Narrowing laccase substrate specificity using active site saturation mutagenesis

Document Type

Article

Publication Date

3-1-2009

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.

Identifier

65549094075 (Scopus)

Publication Title

Combinatorial Chemistry and High Throughput Screening

External Full Text Location

https://doi.org/10.2174/138620709787581675

ISSN

13862073

PubMed ID

19275532

First Page

269

Last Page

274

Issue

3

Volume

12

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