Enzymatic Synthesis of Thymidine Using Bacterial Whole Cells and Isolated Purine Nucleoside Phosphorylase

Abstract

Whole cells of Escherichia coli and the thermostable bacteria, Bacillus stearothermophilus, were used for the efficient synthesis of the biologically and industrially important compound, thymidine, using 2′-deoxyinosine and thymine as substrates. In this conversion, the 2′-deoxyribose moiety of 2′-deoxyinosine was transferred to thymine by the transdeoxyglycosylation activity of these bacterial cells. For example, in the case of Bacillus stearothermophilus, the yield of pure thymidine was 56% (78% conversion). When xanthine oxidase was added to this whole cell process, the product yield increased to 68% (90% conversion). In this transformation, Bacillus stearothermophilus was used at a temperature of 55°C where the solubility of thymine is much higher than at 25°C. The bacterial cells have activity over a broad pH range (approximately 4.0 to 8.0) and the yield of product varied within this pH range with the optimum pH being at 5.2. Both bacterial cells showed a sharp decrease in activity at alkaline pHs. Cells of both bacteria can be used repeatedly without appreciable loss of activity. Thymidine was also produced from thymine and 2′-deoxyinosine using isolated purine nucleoside phos-phorylase. A dramatic increase in conversion occurred with PNPase in the presence of xanthine oxidase.

Key Words:

• Thymidine
• Purine nucleoside phosphorylase
• Xanthine oxidase
• Bacillus stearothermophilus
• Escherichia coli
• Transglycosylation