The behaviour of selected hydrolases and dehydrogenases will be studied in four different non-conventional systems: supercritical carbon dioxide, low water content organic phases, microemulsions and liquid membranes. The reactions under investigation will be various enantio/stereo selective reactions for the production of long chain beta-hydroxylic acids, terpenes, prostaglandin synthons, esters of oligosaccharides and natural glucosides.
Initial objectives for the project are: studies on the enzymatic acylation of natural glycosides in organic solvents and on the behaviour of enzymes in microemulsions; installation of online analysis and comparative studies of various esterification reactions in aqueous systems, liquid membranes, organic solvents, and supercritical carbon dioxide study of native enzymes in hydrolytic and synthetic reactions of sugars and lipids and preparation of new enzymes by immobilization and chemical modification; study of model reactions in different solvents and screening of reactions and enzymes; and development of screening methods, production of raw materials, and development of purification processes for new enzymes.
Lipases form 2 bacteria and 3 fungi: Staphylococcus carnosus (cloned with the enzyme from Staphlyococcus hyicus), Pseudomonas spec, Penicillium simplicissimum and notatum, Rhizopus arrhizus, partially purified or even in an homogeneous state are under study. The purification of lipases to homogeneity suffers from their strong hydrophobic interaction, which reduces the overall yield to 10-20%. Significant amounts of lipases are therefore lost using ultrafiltration which is necessary for final concentration and buffer exchange. Three additional lipases (from geotrichum candidum, penicillium simplicissimum and pseudomonas cepacia) have been purified by differing techniques.
A number of the lipases have been tested in sythetic reactions in microemulsions. Esterifications of various aliphatic alcohols (hexanol, cycloexanol, cholesterol) with natural fatty acids (oleic, lauric, myristic, caprylic, butyric) have been performed and different parameters affecting the enzyme activity were studied. Preliminary results shows that 1,3 regio specific lipases better catalyze the esterification of long chain fatty acids, while non-specific lipases can catalyze the esterification of secondary alcohols with fatty acids. Transesterification between ethyl butyrate and glyc erol using native, chemical modified and immobilized lipase in a two phase system was also investigated. Optimal conversion was obtained in the presence of 5% water. The synthetic activity of lipase was further studied in supercritical carbon dioxide.
Apart form lipases, proteases have also been tested in the nonconventional media systems. Esterification of various natural glycosides by subtilisin in organic solvents have been performed and the various parameters affecting the system as well as the reaction mechanism have been studied. It was found that enzymatic acylation of rutin with fatty acids in organic solvents resulted in a significant change of its cytological properties.
Biotransformation With enzymes and enzyme systems are
of high industrial interest. Normally,aqueous phases are used as reaction media in enzyme technology. Non-conventional media are gaining more and more importance for the production of new interesting products in high enantiomeric and stereo-selective excess. These products are highly interesting for industrial purposes (e.g. Pharmaceuticals). The investigations proposed intend to increase the knowledge, experience, and understanding of enzymes in non-conventional media. The influence of different solvent phases on the enzyme activity and reaction potential Will be studied. From this knowledge optimal reaction conditions and optimal enzyme properties can be derived. The enzyme environment affects the enzyme behavior. Small changes (e.g. Water content, temperature, pressure, or pH-value) can influence the enzymatic reaction tremendously. Enzyme properties can be changed by
protein design, immobilization on solid supports, or chemical modification. Special analysis techniques (e.g. spectroscopic, ESR, NMR) are needed for reaction and enzyme monitoring in the different solvents. Thermodynamic and kinetic studies are necesSary for a process modeling. The integration of different process steps to one single step is another aspect to be investigated in this project.
Funding SchemeCSC - Cost-sharing contracts