Enzymestabilities in SC CO2 and purification methods

The use of enzymes as biocatalysts in supercritical CO 2 is a developing research area with widespread attention, first demonstrated in 1985. Although it has been shown in several studies, that enzymatic catalysis can be performed in supercritical CO 2 over a wide range of pressure and temperature and the main factors causing enzyme inactivation are already known (high temperature, high humidity and pressurization/depressurization steps), until now less data is available on comparison of stabilities of different enzymes. Recently by preliminary studies more information upon stabilities of enzymes under identical conditions were obtained and significant differences in thermostabilities and stabilities after several pressurization/depressurization steps were observed. It is also intended to study the applicabilities of until now not or rarely used enzymes (esterases. cholsterinesterases) as biocatalysts for supercritical CO 2 . By using all new data on stabilities of several enzymes in supercritical CO 2 and the known amino acid sequences it will be tested if proteins with disulfid bridges are more stable than proteins without cysteines. Further it will be investigated, if upon comparison of the stabilities it makes sense to use expensive enzymes with high purity (high activity) instead of cheap enzymes with low purity for enzymatic catalysis. It was observed at the department (unpublished results) that crude enzymes show a significant increase after treatment by super critical fluid extraction (CO2 ). It is an aim to optimize this procedure, that it can be used for the prepurification of enzymes. Therefor it is necessary to combine measurements of enzyme activities with a biophysical characterisation (fluorescence spectroscopy, electronspinresonance-spectroscopy and calorimtric measurements) to give a guarantee, that under the conditions used no changes on the protein itself are occuring. Additionally the feasibility of the following reactions in supercritical carbon dioxide will be tested: 1. Esterification of palmic acid with cholesterol to cholsterylpalmitate (cholesterolesterase) 2. Production of 4-nitrophenylbutyrate with 4-nitrophenol and butyric acid (esterase) 3. Conversion of glycerol and palmic acid to palmitin (lipase)

The use of enzymes as biocatalysts in supercritical carbon dioxide (SC-CO2) is a developing research area with widespread attention, first demonstrated in 1985. Although it has been shown in several studies, that enzymatic catalysis can be performed in SC-CO2 over a wide range of pressure and temperature until not less data is available on comparison of stabilities of different enzymes. The influence of two parameters on enzyme stability in SC-CO2 was investigated in this project. On the one hand, the number of disulphide bonds in the enzyme molecular structure, because a hypothesis was stated in a publication by a German working group, that enzymes with disulphide bonds are more stable. On the other hand, the humidity of the SC-CO2, because water plays an important role in biocatalysis: it is necessary for the catalytic activity, but can denature the enzyme when a certain amount of water is exceeded. Furthermore it was investigated, if upon comparison of the stabilities it makes sense to use expensive enzymes with high purity (high activity) instead of cheap enzymes with low purity for enzymatic catalysis (higher purity may lead to a higher instability of an enzyme preparation). Because of the stability of a number of enzyme preparations, the purification of crude preparation by supercritical fluid extraction using SC-CO2 was also examined. The measurements of enzyme activities were combined with a biophysical characterisation (fluorescence spectroscopy, gel electrophoresis, amount of free amino groups/carbonyl groupshiol groups) to give a guarantee, that under the conditions used no changes on the protein itself were occurring. Additionally the feasibility of the several biocatalytic reactions in SC-CO2 were tested using hydrolytic enzymes (esterases and lipases) for esterification and transesterification.