Exploring the Protein Stability Landscape: Bacillus subtilis Lipase A as a Model for Detergent Tolerance |
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| Authors: | Dr. Alexander Fulton Victorine Josiane Frauenkron‐Machedjou Pia Skoczinski Dr. Susanne Wilhelm Dr. Leilei Zhu Prof. Dr. Ulrich Schwaneberg Prof. Dr. Karl‐Erich Jaeger |
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| Affiliation: | 1. Institut für Molekulare Enzymtechnologie, Heinrich Heine Universit?t Düsseldorf and Forschungszentrum Jülich, Wilhelm‐Johnen‐Strasse, 52426 Jülich (Germany);2. Lehrstuhl für Biotechnologie, RWTH Aachen University, Worringerweg 1, 52074 Aachen (Germany) |
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| Abstract: | A systematic study was conducted with Bacillus subtilis lipase A (BSLA) to determine the effect of every single amino acid substitution on detergent tolerance. BSLA is a minimal α/β‐hydrolase of 181 amino acids with a known crystal structure. It can be expressed in Escherichia coli and is biochemically well characterized. Site saturation mutagenesis resulted in a library of 3439 variants, each with a single amino acid exchange as confirmed by DNA sequencing. The library was tested against four detergents, namely SDS, CTAB, Tween 80, and sulfobetaine. Surface remodeling emerged as an effective engineering strategy to increase tolerance towards detergents. Amino acid residues that significantly affect the tolerance for each of the four detergents were identified. In summary, this systematic analysis provides an experimental dataset to help derive novel protein engineering strategies as well as to direct modeling efforts. |
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| Keywords: | alpha beta hydrolases Bacillus subtilis lipase A detergent effects directed evolution noncovalent interactions |
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