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Engineering Candida antarctica Lipase A for Enantioselective Transformations in Organic Synthesis: Design, Immobilization and Organic Solvent Screening of Smart Enzyme Libraries
Stockholm University, Faculty of Science, Department of Organic Chemistry.
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 [en]
Candida antarctica Lipase A, protein engineering, enzyme libraries, enzyme immobilization, biocatalysis
National Category
Organic Chemistry
Research subject
Organic Chemistry
Identifiers
URN: urn:nbn:se:su:diva-116170ISBN: 978-91-7649-168-3 (print)OAI: oai:DiVA.org:su-116170DiVA: diva2:807420
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
List of papers
1. Combinatorial reshaping of the Candida antarctica lipase A substrate pocket for enantioselectivity using an extremely condensed library
Open this publication in new window or tab >>Combinatorial reshaping of the Candida antarctica lipase A substrate pocket for enantioselectivity using an extremely condensed library
Show others...
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.

Keyword
kinetic resolution, library design, protein design, enzyme catalysis
National Category
Organic Chemistry
Research subject
Organic Chemistry
Identifiers
urn:nbn:se:su:diva-74997 (URN)10.1073/pnas.1111537108 (DOI)000298876500022 ()
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
2. Combinatorial Library Based Engineering of Candida antarctica Lipase A for Enantioselective Transacylation of sec-Alcohols in Organic Solvent
Open this publication in new window or tab >>Combinatorial Library Based Engineering of Candida antarctica Lipase A for Enantioselective Transacylation of sec-Alcohols in Organic Solvent
2015 (English)In: Angewandte Chemie International Edition, ISSN 1433-7851, E-ISSN 1521-3773, Vol. 54, no 14, 4284-4288 p.Article in journal (Refereed) Published
Abstract [en]

A method for determining lipase enantioselectivity in the transacylation of sec-alcohols in organic solvent was developed. The method was applied to a model library of Candida antarctica lipase A (CalA) variants for improved enantioselectivity (E values) in the kinetic resolution of 1-phenylethanol in isooctane. A focused combinatorial gene library simultaneously targeting seven positions in the enzyme active site was designed. Enzyme variants were immobilized on nickel-coated 96-well microtiter plates through a histidine tag (His6 -tag), screened for transacylation of 1-phenylethanol in isooctane, and analyzed by GC. The highest enantioselectivity was shown by the double mutant Y93L/L367I. This enzyme variant gave an E value of 100 (R), which is a dramatic improvement on the wild-type CalA (E=3). This variant also showed high to excellent enantioselectivity for other secondary alcohols tested.

Keyword
biocatalysis, kinetic resolution, lipase A, protein engineering, secondary alcohols
National Category
Organic Chemistry
Research subject
Organic Chemistry
Identifiers
urn:nbn:se:su:diva-116165 (URN)10.1002/anie.201410675 (DOI)000351679600024 ()25676632 (PubMedID)
Funder
Swedish Research Council
Available from: 2015-04-12 Created: 2015-04-12 Last updated: 2017-12-04Bibliographically approved
3. A general protein purification and immobilization method on controlled porosity glass: biocatalytic applications
Open this publication in new window or tab >>A general protein purification and immobilization method on controlled porosity glass: biocatalytic applications
Show others...
2014 (English)In: Chemical Communications, ISSN 1359-7345, E-ISSN 1364-548X, Vol. 50, no 65, 9134-9137 p.Article in journal (Refereed) Published
Abstract [en]

A general combined purification and immobilization method to facilitate biocatalytic process development is presented. The support material, EziG (TM), is based on controlled porosity glass (CPG) or polymer-coated versions thereof (HybCPG) and binds protein affinity tags. Biocatalytic reactions in aqueous and organic media with seven enzymes of biocatalytic interest are shown.

Place, publisher, year, edition, pages
Royal Society of Chemistry, 2014
National Category
Chemical Sciences Organic Chemistry
Research subject
Organic Chemistry
Identifiers
urn:nbn:se:su:diva-107099 (URN)10.1039/c4cc02605e (DOI)000339930400026 ()
Funder
Swedish Research CouncilKnut and Alice Wallenberg Foundation
Note

AuthorCount:7;

Available from: 2014-09-05 Created: 2014-09-03 Last updated: 2017-12-05Bibliographically approved
4. Removing the Active-Site Flap in Lipase A from Candida antarctica Produces a Functional Enzyme without Interfacial Activation
Open this publication in new window or tab >>Removing the Active-Site Flap in Lipase A from Candida antarctica Produces a Functional Enzyme without Interfacial Activation
2016 (English)In: ChemBioChem (Print), ISSN 1439-4227, E-ISSN 1439-7633, Vol. 17, no 2, 141-145 p.Article in journal (Refereed) Published
Abstract [en]

A mobile region is proposed to be a flap that covers the active site of Candida antarctica lipase A. Removal of the mobile region retains the functional properties of the enzyme. Interestingly interfacial activation, required for the wild-type enzyme, was not observed for the truncated variant, although stability, activity, and stereoselectivity were very similar for the wild-type and variant enzymes. The variant followed classical Michaelis-Menten kinetics, unlike the wild type. Both gave the same relative specificity in the transacylation of a primary and a secondary alcohol in organic solvent. Furthermore, both showed the same enantioselectivity in transacylation of alcohols and the hydrolysis of alcohol esters, as well as in the hydrolysis of esters chiral at the acid part.

Keyword
Candida antarctia lipase A, enzyme catalysis, flap, interfacial activation, lid, protein engineering
National Category
Organic Chemistry
Research subject
Organic Chemistry
Identifiers
urn:nbn:se:su:diva-127884 (URN)10.1002/cbic.201500471 (DOI)000369963500005 ()26543016 (PubMedID)
Available from: 2016-06-20 Created: 2016-03-14 Last updated: 2017-10-11Bibliographically approved

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