Anna Joëlle Ruff

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Protein re-engineering by directed evolution has become a standard approach for tailoring enzymes in many fields of science and industry. Advances in screening formats and screening systems are fueling progress and enabling novel directed evolution strategies, despite the fact that the quality of mutant libraries can still be improved significantly.(More)
Mutational events as well as the selection of the optimal variant are essential steps in the evolution of living organisms. The same principle is used in laboratory to extend the natural biodiversity to obtain better catalysts for applications in biomanufacturing or for improved biopharmaceuticals. Furthermore, single mutation in genes of drug-metabolizing(More)
Multi-site-saturation mutagenesis allows altering of "localizable" properties such as activity and selectivity and enables the discovery of cooperative amino acid substitutions which are unlikely to be discovered by saturating single codons individually or iteratively. The herein presented method "OmniChange" does not require any DNA modifying enzyme (e.g.,(More)
During the last decade, directed evolution has become a standard protein engineering strategy to reengineer proteins for industrial applications under high stress conditions (e.g., high temperature, extreme pH, ionic liquids, or organic solvents). The most commonly employed method for diversity generation to improve biocatalysts for these properties is(More)
Fusion protein construction is a widely employed biochemical technique, especially when it comes to multi-component enzymes such as cytochrome P450s. Here we describe a novel method for generating fusion proteins with variable linker lengths, protein fusion with variable linker insertion (P-LinK), which was validated by fusing P450cin monooxygenase (CinA)(More)
Escherichia coli is a common host for recombinant protein production in which production titers are highly dependent on the employed expression system. Promoters are thereby a key element to control gene expression levels. In this study, a novel PLICable promoter toolbox was developed which enables in a single cloning step and after a screening experiment(More)
Sequence Saturation Mutagenesis (SeSaM) is a random mutagenesis method developed to overcome the limitations of existing error-prone PCR (epPCR) protocols. SeSaM is advantageous with respect to (1) elimination of mutagenic "hot spots", (2) increase in frequency of subsequent nucleotide substitutions, (3) control over the mutational bias through the(More)
Zinc-dependent medium chain reductase from Candida parapsilosis can be used in the reduction of carbonyl compounds to pharmacologically important chiral secondary alcohols. To date, the nomenclature of cpADH5 is differing (CPCR2/RCR/SADH) in the literature, and its natural substrate is not known. In this study, we utilized a substrate docking based virtual(More)
A novel whole cell cascade for double oxidation of cyclooctane to cyclooctanone was developed. The one-pot oxidation cascade requires only a minimum of reaction components: resting E. coli cells in aqueous buffered medium (=catalyst), the target substrate and oxygen as environmental friendly oxidant. Conversion of cyclooctane was catalysed with high(More)
Simultaneous multi site-saturation mutagenesis enables to reshape binding pockets especially when cooperative amino acids are targeted, which affect activity and/or selectivity of enzymes. Simultaneous saturation of five positions with OmniChange generates up to 3.2 million different variants in one experiment in a robust and technically simple protocol.(More)