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Immobilized Drosophila melanogaster deoxyribonucleoside kinase (DmdNK) as a high performing biocatalyst for the synthesis of purine arabinonucleotides

Journal article
Authors Immacolata Serra
Silvia Conti
Jure Piškur
Anders R Clausen
Birgitte Munch-Petersen
Marco Terreni
Daniela Ubiali
Published in Advanced Synthesis and Catalysis
Volume 356
Pages 563-570
ISSN 16154150
Publication year 2014
Published at
Pages 563-570
Language en
Links dx.doi.org/10.1002/adsc.201300649
Keywords Biocatalysis, Deoxyribonucleoside kinase, Immobilization, Nucleotides, Phosphorylation
Subject categories Biochemistry and Molecular Biology

Abstract

Fruit fly (Drosophila melanogaster) deoxyribonucleoside kinase (DmdNK; EC: 2.7.1.145) was characterized for its substrate specificity towards natural and non-natural nucleosides, confirming its potential in the enzymatic synthesis of modified nucleotides. DmdNK was adsorbed on a solid ion exchange support (bearing primary amino groups) achieving an expressed activity >98%. Upon cross-linking with aldehyde dextran, expressed activity was 30-40%. Both biocatalysts (adsorbed or cross-linked) were stable at pH 10 and room temperature for 24 h (about 70% of retained activity). The cross-linked DmdNK preparation was used for the preparative synthesis of arabinosyladenine monophosphate (araA-MP) and fludarabine monophosphate (FaraAMP). Upon optimization of the reaction conditions (50 mM ammonium acetate, substrate/ATP ratio= 1:1.25, 2 mM MgCl2, 378C, pH 8) immobilized DmdNK afforded the title nucleotides with high conversion (>90%), whereas with the soluble enzyme lower conversions were achieved (78-87%). Arabinosyladenine monophosphate was isolated in 95% yield and high purity (96.5%). © 2014 Wiley-VCH Verlag GmbH&Co. KGaA, Weinheim.

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