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Title

Directed evolution of a temperature, peroxide and alkaline pH tolerant versatile peroxidase

AuthorsGarcía-Ruiz, Eva ; González-Pérez, David ; Ruiz-Dueñas, F. J. ; Martínez, Ángel T. ; Alcalde Galeote, Miguel
Keywordsα-factor prepro-leader
Directed evolution
Enzyme promiscuity
Saccharomyces cerevisiae
Versatile peroxidase
Issue Date7-Oct-2011
PublisherPortland Press
CitationBiochemical Journal 441(1): 487-498(2012)
AbstractThe VPs (versatile peroxidases) secreted by white-rot fungi are involved in the natural decay of lignin. In the present study, a fusion gene containing the VP from Pleurotus eryngii was subjected to six rounds of directed evolution, achieving a level of secretion in Saccharomyces cerevisiae (21 mg/l) as yet unseen for any ligninolytic peroxidase. The evolved variant for expression harboured four mutations and increased its total VP activity 129-fold. The signal leader processing by the STE13 protease at the Golgi compartment changed as a consequence of overexpression, retaining the additional N-terminal sequence Glu-Ala-Glu-Ala that enhanced secretion. The engineered N-terminally truncated variant displayed similar biochemical properties to those of the non-truncated counterpart in terms of kinetics, stability and spectroscopic features. Additional cycles of evolution raised the T50 8°C and significantly increased the enzyme's stability at alkaline pHs. In addition, the Km for H2O2 was enhanced up to 15-fold while the catalytic efficiency was maintained, and there was an improvement in peroxide stability (with half-lives for H2O2 of 43 min at a H2O2/enzyme molar ratio of 4000:1). Overall, the directed evolution approach described provides a set of strategies for selecting VPs with improvements in secretion, activity and stability
Description12 páginas, 5 figuras, 2 tablas -- PAGS nros. 487-498
Publisher version (URL)http://dx.doi.org/10.1042/BJ20111199
URIhttp://hdl.handle.net/10261/49136
DOI10.1042/BJ20111199
ISSN0264-6021
E-ISSN1470-8728
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