Nucleoside 2'-Deoxyribosyltransferase from Psychrophilic Bacterium Bacillus psychrosaccharolyticus — Preparation of an Immobilized Biocatalyst for the Enzymatic Synthesis of Therapeutic Nucleosides

Abstract

Nucleoside 2'-deoxyribosyltransferase (NDT) from the psychrophilic bacterium Bacillus psychrosaccharolyticus CECT 4074 has been cloned and produced for the first time. A preliminary characterization of the recombinant protein indicates that the enzyme is an NDT type II since it catalyzes the transfer of 2'-deoxyribose between purines and pyrimidines. The enzyme (BpNDT) displays a high activity and stability in a broad range of pH and temperature. In addition, different approaches for the immobilization of BpNDT onto several supports have been studied in order to prepare a suitable biocatalyst for the one-step industrial enzymatic synthesis of different therapeutic nucleosides. Best results were obtained by adsorbing the enzyme on PEI-functionalized agarose and subsequent cross-linking with aldehyde-dextran (20 kDa and 70% oxidation degree). The immobilized OPEN ACCESSMolecules 2014, 19 11232 enzyme could be recycled for at least 30 consecutive cycles in the synthesis of 2'-deoxyadenosine from 2'-deoxyuridine and adenine at 37 °C and pH 8.0, with a 25% loss of activity. High conversion yield of trifluridine (64.4%) was achieved in 2 h when 20 mM of 2'-deoxyuridine and 10 mM 5-trifluorothymine were employed in the transglycosylation reaction catalyzed by immobilized BpNDT at 37 °C and pH 7.5.

2′-Deoxyribosyltransferase from Bacillus psychrosaccharolyticus: A Mesophilic-Like Biocatalyst for the Synthesis of Modified Nucleosides from a Psychrotolerant Bacterium

“…Remarkably, the dependence of its catalytic activity on temperature reveals that BpNDT is not heat-labile (Figure 1), a characteristic of cold-adapted enzymes that results from catalytic centres more mobile or flexible. Indeed, although we have observed that some loops flanking the substrate cavity of BpNDT are more flexible than in the mesophilic LlNDT or LhPDT (Figures 4 and 5), it is obvious from the thermal inactivation experiments that this structural feature does not result in a heat-labile enzyme: BpNDT exhibits highest activity at 40 • C and pH 8 [25]. In fact, it is active under these conditions for a longer period of time than the mesophilic LrNDT: the half-time at 60 • C of BpNDT (21.8 h) is remarkably higher than the one observed for LrNDT (17.9 min) [18].…”

“…Interestingly, B. psychrosaccharolyticus whole cells have been also reported to display nucleoside 2 -deoxyribosyltransferase activity [24] and the analysis of its genome permitted the identification of the ndt gene that codifies a putative NDT [9]. Subsequently, this gene was cloned and overexpressed and the recombinant protein produced and immobilized [25,39].…”

“…Optimal conditions of pH (8.0) and temperature (40 °C) were previously determined by our group [25]. Here, the effects of several cations and other additives as well as ionic strength (I) on BpNDT activity have been studied.…”

“…The gene encoding nucleoside 2 -deoxyribosyltransferase from B. psychrosaccharolyticus CECT 4074 was amplified by the Polymerase Chain Reaction (PCR) with a 6-His tag coding sequence added to its 5 end using pET28a(+)-BpNDT [25] as template. DNA amplification was performed under standard conditions in a Mastercycler (Eppendorf, Hamburg, Germany) thermocycler using Pfu DNA polymerase.…”

Development of an immobilized biocatalyst based on Bacillus psychrosaccharolyticus NDT for the preparative synthesis of trifluridine and decytabine

“…Immobilization often represents a key point to make a step forward in the view of industrial application, since it provides the biocatalyst with enhanced stability and recyclability. Differently from NPs, immobilization of NDTs is still little studied and only a few examples have been reported to date [15,[18][19][20].…”