ABSTRACT
During recent years, structure-based protein design and directed evolution have been widely applied to engineer enzyme activity, specificity, or stability (1). Methodologies such as gene shuffling (2) and combinatorial mutagenesis (3) made it possible to generate diverse molecular repertoires of enzyme variants that were successfully screened for the desired improvements. Screening of large mutant libraries in the range of 104 to 107 different variants is a crucial step in the process and often becomes the limiting factor (4, 5). In cases where the desired enzyme function can be coupled to microbial growth or survival, selection may be applicable. Otherwise, single bacterial cells are clonally expanded and each population is individually tested for the desired activity. Commonly, the bacterial cells are compartmentalized, e.g., by transfer into microtiter plates, followed by cell lysis and testing the lysate for the desired novel or improved function.
