title: Directed evolution of a temperature-, peroxide- and alkaline pH-tolerant versatile peroxidase.
abstract: The 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.
authors: Department of Biocatalysis, Institute of Catalysis, CSIC, Cantoblanco, 28049 Madrid, Spain.
authors: Eva
authors: E
authors: Garcia-Ruiz
authors: David
authors: D
authors: Gonzalez-Perez
authors: Francisco J
authors: FJ
authors: Ruiz-Dueñas
authors: Angel T
authors: AT
authors: Martínez
authors: Miguel
authors: M
authors: Alcalde
mesh_terms: D001665:Binding Sites; D019020:Directed Molecular Evolution; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism*; D015966:Gene Expression Regulation, Fungal / Q000502:physiology; D006861:Hydrogen Peroxide / Q000494:pharmacology*; D006863:Hydrogen-Ion Concentration; D008345:Manganese / Q000378:metabolism; D008958:Models, Molecular; D010544:Peroxidases / Q000145:classification / Q000235:genetics / Q000378:metabolism*; D020076:Pleurotus / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D013696:Temperature
chemical_list: D005656:Fungal Proteins; D008345:Manganese; D006861:Hydrogen Peroxide; D010544:Peroxidases