LIPASES CAN BE USED AS CATALISTS FOR (TRANS-)ESTERIFICATION. THEY WILL THUS BE OF IMPORTANCE IN THE OILS AND FAT INDUSTRY, REPLACING SOME CHEMICAL PROCESSES WITH MILDER, MORE NATURAL AND MORE EFFICIENT TECHNIQUES. LIPASES WITH NOVEL TYPES OF SPECIFICITY WILL GIVE ADDITIONAL OPPORTUNITIES BY EXTENDING THE RANGE OF TRIACYLGLYCEROL MIXTURES WHICH CAN BE PRODUCED.
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.
THE WORK AT THE GENETIC LEVEL WILL CONSIST OF :
- SUBCLONING THE LIPASE GENES, CONSTRUCTION OF PHYSICAL AND GENETIC MAPS,
- SEQUENCING OF THE STAPHYLOCOCCAL LIPASE GENE;
- INCREASE LIPASE PRODUCTION USING ENHANCED PROMOTOR ACTIVITY;
- SITE SPECIFIC ALTERATIONS OF THE PRIMARY SEQUENCE OF THE LIPASES;
AND AT THE BIOCHEMICAL LEVEL OF :
- ISOLATION AND PURIFICATION OF THE VARIOUS (PHOSPHO-)LIPASES;
- DETERMINATION OF ENZYME CHARACTERISTICS (SUBSTRATE SPECIFICITY, MOLECULAR WEIGHT, PH- AND TEMPERATURE PROFILES);
- DETERMINATION OF THE CATALYTIC CENTRE(S).