Synthesis of 5-methyluridine by a thermophile, Bacillus stearothermophilus JTS 859.

Abstract

Manythermophiles, which can grow at 65°C, were examined as to their ability to produce 5-methyluridine from inosine and thymine (5-methyluracil) in the presence of phosphate and cells as enzymesources. Bacillus stearothermophilus JTS 859 was selected as a strain that synthesized 5-methyluridine efficiently. The reaction is supposed to be carried out by a combination of a thermostable purine nucleoside phosphorylase and a thermostable pyrimidine nucleoside phosphorylase. Their halflives were 7200hr and 400hr at 63 C, and 148hr and 14hr at 70 C, respectively.Nucleosides such as inosine, adenosine, uridine and cytidine are produced by a fermentation method, but thymidine has not been produced by such a method. Thymidine is practically produced by hydrolysis and separation from deoxyribonucleic acid derived from natural sources such as milt. As an alternative to this method, we developed a method for the practical production of thymidine from non-natural sources. Wetried to synthesize 5-methyluridine (5MU) from thymine (5-methyluracil) and inosine enzymatically at first by means of the ribosyl transfer reaction, as shown in the following chemical equations, and to chemically reduce the hydroxy group at the 2'-position on the ribose ring of 5MUto yield thymidine.Inosine + Phosphate-åº Ribose-1 -phosphate + Hypoxanthine (1)Ribose-1 -phosphate + Thymine-åº 5-Methyluridine + PhosphateWeselected inosine as a ribose donor because it it produced abundantly in industrial fermentations. This paper describes the synthesis of 5-methyluridine using thermostable enzymes produced by a thermophile. 197The ribosyl transfer reaction has been studied for a long time.1~4) This reaction is carried out by purine nucleoside phosphorylase, pyrimidine nucleoside phosphorylase, uridine phosphorylase, thymidine phosphorylase and a combination of these enzymes. The substrate specificities of these enzymes are not strict, therefore, the ribosyl transfer reaction has been applied for the synthesis of nucleoside analogues, 5-fluorouridine, 5-bromouridine and so on.5~7) It is necessary that the enzymes used for practical application should be stable for a long time. Enzymes derived from mesophiles have been used for practical application.As for thermostable enzymes derived from thermophiles, only pyrimidine nucleoside phosphorylase from Bacillus stearothermophilus NCA10 has been reported.8)The application of thermostable enzymes has several advantages.9) The reaction can be carried out at high temperature for a long time and the reaction rate is also high. The higher solubility of the reactants allows operation with higher concentrations of reactants. The solubility of 5MUis 83g/1 at 25°C and above 500 g/1 at 63°C. Thermostable enzyme reactors are resistant to bacterial contamination due to the high operation temperature. This is impor-

Biocatalytic routes to anti-viral agents and their synthetic intermediates

“…57,58 Like the previously mentioned nucleoside analogues, 17 has been obtained through the use of isolated phosphorylases or whole cells overexpressing such enzymes (Scheme 5). [59][60][61][62] Arguably, hydrolases are the most commonly applied enzymes in the industrial synthesis of small-molecules. 63 This class comprises enzymes capable of cleaving bonds through attack of a water molecule.…”

Cloning, purification and characterisation of a recombinant purine nucleoside phosphorylase from Bacillus halodurans Alk36

“…In addition, thermophiles have been shown to harbour enzymes that exhibit a much greater thermostability. For example, the thermophile Geobacillus stearothermophilus (previously Bacillus stearothermophilus ) has two purine nucleoside phosphorylases which have been characterised (Hori et al 1989a, b; Saunders et al 1969) and applied in the synthesis of 5-methyluridine (Hori et al 1989c, 1991). However, although these enzymes are thermostable, they have low levels of expression in the wild type.…”

Purification and Characterization of Second Thermostable Purine Nucleoside Phosphorylase inBacillus stearothermophilusJTS 859

“…We have studied the practical application of ribosyl transfer reaction by thermostable PUNP and pyrimidine nucleoside phosphorylase (PYNP) (EC 2.4.2.2) at high temperatures. 5 ) We reported the purification and the characterization of a thermostable PUNP, PUNPI from Bacillus stearothermophilus JTS 859, and also the existence of another PUNP, PUNPII, in our previous paper. 6 ) This paper describes the purification and the characterization of PUNPII.…”