Laboratory evolution of laccase for substrate specificity

Nirupama Gupta, Frederick S. Lee, Edgardo T. Farinas

Research output: Contribution to journalArticlepeer-review

46 Scopus citations


A laccase, CotA, from Bacillus subtilis was engineered using a combination of rational and directed evolution approaches. CotA is a generalist, an enzyme with broad specificity, and it was optimized to be a specialist, an enzyme with narrowed specificity. Wild-type CotA oxidizes ABTS (ABTS=diammonium 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonate) and SGZ (SGZ=4-hydroxy-3,5-dimethoxy-benzaldehyde azine), and it was engineered for increased specificity for ABTS. Based on the ABTS-bound crystal structure of CotA, 19 amino acids are within 6Å of ABTS, and they were simultaneously randomized. A mutant was identified that was 132 times more specific for ABTS. Unexpectedly, the variant was found to acquire enhanced thermal stability. The half-life for the heat inactivation (t1/2) at 80°C was increased by 62min for the mutant. Laccases have several applications in biotechnology, which include pulp bleaching, biosensors, bioremediation, and biofuel cells. The substrate specificity of CotA is moldable and the enzyme can be tailored to oxidize a variety of target molecules for specific purposes.

Original languageEnglish (US)
Pages (from-to)230-234
Number of pages5
JournalJournal of Molecular Catalysis B: Enzymatic
Issue number3-4
StatePublished - 2010

All Science Journal Classification (ASJC) codes

  • Catalysis
  • Bioengineering
  • Biochemistry
  • Process Chemistry and Technology


  • Directed evolution
  • Laccase
  • Protein engineering
  • Substrate specificity
  • Thermal stability


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