Characterization and application of biocatalysts in extreme environments

The effects of elevated pressures on protein function and stability are reviewed. Equations describing protein unfolding in the temperature-pressure plane are derived and discussed in light of a growing body of empirical results collected over the last 50 years. The relevance of thermostable and barostable enzymes to commercial processes are also discussed. Examples of high temperature and/or high pressure bioprocesses in industry are emphasized, and the primary considerations motivating their use are reviewed.;The discovery, isolation and characterization of a hyperthermophilic serine protease from Methanococcus jannaschii, a deep-sea hydrothermal vent archaeon, are described. This protease is among the most thermophilic enzymes reported, with a half-life of 45 min at its optimum temperature of 116;High-pressure kinetic studies were also used to probe the transition state for subtilisin-catalyzed transesterification in solvents spanning a wide range of dielectric constants. These studies indicate that the catalytic triad of subtilisin is preserved in polar and apolar solvents, the effective dielectric of the active site polypeptide is low, and the active site is occupied by solvent. Kirkwood's electrostatic model described the solvent effects and allowed semiquantitative description of the dipole generated during formation of the transition state of the native enzyme and an active-site mutant.;Subtilisin BPN' solubilized in organic solvents by ion pairing with anionic surfactant (Aerosol OT) was examined in the absence of reverse micelles. A model based on kinetic and spectroscopic (CD and EPR) studies is proposed to explain the exceptional activity of organic solvent-solubilized subtilisin BPN', and its solvent- and thermally-induced inactivation. The first demonstration of enzyme renaturation in bulk organic solvent is presented.;Strain 0420-1, isolated from a deep-sea hydrothermal vent, was grown aerobically at room temperature in artificial seawater containing up to 1000 ppm cadmium. Upon reaching stationary phase in medium containing 100 ppm cadmium, strain 0420-1 removed ca. 99% of the cadmium from solution by a combination of precipitation and adsorption.