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Institute: search.filters.UBSInstitut.Institut für Technische BiochemieInstitute: search.filters.UBSInstitut.Sonstige EinrichtungAuthor: search.filters.author.Schmid, Rolf D.Author: search.filters.author.Schmitt, Jutta

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    Directed evolution of a bacterial alpha-amylase : towards enhanced pH-performance and higher specific activity
    (2003) Bessler, Cornelius; Schmitt, Jutta; Maurer, Karl-Heinz; Schmid, Rolf D.
    Alpha-Amylases, in particular, microbial Alpha-amylases are used widely in industrial processes such as starch liquefaction and pulp processes and more recently in detergency. Following the need for Alpha-amylases adapted to latter, we enhanced the alkali-activity of the Alpha-amylase from Bacillus amyloliquefaciens (BAA). The genes coding for the wild type BAA and the mutants BAA S201N and BAA N297D were subjected to error prone PCR and gene shuffling. For the screening of mutants we developed a novel, reliable assay suitable for high throughput screening based on the Phadebas® assay. One mutant (BAA 42) has an optimal activity at pH 7, corresponding to a shift of one pH unit compared to the wild type. BAA 42 is active over a broader pH-range than the wild type resulting in a fivefold higher activity at pH 10. In addition, the activity in periplasmic extracts and the specific activity increased 4 and 1.5 fold, respectively. Another mutant (BAA 29) possesses a wild type like pH-profile but reveals a 40-fold higher activity in periplasmic extracts and a nine fold higher specific activity. The comparison of the amino acid sequences of these two mutants with other homologous microbial Alpha-amylases revealed the mutation of the highly conserved residues W194R, S197P and A230V. In addition, three further mutations were found K406R, N414S and E356D, the latter being present in other bacterial Alpha-amylases.
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    Blocking the tunnel: engineering of Candida rugosa lipase mutants with short chain length specificity
    (2002) Schmitt, Jutta; Brocca, Stefania; Schmid, Rolf D.; Pleiss, Jürgen
    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.