Engineering a bacterial DyP-Type peroxidase for enhanced oxidation of lignin-related phenolics at alkaline pH
Autor(a) principal: | |
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Data de Publicação: | 2017 |
Outros Autores: | , , , |
Tipo de documento: | Artigo |
Idioma: | eng |
Título da fonte: | Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) |
Texto Completo: | http://hdl.handle.net/10400.21/9778 |
Resumo: | Dye-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. |
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Engineering a bacterial DyP-Type peroxidase for enhanced oxidation of lignin-related phenolics at alkaline pHDirected evolutionDye-decolorizing peroxidasesLigninolytic enzymesEnzyme specificityEpistasisPseudomonas putida MET 94Evolução dirigidaPeroxidases descolorantesDye-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.American Chemical SocietyRCIPLBrissos, VâniaTavares, DiogoSousa, Ana CatarinaRobalo, Maria PaulaMartins, Lígia O.2019-03-26T09:06:51Z2017-052017-05-01T00:00:00Zinfo:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/articleapplication/pdfhttp://hdl.handle.net/10400.21/9778engBRISSOS, 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. 3454-34652155-543510.1021/acscatal.6b03331metadata only accessinfo:eu-repo/semantics/openAccessreponame:Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)instname:Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informaçãoinstacron:RCAAP2023-08-03T09:58:53Zoai:repositorio.ipl.pt:10400.21/9778Portal AgregadorONGhttps://www.rcaap.pt/oai/openaireopendoar:71602024-03-19T20:18:17.269593Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) - Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informaçãofalse |
dc.title.none.fl_str_mv |
Engineering a bacterial DyP-Type peroxidase for enhanced oxidation of lignin-related phenolics at alkaline pH |
title |
Engineering a bacterial DyP-Type peroxidase for enhanced oxidation of lignin-related phenolics at alkaline pH |
spellingShingle |
Engineering a bacterial DyP-Type peroxidase for enhanced oxidation of lignin-related phenolics at alkaline pH Brissos, Vânia Directed evolution Dye-decolorizing peroxidases Ligninolytic enzymes Enzyme specificity Epistasis Pseudomonas putida MET 94 Evolução dirigida Peroxidases descolorantes |
title_short |
Engineering a bacterial DyP-Type peroxidase for enhanced oxidation of lignin-related phenolics at alkaline pH |
title_full |
Engineering a bacterial DyP-Type peroxidase for enhanced oxidation of lignin-related phenolics at alkaline pH |
title_fullStr |
Engineering a bacterial DyP-Type peroxidase for enhanced oxidation of lignin-related phenolics at alkaline pH |
title_full_unstemmed |
Engineering a bacterial DyP-Type peroxidase for enhanced oxidation of lignin-related phenolics at alkaline pH |
title_sort |
Engineering a bacterial DyP-Type peroxidase for enhanced oxidation of lignin-related phenolics at alkaline pH |
author |
Brissos, Vânia |
author_facet |
Brissos, Vânia Tavares, Diogo Sousa, Ana Catarina Robalo, Maria Paula Martins, Lígia O. |
author_role |
author |
author2 |
Tavares, Diogo Sousa, Ana Catarina Robalo, Maria Paula Martins, Lígia O. |
author2_role |
author author author author |
dc.contributor.none.fl_str_mv |
RCIPL |
dc.contributor.author.fl_str_mv |
Brissos, Vânia Tavares, Diogo Sousa, Ana Catarina Robalo, Maria Paula Martins, Lígia O. |
dc.subject.por.fl_str_mv |
Directed evolution Dye-decolorizing peroxidases Ligninolytic enzymes Enzyme specificity Epistasis Pseudomonas putida MET 94 Evolução dirigida Peroxidases descolorantes |
topic |
Directed evolution Dye-decolorizing peroxidases Ligninolytic enzymes Enzyme specificity Epistasis Pseudomonas putida MET 94 Evolução dirigida Peroxidases descolorantes |
description |
Dye-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. |
publishDate |
2017 |
dc.date.none.fl_str_mv |
2017-05 2017-05-01T00:00:00Z 2019-03-26T09:06:51Z |
dc.type.status.fl_str_mv |
info:eu-repo/semantics/publishedVersion |
dc.type.driver.fl_str_mv |
info:eu-repo/semantics/article |
format |
article |
status_str |
publishedVersion |
dc.identifier.uri.fl_str_mv |
http://hdl.handle.net/10400.21/9778 |
url |
http://hdl.handle.net/10400.21/9778 |
dc.language.iso.fl_str_mv |
eng |
language |
eng |
dc.relation.none.fl_str_mv |
BRISSOS, 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. 3454-3465 2155-5435 10.1021/acscatal.6b03331 |
dc.rights.driver.fl_str_mv |
metadata only access info:eu-repo/semantics/openAccess |
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metadata only access |
eu_rights_str_mv |
openAccess |
dc.format.none.fl_str_mv |
application/pdf |
dc.publisher.none.fl_str_mv |
American Chemical Society |
publisher.none.fl_str_mv |
American Chemical Society |
dc.source.none.fl_str_mv |
reponame:Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) instname:Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informação instacron:RCAAP |
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Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) |
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Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) - Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informação |
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