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Combinatorial reshaping of the Candida antarctica lipase A substrate pocket for enantioselectivity using an extremely condensed library
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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2012 (English)In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 109, no 1, 78-83 p.Article in journal (Refereed) Published
Abstract [en]

A highly combinatorial structure-based protein engineering method for obtaining enantioselectivity is reported that results in a thorough modification of the substrate binding pocket of Candida antarctica lipase A (CALA). Nine amino acid residues surrounding the entire pocket were simultaneously mutated, contributing to a reshaping of the substrate pocket to give increased enantioselectivity and activity for a sterically demanding substrate. This approach seems to be powerful for developing enantioselectivity when a complete reshaping of the active site is required. Screening toward ibuprofen ester 1, a substrate for which previously used methods had failed, gave variants with a significantly increased enantioselectivity and activity. Wild-type CALA has a moderate activity with an E value of only 3.4 toward this substrate. The best variant had an E value of 100 and it also displayed a high activity. The variation at each mutated position was highly reduced, comprising only the wild type and an alternative residue, preferably a smaller one with similar properties. These minimal binary variations allow for an extremely condensed protein library. With this highly combinatorial method synergistic effects are accounted for and the protein fitness landscape is explored efficiently.

Place, publisher, year, edition, pages
2012. Vol. 109, no 1, 78-83 p.
Keyword [en]
kinetic resolution, library design, protein design, enzyme catalysis
National Category
Organic Chemistry
Research subject
Organic Chemistry
Identifiers
URN: urn:nbn:se:su:diva-74997DOI: 10.1073/pnas.1111537108ISI: 000298876500022OAI: oai:DiVA.org:su-74997DiVA: diva2:513486
Funder
Swedish Research Council, 621-2010-4737Knut and Alice Wallenberg FoundationSwedish Foundation for Strategic Research
Available from: 2012-04-02 Created: 2012-04-02 Last updated: 2017-12-07Bibliographically approved
In thesis
1. 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
2. Engineering Candida antarctica Lipase A for Enantioselective Transformations in Organic Synthesis: Design, Immobilization and Organic Solvent Screening of Smart Enzyme Libraries
Open this publication in new window or tab >>Engineering Candida antarctica Lipase A for Enantioselective Transformations in Organic Synthesis: Design, Immobilization and Organic Solvent Screening of Smart Enzyme Libraries
2015 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The use of enzymes as catalysts in organic synthesis constitutes an attractive alternative to conventional chemical catalysis. Enzymes are non-toxic and biodegradable and they can operate under mild reaction conditions. Furthermore, they often display high chemo-, regio- and stereoselectivity, enabling specific reactions with single product outcome.

By the use of protein engineering, enzymes can be altered for the specific needs of the researcher. The major part of this thesis describes engineering of lipase A from Candida antarctica (CalA), for improved enantioselectivity in organic synthetic transformations.

The first part of the thesis describes a highly combinatorial method for the introduction of mutation sites in an enzyme library. By the simultaneous introduction of nine mutations, we found an enzyme variant with five out of the nine possible mutations. This quintuple variant had an enlarged active site pocket and was enantioselective and active for our model substrate, an ibuprofen ester. This is a bulky substrate for which the wild-type enzyme shows no enantioselectivity and very poor activity.

In the second part of the thesis, we continued our approach of combinatorial, focused enzyme libraries. This time we aimed at decreasing the alcohol pocket of CalA, in order to increase the enantioselectivity for small and medium-sized secondary alcohols. The enzyme library was bound on microtiter plates and screened by a transacylation reaction in organic solvent. This library yielded an enzyme variant with high enantioselectivity for the model substrate 1-phenyl ethanol, and high to excellent selectivity for other alcohols tested. Screening in organic solvent is advantageous since a potential hit is more synthetically useful.

In the third part of the thesis, we used manipulated beads of controlled porosity glass (EziG™) for enzyme immobilization, and demonstrated the generality of this carrier for several enzyme classes. EziG™ allowed fast enzyme immobilization with simultaneous purification and yielded active biocatalysts in all cases.

The last project describes the function of the proposed active site flap in CalA. In our study, we removed this motif. The engineered variant was compared to the wild-type enzyme by testing the amount of interfacial activation and the selectivity for certain alcohols. We showed that the motif is indeed controlling the entrance to the active site and that the flap is not part of the enantioselectivity determining machinery. 

Place, publisher, year, edition, pages
Stockholm: Department of Organic Chemistry, Stockholm University, 2015. 59 p.
Keyword
Candida antarctica Lipase A, protein engineering, enzyme libraries, enzyme immobilization, biocatalysis
National Category
Organic Chemistry
Research subject
Organic Chemistry
Identifiers
urn:nbn:se:su:diva-116170 (URN)978-91-7649-168-3 (ISBN)
Public defence
2015-05-28, 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 4: Manuscript.

Available from: 2015-05-06 Created: 2015-04-12 Last updated: 2017-10-11Bibliographically approved

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Sandström, Anders G.Wikmark, YlvaEngström, KarinBäckvall, Jan-E.
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