Improved Enantioselectivity of Subtilisin Carlsberg towards Secondary Alcohols by Protein Engineering

Robin Dorau, Tamas Görbe, Maria Svedendahl Humble*

*Corresponding author for this work

Research output: Contribution to journalJournal articleResearchpeer-review

Abstract

Generally, the catalytic activity of subtilisin Carlsberg (SC) for transacylation reactions with secondary alcohols in organic solvent is low. Enzyme immobilization and protein engineering was performed to improve the enantioselectivity of SC towards secondary alcohols. Possible amino-acid residues for mutagenesis were found by combining available literature data with molecular modeling. SC variants were created by site-directed mutagenesis and were evaluated for a model transacylation reaction containing 1-phenylethanol in THF. Variants showing high E values (>100) were found. However, the conversions were still low. A second mutation was made, and both the E values and conversions were increased. Relative to that shown by the wild type, the most successful variant, G165L/M221F, showed increased conversion (up to 36%), enantioselectivity (E values up to 400), substrate scope, and stability in THF.
Original languageEnglish
JournalChembiochem
Volume19
Issue number4
Pages (from-to)338-346
ISSN1439-4227
DOIs
Publication statusPublished - 2018

Keywords

  • Biocatalysis
  • Molecular modeling
  • Enzymes
  • Immobilization
  • Kinetic resolution
  • Transacylation

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