| 1. |
- Zumarraga, Miren, et al.
(författare)
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Combinatorial saturation mutagenesis of the Myceliophthora thermophila laccase T2 mutant : the connection between the C-terminal plug and the conserved 509VSG511 tripeptide
- 2008
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Ingår i: Combinatorial chemistry & high throughput screening. - : Bentham Science Publishers Ltd.. - 1386-2073 .- 1875-5402. ; 11:10, s. 807-816
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Tidskriftsartikel (refereegranskat)abstract
- A mutant laccase from the Ascomycete Myceliophthora thermophila has been submitted to iterative cycles of combinatorial satn. mutagenesis through in vivo overlap extension in Saccharomyces cerevisiae. Over 180,000 clones were explored, among which the S510G mutant revealed a direct interaction between the conserved 509VSG511 tripeptide, located in the neighborhood of the T1 site, and the C-terminal plug. The KmO2 value of the mutant increased 1.5-fold, and the electron transfer pathway between the reducing substrate and the T1 copper ion was altered, improving the catalytic efficiency towards non-phenolic and phenolic substrates by about 3- and 8-fold. Although the geometry at the T1 site was perturbed by the mutation, paradoxically the laccase redox potential was not significantly altered. Together, the results obtained in this study suggest that the 509VSG511 tripeptide may play a hitherto unrecognized role in regulating the traffic of oxygen through the C-terminal plug, the latter blocking access to the T2/T3 copper cluster in the native enzyme.
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| 2. |
- Zumarraga, Miren, et al.
(författare)
-
In vitro evolution of a fungal laccase in high concentrations of organic cosolvents
- 2007
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Ingår i: Chemistry and Biology. - : Elsevier BV. - 1879-1301 .- 1074-5521. ; 14:9, s. 1052-1064
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Tidskriftsartikel (refereegranskat)abstract
- Fungal laccases are remarkable green catalysts that have a broad substrate specificity and many potential applications in bioremediation, lignocellulose processing, organic synthesis, and more. However, most of these transformations must be carried out at high concentrations of organic cosolvents in which laccases undergo unfolding, thereby losing their activity. We have tailored a thermostable laccase that tolerates high concentrations of cosolvents, the genetic product of five rounds of directed evolution expressed in Saccharomyces cerevisiae. This evolved laccase-R2 variant-was capable of resisting a wide array of cosolvents at concentrations as high as 50% (v/v). Intrinsic laccase features such as the redox potential and the geometry of catalytic coppers varied slightly during the course of the molecular evolution. Some mutations at the protein surface stabilized the laccase by allowing additional electrostatic and hydrogen bonding to occur.
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