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Please use this identifier to cite or link to this item: http://hdl.handle.net/10261/49942
Title: Engineering Platforms for Directed Evolution of Laccase from Pycnoporus cinnabarinus
Authors: Camarero, Susana; Pardo, Isabel; Cañas Portilla, Ana Isabel; Molina, Pedro I.; Record, Éric; Martínez Ferrer, Ángel Tomás; Martínez, María Jesús; Alcalde Galeote, Miguel
Issue Date: Mar-2012
Publisher: American Society for Microbiology
Citation: Applied and Environmental Microbiology 78(5): 1370–1384(2012)
Abstract: While the Pycnoporus cinnabarinus laccase (PcL) is one of the most promising high-redox-potential enzymes for environmental biocatalysis, its practical use has to date remained limited due to the lack of directed evolution platforms with which to improve its features. Here, we describe the construction of a PcL fusion gene and the optimization of conditions to induce its functional expression in Saccharomyces cerevisiae, facilitating its directed evolution and semirational engineering. The native PcL signal peptide was replaced by the α-factor preproleader, and this construct was subjected to six rounds of evolution coupled to a multiscreening assay based on the oxidation of natural and synthetic redox mediators at more neutral pHs. The laccase total activity was enhanced 8,000-fold: the evolved α-factor preproleader improved secretion levels 40-fold, and several mutations in mature laccase provided a 13.7-fold increase in kcat. While the pH activity profile was shifted to more neutral values, the thermostability and the broad substrate specificity of PcL were retained. Evolved variants were highly secreted by Aspergillus niger (∼23 mg/liter), which addresses the potential use of this combined-expression system for protein engineering. The mapping of mutations onto the PcL crystal structure shed new light on the oxidation of phenolic and nonphenolic substrates. Furthermore, some mutations arising in the evolved preproleader highlighted its potential for heterologous expression of fungal laccases in yeast (S. cerevisiae)
Description: 15 páginas, 6 figuras, 3 tablas -- PAGS nros. 1370-1384
Publisher version (URL): http://dx.doi.org/10.1128/​AEM.07530-11
URI: http://hdl.handle.net/10261/49942
ISSN: 0099-2240
DOI: 10.1128/​AEM.07530-11
E-ISSN: 1098-5336
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