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Exploring the structure-function relationship of pseudomonas and bacillus lipases

Objective

The structural and functional characterization of 10 to 15 lipases. The aim of the project is to acquire so much new knowledge about a sufficient number of these enzymes that it will be possible to understand why they are lipases and how they function as such.
Pseudomonas aeruginosa lipase has been purified and enzymatic characterization carried out. A model of the 3-dimensional structure has been prepared using existing lipase structures and amino acid sequence homologies. The amino acid sequence of Bacillus subtilis lipase has been completed and verified. The gene is coding for a precursor lipase molecule consisting of a leader of 32 amino acids and a mature lipase of 184 amino acids. Purification and enzymatic characterization of the lipase has been carried out.
The project aims at elucidating the 3-dimensional structure and the major kinetic properties of lipases from Pseudomonas and Bacillus species. The research involves collaboration between four laboratories in Germany (Professor Winkler/Dr Jager: Ruhr Universitat Bochum), Belgium (Professor Colson: Universite Catholique de Louvain-la-Neuve) and the Netherlands (Dr Misset: Gist-brocades and Dr B. Dijkstra: State University Groningen). From culture filtrates of the mentioned microorganisms (supplied by Misset), the enzymes are purified and (kinetically) characterized (Winkler: Pseudomonas and Colson: Bacillus) and subsequently subjected to crystallization in order to determine the 3-dimensional structure by X-ray crystallography (Dijkstra). Furthermore, samples of the purified lipases will be kinetically studied by the group of Dr Verger (Marseille) using monolayer and oil-droplet techniques.
Site directed mutagenesis will be used in order to identify amino acids which are important for the catalytic function of the lipases.

Funding Scheme

CSC - Cost-sharing contracts

Coordinator

Gist - Brocades BV
Address
1,Wateringsweg
2611 XT Delft
Netherlands

Participants (3)

RUHR-UNIVERSITY BOCHUM
Germany
Address
Universitaetsstrasse 150
44780 Bochum
Rijksuniversiteit Groningen
Netherlands
Address
16,Nijenborgh
9747 AG Groningen
UNIVERSITE CATHOLIQUE DE LOUVAIN
Belgium
Address
5/3,Place Croix Du Sud 5/3
1348 Louvain-la-neuve