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dc.contributor.authorOrrego, Alejandro H.-
dc.contributor.authorRomero-Fernández, Maria-
dc.contributor.authorMillán-Linares, María del Carmen-
dc.contributor.authorYust, María del Mar-
dc.contributor.authorGuisán, José Manuel-
dc.contributor.authorRocha-Martín, Javier-
dc.identifier.citationCatalysts 8(8): 333 (2018)-
dc.description.abstractEnzyme immobilization by multipoint covalent attachment on supports activated with aliphatic aldehyde groups (e.g., glyoxyl agarose) has proven to be an excellent immobilization technique for enzyme stabilization. Borohydride reduction of immobilized enzymes is necessary to convert enzyme–support linkages into stable secondary amino groups and to convert the remaining aldehyde groups on the support into hydroxy groups. However, the use of borohydride can adversely affect the structure–activity of some immobilized enzymes. For this reason, 2-picoline borane is proposed here as an alternative milder reducing agent, especially, for those enzymes sensitive to borohydride reduction. The immobilization-stabilization parameters of five enzymes from different sources and nature (from monomeric to multimeric enzymes) were compared with those obtained by conventional methodology. The most interesting results were obtained for bacterial (R)-mandelate dehydrogenase (ManDH). Immobilized ManDH reduced with borohydride almost completely lost its catalytic activity (1.5% of expressed activity). In contrast, using 2-picoline borane and blocking the remaining aldehyde groups on the support with glycine allowed for a conjugate with a significant activity of 19.5%. This improved biocatalyst was 357-fold more stable than the soluble enzyme at 50 ◦C and pH 7. The results show that this alternative methodology can lead to more stable and active biocatalysts.-
dc.description.sponsorshipThis research was funded by the Spanish Ministry of Economy, Industry and Competitiveness (projects BIO2012-36861 and CTQ2015-70348) and the EU FP7 project SuSy (Sucrose Synthase as Cost-Effective Mediator of Glycosylation Reactions, CKBBE/3293). Javier Rocha-Martin is grateful for the Juan de la Cierva fellowship (IJCI-2014-19260) funded by the Spanish Ministry of Economy, Industry and Competitiveness. The authors would like to thank the Spanish Ministry of Economy, Industry and Competitiveness for financial support awarded María Fernández-Romero (BES-2013-065396).-
dc.publisherMultidisciplinary Digital Publishing Institute-
dc.relation.isversionofPublisher's version-
dc.subjectEnzyme immobilization-
dc.subject2-picoline borane-
dc.subjectGlyoxyl agarose-
dc.subjectSchiff base-
dc.subjectEnzyme stabilization-
dc.titleStabilization of Enzymes by Multipoint Covalent Attachment on Aldehyde-Supports: 2-Picoline Borane as an Alternative Reducing Agent-
dc.description.peerreviewedPeer reviewed-
dc.contributor.funderMinisterio de Economía, Industria y Competitividad (España)-
dc.contributor.funderEuropean Commission-
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