Protein engineering of a Pseudomonas fluorescens esterase : alteration of substrate specificity and stereoselectivity The aim of this thesis was to validate a method called OSCARR for One pot Simple Cassette Randomization and Recombination for focused directed evolution which had been developed by Dr. Hidalgo. It is based upon the megaprimer PCR method using outer primers differing in TM and including asymmetric cycles before the addition of the forward primer to generate more mutated megaprimer. As mutation carrying primers spiked oligonucleotides are employed. These spiked oligonucleotides are designed using an algorithm and have strictly defined composition of nucleotides at each position. An OSCARR library of the Pseudomonas fluorescens esterase I (PFE I) of approximately 8000 clones was generated and screened for altered chain length selectivity. Two mutants with higher activity towards medium chain length p nitrophenyl esters were identified both carried the mutation F126I which causes the substrate entrance tunnel to be widened thus facilitating access of bulkier substrates to the active site. One mutant carried the additional mutation G120S which completes a catalytic tetrad which is observed mainly in proteases... Gerichtete Evolution Arylesterase Substratspezifität Enantioselektivität Proteindesign gamma Lactamase