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Engineering of Candida antarctica Lipase A and B
Stockholm University, Faculty of Science, Department of Organic Chemistry.
2019 (English)Licentiate thesis, comprehensive summary (Other academic)
Abstract [en]

In organic chemistry, it is often desirable to have an efficient process yieldingpure product and not too much waste. When applicable, it is also desirable tohave a high enantioselective outcome. Enzymes are often displaying high regio-,chemo- and stereoselectivity. Furthermore, they are biodegradable, and theywork under mild conditions regarding temperature and solvent. Using enzymesas catalysts in organic synthesis is therefore an attractive approach.

Enzymes have evolved over thousands of years to catalyze specific reactionsand they therefore often have a quite specific substrate scope. It is not alwayspossible to find the exact match between the desired reaction and an enzyme.Furthermore, reaction conditions such as high temperatures, the need of organicsolvents, or the substrate might be non-compatible with the enzyme. Directedevolution and protein engineering have been developed as methods forintroducing changes in already existing enzymes. By changing the genotype ofan enzyme, new enzyme variants can be obtained that are able to perform newreactions, tolerate new reaction conditions, and accepting a broader scope ofsubstrates. Immobilization of enzymes is also a strategy that has proven toincrease the stability and performance of an enzyme, allowing the use of highertemperatures and organic solvents.

In this thesis, two lipases from Candida antarctica, denoted Lipase A (Cal A)and Lipase B (Cal B), have been studied. For both lipases, protein engineeringhas been used in order to improve the enzymes regarding activity andenantioselectivity for applications in organic synthesis.

In the first part, a semi-rational protein engineering approach was used to createa highly condensed combinatorial library of Cal A variants for resolution ofchiral tertiary alcohols. Tertiary alcohols are sterically demanding substrates,and only a few reports of enzymes being active towards them are known.Exploring new enzyme variants that show activity towards tertiary alcohols istherefore of importance. To improve wild-type Cal A, seven positions in theactive site of Cal A were chosen and simultaneously altered. In the library, avariant with one single mutation was found to have promising activity. Thisvariant showed retained enantioselectivity and a 10-fold increase in activity(rate) towards the target tertiary alcohol.

The second part consists of a study aiming at reversing the enantioselectivity ofCal B towards secondary alcohols with long alkyl chains. Also here, acondensed and highly combinatorial library was created where a cavity was 

constructed in the enzyme active site. This project is still ongoing, with the aimof finding a high performing Cal B variant.

Place, publisher, year, edition, pages
Stockholm: Stockholms universitets förlag, 2019. , p. 60
National Category
Chemical Sciences
Identifiers
URN: urn:nbn:se:su:diva-165595OAI: oai:DiVA.org:su-165595DiVA, id: diva2:1284867
Presentation
2019-03-01, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 C, Stockholm, 10:00 (English)
Funder
Knut and Alice Wallenberg FoundationAvailable from: 2019-02-04 Created: 2019-02-01 Last updated: 2019-02-04Bibliographically approved

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