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  • 1.
    Sandström, Anders G.
    Stockholm University, Faculty of Science, Department of Organic Chemistry.
    Highly Combinatorial Reshaping of the Candida antarctica lipase A Substrate Pocket Using an Extremely Condensed LibraryManuscript (preprint) (Other academic)
    Abstract [en]

    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.

  • 2.
    Sandström, Anders G.
    Stockholm University, Faculty of Science, Department of Organic Chemistry.
    Protein Engineering of Candida antarctica Lipase A: Enhancing Enzyme Properties by Evolutionary and Semi-Rational Methods2010Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Enzymes are gaining increasing importance as catalysts for selective transformations in organic synthetic chemistry. The engineering and design of enzymes is a developing, growing research field that is employed in biocatalysis. In the present thesis, combinatorial protein engineering methods are applied for the development of Candida antarctica lipase A (CALA) variants with broader substrate scope and increased enantioselectivity. Initially, the structure of CALA was deduced by manual modelling and later the structure was established by X-ray crystallography. The elucidation of the structure of CALA revealed several biocatalytically interesting features. With the knowledge derived from the enzyme structure, enzyme variants were produced via iterative saturation mutagenesis (ISM), a powerful protein engineering approach. Several of these variants were highly active and enantioselective towards bulky esters. Furthermore, an extensively combinatorial protein engineering approach was developed and investigated. A CALA variant with a spacious substrate binding pocket that can accommodate an unusually bulky substrate, an ester derivate of the non-steroidal anti-inflammatory drug (S)-ibuprofen, was obtained with this approach.

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