PEDS Advance Access originally published online on February 19, 2007
Protein Engineering Design and Selection 2007 20(3):125-131; doi:10.1093/protein/gzm003
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Highly enantioselective kinetic resolution of two tertiary alcohols using mutants of an esterase from Bacillus subtilis
1 Institute of Biochemistry, Department of Biotechnology and Enzyme Catalysis, Greifswald University, Felix-Hausdorff-Str. 4, D-17487 Greifswald, Germany 2 Royal Institute of Technology (KTH), School of Biotechnology, Department of Biochemistry, AlbaNova University Center, 10691 Stockholm, Sweden
4 To whom correspondence should be addressed. E-mail: uwe.bornscheuer{at}uni-greifswald.de
Enzyme-catalyzed kinetic resolutions of secondary alcohols are a standard procedure today and several lipases and esterases have been described to show high activity and enantioselectivity. In contrast, tertiary alcohols and their esters are accepted only by a few biocatalysts. Only lipases and esterases with a conserved GGG(A)X-motif are active, but show low activity combined with low enantioselectivity in the hydrolysis of tertiary alcohol esters. We show in this work that the problematic autohydrolysis of certain compounds can be overcome by medium and substrate engineering. Thus, 3-phenylbut-1-yn-3-yl acetate was hydrolyzed by the esterase from Bacillus subtilis (BS2, mutant Gly105Ala) with an enantioselectivity of E = 56 in the presence of 20% (v/v) DMSO compared to E = 28 without a cosolvent. Molecular modeling was used to study the interactions between BS2 and tertiary alcohol esters in their transition state in the active site of the enzyme. Guided by molecular modeling, enzyme variants with highly increased enantioselectivity were created. For example, a Glu188Asp mutant converted the trifluoromethyl analog of 3-phenylbut-1-yn-3-yl acetate with an excellent enantioselectivity (E > 100) yielding the (S)-alcohol with > 99%ee. In summary, protein engineering combined with medium and substrate engineering afforded tertiary alcohols of very high enantiomeric purity.
Keywords: tertiary alcohol/enantioselectivity/enzyme catalysis/esterase/rational protein design
3 These authors contributed equally to this work.
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