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Prediction of the Candida antarctica lipase A protein structure by comparative modeling and site-directed mutagenesis
Stockholm University, Faculty of Science, Department of Organic Chemistry.
Max-Planck-Institut für Kohlenforschung, Kaiser-Wilhelm-Platz 1, 45470 Mülheim an der Ruhr, Germany.
Stockholm University, Faculty of Science, Department of Organic Chemistry.
Stockholm University, Faculty of Science, Department of Organic Chemistry.
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2007 (English)In: ChemBioChem (Print), ISSN 1439-4227, E-ISSN 1439-7633, Vol. 8, no 12, 1409-1415 p.Article in journal (Refereed) Published
Abstract [en]

A number of model structures of the CalA suggested by comparative modeling were tested by site-directed mutagenesis. Enzyme variants were created where amino acids predicted to play key roles for the lipase activity in the different models were replaced by an inert amino acid (alanine). The results from activity measurements of the overproduced and purified mutant enzymes indicate a structure where the active site consists of amino acid residues Ser184, His366, and Asp334 and in which there is no lid. This model can be used for future targeted modifications of the enzyme to obtain new substrate acceptance, better thermostability, and higher enantioselectivity.

Place, publisher, year, edition, pages
2007. Vol. 8, no 12, 1409-1415 p.
Keyword [en]
Candida antarctica lipase A, chiral resolution, enzyme models, hydrolases, protein models
Identifiers
URN: urn:nbn:se:su:diva-18160DOI: 10.1002/cbic.200700179ISI: 000248851700013OAI: oai:DiVA.org:su-18160DiVA: diva2:184683
Available from: 2007-10-17 Created: 2007-10-17 Last updated: 2010-12-15Bibliographically approved
In thesis
1. Protein Engineering of Candida antarctica Lipase A: Enhancing Enzyme Properties by Evolutionary and Semi-Rational Methods
Open this publication in new window or tab >>Protein Engineering of Candida antarctica Lipase A: Enhancing Enzyme Properties by Evolutionary and Semi-Rational Methods
2010 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Enzymes are gaining increasing importance as catalysts for selective transformations in organic synthetic chemistry. The engineering and design of enzymes is a developing, growing research field that is employed in biocatalysis. In the present thesis, combinatorial protein engineering methods are applied for the development of Candida antarctica lipase A (CALA) variants with broader substrate scope and increased enantioselectivity. Initially, the structure of CALA was deduced by manual modelling and later the structure was established by X-ray crystallography. The elucidation of the structure of CALA revealed several biocatalytically interesting features. With the knowledge derived from the enzyme structure, enzyme variants were produced via iterative saturation mutagenesis (ISM), a powerful protein engineering approach. Several of these variants were highly active and enantioselective towards bulky esters. Furthermore, an extensively combinatorial protein engineering approach was developed and investigated. A CALA variant with a spacious substrate binding pocket that can accommodate an unusually bulky substrate, an ester derivate of the non-steroidal anti-inflammatory drug (S)-ibuprofen, was obtained with this approach.

Place, publisher, year, edition, pages
Stockholm: Department of Organic Chemistry, Stockholm University, 2010. 70 p.
Keyword
lipase, protein engineering, directed evolution, kinetic resolution, structural biology
National Category
Biocatalysis and Enzyme Technology
Research subject
Organic Chemistry
Identifiers
urn:nbn:se:su:diva-49248 (URN)978-91-7447-202-8 (ISBN)
Public defence
2011-01-28, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, Stockholm, 13:00 (English)
Opponent
Supervisors
Note
At the time of the doctoral defence the following paper was unpublished and had a status as follows: Paper nr. 5: ManuscriptAvailable from: 2011-01-03 Created: 2010-12-13 Last updated: 2011-02-21Bibliographically approved

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