2024-03-29T13:49:00Zhttp://digital.csic.es/dspace-oai/requestoai:digital.csic.es:10261/1743662021-12-27T16:27:06Zcom_10261_101com_10261_5col_10261_354
2019-01-18T15:02:46Z
urn:hdl:10261/174366
Directed evolution of unspecific peroxygenase from Agrocybe aegerita
Molina-Espeja, Patricia
García-Ruiz, Eva
González-Pérez, David
Ullrich, René
Hofrichter, Martin
Alcalde Galeote, Miguel
European Commission
Ministerio de Economía y Competitividad (España)
Molina-Espeja, Patricia [0000-0002-2590-0932]
Alcalde, Miguel [0000-0001-6780-7616]
Garcia-Ruiz, Eva [0000-0001-7965-9948]
Unspecific peroxygenase (UPO)
Agrocybe aegerita
enzymes
Unspecific peroxygenase (UPO) represents a new type of heme-thiolate enzyme with self-sufficient mono(per)oxygenase activity and many potential applications in organic synthesis. With a view to taking advantage of these properties, we subjected the Agrocybe aegerita UPO1-encoding gene to directed evolution in Saccharomyces cerevisiae. To promote functional expression, several different signal peptides were fused to the mature protein, and the resulting products were tested. Over 9,000 clones were screened using an ad hoc dual-colorimetric assay that assessed both peroxidative and oxygen transfer activities. After 5 generations of directed evolution combined with hybrid approaches, 9 mutations were introduced that resulted in a 3,250-fold total activity improvement with no alteration in protein stability. A breakdown between secretion and catalytic activity was performed by replacing the native signal peptide of the original parental type with that of the evolved mutant; the evolved leader increased functional expression 27-fold, whereas an 18-fold improvement in the kcat/Km value for oxygen transfer activity was obtained. The evolved UPO1 was active and highly stable in the presence of organic cosolvents. Mutations in the hydrophobic core of the signal peptide contributed to enhance functional expression up to 8 mg/liter, while catalytic efficiencies for peroxidative and oxygen transfer reactions were increased by several mutations in the vicinity of the heme access channel. Overall, the directed-evolution platform described is a valuable point of departure for the development of customized UPOs with improved features and for the study of structure-function relationships. © 2014, American Society for Microbiology.
2019-01-18T15:02:46Z
2019-01-18T15:02:46Z
2014
2019-01-18T15:02:47Z
artículo
Applied and Environmental Microbiology 80: 3496- 3507 (2014)
1098-5336
http://hdl.handle.net/10261/174366
10.1128/AEM.00490-14
1098-5336
http://dx.doi.org/10.13039/501100003329
http://dx.doi.org/10.13039/501100000780
24682297
eng
Postprint
https://doi.org/doi:10.1128/AEM.00490-14
Sí
info:eu-repo/grantAgreement/EC/FP7/26537
info:eu-repo/grantAgreement/MINECO/Plan Estatal de Investigación Científica y Técnica y de Innovación 2013-2016/BIO2010-19697
info:eu-repo/grantAgreement/EC/FP7/613549
Copyright © 2014, American Society for Microbiology. All Rights Reserved.
openAccess
American Society for Microbiology