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CONVERSION OF PANCREATIC PHOSPHOLIPASE A2 INTO TRIGLYCERIDE DEGRADING LIPASE

Objective

CONSTRUCTION OF STABLE, SPECIFIC FAT-DEGRADING ENZYMES WHICH MAY BE OF GREAT INTEREST AS ADDITIVES FOR DETERGENT OR AFTER IMMOBILIZATION ON SOLID SUPPORT COULD FORM AN EXCELLENT TOOL FOR THE SYNTHESIS OF LIPIDS OR PHOSPHOLIPIDS IN ENZYME REACTORS.
Automatic tensiometry is achieved by using the drop method for measuring surface tensions. The apparatus analyses and numerates, in real time, the shape of the drop using a charge coupled device (CCD) detector. The surface tension, the area and volume of the drop are calculated. The volume of the drop may be regulated on demand, in order to maintain the surface tension at a predetermined value.

Most living organisms contain enzymes which selectively cleave certain bonds in fat thereby rendering them water soluble. Such enzymes may have industrial applications in the biotechnological production of water insoluble fine chemicals and as additives in washing detergents. This requires stable enzymes of a well defined (tailored) specificty.

A deeper insight into the specificity of (phospho)lipases, which degrade such related compounds as phospholipids and triglycerides, could lead to the design of enzymes with altered specificity for lipid molecules. As a starting point, the 3-dimensional model of the stable and high specific porcine pancreatic phospholipase A2 was used. Attempts were made to introduce by protein engineering changes that affect specificity, but not stability. For a rational design of these changes, more 3-dimensional structures are required. Thus a bacterial aspecific (phospho)lipase was isolated, characterized at the biochemical level and attempts were made to obtain its 3-dimensional structure.

The straphylococcal lipase was produced, purified and characterized. The enzyme can be described as a phospholipase A1 with significant lipase activity. Mutants were produced allowing for a more simplified purification and the introduction of a unique thiol group into the active site. Both mutations may increase the chance to solve its 3-dimensional structure. Several mutants of pancreatic phospholipase A2 were produced and characterized. 2 mutations are of special interest: mutant 1 has increase activity on neutral, but reduced activity on negatively charged phospholipids; mutant 2 has a changed chiral specificity in addition to elevated activity on uncharged phospholipids.
TRACING CATALYTIC ACTIVITIES OF GENETICALLY MODIFIED (PHOSPHO) LIPASES USING LIPID MONOLYERS AS SUBSTRATES. DURING THE COURSE OF THE REACTION SEVERAL PHYSICOCHEMICAL PARAMETERS WILL BE MONITORED: SURFACE PRESSURE, SURFACE POTENTIAL, RADIOACTIVITY.
THE QUALITY OF INTERFACE CONTROLLED BY THE NATURE, DENSITY AND ORIENTATION OF THE LIPIDS WILL BE MODIFIED. THE ZERO-ORDER THROUGH TECHNIQUE WILL BE USED TO STUDY THE HYDROLYSIS OF MIXED MONOMOLECULAR FILMS AT CONSTANT SURFACE DENSITY AND CONSTANT LIPID COMPOSITION.

Funding Scheme

CSC - Cost-sharing contracts

Coordinator

Centre National de la Recherche Scientifique (CNRS)
Address
31 Chemin Joseph Aiguier
13402 Marseille
France

Participants (1)

STATE UNIVERSITY OF UTRECHT
Netherlands
Address

Utrecht