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Appl Microbiol Biotechnol (2011) 91:317–327 DOI 10.1007/s00253-011-3221-7

BIOTECHNOLOGICALLY RELEVANT ENZYMES AND PROTEINS

Enzymatic synthesis of nucleoside analogues using immobilized 2′-deoxyribosyltransferase from Lactobacillus reuteri
Jesús Fernández-Lucas & Alba Fresco-Taboada & Carmen Acebal & Isabel de la Mata & Miguel Arroyo

Received: 12 January 2011 / Revised: 23 February 2011/ Accepted: 2 March 2011 / Published online: 8 April 2011 # Springer-Verlag 2011

Abstract Covalent attachment of recombinant Lactobacillus reuteri 2′-deoxyribosyltransferase to Sepabeads EC-EP303 leads to the immobilized biocatalyst SLrNDT4, which displayed an enzymatic activity of 65.4 IU/g of wet biocatalyst in 2′-deoxyadenosine synthesis from 2′-deoxyuridine and adenine at 40°C and pH 6.5.Response surface methodology was employed for the optimization of SLrNDT4 activity. Optimal conditions for SLrNDT4 highest activity were observed at 40°C and pH 6.5. Immobilized biocatalyst retained 50% of its maximal activity after 17.9 h at 60°C, whereas 96% activity was observed after storage at 40°C for 110 h. This novel immobilized biocatalyst has been successfully employed in the enzymaticsynthesis of different natural and therapeutic nucleosides effective against cancer and viral diseases. Among these last products, enzymatic synthesis of therapeutic nucleosides such as 5-ethyl-2′-deoxyuridine and 5-trifluorothymidine has been carried out for the first time. Importantly for its potential application, SLrNDT4 could be recycled for 26 consecutive batch reactions in the synthesis of2,6-diaminopurine2′-deoxyriboside with negligible loss of catalytic activity.

Keywords 2′-Deoxyribosyltransferase . Nucleoside synthesis . Lactobacillus reuteri . Immobilization . Sepabeads

Introduction Modified nucleosides are important molecules which display antiviral and antitumoral activity and can be used as starting materials for antisense oligonucleotides (De Clercq 2010; Robak et al.2006). In all cases, acquired resistance and/or non-desired side effects of these modified nucleosides are major problems that have encouraged the development of new analogues with therapeutic activity. Nucleoside analogues have been traditionally synthesized by different chemical methods, which often require timeconsuming multistep processes including protection– deprotection reactions on theheterocycle base and/or the pentose moiety to allow the modification of naturally occurring nucleosides (Boryski 2008). In this sense, an enzymatic process for the synthesis of modified nucleosides is an interesting alternative to the known chemical procedures. This biotechnological approach shows many advantages such as very mild reaction conditions, high stereo- and regioselectivity, and anenvironment-friendly technology (Lewkowicz and Iribarren 2006; Li et al. 2010; Mikhailopulo 2007). In general, nucleoside phosphorylases and N-deoxyribosyltransferases have been used in nucleoside synthesis by mediating the transfer of glycosyl residues to acceptor bases. Such biotransformations have either been accomplished by employing isolated enzymes (Christoffersen et al. 2010; Okuyama et al. 2003)or whole cells of microorganisms containing high percentages of the required enzyme (Bentancor et al. 2004; Fernández-Lucas et al. 2007, 2008; Médici et al. 2006; Trelles et al. 2003).

J. Fernández-Lucas : A. Fresco-Taboada : C. Acebal : I. de la Mata : M. Arroyo (*) Departamento de Bioquímica y Biología Molecular I, Facultad de Ciencias Biológicas, Universidad Complutense de Madrid, c/JoséAntonio Novais 2, 28040 Madrid, Spain e-mail: arroyo@bbm1.ucm.es J. Fernández-Lucas Departamento de Química Orgánica y Farmacéutica, Facultad de Farmacia, Universidad Complutense de Madrid, Avda. Complutense s/n, 28040 Madrid, Spain

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Appl Microbiol Biotechnol (2011) 91:317–327

N-deoxyribosyltransferases (EC 2.4.2.6) catalyze the cleavage of the glycosidic bond of a 2′-β-deoxynucleoside...