Saturation mutagenesis constitutes a powerful method in the directed evolution of enzymes. Traditional protocols of whole plasmid amplification such as Stratagene's QuikChange™ sometimes fail when the templates are difficult to amplify. In order to overcome such restrictions, we have devised a simple two-primer, two-stage polymerase chain reaction (PCR) method which constitutes an improvement over existing protocols. In the first stage of the PCR, both the mutagenic primer and the antiprimer that are not complementary anneal to the template. In the second stage, the amplified sequence is used as a megaprimer. Sites composed of one or more residues can be randomized in a single PCR reaction, irrespective of their location in the gene sequence.The method has been applied to several enzymes successfully, including P450-BM3 from Bacillus megaterium, the lipases from Pseudomonas aeruginosa and Candida antarctica and the epoxide hydrolase from Aspergillus niger. Here, we show that megaprimer size as well as the direction and design of the antiprimer are determining factors in the amplification of the plasmid. Comparison of the results with the performances of previous protocols reveals the efficiency of the improved method.

Mutants of the lipase from Bacillus subtilis, previously engineered for enhanced thermostability using directed evolution based on the B-FIT method, show significantly increased tolerance to hostile organic solvents.

In rare but nevertheless important cases it is of practical interest to decrease the thermostability of an enzyme, that is, to increase thermolability in a controlled manner. In the present model study, this unconventional goal has been reached by applying directed evolution to the lipase from Pseudomonas aeruginosa (PAL). By utilizing the B-factor iterative test (B-FIT), previously developed to increase the thermostability of enzymes, it was possible to reduce the T(15)(50) value from 71.6 degrees C in the case of wild type (WT-PAL) to 35.6 degrees C (best mutant) without affecting the catalytic profile in terms of substrate acceptance or enantioselectivity at room temperature. Accordingly, saturation mutagenesis was performed at sites in PAL, which on the basis of its X-ray structure, have the lowest B-factors indicative of high rigidity. Focused mutations were introduced which can be expected to decrease rigidity, the ensuing increased flexibility leading to higher thermolability without changing the actual catalytic profile.