| Autor: |
Sandström AG; Department of Organic Chemistry, Arrhenius Laboratory, Stockholm University, SE-106 91 Stockholm, Sweden., Wikmark Y, Engström K, Nyhlén J, Bäckvall JE |
| Jazyk: |
angličtina |
| Zdroj: |
Proceedings of the National Academy of Sciences of the United States of America [Proc Natl Acad Sci U S A] 2012 Jan 03; Vol. 109 (1), pp. 78-83. Date of Electronic Publication: 2011 Dec 16. |
| DOI: |
10.1073/pnas.1111537108 |
| Abstrakt: |
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. |
| Databáze: |
MEDLINE |
| Externí odkaz: |
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