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dc.contributor.authorMaté, Diana M.-
dc.contributor.authorGonzález-Pérez, David-
dc.contributor.authorKittl, Roman-
dc.contributor.authorLudwig, Roland-
dc.contributor.authorAlcalde Galeote, Miguel-
dc.identifier.citationBMC Biotechnology 13:38 (2013)es_ES
dc.description12 páginas, 6 figurases_ES
dc.description.abstract[Background] Basidiomycete high-redox potential laccases (HRPLs) working in human physiological fluids (pH 7.4, 150 mM NaCl) arise great interest in the engineering of 3D-nanobiodevices for biomedical uses. In two previous reports, we described the directed evolution of a HRPL from basidiomycete PM1 strain CECT 2971: i) to be expressed in an active, soluble and stable form in Saccharomyces cerevisiae, and ii) to be active in human blood. In spite of the fact that S. cerevisiae is suited for the directed evolution of HRPLs, the secretion levels obtained in this host are not high enough for further research and exploitation. Thus, the search for an alternative host to over-express the evolved laccases is mandatory.es_ES
dc.description.abstract[Results] A blood-active laccase (ChU-B mutant) fused to the native/evolved α-factor prepro-leader was cloned under the control of two different promoters (PAOX1 and PGAP) and expressed in Pichia pastoris. The most active construct, which contained the PAOX1 and the evolved prepro-leader, was fermented in a 42-L fed-batch bioreactor yielding production levels of 43 mg/L. The recombinant laccase was purified to homogeneity and thoroughly characterized. As happened in S. cerevisiae, the laccase produced by P. pastoris presented an extra N-terminal extension (ETEAEF) generated by an alternative processing of the α-factor pro-leader at the Golgi compartment. The laccase mutant secreted by P. pastoris showed the same improved properties acquired after several cycles of directed evolution in S. cerevisiae for blood-tolerance: a characteristic pH-activity profile shifted to the neutral-basic range and a greatly increased resistance against inhibition by halides. Slight biochemical differences between both expression systems were found in glycosylation, thermostability and turnover numbers.-
dc.description.abstract[Conclusions] The tandem-yeast system based on S. cerevisiae to perform directed evolution and P. pastoris to over-express the evolved laccases constitutes a promising approach for the in vitro evolution and production of these enzymes towards different biocatalytic and bioelectrochemical applications.-
dc.description.sponsorshipThis study is based upon a work funded by European Union Projects (grant numbers NMP4-SL-2009-229255-3D-Nanobiodevice, FP7-KBBE-2010-4-26537-Peroxicats and COST Action CM0701) and a Spanish National Project (Evofacel, BIO2010-19697) and by the Austrian Science Fund (FWF, P25148-B20). We thank Prof. S. Shleev from Malmö University (Sweden) for carrying out the measurements of the laccase activity in human plasma and blood. D.M.M. is grateful to the CSIC for a JAE fellowship.es_ES
dc.publisherBioMed Centrales_ES
dc.relation.isversionofPublisher’s version-
dc.subjectHigh-redox potential laccasees_ES
dc.subjectFunctional expressiones_ES
dc.subjectPichia pastorises_ES
dc.subjectSaccharomyces cerevisiaees_ES
dc.subjectDirected evolutiones_ES
dc.subjectHalide inhibitiones_ES
dc.subjectHydroxyl inhibitiones_ES
dc.subjectBlood tolerancees_ES
dc.titleFunctional expression of a blood tolerant laccase in Pichia pastorises_ES
dc.description.peerreviewedPeer reviewedes_ES
dc.contributor.funderEuropean Commission-
dc.contributor.funderAustrian Science Fund-
dc.contributor.funderMinisterio de Economía y Competitividad (España)-
dc.contributor.funderConsejo Superior de Investigaciones Científicas (España)-
dc.contributor.funderMalmö University-
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