Efficient Synthesis of Purine Nucleoside Analogs by a New Trimeric Purine Nucleoside Phosphorylase from Aneurinibacillus migulanus AM007

Purine nucleoside phosphorylases (PNPs) are promising biocatalysts for the synthesis of purine nucleoside analogs. Although a number of PNPs have been reported, the development of highly efficient enzymes for industrial applications is still in high demand. Herein, a new trimeric purine nucleoside phosphorylase (AmPNP) fromAneurinibacillus migulanusAM007 was cloned and heterologously expressed inEscherichia coliBL21(DE3). TheAmPNP showed good thermostability and a broad range of pH stability. The enzyme was thermostable below 55 °C for 12 h (retaining nearly 100% of its initial activity), and retained nearly 100% of the initial activity in alkaline buffer systems (pH 7.0–9.0) at 60 °C for 2 h. Then, a one-pot, two-enzyme mode of transglycosylation reaction was successfully constructed by combining pyrimidine nucleoside phosphorylase (BbPyNP) derived fromBrevibacillus borstelensisLK01 andAmPNP for the production of purine nucleoside analogs. Conversions of 2,6-diaminopurine ribonucleoside (1), 2-amino-6-chloropurine ribonucleoside (2), and 6-thioguanine ribonucleoside (3) synthesized still reached >90% on the higher concentrations of substrates (pentofuranosyl donor: purine base; 20:10 mM) with a low enzyme ratio ofBbPyNP:AmPNP (2:20 μg/mL). Thus, the new trimericAmPNP is a promising biocatalyst for industrial production of purine nucleoside analogs.Keywords:trimeric nucleoside phosphorylase;thermostability;purine nucleoside analogs;enzyme ratio