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Highly enantioselective kinetic resolution of two tertiary alcohols using mutants of an esterase from Bacillus subtilis
KTH, School of Biotechnology (BIO), Biochemistry.
KTH, School of Biotechnology (BIO), Biochemistry.
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2007 (English)In: Protein Engineering Design & Selection, ISSN 1741-0126, E-ISSN 1741-0134, Vol. 20, no 3, 125-131 p.Article in journal (Refereed) Published
Abstract [en]

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.

Place, publisher, year, edition, pages
2007. Vol. 20, no 3, 125-131 p.
Keyword [en]
tertiary alcohol, enantioselectivity, enzyme catalysis, esterase, rational protein design, des molecules conjuguees, candida-antarctica, oxyanion hole, acetate esters, nucleic-acids, force-field, lipase-b, enzyme, transesterification, stereocenters
National Category
Biochemistry and Molecular Biology
Identifiers
URN: urn:nbn:se:kth:diva-16506DOI: 10.1093/protein/gzm003ISI: 000245354800004Scopus ID: 2-s2.0-34047217953OAI: oai:DiVA.org:kth-16506DiVA: diva2:334548
Note
QC 20100525Available from: 2010-08-05 Created: 2010-08-05 Last updated: 2011-02-07Bibliographically approved
In thesis
1. Molecular modelling - understanding and prediction of enzyme selectivity.
Open this publication in new window or tab >>Molecular modelling - understanding and prediction of enzyme selectivity.
2009 (English)Licentiate thesis, comprehensive summary (Other academic)
Abstract [en]

Molecular modelling strategies for evaluation of enzyme selectivity wereinvestigated with a focus on principles of how molecular interactionscould be evaluated to provide information about selectivity. Althoughmolecular modelling provides tools for evaluation of geometrical andenergy features of molecular systems, no general strategies for evaluationof enzyme selectivity exist. Geometrical analyses can be based uponinspection and reasoning about molecular interactions, which provide aneasily accessible way to gain information, but suffer from the risk of biasput in by the modeller. They can also be based on geometrical features ofmolecular interactions such as bond lengths and hydrogen-bond formation.Energy analyses are appealing for their modeller independenceand for the possibility to predict not only stereopreference, but also itsmagnitude.In this thesis, four examples of enantio- or regioselective serinehydrolase-catalysed reaction systems are presented together with developedmodelling protocols for explanation, prediction or enhancement ofselectivity. Geometrical as well as energy-based methodology were used,and provided an understanding of the structural basis of enzymeselectivity. In total, the protocols were successful in making qualitative explanationsand predictions of stereoselectivity, although quantitative determinationswere not achieved.

Place, publisher, year, edition, pages
Stockholm: KTH, 2009. 27 p.
Series
Trita-BIO-Report, ISSN 1654-2312 ; 2009:11
Keyword
molecular modelling; regioselectivity; enantioselectivity; molecular dynamics; serine hydrolase; stereospecificity
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-10532 (URN)978-91-7415-325-5 (ISBN)
Presentation
(English)
Supervisors
Available from: 2009-05-26 Created: 2009-05-25 Last updated: 2010-10-12Bibliographically approved

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