Colorimetric Assays for Screening Laccases

  • Miguel Alcalde
  • Thomas Bulter
Part of the Methods in Molecular Biology™ book series (MIMB, volume 230)


In this chapter, we describe colorimetric methods for screening laccases using natural and nonnatural substrates. Laccases (EC are blue-copper enzymes that oxidize phenols, polyphenols, and anilines (1,2). The catalytic capabilities of laccase can be greatly enhanced by the addition of suitable mediator compounds. In the presence of some of its primary substrates (such as 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid [ABTS] or 1-hydroxy-benzothiazole [HBT], laccase can catalyze the oxidation of nonnatural substrates, including polycyclic aromatic hydrocarbons (PAHs), a class of highly toxic organic pollutants widely distributed in terrestrial and aquatic environments (3, 4, 5, 6, 7). The mechanism of oxidation by laccase-mediator systems (LMS) is still under discussion. In spite of enhancing the range of compounds amenable to oxidation by laccases, mediators have several disadvantages: they are expensive, poisonous, and show side reactions with substrates and products, leading to reduced yield and impurity of the products. Inactivation of laccase by free radicals of the mediators is an additional drawback (8,9). To optimize laccases for mediated applications or make them independent of mediators using directed evolution requires mediator-dependent screens.


Laccase Activity Directed Evolution Trametes Versicolor Nonnatural Substrate Endpoint Assay 
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Copyright information

© Humana Press Inc., Totowa, NJ 2003

Authors and Affiliations

  • Miguel Alcalde
    • 1
  • Thomas Bulter
    • 2
  1. 1.Division of Chemistry and Chemical EngineeringCalifornia Institute of TechnologyPasadena
  2. 2.Department of Chemical EngineeringUniversity of California at Los AngelesLos Angeles

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