An Inferred Ancestral CotA Laccase with Improved Expression and Kinetic Efficiency
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Published:2023-06-30
Issue:13
Volume:24
Page:10901
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ISSN:1422-0067
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Container-title:International Journal of Molecular Sciences
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language:en
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Short-container-title:IJMS
Author:
Lei Lei1, Zhao Lijun1, Hou Yiqia1, Yue Chen1, Liu Pulin1, Zheng Yanli1, Peng Wenfang2ORCID, Yang Jiangke1
Affiliation:
1. School of Life Science and Technology, Wuhan Polytechnic University, Wuhan 430023, China 2. State Key Laboratory of Biocatalysis and Enzyme Engineering, College of Life Science, Hubei University, Wuhan 430062, China
Abstract
Laccases are widely used in industrial production due to their broad substrate availability and environmentally friendly nature. However, the pursuit of laccases with superior stability and increased heterogeneous expression to meet industry demands appears to be an ongoing challenge. To address this challenge, we resurrected five ancestral sequences of laccase BsCotA and their homologues. All five variants were successfully expressed in soluble and functional forms with improved expression levels in Escherichia coli. Among the five variants, three exhibited higher catalytic rates, thermal stabilities, and acidic stabilities. Notably, AncCotA2, the best-performing variant, displayed a kcat/KM of 7.5 × 105 M−1·s−1, 5.2-fold higher than that of the wild-type BsCotA, an improved thermo- and acidic stability, and better dye decolorization ability. This study provides a laccase variant with high application potential and presents a new starting point for future enzyme engineering.
Funder
National Natural Science Foundation of China Hubei Province key research and development program Opening Funding Project of the State Key Laboratory of Biocatalysis and Enzyme Engineering
Subject
Inorganic Chemistry,Organic Chemistry,Physical and Theoretical Chemistry,Computer Science Applications,Spectroscopy,Molecular Biology,General Medicine,Catalysis
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