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.
Research has been carried out into the structure, the interfacial binding and the catalytic mechanism of triacylglycerol lipases and colipases of the digestive tract and exocellular fungal lipase (PGS-lipase). Results were as follows:
16 cutinase mutants have been produced for crystallization purposes;
the 3-dimensional structure of a recombinant cutinase has been solved at 1.25 angstrom resolution;
preparation of 160 mg of pure porcine procolipase and trypsin activated colipase for nuclear magnetic resonance (NMR) and X-ray structural studies;
epitope mapping specificity of 8 antiporcine procolipase monoclonal antibodies;
the spatial interactions between histidine and tyrosine residues is a common feature for pig, horse and ox colipases, as shown by high resolution proton NMR;
a large number of 2-dimensional NMR spectra at 500 and 600 MHz have been recorded for procolipase;
isoform purifcation of gastric lipases;
the stability of cutinase in reversed micelles of AOT/isooctane could be increased 45-fold by encapsulating the lipase with hexanol;
the crucial role of water was evidenced by NMR studies of cutinase catalyzed synthesis of ester in liquid solid media;
transesterification reactions catalyzed by lipases were observed in gas phase;
6 isomeric pseudoglycerides : didecanoyl-deoxyamino-O-methyl glycerol, were synthesized in optically pure form;
the steroselectivity of 24 lipases of animal and microbial origin towards homogeneous prochiral triglycerides was determined.
The aim of this part of the T project is to elucidate the structure function relationship of triacylglycerol lipases and colipases of the digestive tract and exocellular fungal lipases (PGS lipase) with special reference to the delineation of the particular domains involved in lipid protein interaction, and to the identification of residues essential for catalysis. The research will include crystallization studies and the preparation of heavy atom derivatives for the X-ray diffraction analysis of several native and mutated gastric lipases as well as native and mutated PGS-lipases.
2-dimensional and 3-dimensional NMR spectroscopy will be used to elucidate the structure of pancreatic colipases in solution and to investigate the interactions of these colipases in solution with lipid micelles and pancreatic lipase. The kinetics of the lipase hydration state will also be studied using NMR spectroscopy. The topology of the binding domains of colipase will be approached using epitope mapping with monoclonal antibodies as well as site directed mutagenesis. Monomolecular film technology will be used to show the pancreatic colipase epitopes involved in the lipid binding site and those responsible for the interfacial anchoring of pancreatic lipase. Reversed micellar systems will be used for kinetic and spectroscopic studies of pure lipases. The catalytic activity of lipase crystals will be monitored in solid gas biphasic systems.
Development of the so called oil drop method based on changes in interfacial tension of a triglyceride drop immersed in a lipase solution.
Studies on new lipase substrates and substrate analogues forming mixed monomolecular films. Studies of lipase stereoselectivity on chiral and prochiral substrate analogues.
Biochemical and kinetic investigations on native gastric lipases.
Various stages in the purification, crystallization and cloning of the selected lipases have already been reached by some of the partners. Our aim is either to create or reinforce close collaboration between the participants, whose fields of specialization are all complementary.
Funding SchemeCSC - Cost-sharing contracts
3584 CH Utrecht
3584 CH Utrecht