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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. F126I had a stronger influence on chain-length specificity, so the further amino acids which form the “bottleneck” to the active site were mutated to further widen the entrance, and mutants with improved activity were found. The bottleneck mutants which consist of single, double, triple and quadruple mutants which are mostly combinations of F126L, F144L, F159L and I225L were then assayed for altered enantioselectivity against chiral acids and secondary alcohols. For substrates 1-phenyl-1-propyl acetate (2), 1-phenyl-2-propyl acetate (3) and 1-phenyl ethyl acetate (4), mutants with increased enantioselectivity were found. I225L plays a crucial role, as it is vital for enantioselectivity against 3, but destroys selectivity against 2, both facts obvious from the comparison of the triple mutant without I225L (mutant T3) and the corresponding quadruple mutant including I225L (mutant Q). However, the single mutant I225L alone does not possess high selectivity against 3, so synergistic effects play an important role. The PFE I wild type already possesses a good enantioselectivity in the hydrolysis of 4, but all mutants which were analyzed in detail surpass the wild type. The program YASARA was then used to calculate docking solutions for both enantiomers of 2 and 3 into the wild type and the best mutant. The results revealed that the mutants’ widened bottleneck allows the phenyl moiety of the substrates to point towards the access tunnel, while only (R)-2 does so in the wild type. Residues 126 and 144 do not come very close to the substrate and are more likely to influence substrate diffusion. Another goal was to find a way to confer promiscuous amidase activity upon the PFE I. In the search for structural homologues, a close structural neighbour with amidase activity was found. The --lactamase from Aureobacterium sp. was named after its activity toward the Vince lactam 2-azabicyclo[2.2.1]hept-5-en-3-one. Biocatalysis experiments with the PFE I and its mutants revealed an excellent enantioselectivity against the ( )-lactam. Specific activities were determined for purified proteins, and the activity of some mutants was within the same order of magnitude as lactamase’s activity.