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

Abstract

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) from Aneurinibacillus migulanus AM007 was cloned and heterologously expressed in Escherichia coli BL21(DE3). The AmPNP 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 from Brevibacillus borstelensis LK01 and AmPNP 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 of BbPyNP: AmPNP (2:20 μg/mL). Thus, the new trimeric AmPNP is a promising biocatalyst for industrial production of purine nucleoside analogs.

Sustainable Flow‐Synthesis of (Bulky) Nucleoside Drugs by a Novel and Highly Stable Nucleoside Phosphorylase Immobilized on Reusable Supports

“…The phosphate concentration was therefore set at 20 m m , which showed the best transglycosylation yield (Table S2). Regarding the sugar donor (inosine), other works, however, stated in general the use of 1–4 equivalents of different sugar donors, with yields spanning greatly from low percent to high eighties, with the highest reported the synthesis of adenosine using uridine [7,9,11,25,35] . Our results are indeed in agreement, and as high as 20 equivalents (100 m m ) of inosine were required to observe total conversion at 5 m m scale in batch conditions and after 24 h (Table S3).…”

“…Thermostable enzymes certainly have a higher longevity, which is beneficial to achieve a longer operational stability under continuous flow processes. Such stability is comparable to the PNP from A. migulanus and very superior to the PNP from B. stearothermophilus [25,34] . The temperature stability was further improved after immobilization (Figure S12C).…”

Genome-Driven Discovery of Enzymes with Industrial Implications from the Genus Aneurinibacillus

“…Examples of nucleoside (both purine and pyrimidine) phosphorylase enzymes representing EC:2.4.2.1 and EC:2.4.2.2 were also found in the pan-genome of Aneurinibacillus strains. Recently, a new thermostable trimeric purine nucleoside phosphorylase was cloned and characterized from Aneurinibacillus migulanus AM007 [38]. Such enzymes are used as biocatalysts for the synthesis of pentose-1-phosphates and nucleoside analogues that represent an important source of many drugs including antiviral and anticancer drugs [79].…”

“…However, only a limited number of genomes were used in such studies. This limits our understanding of the complete potential of microorganisms, which have shown great capabilities for diverse types of enzyme activities, or the production of bioactive compounds [16,22,[37][38][39][40]. Therefore, in this work we apply genome mining approaches to investigate the biosynthetic potential as well as explore the diverse sets of enzymes for biomass degradation and heavy metal resistance present in Aneurinibacillus genomes.…”

Green Production of Cladribine by Using Immobilized 2′-Deoxyribosyltransferase from Lactobacillus delbrueckii Stabilized through a Double Covalent/Entrapment Technology

“…Traditionally, cladribine is synthesized by chemical methods that require multiple reaction steps, the use of organic solvents, and removal of protecting groups, causing the accumulation of racemic mixtures that affect further purification [3,4]. However, the use of bioprocesses catalyzed by whole cells or enzymes has emerged as a green synthetic alternative with multiple advantages, such as mild reaction conditions, high efficiency, and regio-, stereo-, enantioselectivity [5][6][7][8][9][10][11][12][13][14][15]. In this sense, the transglycosylation reaction catalyzed by nucleoside phosphorylases (NPs) or 2 -deoxyribosyltransferases (NDTs) is the most studied biocatalytic methodology for the synthesis of nucleoside analogs (NAs) up to date [5][6][7][8][9][10][11][12][13][14][15].…”