Rational strategies for directed evolution of biocatalysts -: Application to Candida antarctica lipase B (CALB)

Provided that the industrial constraints have been properly defined, the directed evolution technologies available today enable one to design tailor-made enzymes and biocatalytic routes for chemical processes. Family shuffling has proved to be a successful strategy using traditional recombination protocols. However, when starting from a single gene, the first step is to create the appropriate population of parental genes to ensure an efficient recombination during gene shuffling. Our recent work focused on the determination of rational directed evolution strategies that can be applied for the creation of an improved biocatalyst within the requested industrial timelines. For this purpose, we have developed a rational approach that first explores the "protein plasticity" (ability of the protein to accept mutations with a limited loss of activity) of the enzyme, which enables us to estimate (i) the "optimal mutation load" (number of mutations introduced per gene that gives the highest frequency of improved variants) and (ii) the "ad minima size sample" (minimal number of clones to be screened) that can be used to rapidly improve this enzyme. We have then applied this approach to create in a few weeks variants of the well known lipase B with a seven fold improvement in thermostability.