A thermostable pyrimidine nucleoside phosphorylase has been purified from Bacillus stearothevmophilus JTS 859. The enzyme had a molecular weight of 85, 000, consisting of 2 identical subunits (Mw 54, 000). Its isoelectric point was 4.8. The Michaelis constants for 5-methyluridine, uridine, thymidine, and 2'-deoxyuridine were 0.32, 0.19, 0.46, and 0.58 mM, respectively. The optimal temperature of the reaction was 70°C. The enzyme was found not to be a SH-enzyme based on the following three points. First, the enzyme reaction was not inhibited by PC MB or iodoacetic acid. Second, the optimal pH of the reaction was broad, from 7.0 to 11.5. Third, the enzyme did not contain cysteine. The half-life of the enzyme was 15.1 hr in 20mM potassium phosphate and ImM 5-methyluridine (pH 7.0) at 70°C. Due to the high optimal temperature, the broad optimal pH range, and the long half-life of the enzyme, the enzyme is useful for practical applications.
Read full abstract