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

Cite this

Dorau, Robin ; Görbe, Tamas ; Svedendahl Humble, Maria. / Improved Enantioselectivity of Subtilisin Carlsberg towards Secondary Alcohols by Protein Engineering. In: Chembiochem. 2018 ; Vol. 19, No. 4. pp. 338-346.
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title = "Improved Enantioselectivity of Subtilisin Carlsberg towards Secondary Alcohols by Protein Engineering",
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.",
keywords = "Biocatalysis, Molecular modeling, Enzymes, Immobilization, Kinetic resolution, Transacylation",
author = "Robin Dorau and Tamas G{\"o}rbe and {Svedendahl Humble}, Maria",
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Improved Enantioselectivity of Subtilisin Carlsberg towards Secondary Alcohols by Protein Engineering. / Dorau, Robin; Görbe, Tamas; Svedendahl Humble, Maria.

In: Chembiochem, Vol. 19, No. 4, 2018, p. 338-346.

Research output: Contribution to journalJournal articleResearchpeer-review

TY - JOUR

T1 - Improved Enantioselectivity of Subtilisin Carlsberg towards Secondary Alcohols by Protein Engineering

AU - Dorau, Robin

AU - Görbe, Tamas

AU - Svedendahl Humble, Maria

PY - 2018

Y1 - 2018

N2 - 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.

AB - 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.

KW - Biocatalysis

KW - Molecular modeling

KW - Enzymes

KW - Immobilization

KW - Kinetic resolution

KW - Transacylation

U2 - 10.1002/cbic.201700408

DO - 10.1002/cbic.201700408

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VL - 19

SP - 338

EP - 346

JO - ChemBioChem

JF - ChemBioChem

SN - 1439-4227

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ER -