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)
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.
Candida antarctica Lipase A, protein engineering, enzyme libraries, enzyme immobilization, biocatalysis
Research subject Organic Chemistry
IdentifiersURN: urn:nbn:se:su:diva-116170ISBN: 978-91-7649-168-3OAI: oai:DiVA.org:su-116170DiVA: diva2:807420
2015-05-28, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, Stockholm, 10:00 (English)
Kazlauskas, Romas, Professor
Bäckvall, Jan-Erling, Professor
At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 4: Manuscript.2015-05-062015-04-122015-05-07Bibliographically approved
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