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Combinatorial Saturation Mutagenesis by in vivo Overlap Extension for the Engineering of Fungal Laccases

AutorAlcalde Galeote, Miguel ; Zumárraga, Miren ; Polaina Molina, Julio ; Ballesteros Olmo, Antonio ; Plou Gasca, Francisco José
Palabras claveSaccharomyces cerevisiae
Redox potential
Terminal plug
Fecha de publicación2006
EditorBentham Science Publishers
CitaciónCombinatorial Chemistry and High Throughput Screening 9, 719-727 (2006)
ResumenCombinatorial saturation mutagenesis -CSM- is a valuable tool for improving enzymatic properties from hot-spot residues discovered by directed enzyme evolution or performing semi-rational studies. CSM coupled to a reliable high-throughput screening assay -CV below 10 %- has been used to enhance turnover rates in the fungal laccase variant T2 from Myceliophthora thermophila. The influence of the highly conserved pentapeptide 509-513 on the redox potential of blue-copper containing enzymes is well described. We focused combinatorial saturation mutagenesis in residues Ser510 and Leu513. Libraries were constructed in Saccharomyces cerevisiae by in vivo overlap extension -IVOE- of the PCR products. This methodology provides a simple manner to build CSM libraries avoiding extra PCR reactions, by-products formation and in vitro ligation steps. After exploring more than 1,700 clones, mutant (7E1) with 3-fold better kinetics than parent type was found. 7E1 showed one synonymous mutation (L513L, CGT/TTG) and beneficial mutation S510G (TCG/GGG) which can not be achieved by conventional error-prone PCR techniques. Mutation S510G seems to affect the C-terminal plug, which modulates the transit of water and oxygen to the trinuclear copper cluster.
Versión del editor1386-2073/06
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