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Hoffmann, R. A wiki for the life sciences where authorship matters. Nature Genetics (2008)
 
 
 

Combinatorial saturation mutagenesis by in vivo overlap extension for the engineering of fungal laccases.

Combinatorial 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 -coefficient of variance 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 approximately 3-fold higher kinetics than parent type was found. 7E1 showed one synonymous mutation (L513L, CGT/TTG) and one beneficial mutation S510G (TCG/GGG) that 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.[1]

References

  1. Combinatorial saturation mutagenesis by in vivo overlap extension for the engineering of fungal laccases. Alcalde, M., Zum??rraga, M., Polaina, J., Ballesteros, A., Plou, F.J. Comb. Chem. High Throughput Screen. (2006) [Pubmed]
 
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