Iterative saturation mutagenesis (ISM) for rapid directed evolution of functional enzymes

Iterative saturation mutagenesis ( ISM) is a new and efficient method for the directed evolution of functional enzymes. It reduces the necessary molecular biological work and the screening effort drastically. It is based on a Cartesian view of the protein structure, performing iterative cycles of saturation mutagenesis at rationally chosen sites in an enzyme, a given site being composed of one, two or three amino acid positions. The basis for choosing these sites depends on the nature of the catalytic property to be improved, e. g., enantioselectivity, substrate acceptance or thermostability. In the case of thermostability, sites showing highest B-factors ( available from X-ray data) are chosen. The pronounced increase in thermostability of the lipase from Bacillus subtilis ( Lip A) as a result of applying ISM is illustrated here.