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Directed evolution of an enantioselective lipase with broad substrate scope for hydrolysis of α-substituted esters
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.
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.

Place, publisher, year, edition, pages
2010. Vol. 132, no 20, 7038-7042 p.
National Category
Organic Chemistry
Identifiers
URN: urn:nbn:se:su:diva-45970DOI: 10.1021/ja100593jOAI: oai:DiVA.org:su-45970DiVA: diva2:370584
Funder
Swedish Research CouncilKnut and Alice Wallenberg Foundation
Available from: 2010-11-17 Created: 2010-11-17 Last updated: 2017-12-12Bibliographically 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
2. Enantioselective biotransformations using engineered lipases from Candida antarctica
Open this publication in new window or tab >>Enantioselective biotransformations using engineered lipases from Candida antarctica
2012 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Enzymes are attractive catalysts in organic synthesis since they are efficient, selective and environmentally friendly. A large number of enzyme-catalyzed transformations have been described in the literature. If no natural enzyme can carry out a desirable reaction, one possibility is to modify an existing enzyme by protein engineering and thereby obtain a catalyst with the desired properties. In this thesis, the development of enantioselective enzymes and their use in synthetic applications is described. 

In the first part of this thesis, enantioselective variants of Candida antarctica lipase A (CALA) towards α-substituted p-nitrophenyl esters were developed by directed evolution. A highly selective variant of CALA towards p-nitrophenyl 2-phenylpropanoate was developed by pairwise randomization of amino acid residues close to the active site. The E value of this variant was 276 compared to 3 for the wild type.

An approach where nine residues were altered simultaneously was used to discover another highly enantioselective CALA variant (E = 100) towards an ibuprofen ester. The sterical demands of this substrate made it necessary to vary several residues at the same time in order to reach a variant with improved properties.

In the second part of the thesis, a designed variant of Candida antarctica lipase B (CALB) was employed in kinetic resolution (KR) and dynamic kinetic resolution (DKR) of secondary alcohols. The designed CALB variant (W104A) accepts larger substrates compared to the wild type, and by the application of CALB W104A, the scope of these resolutions was extended.

First, a DKR of phenylalkanols was developed using CALB W104A. An enzymatic resolution was combined with in situ racemization of the substrate, to yield the products in up to 97% ee. Secondly, the KR of diarylmethanols with CALB W104A was developed. By the use of diarylmethanols with two different aryl groups, highly enantioselective transformations were achieved.

Place, publisher, year, edition, pages
Stockholm: Department of Organic Chemistry, Stockholm University, 2012. 55 p.
Keyword
protein engineering, directed evolution, kinetic resolution, dynamic kinetic resolution, biotransformation, lipase
National Category
Organic Chemistry
Research subject
Organic Chemistry
Identifiers
urn:nbn:se:su:diva-75000 (URN)978-91-7447-468-8 (ISBN)
Public defence
2012-05-11, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, Stockholm, 10:00 (English)
Opponent
Supervisors
Note

At the time of the doctoral defense, the following paper was unpublished and had a status as follows:  Paper 5: Submitted.

Available from: 2012-04-19 Created: 2012-04-02 Last updated: 2013-01-14Bibliographically approved

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