Journal of Molecular Catalysis B: Enzymatic 115 (2015) 119–127 Contents lists available at ScienceDirect Journal of Molecular Catalysis B: Enzymatic j ourna l ho me pa ge: www.elsevier.com/locate/molcatb Immobilization of thermostable nucleoside phosphorylases on MagReSyn ® epoxide microspheres and their application for the synthesis of 2,6-dihalogenated purine nucleosides Xinrui Zhou a , Igor A. Mikhailopulo b , Mariano Nicolas Cruz Bournazou a , Peter Neubauer a,∗ a Chair of Bioprocess Engineering, Institute of Biotechnology, Technische Universität Berlin, Ackerstr. 76, ACK24, 13355 Berlin, Germany b Institute of Bioorganic Chemistry, National Academy of Sciences of Belarus, Acad. Kuprevicha 5/2, 220141 Minsk, Belarus a r t i c l e i n f o Article history: Received 18 December 2014 Received in revised form 16 February 2015 Accepted 17 February 2015 Available online 25 February 2015 Keywords: Immobilization Magnetic microspheres Nucleoside phosphorylases a b s t r a c t Immobilization of enzymes has been considered as an efficient approach to facilitate enzyme recovery and to improve biocatalyst stability. However, multimeric nucleoside phosphorylases, useful for the synthesis of modified nucleosides, encounter several challenges to their immobilization, including requirement for high enzymatic load, and poor retention of enzyme activity. In this study, multimeric enzymes pyrimidine nucleoside phosphorylase (PyNP) and purine nucleoside phosphorylase (PNP), from Thermus thermophilus and Geobacillus thermoglucosidasius, respectively, were successfully immobilized on the magnetic beads with cross-linked polyethyleneimine and epoxide functional groups, resulting in high enzyme loading (up to 0.4 and 1.3 g per g dry beads), high enzyme activity maintenance (41% and 83% under the highest enzyme loading), and improved enzyme stability. The screening of the immobilization conditions showed that binding buffer pH, enzyme loading amount, binding temperature and binding time are important factors for the immobilization yield. The application of the immobilized enzymes in the synthesis of 2,6- dihalogenated purine nucleosides achieved with high substrate conversion (78.5–85.5%) as well as high productivity (1.5–2.0 g L -1 h -1 ). © 2015 Elsevier B.V. All rights reserved. 1. Introduction Nucleoside phosphorylases (NPs) are efficient biocatalysts, which have been extensively studied especially for the synthesis of modified nucleosides of pharmaceutical and biological inter- est [1,2]. Thermostable NPs from thermophilic microorganisms, retaining the high regio- and stereo-selectivity of the mesophilic NPs, have further advantages that are not only associated with an increased thermostability but also with a broader substrate spec- trum and a wider tolerance to many stresses [3–7]. Among them, purine nucleoside phosphorylase (PNP, EC 2.4.2.1) from Geobacillus thermoglucosidasius (GtPNP) [3] and pyrimidine nucleoside phos- phorylase (PyNP, EC 2.4.2.2) from Thermus thermophiles (TtPyNP) [4] have shown such promising properties. However, the majority of modified nucleosides are not currently synthesized by biocat- alysts yet, but rather by traditional chemical synthesis, which consists of laborious multistage processes and often leads to low yields due to the poor regio- and stereo-selectivity resulting in the ∗ Corresponding author. Tel.: +49 3031472269. E-mail address: peter.neubauer@tu-berlin.de (P. Neubauer). formation of unwanted isomers. It is expected that with the devel- opment of biocatalysts, enzymatic synthesis could be adopted for the biocatalytic synthesis of valuable pharmaceutical intermedi- ates for their viable economic industrial production. Immobilization has been recognized as a powerful method to realize the application of enzymes under the harsh conditions required in some industrial processes. More importantly, immobi- lized enzymes can be easily recovered for a potential reuse, which permits the reduction of biocatalyst cost, simplifies the design of the reactor and allows to realize in some cases a better control of the reaction [8,9]. Therefore, it is highly desirable to immobilize NPs even if they are thermostable. There are several reports on the immobilization of NPs on different supports. For instance, thermostable GsPNP and GsPyNP from G. stearothermophilus (previously Bacillus stearothermophilus) were immobilized on anion exchange resins (DEAE-Toyopearl 650 M) [10], or on the aminopropylated macroporous glass AP- CPG-170 [6]; uridine phosphorylase (UP) and PNP from E. coli were immobilized on epoxy-activated resin Sepabead EC-EP [11]; EcTP from E. coli, BsPNP and BsPyNP from Bacillus subtilis, AhPNP from Aeromonas hydrophila, were immobilized and compared with http://dx.doi.org/10.1016/j.molcatb.2015.02.009 1381-1177/© 2015 Elsevier B.V. All rights reserved.