is to provide 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 for producing nucleoside-5′-phosphate esters.
SUMMARY OF THE INVENTION
As a result of various investigations made by the present inventors in order to develop a method for producing nucleoside-5′-phosphate esters which is more efficient than the conventional methods, it has been found that nucleoside-5′-phosphate esters may be efficiently produced in high yield by using an acid phosphatase to catalyze the transfer of a phosphate group from a phosphate group donor to a nucleoside at a pH 3.0 to 5.5. 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 an increased affinity for the nucleoside as compared with the wild type acid phosphatase by introducing a mutation in an acid phosphatase derived from a bacterium belonging to the genus Escherichia. Moreover, the present inventors have discovered that the yield of nucleoside-5′-phosphate ester is remarkably improved by expressing the gene in large quantities using genetic engineering techniques.
Further, the present inventors have conducted experiments to prepare a mutant acid phosphatase with an increased temperature stability, in order to conduct the phosphate transfer reaction catalyzed by the acid phosphatase at a higher temperature. Conducting the reaction at higher temperature leads to more effective production of nucleoside-5′-phosphate esters because the reaction speed is elevated and the concentration of the nucleoside in the reaction solution may be higher. Also, the present inventors have succeeded in the preparation of a mutant acid phosphatase which has an increased temperature stability as compared to the mutant acid phosphatases described in Example 19 which may be used in a reactions at a high temperature.
Accordingly, the present invention provides a method for producing nucleoside-5′-phosphate esters by contacting (1) a nucleoside, (2) a phosphate group donor, and (3) an acid phosphatase having an increased affinity for the nucleoside and/or an increased temperature stability at a pH of 3.0 to 5.5, to produce a nucleoside-5′-phosphate ester, followed by isolating the nucleoside-5′-phosphate ester. In a preferred embodiment, the nucleoside and the phosphate group donor are cultured in the presence a microorganism transformed with a recombinant DNA comprising a gene encoding the acid phosphatase.
The objects of the present invention may also be accomplished with mutant acid phosphatases having an increased affinity for the nucleoside and/or an increased temperature stability, genes coding for the acid phosphatases, recombinant DNAs containing the genes, and microorganisms harboring, i.e., transformed with, the recombinant DNA.


REFERENCES:
patent: 39 29858 (1964-12-01), None
patent: 42 1186 (1967-01-01), None
patent: 53 56390 (1978-05-01), None
patent: 56 82098 (1981-07-01), None
patent: 63 230094 (1988-09-01), None
patent: 07 231793 (1995-09-01), None
Abstract for Congress of the Society for Fermentation and Bioengineering, p. 356, Oct. 10, 1994, 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, Mihara et al, “Enzymatic Phosphorylation Reaction of Nucleoside using Pyrophosphate as a Phosphate Donor” (With English Translation).

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