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Improved Enantioselectivity of Subtilisin Carlsberg Towards Secondary Alcohols by Protein Engineering
Stockholm University, Faculty of Science, Department of Organic Chemistry.
2018 (English)In: ChemBioChem (Print), ISSN 1439-4227, E-ISSN 1439-7633, Vol. 19, no 4, p. 338-346Article in journal (Refereed) Published
Abstract [en]

Generally, the catalytic activity of subtilisin Carlsberg (SC) for transacylation reactions with secondary alcohols in organic solvent is low. Enzyme immobilization and protein engineering was performed to improve the enantioselectivity of SC towards secondary alcohols. Possible amino-acid residues for mutagenesis were found by combining available literature data with molecular modeling. SC variants were created by site-directed mutagenesis and were evaluated for a model transacylation reaction containing 1-phenylethanol in THF. Variants showing high E values (>100) were found. However, the conversions were still low. A second mutation was made, and both the E values and conversions were increased. Relative to that shown by the wild type, the most successful variant, G165L/M221F, showed increased conversion (up to 36 %), enantioselectivity (E values up to 400), substrate scope, and stability in THF.

Place, publisher, year, edition, pages
2018. Vol. 19, no 4, p. 338-346
Keywords [en]
biocatalysis, enzymes, immobilization, kinetic resolution, molecular modeling, transacylation
National Category
Organic Chemistry
Research subject
Organic Chemistry
Identifiers
URN: urn:nbn:se:su:diva-153920DOI: 10.1002/cbic.201700408ISI: 000425511500006OAI: oai:DiVA.org:su-153920DiVA, id: diva2:1188569
Available from: 2018-03-07 Created: 2018-03-07 Last updated: 2018-03-20Bibliographically approved
In thesis
1. Heterogeneous catalysis in racemization and kinetic resolution along a journey in protein engineering
Open this publication in new window or tab >>Heterogeneous catalysis in racemization and kinetic resolution along a journey in protein engineering
2018 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The first part of my thesis concerns the use of heterogeneous acidic resins for racemization of tert-alcohols without any side-product formation. The focus was to develop a system which can be further extended to a DKR protocol consisting of an enzymatic KR reaction. Based on our knowledge of the resins, an unexpected migratory DKR protocol turned out to be an efficient method for the synthesis of carbocyclic allylic carbinols.

The development of enzyme and metal catalyst hybrids was already an ongoing theme in our group. A supporter-free biohybrid catalyst was developed which can be used in several different types of reactions. The Pd(0)-CalB CLEA catalyst was applied in a two-step-cascade transformation and in the DKR of benzylic primary amines. The catalyst was characterized by different analytical techniques, to understand its composition and structure.

The enzymes have always been the main focus of the studies and therefore wild type enzymes were initially utilized. However, these natural biocatalysts are associated with certain limitations. In contrast, protein engineering allows for enzymes to be modified and optimized. We have used the technique to create a subtilisin Carlsberg mutant, which was studied both by modeling and in vitro. The mutant was found to catalyze the (S)-selective transesterification of sec-alcohols containing long aliphatic carbon chains, and it also exhibited higher performance in organic solvent.

The last project concerned the protein engineering of CalA enzyme towards tert-alcohols. The kinetic resolution of tert-alcohols with this enzyme is very slow but it occurs with good enantioselectivity. The aim was therefore to improve the activity of CalA via protein engineering. Seven amino acids were mutated close to the active site and a library was created based on our prediction. Throughout the screening, a few variants showed higher activity, which were sequenced and further analyzed in the transesterification of tert-alcohols.

Place, publisher, year, edition, pages
Stockholm: Department of Organic Chemistry, Stockholm University, 2018. p. 81
Keywords
racemization, protein engineering, kinetic resolution, heterogeneous catalysis, biohybrid catalyst
National Category
Organic Chemistry
Research subject
Organic Chemistry
Identifiers
urn:nbn:se:su:diva-153921 (URN)978-91-7797-185-6 (ISBN)978-91-7797-186-3 (ISBN)
Public defence
2018-04-26, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, Stockholm, 10:00 (English)
Opponent
Supervisors
Note

At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 4: Manuscript. Paper 6: Manuscript.

Available from: 2018-04-03 Created: 2018-03-08 Last updated: 2018-03-23Bibliographically approved

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