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.
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
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
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
analogues, will show the changes in enzyme structure resulting from the
mutations, and the changes in chiral substrate binding. With the recent availability
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
stability, pH profile, substrate concentration, product and/or substrate
inhibition and immobilisation parameters will be tested and translated
opportunities. Finally the behaviour of the biocatalysts in a pilot
conversion on the selected model chiral substrates (in lipid or ester form)
1. periplasmic compartmentalisation achieved.
5. chirality assays for lipase in place
7. cocrystals of lipase with novel model substrates.
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
2. enantioselective lipase variants are enriched by repeated rounds of growth
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.
Wim J. Quax
Department of Pharmaceutical Biology
University of Groningen
9713 AV Groningen, The Netherlands
Tel: +31-50-363 2558
Fax: +31-50-363 3000
Karl Erich Jaeger
Lehrstuhl Biologie der Mikroorganismen
University of Bochum
44780 Bochum, Germany
Tel: +49-234-322 3101
Fax: +49-234-321 4425
Laboratoire de Liplolyse Enzymatique - UPR 9025
Centre National de la Recherche Scientifique
13402 Marseille, France
Tel: +33-491-164 093
Fax: +33-491-715 857
Corporate Research Division
Dragoco Gerberding and Co. AG
37601 Holzminden, Germany
Tel: +49-5531-971 270
Fax: +40-5531-971 158
Laboratory of Biophysical Chemistry
University of Groningen
9747 AG Groningen, The Netherlands
Tel: +31-50-363 4381
Fax: +31-50-363 4800
Manfred T. Reetz
Department of Synthetic Organic Chemistry
Max-Planck-Institut für Kohlenforschung
45470 Mülheim an der Ruhr, Germany
Tel: +49-208-306 2000
Fax: +49-208-306 2985