Narrowing laccase substrate specificity using active site saturation mutagenesis

Nirupama Gupta, Edgardo T. Farinas

Research output: Contribution to journalArticlepeer-review

19 Scopus citations

Abstract

The laccase CotA from Bacillus subtilis was converted from a generalist, an enzyme with broad specificity, to a specialist, an enzyme with narrowed specificity. Laccases are members of the multicopper oxidase family and have many applications in biotechnology. To date, it has not been demonstrated that substrate specificity can be tapered for a laccase. 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 by combining rational and directed evolution approaches. The wild-type was evolved by simultaneously randomizing 19 amino acids found in the substrate-binding pocket. A mutant was identified that had a catalytic efficiency, (kcat/KM)ATBS /(kcat/KM)SGZ, 7.0 times greater when compared to the wild-type after one round of screening. This illustrates that the substrate-binding pocket is highly evolvable for specificity.

Original languageEnglish (US)
Pages (from-to)269-274
Number of pages6
JournalCombinatorial Chemistry and High Throughput Screening
Volume12
Issue number3
DOIs
StatePublished - Mar 1 2009

All Science Journal Classification (ASJC) codes

  • Drug Discovery
  • Computer Science Applications
  • Organic Chemistry

Keywords

  • Directed evolution
  • Laccase
  • Saturation mutagenesis
  • protein engineering
  • substrate specificity

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