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Autor(en): Schmitt, Jutta
Brocca, Stefania
Schmid, Rolf D.
Pleiss, Jürgen
Titel: Blocking the tunnel: engineering of Candida rugosa lipase mutants with short chain length specificity
Erscheinungsdatum: 2002
Dokumentart: Preprint
Erschienen in: Protein engineering, design, and selection 15 (2002), S. 595-601. URL
Zusammenfassung: The molecular basis of chain length specificity of Candida rugosa lipase 1 was investigated by molecular modelling and site-directed mutagenesis. The synthetic lip1 gene and the lipase mutants were expressed in Pichia pastoris and assayed for their chain length specificity in single substrate assays using triglycerides as well as in a competitive substrate assay using a randomized oil. Mutation of amino acids at different locations inside the tunnel (P246F, L413F, L410W, L410F/S300E, L410F/S365L) resulted in mutants with a different chain length specificity. Mutants P246F and L413F have a strong preference for short chain lengths whereas substrates longer than C10 are hardly hydrolyzed. Increasing the bulkiness of the amino acid at position 410 led to mutants that show a strong discrimination of chain lengths longer than C14. The results obtained can be explained by a simple mechanical model: the activity for a fatty acid sharply decreases as it becomes long enough to reach the mutated site. In contrast, a mutation at the entrance of the tunnel (L304F) has a strong impact on C4 and C6 substrates. This mutant is nevertheless capable to hydrolyze chain lengths longer than C8.
Enthalten in den Sammlungen:03 Fakultät Chemie

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