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.

Selective catalysts for sustainable oxidation of alkanes are highly demanded because of the abundance of these molecules in the environment, the possibility to transform them into higher-value compounds, such as chemicals or synthetic fuels, and the fact that, kinetically speaking, this is a difficult reaction. Numerous chemical and biological catalysts have been developed in the lasts decades for this purpose, rendering the overview over this field of chemistry difficult. After giving a definition of the ideal catalyst for alkane oxyfunctionalization, this review aims to present the catalysts available today that are closest to ideal.