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Directed evolution of Candida antarctica lipase A using an episomaly replicating yeast plasmid
Stockholm University, Faculty of Science, Department of Organic Chemistry.
Stockholm University, Faculty of Science, Department of Organic Chemistry.
Stockholm University, Faculty of Science, Department of Organic Chemistry.
Stockholm University, Faculty of Science, Department of Organic Chemistry.
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2009 (English)In: Protein Engineering Design & Selection, ISSN 1741-0126, E-ISSN 1741-0134, Vol. 22, no 7, 413-420 p.Article in journal (Refereed) Published
Abstract [en]

We herein report the first directed evolution of Candida antarctica lipase A (CalA), employing a combinatorial active-site saturation test (CAST). Wild-type CalA has a modest E-value of 5.1 in kinetic resolution of 4-nitrophenyl 2-methylheptanoate. Enzyme variants were expressed in Pichia pastoris by using the episomal vector pBGP1 which allowed efficient secretory expression of the lipase. Iterative rounds of CASTing yielded variants with good selectivity toward both the (S)- and the (R)-enantiomer. The best obtained enzyme variants had E-values of 52 (S) and 27 (R).

Place, publisher, year, edition, pages
Oxford University Press , 2009. Vol. 22, no 7, 413-420 p.
National Category
Organic Chemistry
Identifiers
URN: urn:nbn:se:su:diva-30458DOI: 10.1093/protein/gzp019ISI: 000267226800004OAI: oai:DiVA.org:su-30458DiVA: diva2:272351
Available from: 2009-10-15 Created: 2009-10-15 Last updated: 2010-12-15Bibliographically approved
In thesis
1. Theoretical modeling of metal- and enzyme catalyzed transformations
Open this publication in new window or tab >>Theoretical modeling of metal- and enzyme catalyzed transformations
2010 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

This thesis is focused on describing and predicting catalytic reactions. The major part of the work is based on density functional theory (DFT). In some cases where the size of the investigated system precluded the use of more accurate methods molecular dynamics was employed. In several cases the proposed mechanism was later tested in the laboratory. A few examples where the predictions were confirmed are:

  • The formation of an acyl intermediate in the activation of a ruthenium catalyst used for racemizing alcohols. This intermediate was observed by both NMR and in situ FT-IR.
  • The improvement of the substrate specificity and catalytic activity of Candida antarctica lipase A by modifying amino acids close to the active site.
  • The improved specificity of Candida antarctica lipase B toward δ-substituted secondary alcohols by an enzyme variant where the alanine in position 281 was exchanged for a serine.

In other cases experimental results were complemented with a theoretical investigation, for example:

  • The observed second order rate constant for a ruthenium based catalyst used for water oxidation was explained and a novel intramolecular mechanism based on a high valent ruthenium dimer was suggested.
  • The effects of electron withdrawing/donating axial ligands on the performance of ruthenium catalyzed water oxidation were addressed.
  • Mechanisms of H2 activation by Lewis acid/Lewis base adducts were rationalized. One example of the predictive power of computational chemistry is the mechanism of hydrogen uptake by phosphanylboranes; the potential energy barrier for the transition state could be predicted within a few kcal/mol based on the orbital energies of the starting material.
Place, publisher, year, edition, pages
Stockholm: Department of Organic Chemistry, Stockholm University, 2010. 96 p.
Keyword
density functinal theory, computational chemistry, directed evolution, enzyme, mechanistic studies, catalysis, ruthenium, hydrogen transfer, racemization, artificial photosynthesis, frustrated lewis pairs, hydrogen storage
National Category
Organic Chemistry
Research subject
Organic Chemistry
Identifiers
urn:nbn:se:su:diva-38344 (URN)978-91-7447-063-5 (ISBN)
Public defence
2010-05-12, Magnelisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, Stockholm, 13:00 (English)
Opponent
Supervisors
Note
At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 2: Submitted. Paper 3: Submitted. Paper 8: In press.Available from: 2010-04-20 Created: 2010-04-08 Last updated: 2010-05-28Bibliographically approved
2. 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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Sandström, Anders G.Engström, KarinNyhlén, JonasBäckvall, Jan-Erling
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