Objective
The structural and functional characterisation of 10-15 lipases is under investigation. 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.
The project focussed on the purification, X-ray structure elucidation, protein engineering and mechanistic investigation of various fungal and bacterial lipases. These included lipases of Geotrichum candidum, Candida cylindracea, Rhizopus, Pseudomonas, Penicillium, Chromobacterium and a thermophilic lipase of Bacillus stearothermophilus. Purification protocols are available for most lipases under study. Diffracting crystals were obtained for 4, needle form crystals obtained for 2 lipases and 1 lipase structure was obtained by modelling. Two lipases have been cloned. The stereoselectivity, towards prochiral trigylcerides, of 24 purified lipases has been investigated. Most of the lipases showed stereopreference for the sn-1 position and hydrolysis of the sn-2 bond was catalyzed by few lipases.
The project aims at the structure of microbial lipases, of different substrate specificity, using X-ray analysis. This will allow formulation of the catalytic mechanism and specificity when combined with protein chemistry and site-directed mutagenesis.4 classes of lipase (specific towards unsaturated fatty acids, 1,3-regio-specific, non-specific, thermophilic) are proposed. Three distinctly different Geotrichum lipases will be investigated: Unilever 'Geotrichum B' - which has a unique specificity for d9-unsaturated fatty acids, Unilever 'Geotrichum A' - which has little specificity for this substrate, and Geotrichum candidum ATCC 34614 (
'Geotrichum C'), which has moderate specificity. The lipase from Rhizopus arrhizus, Candida cylindracea ATCC 14830
and pseudomonas spec. ATCC 21808 will serve as examples for 1,3-regio-specific, non-specific and non-specific thermostable lipases. The proposed lipases have, to different extents, been purified, crystallized or cloned by members of the project group.The project now aims at a concerted effort to establish the following results, in the framework of the targets defined above:
a) purification to homogeneity of the enzymes in at least 100 mg quantities,
b crystallization, and heavy metal substitution,
c) cloning, expression, secretion and sequencing in E.coli and subsequently in Aspergillus or Saccharomyces,
d) determination of detailed kinetic,
e) chemical modification followed by protein sequencing, and
f) site directed mutagenesis/computer modelling ('Protein Engineering').
Fields of science (EuroSciVoc)
CORDIS classifies projects with EuroSciVoc, a multilingual taxonomy of fields of science, through a semi-automatic process based on NLP techniques. See: The European Science Vocabulary.
CORDIS classifies projects with EuroSciVoc, a multilingual taxonomy of fields of science, through a semi-automatic process based on NLP techniques. See: The European Science Vocabulary.
- natural sciences biological sciences biochemistry biomolecules lipids
- natural sciences biological sciences biochemistry biomolecules proteins enzymes
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Funding scheme (or “Type of Action”) inside a programme with common features. It specifies: the scope of what is funded; the reimbursement rate; specific evaluation criteria to qualify for funding; and the use of simplified forms of costs like lump sums.
Coordinator
38124 Braunschweig
Germany
The total costs incurred by this organisation to participate in the project, including direct and indirect costs. This amount is a subset of the overall project budget.