Patent ID: 6015697
Filing Date: 2000-01-18
Classification: C12N,C12P

Abstract:
A method for producing a nucleoside -5'-phosphate ester comprising the steps of effecting a transphosphorylation reaction by reacting (1) a nucleoside selected from the group consisting of inosine, guano sine, adenosine, xanthosine, purine riboside, 6-methoxypurine riboside, 2,6-diaminopurine riboside, 6-fluoropurine riboside, 6-thiopurine riboside, 2-amino-6-thiopurine riboside, mercaptoguanosines uridine, cytidine, 5-aminouridine, 5-hydroxyuridine, 5-bromouridine and 6-azauridine and (2) a phosphate group donor selected from the group consisting of polyphosphoric acid or salt thereof, phenylphosphoric acid or salt thereof, carbamyl phosphate or salt thereof and acetylphosphoric acid or salt thereof in the presence of an acid phosphatase, in solution at a pH of 3.0 to 5.5 to produce a nucleoside -5-phosphate ester and isolating the nucleoside -5'-phosphate, wherein the acid phosphatase comprises an amino acid sequence which is an amino acid sequence selected from the group consisting of SEQ ID NO: 4, 8, 24, 26 and 28 and said acid phosphatase has a mutation which decreases the K.sub.m value for the nucleoside and/or increases the temperature stability as compared to said amino acid sequence selected from the group consisting of SEQ ID NO: 4, 8, 24, 26 and 28, wherein the K.sub.m value for the nucleoside is determined by measuring the transphosphorylation activity of the acid phosphatase at 30.degree. C. and pH 4.0 in the presence of 100 .mu.mol/ml of sodium pyrophosphate.