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2000
Volume 9, Issue 10
  • ISSN: 1386-2073
  • E-ISSN: 1875-5402

Abstract

Combinatorial saturation mutagenesis -CSM- is a valuable tool for improving enzymatic properties from hotspot 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 ∼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.

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/content/journals/cchts/10.2174/138620706779026079
2006-12-01
2025-07-04
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