Journal
CHEMBIOCHEM
Volume 16, Issue 6, Pages 937-945Publisher
WILEY-V C H VERLAG GMBH
DOI: 10.1002/cbic.201402682
Keywords
BSLA; directed evolution; ionic liquids; protein engineering; site saturation mutagenesis
Funding
- Deutsche Forschungsgemeinschaft (DFG) [GRK 1166]
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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.
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