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.

Efficiency counts: A new method of directed evolution for increasing the thermostability of proteins saves time and effort. In an illustrative study, iterative cycles of saturation mutagenesis were performed at those sites in a lipase that display high B factors.

CASTing for success: The traditional problem of expanding the range of substrate acceptance of enzymes can be solved by creating focused libraries of mutants resulting from randomization of pairs of properly chosen amino acids around the active site (see example with the lipase from Pseudomonas aeruginosa, amino acid pairs are shown in the same color). CAST=combinatorial active‐site saturation test.