title: Biosynthesis of an antiviral compound using a stabilized phosphopentomutase by multipoint covalent immobilization.
abstract: Ribavirin is a synthetic guanosine analogue with a broad-spectrum of antiviral activity. It is clinically effective against several viruses, such as respiratory syncytial virus, several hemorrhagic fever viruses and HCV when combined with pegylated interferon-α. Phosphopentomutase (PPM) catalyzes the transfer of intramolecular phosphate (from C1 to C5) on ribose, and is involved in pentose phosphate pathway and in purine metabolism. Reactions catalyzed by this enzyme are useful for nucleoside analogues production. However, out of its natural environment PPM is unstable and its stability is affected by parameters such as pH and temperature. Therefore, to irreversibly immobilize this enzyme, it needs to be stabilized. In this work, PPM from Escherichia coli ATCC 4157 was overexpressed, purified, stabilized at alkaline pH and immobilized on several supports. The activity of different additives as stabilizing agents was evaluated, and the best result was found using 10% (v/v) glycerol. Under this condition, PPM maintained 86% of its initial activity at pH 10 after 18h incubation, which allowed further covalent immobilization of this enzyme on glyoxyl-agarose with a high yield. This is the first time that PPM has been immobilized by multipoint covalent attachment on glyoxyl support, this derivative being able to biosynthesize ribavirin from α-d-ribose-5-phosphate.
authors.affiliation: Sustainable Biotechnology Laboratory (LIBioS), National University of Quilmes, Roque Sáenz Peña 352, Bernal B1876BXD, Argentina; National Scientific and Technical Research Council (CONICET), Godoy Cruz 2290, CABA C1425FQB, Argentina.
authors.forename: Cintia W
authors.initials: CW
authors.lastname: Rivero
authors.affiliation: Sustainable Biotechnology Laboratory (LIBioS), National University of Quilmes, Roque Sáenz Peña 352, Bernal B1876BXD, Argentina; National Scientific and Technical Research Council (CONICET), Godoy Cruz 2290, CABA C1425FQB, Argentina.
authors.forename: Eliana C
authors.initials: EC
authors.lastname: De Benedetti
authors.affiliation: Heterogeneous Biocatalysis Group, CIC Biomagune, Edificio Empresarial "C", Paseo Miramón 182, Donostia-San Sebastián 20009, Spain; Ikerbasque, Basque Foundation for Science, Alda. Urquijo, Urkixo Zumarkalea 36, Bilbao 48011, Spain.
authors.forename: Fernando López
authors.initials: FL
authors.lastname: Gallego
authors.affiliation: Research Institute of Food Science (CIAL-CSIC), Campus UAM-Cantoblanco, Madrid 28049, Spain; Department of Engineering and Technologies, Polytechnic Institute of Science and Technology, Av. Luanda Sul, Rua Lateral Via S10, Talatona, Luanda Sul, Angola.
authors.forename: Benevides C
authors.initials: BC
authors.lastname: Pessela
authors.affiliation: Institute of Catalysis and Petrochemistry (ICP-CSIC), Campus UAM-Cantoblanco, Madrid 28049, Spain.
authors.forename: José M
authors.initials: JM
authors.lastname: Guisán
authors.affiliation: Sustainable Biotechnology Laboratory (LIBioS), National University of Quilmes, Roque Sáenz Peña 352, Bernal B1876BXD, Argentina; National Scientific and Technical Research Council (CONICET), Godoy Cruz 2290, CABA C1425FQB, Argentina. Electronic address: jtrelles@unq.edu.ar.
authors.forename: Jorge A
authors.initials: JA
authors.lastname: Trelles
mesh_terms: D000998:Antiviral Agents / Q000378:metabolism*; D004795:Enzyme Stability; D004800:Enzymes, Immobilized / Q000737:chemistry / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D005079:Excipients; D006863:Hydrogen-Ion Concentration; D008958:Models, Molecular; D010770:Phosphotransferases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D012254:Ribavirin / Q000378:metabolism*; D013696:Temperature
chemical_list: D000998:Antiviral Agents; D004800:Enzymes, Immobilized; D029968:Escherichia coli Proteins; D005079:Excipients; D012254:Ribavirin; D010770:Phosphotransferases; C022375:phosphopentomutase
keywords: Escherichia coli; Glyoxyl-agarose; Ribavirin; Stabilizing agents