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Protein Engineering of Candida antarctica Lipase A: Enhancing Enzyme Properties by Evolutionary and Semi-Rational Methods
Stockholm University, Faculty of Science, Department of Organic Chemistry. (Jan-Erling Bäckvall)
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 [en]
lipase, protein engineering, directed evolution, kinetic resolution, structural biology
National Category
Biocatalysis and Enzyme Technology
Research subject
Organic Chemistry
Identifiers
URN: urn:nbn:se:su:diva-49248ISBN: 978-91-7447-202-8 (print)OAI: oai:DiVA.org:su-49248DiVA: diva2:377788
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At the time of the doctoral defence the following paper was unpublished and had a status as follows: Paper nr. 5: ManuscriptAvailable from: 2011-01-03 Created: 2010-12-13 Last updated: 2011-02-21Bibliographically approved
List of papers
1. X-Ray structure of Candida antarctica lipase A shows a novel lid structure and a likely mode of interfacial activation
Open this publication in new window or tab >>X-Ray structure of Candida antarctica lipase A shows a novel lid structure and a likely mode of interfacial activation
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2008 (English)In: Journal of Molecular Biology, ISSN 0022-2836, Vol. 376, no 1, 109-119 p.Article in journal (Refereed) Published
Abstract [en]

In nature, lipases (EC 3.1.1.3) catalyze the hydrolysis of triglycerides to form glycerol and fatty acids. Under the appropriate conditions, the reaction is reversible, and so biotechnological applications commonly make use of their capacity for esterification as well as for hydrolysis of a wide variety of compounds. In the present paper, we report the X-ray structure of lipase A from Candida antarctica, solved by single isomorphous replacement with anomalous scattering, and refined to 2.2-Å resolution. The structure is the first from a novel family of lipases. Contrary to previous predictions, the fold includes a well-defined lid as well as a classic α/β hydrolase domain. The catalytic triad is identified as Ser184, Asp334 and His366, which follow the sequential order considered to be characteristic of lipases; the serine lies within a typical nucleophilic elbow. Computer docking studies, as well as comparisons to related structures, place the carboxylate group of a fatty acid product near the serine nucleophile, with the long lipid tail closely following the path through the lid that is marked by a fortuitously bound molecule of polyethylene glycol. For an ester substrate to bind in an equivalent fashion, loop movements near Phe431 will be required, suggesting the primary focus of the conformational changes required for interfacial activation. Such movements will provide virtually unlimited access to solvent for the alcohol moiety of an ester substrate. The structure thus provides a basis for understanding the enzyme's preference for acyl moieties with long, straight tails, and for its highly promiscuous acceptance of widely different alcohol and amine moieties. An unconventional oxyanion hole is observed in the present structure, although the situation may change during interfacial activation

Place, publisher, year, edition, pages
Elsevier, 2008
Keyword
lipase; interfacial activation; hydrolase; X-ray structure; substrate specificity
National Category
Organic Chemistry
Identifiers
urn:nbn:se:su:diva-13671 (URN)doi:10.1016/j.jbm.2007.10.079 (DOI)000253181500011 ()
Available from: 2008-12-03 Created: 2008-12-03 Last updated: 2010-12-15Bibliographically approved
2. Prediction of the Candida antarctica lipase A protein structure by comparative modeling and site-directed mutagenesis
Open this publication in new window or tab >>Prediction of the Candida antarctica lipase A protein structure by comparative modeling and site-directed mutagenesis
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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.

Keyword
Candida antarctica lipase A, chiral resolution, enzyme models, hydrolases, protein models
Identifiers
urn:nbn:se:su:diva-18160 (URN)10.1002/cbic.200700179 (DOI)000248851700013 ()
Available from: 2007-10-17 Created: 2007-10-17 Last updated: 2010-12-15Bibliographically approved
3. Directed evolution of Candida antarctica lipase A using an episomaly replicating yeast plasmid
Open this publication in new window or tab >>Directed evolution of Candida antarctica lipase A using an episomaly replicating yeast plasmid
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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
National Category
Organic Chemistry
Identifiers
urn:nbn:se:su:diva-30458 (URN)10.1093/protein/gzp019 (DOI)000267226800004 ()
Available from: 2009-10-15 Created: 2009-10-15 Last updated: 2010-12-15Bibliographically approved
4. Directed evolution of an enantioselective lipase with broad substrate scope for hydrolysis of α-substituted esters
Open this publication in new window or tab >>Directed evolution of an enantioselective lipase with broad substrate scope for hydrolysis of α-substituted esters
2010 (English)In: Journal of the American Chemical Society, ISSN 0002-7863, E-ISSN 1520-5126, Vol. 132, no 20, 7038-7042 p.Article in journal (Refereed) Published
Abstract [en]

A variant of Candida antarctica lipase A (CalA) was developed for the hydrolysis of α-substituted p-nitrophenyl esters by directed evolution. The E values of this variant for 7 different esters was 45−276, which is a large improvement compared to 2−20 for the wild type. The broad substrate scope of this enzyme variant is of synthetic use, and hydrolysis of the tested substrates proceeded with an enantiomeric excess between 95−99%. A 30-fold increase in activity was also observed for most substrates. The developed enzyme variant shows (R)-selectivity, which is reversed compared to the wild type that is (S)-selective for most substrates.

National Category
Organic Chemistry
Identifiers
urn:nbn:se:su:diva-45970 (URN)10.1021/ja100593j (DOI)
Funder
Swedish Research CouncilKnut and Alice Wallenberg Foundation
Available from: 2010-11-17 Created: 2010-11-17 Last updated: 2012-04-02Bibliographically approved
5. Highly Combinatorial Reshaping of the Candida antarctica lipase A Substrate Pocket Using an Extremely Condensed Library
Open this publication in new window or tab >>Highly Combinatorial Reshaping of the Candida antarctica lipase A Substrate Pocket Using an Extremely Condensed Library
(English)Manuscript (preprint) (Other academic)
Abstract [en]

A highly combinatorial structure based protein engineering method is demonstrated resulting in a thorough modification of the binding pocket of Candida antarctica lipase A (CALA). Nine amino acid sites surrounding the entire pocket were simultaneously mutated, contributing to a sculpting of the substrate pocket toward a sterically demanding substrate, an ibuprofen ester. The best variant was highly active and displayed remarkable increase in enantioselectivity toward the substrate, with an E-value of 101, compared to the wild type CALA that poor activity and possesses an E-value of 3.4. The potential mutations introduced were a highly reduced set of amino acids, containing only the wild type residue and an alternative residue, preferably a smaller one with similar properties. These minimal ‘binary’ sets allow for extremely condensed protein libraries. The choice of amino acid sites were based on a computer model, with the substrate forcibly bound in the active site. This highly combinatorial method can be used to obtain tailor-made enzymes that are active toward substrates that are not normally accepted by the enzyme. When multiple sites are altered simultaneously, there is a higher possibility of obtaining positive synergistic effects, and the protein fitness landscape is explored efficiently.

Keyword
lipase, protein engineering, directed evolution, kinetic resolution
National Category
Biocatalysis and Enzyme Technology
Research subject
Organic Chemistry
Identifiers
urn:nbn:se:su:diva-49247 (URN)
Available from: 2010-12-14 Created: 2010-12-13 Last updated: 2010-12-15Bibliographically approved

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