Highly Combinatorial Reshaping of the Candida antarctica lipase A Substrate Pocket Using an Extremely Condensed Library
(English)Manuscript (preprint) (Other academic)
A highly combinatorial structure based protein engineering method is demonstrated resulting in a thorough modification of the binding pocket of Candida antarctica lipase A (CALA). Nine amino acid sites surrounding the entire pocket were simultaneously mutated, contributing to a sculpting of the substrate pocket toward a sterically demanding substrate, an ibuprofen ester. The best variant was highly active and displayed remarkable increase in enantioselectivity toward the substrate, with an E-value of 101, compared to the wild type CALA that poor activity and possesses an E-value of 3.4. The potential mutations introduced were a highly reduced set of amino acids, containing only the wild type residue and an alternative residue, preferably a smaller one with similar properties. These minimal ‘binary’ sets allow for extremely condensed protein libraries. The choice of amino acid sites were based on a computer model, with the substrate forcibly bound in the active site. This highly combinatorial method can be used to obtain tailor-made enzymes that are active toward substrates that are not normally accepted by the enzyme. When multiple sites are altered simultaneously, there is a higher possibility of obtaining positive synergistic effects, and the protein fitness landscape is explored efficiently.
lipase, protein engineering, directed evolution, kinetic resolution
Biocatalysis and Enzyme Technology
Research subject Organic Chemistry
IdentifiersURN: urn:nbn:se:su:diva-49247OAI: oai:DiVA.org:su-49247DiVA: diva2:376887