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Engineering a bacterial DyP-Type peroxidase for enhanced oxidation of lignin-related phenolics at alkaline pH

2017-05Journal article Restricted access
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dc.contributor.authorBrissos, Vânia
dc.contributor.authorTavares, Diogo
dc.contributor.authorSousa, Ana Catarina
dc.contributor.authorRobalo, Maria Paula
dc.contributor.authorMartins, Lígia O.
dc.date.accessioned2019-03-26T09:06:51Z
dc.date.available2019-03-26T09:06:51Z
dc.date.issued2017-05
dc.description.abstractDye-decolorizing peroxidases (DyPs) are a family of microbial heme-containing peroxidases that show important properties for lignocellulose biorefineries due to their ability to oxidize lignin-related compounds. Directed evolution was used to improve the efficiency of the bacterial PpDyP from Pseudomonas putida MET94 for phenolic compounds. Three rounds of random mutagenesis by error prone PCR of the ppDyP gene followed by high-throughput screening allow identification of the 6E10 variant showing a 100-fold enhanced catalytic efficiency (k(ca)t/K-m) for 2,6-dimethoxyphenol (DMP), similar to that exhibited by fungal lignin peroxidases (similar to 10(5) M-1 s(-1)). The evolved variant showed additional improved efficiency for a number of syringyl-type phenolics, guaiacol, aromatic amines, Kraft lignin, and the lignin phenolic model dimer guaiacylglycerol-beta-guaiacyl ether. Importantly, variant 6E10 displayed optimal pH at 8.5, an upshift of 4 units in comparison to the wild type, showed resistance to hydrogen peroxide inactivation, and was produced at 2-fold higher yields. The acquired mutations in the course of the evolution affected three amino acid residues (E188K, A142V, and H125Y) situated at the surface of the enzyme, in the second shell of the heme cavity. Biochemical analysis of hit variants from the laboratory evolution, and single variants constructed using site-directed mutagenesis, unveiled the critical role of acquired mutations from the catalytic, stability, and structural viewpoints. We show that epistasis between A142V and E188K mutations is crucial to determine the substrate specificity of 6E10. Evidence suggests that ABTS and DMP oxidation occurs at the heme access channel. Details of the catalytic cycle of 6E10 were elucidated through transient kinetics, providing evidence for the formation of a reversible enzyme hydrogen peroxide complex (Compound 0) barely detected in the majority of heme peroxidases studied to date.pt_PT
dc.description.versioninfo:eu-repo/semantics/publishedVersionpt_PT
dc.identifier.citationBRISSOS, Vânia; [et al] – Engineering a bacterial DyP-Type peroxidase for enhanced oxidation of lignin-related phenolics at alkaline pH. ACS Catalysis. ISSN 2155-5435. Vol. 7, N.º 5 (2017), pp. 3454-3465pt_PT
dc.identifier.doi10.1021/acscatal.6b03331pt_PT
dc.identifier.issn2155-5435
dc.identifier.urihttp://hdl.handle.net/10400.21/9778
dc.language.isoengpt_PT
dc.peerreviewedyespt_PT
dc.publisherAmerican Chemical Societypt_PT
dc.relationPTDC/BBB-EBB/0122/2014 - FCTpt_PT
dc.relationSFRH/BPD/109431/2015 - FCTpt_PT
dc.subjectDirected evolutionpt_PT
dc.subjectDye-decolorizing peroxidasespt_PT
dc.subjectLigninolytic enzymespt_PT
dc.subjectEnzyme specificitypt_PT
dc.subjectEpistasispt_PT
dc.subjectPseudomonas putida MET 94pt_PT
dc.subjectEvolução dirigidapt_PT
dc.subjectPeroxidases descolorantespt_PT
dc.titleEngineering a bacterial DyP-Type peroxidase for enhanced oxidation of lignin-related phenolics at alkaline pHpt_PT
dc.typejournal article
dspace.entity.typePublication
oaire.awardURIinfo:eu-repo/grantAgreement/FCT/3599-PPCDT/RECI%2FQEQ-QIN%2F0189%2F2012/PT
oaire.awardURIinfo:eu-repo/grantAgreement/FCT/5876/UID%2FMulti%2F04551%2F2013/PT
oaire.citation.endPage3465pt_PT
oaire.citation.issue5pt_PT
oaire.citation.startPage3454pt_PT
oaire.citation.titleACS Catalysispt_PT
oaire.citation.volume7pt_PT
oaire.fundingStream3599-PPCDT
oaire.fundingStream5876
person.familyNameBrissos
person.familyNameSousa
person.familyNameRobalo
person.familyNameMartins
person.givenNameVânia
person.givenNameAna Catarina
person.givenNameMaria Paula
person.givenNameLígia O.
person.identifier319225
person.identifier.ciencia-id5114-E972-5B61
person.identifier.ciencia-idE81E-0596-BF29
person.identifier.ciencia-idE11C-A704-9C18
person.identifier.ciencia-id731B-B843-C1DF
person.identifier.orcid0000-0002-8171-0113
person.identifier.orcid0000-0001-6133-7406
person.identifier.orcid0000-0002-8200-6910
person.identifier.orcid0000-0003-0082-9591
person.identifier.ridM-5789-2013
person.identifier.ridU-3597-2017
person.identifier.ridB-2002-2012
person.identifier.ridC-2513-2009
person.identifier.scopus-author-id24079497500
person.identifier.scopus-author-id9269662100
person.identifier.scopus-author-id7102109314
project.funder.identifierhttp://doi.org/10.13039/501100001871
project.funder.identifierhttp://doi.org/10.13039/501100001871
project.funder.nameFundação para a Ciência e a Tecnologia
project.funder.nameFundação para a Ciência e a Tecnologia
rcaap.rightsclosedAccesspt_PT
rcaap.typearticlept_PT
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