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Apparent kinetic constants kcat and Km were determined for tyrocidine thioesterase (TycC TE) using randomized peptide N-acetylcysteamine thioesters as substrate analogues. The enzyme has been found to be adequately active for the synthesis of positional-scanning libraries for novel antibiotic screening with reduced kcat/Km in the range of 2 to 82 folds lower than that of the wild-type sequence