2. Academic Validation
  3. Colorimetric High-Throughput Screening Assays for the Directed Evolution of Fungal Laccases

Colorimetric High-Throughput Screening Assays for the Directed Evolution of Fungal Laccases

  • Methods Mol Biol. 2018;1685:247-254. doi: 10.1007/978-1-4939-7366-8_14.
Isabel Pardo 1,  Susana Camarero 2
Affiliations

Affiliations

  • 1 Centro de Investigaciones Biológicas, CSIC, Ramiro de Maeztu, 9, 28040, Madrid, Spain.
  • 2 Centro de Investigaciones Biológicas, CSIC, Ramiro de Maeztu, 9, 28040, Madrid, Spain. susanacam@cib.csic.es.
PMID: 29086313 DOI: 10.1007/978-1-4939-7366-8_14
Abstract

In this chapter we describe several high-throughput screening assays for the evaluation of mutant libraries for the directed evolution of Fungal laccases in the yeast Saccharomyces cerevisiae. The assays are based on the direct oxidation of three syringyl-type Phenols derived from lignin (sinapic acid, acetosyringone, and syringaldehyde), an artificial laccase mediator (violuric acid), and three organic synthetic dyes (Methyl Orange, Evans Blue, and Remazol Brilliant Blue). While the assays with the natural Phenols can be used for laccases with low redox potential, the rest are exclusive for high-redox potential laccases. In fact, the violuric acid assay is devised as a method to ascertain that the high-redox potential of laccase is not lost during directed evolution.

Keywords

Colorimetric assays; Directed evolution; High-throughput screening; Laccase; Mutant libraries; Synthetic organic dyes; Syringyl-type phenols; Violuric acid.

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