ABSTRACT

The European Pharmacopoeia mentions around 1600 different synthetic pharmaceuticals of which around 40% display chirality. Despite the fact that the "wrong" stereo-isomer only contributes to unwanted side effects, the majority of chiral drugs are administered as a racemate. As a result of more stringent safety precautions there is now a strong need for efficient and clean enantioselective catalysts. Lipases are very promising candidates, however, most of them have evolved under natural conditions different from the industrial applications. This proposal aims to develop novel tools for directed evolution of enzymes in the laboratory and to provide novel lipase variants as enantioselective biocatalysts for important synthons. Both phage display and periplasmic display will be explored as selection techniques. The model proteins are Bacillus subtilis and Pseudomonas aeruginosa lipase, whose 3D structures have recently been solved.

OBJECTIVES

The project's technological aims are to:

• provide directed evolution systems that can deliver enantioselective biocatalysts of utmost specificity.
• create a phage display selection system for Bacillus subtilis lipase variants.
• apply directed evolution of Pseudomonas and Bacillus lipase variants using periplasmic compartmentalisation to obtain enantiopure products.
• rationalise the observed phenotypes by high-resolution X-ray crystallography of interesting lipase variants.

For the exploitation and dissemination of results work package WP6 defines:

• the participation of a fine chemicals industry in the partnership.
• the establishment of a contract research and resource service body.
• the establishment of two start-up companies developing (protected) tools for chiral resolutions of pharmaceuticals.

DESCRIPTION OF THE WORK

The activities have been divided into explorative work packages (WP1 and WP2), standardisation work packages (WP3) and application work packages (WP4 and WP5).

WP1 will deal with in vivo selection of novel lipase activities based on growth selections. Coupling of phenotype to genotype will be ascertained by periplasmic display of the lipase. A novel approach of suicide selection will be employed.

WP2 deals with in vitro selection of lipases displayed at the surface of phage fd in a fusion with coat proteins. This allows the selection of lipase variants on immobilised transition state analogues of otherwise non-diffusible or toxic compounds. Moreover the phage display system allows for selecting really large libraries of lipase variant (>10exp12), which goes beyond any high-throughput screening.

WP3 develops the assays and screens for further characterisation of the selected variants. A novel substrate, parinaric acid, which shows natural fluorescence, will be used for medium throughput microtitre screens.

WP4 rationalises the observed changes in enantioselectivity for the substrates that are the focus of this proposal. Comparison of the variants' structures with those of the wild type, both with and without bound chiral substrates or substrate analogues, will show the changes in enzyme structure resulting from the mutations, and the changes in chiral substrate binding. With the recent availability of the 3D structures of these lipases this offers a unique learning opportunity for protein engineering.

WP5 will provide the boundary industrial conditions towards which the lipases will have to be evolved. Robustness of the biocatalysts as defined by temperature stability, pH profile, substrate concentration, product and/or substrate inhibition and immobilisation parameters will be tested and translated into improvement opportunities. Finally the behaviour of the biocatalysts in a pilot conversion on the selected model chiral substrates (in lipid or ester form) will be assessed.

DELIVERABLES

Year 1

1. periplasmic compartmentalisation achieved.

5. chirality assays for lipase in place

7. cocrystals of lipase with novel model substrates.

Year 2

3. selection of phage-bound lipases on model compounds achieved.

9. immobilised lipase available for mimicking process conditions.

10. decision on the commercialisation strategy and the distribution among partners.

Year 3

2. enantioselective lipase variants are enriched by repeated rounds of growth selection.

4. optimal enantioselectivity evolved via in vitro selection.

6. kinetic resolution of racemic mixtures by lipases and variants.

8. rationalisation of enantioselectivity and 3D structures.

CONSORTIUM