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Defining Substrate Specificities for Lipase and Phospholipase Candidates
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Defining Substrate Specificities for Lipase and Phospholipase Candidates

Published on: November 23, 2016

Narrowing laccase substrate specificity using active site saturation mutagenesis.

Nirupama Gupta1, Edgardo T Farinas

  • 1Department of Chemistry and Environmental Science, New Jersey Institute of Technology, Newark, New Jersey, NJ 07102, USA.

Combinatorial Chemistry & High Throughput Screening
|March 12, 2009
PubMed
Summary
This summary is machine-generated.

Researchers engineered the Bacillus subtilis laccase CotA from a generalist to a specialist enzyme. This study demonstrates that laccase substrate specificity can be successfully narrowed through protein evolution.

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Last Updated: Jun 25, 2026

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In Vitro Directed Evolution of a Restriction Endonuclease with More Stringent Specificity
09:16

In Vitro Directed Evolution of a Restriction Endonuclease with More Stringent Specificity

Published on: March 25, 2020

Area of Science:

  • Biochemistry
  • Enzymology
  • Protein Engineering

Background:

  • Laccases, belonging to the multicopper oxidase family, possess broad applications in biotechnology.
  • Tapering substrate specificity for laccases has not been previously demonstrated.
  • Wild-type CotA from Bacillus subtilis exhibits broad substrate specificity, oxidizing both ABTS and SGZ.

Purpose of the Study:

  • To engineer the laccase CotA for increased specificity towards ABTS.
  • To demonstrate the evolvability of the substrate-binding pocket for enhanced specificity.

Main Methods:

  • Combined rational and directed evolution approaches.
  • Simultaneously randomized 19 amino acids within the substrate-binding pocket of wild-type CotA.
  • Screened for mutants with improved substrate specificity.

Main Results:

  • Identified a mutant with a 7.0-fold greater catalytic efficiency for ABTS over SGZ compared to wild-type CotA.
  • This improvement was achieved after a single round of screening.
  • The results highlight the high evolvability of the laccase substrate-binding pocket.

Conclusions:

  • The substrate-binding pocket of laccase CotA is highly evolvable for specificity.
  • It is possible to convert a generalist laccase into a specialist enzyme through protein engineering.
  • This work opens avenues for tailoring laccases for specific biotechnological applications.