| Summary: | 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 (<i>Am</i>PNP) from <i>Aneurinibacillus migulanus</i> AM007 was cloned and heterologously expressed in <i>Escherichia coli</i> BL21(DE3). The <i>Am</i>PNP 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 (<i>Bb</i>PyNP) derived from <i>Brevibacillus borstelensis</i> LK01 and <i>Am</i>PNP for the production of purine nucleoside analogs. Conversions of 2,6-diaminopurine ribonucleoside (<b>1</b>), 2-amino-6-chloropurine ribonucleoside (<b>2</b>), and 6-thioguanine ribonucleoside (<b>3</b>) synthesized still reached >90% on the higher concentrations of substrates (pentofuranosyl donor: purine base; 20:10 mM) with a low enzyme ratio of <i>Bb</i>PyNP: <i>Am</i>PNP (2:20 μg/mL). Thus, the new trimeric <i>Am</i>PNP is a promising biocatalyst for industrial production of purine nucleoside analogs.
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