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)
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)
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)
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)
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)
The quality of amino acid substitution patterns in random mutagenesis libraries is decisive for the success in directed evolution campaigns. In this manuscript, we provide a detailed analysis of the amino acid substitutions by analyzing 3000 mutations of three random mutagenesis libraries (1000 mutations each; epPCR with a low-mutation and a high-mutation(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)
Advances in directed monooxygenase evolution: From diversity generation and flow cytometry screening to tailor-made monooxygenases Directed Evolution became a powerful tool for proteins engineers to generate tailor-made biocatalyst. Directed protein evolution consist of the following three consecutive main steps, which are performed in iterative cycles;(More)
The aromatic hydroxylation of pseudocumene (1a) and mesitylene (1b) with P450 BM3 yields key phenolic building blocks for α-tocopherol synthesis. The P450 BM3 wild-type (WT) catalyzed selective aromatic hydroxylation of 1b (94%), whereas 1a was hydroxylated to a large extent on benzylic positions (46-64%). Site-saturation mutagenesis generated a new P450(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)