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Towards Understanding Directed Evolution: More than Half of All Amino Acid Positions Contribute to Ionic Liquid Resistance of Bacillus subtilis Lipase A
Author(s) -
FrauenkronMachedjou Victorine Josiane,
Fulton Alexander,
Zhu Leilei,
Anker Carolin,
Bocola Marco,
Jaeger KarlErich,
Schwaneberg Ulrich
Publication year - 2015
Publication title -
chembiochem
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.05
H-Index - 126
eISSN - 1439-7633
pISSN - 1439-4227
DOI - 10.1002/cbic.201402682
Subject(s) - saturated mutagenesis , directed evolution , bacillus subtilis , protein engineering , ionic liquid , amino acid , biocatalysis , lipase , mutagenesis , chemistry , enzyme , mutant , biochemistry , stereochemistry , gene , biology , catalysis , genetics , bacteria
Ionic liquids (ILs) are attractive (co‐)solvents for biocatalysis. However, in high concentration (>10 % IL), enzymes usually show decreased activity. No general principles have been discovered to improve IL resistance of enzymes by protein engineering. We present a systematic study to elucidate general engineering principles by site saturation mutagenesis on the complete gene bsla . Screening in presence of four [BMIM]‐based ILs revealed two unexpected lessons on directed evolution: 1) resistance improvement was obtainable at 50–69 % of all amino acid positions, thus explaining the success of small sized random mutant libraries; 2) 6–13 % of substitutions led to improved resistance. Among these, 66–95 % were substitutions by chemically different amino acids (e.g., aromatic to polar/aliphatic/charged amino acids), thus indicating that mutagenesis methods introducing such changes should, at least for lipases like BSLA, be favored to improve IL resistance.