Combination of computational prescreening and experimental library construction can accelerate enzyme optimization by directed evolution

Chiral compounds can be produced efficiently by using biocatalysts. However, wild-type enzymes often do not meet the requirements of a production process, making optimization by rational design or directed evolution necessary. Here, we studied the lipase-catalyzed hydrolysis of the model substrate 1...

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Bibliographic Details
Published in:Protein engineering, design and selection design and selection, 2005-11, Vol.18 (11), p.509-514
Main Authors: Funke, Susanne Aileen, Otte, Nikolaj, Eggert, Thorsten, Bocola, Marco, Jaeger, Karl-Erich, Thiel, Walter
Format: Article
Language:English
Subjects:
Amino Acid Substitution - genetics
Bacillus subtilis
Bacillus subtilis - chemistry
Bacillus subtilis - enzymology
Bacillus subtilis - genetics
Computational Biology - methods
directed evolution
Directed Molecular Evolution - methods
enantioselectivity
Gene Library
Hydrolysis
Lipase - chemistry
Lipase - genetics
MM calculation
Models, Chemical
molecular modeling
Mutagenesis
Protein Structure, Secondary
saturation mutagenesis
Substrate Specificity
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Summary:Chiral compounds can be produced efficiently by using biocatalysts. However, wild-type enzymes often do not meet the requirements of a production process, making optimization by rational design or directed evolution necessary. Here, we studied the lipase-catalyzed hydrolysis of the model substrate 1-(2-naphthyl)ethyl acetate both theoretically and experimentally. We found that a computational equivalent of alanine scanning mutagenesis based on QM/MM methodology can be applied to identify amino acid positions important for the activity of the enzyme. The theoretical results are consistent with concomitant experimental work using complete saturation mutagenesis and high-throughput screening of the target biocatalyst, a lipase from Bacillus subtilis. Both QM/MM-based calculations and molecular biology experiments identify histidine 76 as a residue that strongly affects the catalytic activity. The experiments demonstrate its important influence on enantioselectivity.
ISSN:1741-0126
1741-0134
DOI:10.1093/protein/gzi062