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Molecules 2016, 21(12), 1621; doi:10.3390/molecules21121621

Immobilization of Neutral Protease from Bacillus subtilis for Regioselective Hydrolysis of Acetylated Nucleosides: Application to Capecitabine Synthesis

1
Department of Drug Sciences, University of Pavia, via Taramelli 12, I-27100 Pavia, Italy
2
Department of Food, Environmental and Nutritional Sciences—DeFENS, University of Milano, via L. Mangiagalli 25, I-20133 Milano, Italy
3
Department for Innovation in Biological, Agro-food and Forest Systems, University of Tuscia, via S. Camillo de Lellis, I-01100 Viterbo, Italy
*
Author to whom correspondence should be addressed.
Academic Editor: Roberto Fernàndez-Lafuente
Received: 10 September 2016 / Revised: 19 November 2016 / Accepted: 21 November 2016 / Published: 25 November 2016
(This article belongs to the Special Issue Enzyme Immobilization 2016)
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Abstract

This paper describes the immobilization of the neutral protease from Bacillus subtilis and its application in the regioselective hydrolysis of acetylated nucleosides, including building blocks useful for the preparation of anticancer products. Regarding the immobilization study, different results have been obtained depending on the immobilization procedure. Epoxy hydrophobic carriers gave a poorly stable derivative that released almost 50% of the immobilized protein under the required reaction conditions. On the contrary, covalent immobilization on a differently activated hydrophilic carrier (agarose) resulted in very stable enzyme derivatives. In an attempt to explain the obtained enzyme immobilization results, the hypothetical localization of lysines on the enzyme surface was predicted in a 3D structure model of B. subtilis protease N built in silico by using the structure of Staphylococcus aureus metalloproteinase as the template. The immobilized enzyme shown a high regioselectivity in the hydrolysis of different peracetylated nucleosides. A stable enzyme derivative was obtained and successfully used in the development of efficient preparative bioprocesses for the hydrolysis of acetylated nucleosides, giving new intermediates for the synthesis of capecitabine in high yield. View Full-Text
Keywords: protease N; immobilization; nucleosides; regioselective hydrolysis; anticancer products; capecitabine protease N; immobilization; nucleosides; regioselective hydrolysis; anticancer products; capecitabine
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MDPI and ACS Style

Bavaro, T.; Cattaneo, G.; Serra, I.; Benucci, I.; Pregnolato, M.; Terreni, M. Immobilization of Neutral Protease from Bacillus subtilis for Regioselective Hydrolysis of Acetylated Nucleosides: Application to Capecitabine Synthesis. Molecules 2016, 21, 1621.

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