Activation and denaturation of hydrolases in dry and humid supercritical carbon dioxide (SC-CO2)

The effects of enzyme treatment with dry and humid supercritical carbon dioxide (SC-CO(2)) were investigated using hydrolases (EC 3.1.1.1 and EC 3.1.1.3). A crude and a purified preparation of esterase EP10 from Burkholderia gladioli was incubated in SC-CO(2) for long term and repeated high pressure treatments. Concerning the crude preparation, incubation for 24 h in SC-CO(2) at 150 bar and 35 degreesC had no effect on enzyme activity while incubation at 75 degreesC led to a distinct loss of residual activity. After 30 pressurization and depressurization steps at 35 degreesC and 150 bar, the crude enzyme preparation showed an activity increase. Using the purified enzyme preparation of esterase EP10 from B. gladioli, no significant effects could be observed. Fluorescence spectra indicated no conformational change before and after treatment with SC-CO(2). Treatment of a preparation of esterase from porcine liver with wet and dry SC-CO(2) at 200 bar at different temperatures showed a significant denaturing influence of the dissolved water on residual activities of the enzyme at temperatures of more than 40 degreesC. Lipase from Candida rugosa and esterase from porcine liver were treated at 150 and 300 bar at a constant temperature of 40 degreesC and an incubation time of 22 h. During these treatments, different amounts of water were introduced into the SC-CO(2). The results showed an increase of the water content in the treated enzyme preparation while the enzyme activity remained stable till the maximum amount of water soluble in this medium was injected into the SC-CO(2). (C) 2000 Elsevier Science B.V. All rights reserved.