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.
Keywords: Laccase, directed evolution, saturation mutagenesis, substrate specificity, protein engineering
Combinatorial Chemistry & High Throughput Screening
Title: Narrowing Laccase Substrate Specificity Using Active Site Saturation Mutagenesis
Volume: 12 Issue: 3
Author(s): Nirupama Gupta and Edgardo T. Farinas
Affiliation:
Keywords: Laccase, directed evolution, saturation mutagenesis, substrate specificity, protein engineering
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.
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Cite this article as:
Gupta Nirupama and Farinas T. Edgardo, Narrowing Laccase Substrate Specificity Using Active Site Saturation Mutagenesis, Combinatorial Chemistry & High Throughput Screening 2009; 12 (3) . https://dx.doi.org/10.2174/138620709787581675
DOI https://dx.doi.org/10.2174/138620709787581675 |
Print ISSN 1386-2073 |
Publisher Name Bentham Science Publisher |
Online ISSN 1875-5402 |
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