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12 páginas, 5 figuras, 2 tablas -- PAGS nros. 487-498, 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

40 páginas, 6 figuras y 4 tablas, During directed evolution to functionally express the high redox potential laccase from the PM1 basidiomycete in Saccharomyces cerevisiae (Mate et al. 2010), the characteristic maximum absorption at the T1 copper site (Abs610T1Cu) was quenched, switching the typical blue colour of the enzyme to yellow. To determine the molecular basis of this colour change, we characterized the original wild-type laccase and its evolved mutant. Peptide printing and Maldi-TOF analysis confirmed the absence of contaminating protein traces that could mask the Abs610T1Cu, while conservation of the redox potential at the T1 site was demonstrated by spectroelectrochemical redox titrations. Both wild-type and evolved laccases were capable of oxidizing a broad range of substrates (ABTS, guaiacol, DMP, synapic acid) and they displayed similar catalytic efficiencies. The laccase mutant could only oxidize high redox potential dyes (Poly R478, Reactive Black 5, Azure B) in the presence of exogenous mediators, indicating that the yellow enzyme behaves like a blue laccase. The main consequence of over-expressing the mutant laccase was the generation of a six-residue N-terminal acidic extension, which was associated with the failure of the STE13 protease in the Golgi compartment giving rise to alternative processing. Removal of the N-terminal tail had a negative effect on laccase stability, secretion and its kinetics, although the truncated mutant remained yellow. The results of CD spectra analysis suggested that polyproline helixes were formed during the directed evolution altering spectral properties. Moreover, introducing the A461T and S426N mutations in the T1 environment during the first cycles of laboratory evolution appeared to mediate the alterations to Abs610T1Cu by affecting its coordinating sphere. This laccase mutant is a valuable departure point for further protein engineering towards different fates., We thank Dr Francisco J. Plou from the Institute of Catalysis (CSIC, Spain) for assistance with the HPLC purification. This study was based on work funded by EU Project FP7 (3D-Nanobiodevice, NMP4-SL-2009-229255), COST Action CM0701, Swedish Research Council (2009-3266) and the Spanish National project (Evofacel, BIO2010-19697). D.M.M. was supported by a JAE fellowship (CSIC) and E.G.R by a 3D-Nanobiodevices contract.

28 p.-4 fig.-1 fig.supl., DNA recombination methods are useful tools to generate diversity in directed evolution protein engineering studies. We have designed an array of chimeric laccases with high-redox potential by in vitro and in vivo DNA recombination of two fungal laccases (from Pycnoporus cinnabarinus and PM1 basidiomycete), which were previously tailored by laboratory evolution for functional expression in Saccharomyces cerevisiae. The laccase fusion genes (including the evolved α-factor prepro-leaders for secretion in yeast) were subjected to a round of family shuffling to construct chimeric libraries and the best laccase hybrids were identified in dual high-throughput screening assays. Using this approach, we identified chimeras with up to six crossover events in the whole sequence, and we obtained active hybrid laccases with combined characteristics in terms of pH activity and thermostability, This work was supported by the National projects (PIE 200920I207 and BIO2010-19697) and EU Project NMP4-SL-2009-229255.