This study examines the apparent kinetics of immobilized lipase-catalyzed synthesis of isoamyl acetate at high hydrostatic pressure (HHP) in hexane. HHP reduced thermal inactivation of lipase by up to 152% after 4 h at 80°C and 400 MPa when compared to incubations at low pressure. No significant differences were found in activation energy (Ea) at different pressures, regardless of the pressurization and heating sequence, and were between 35.7 ± 3.5 and 47.8 ± 8.2 kJ mol⁻¹, depending on the method. In all methods utilized, activity at 63.5 and 80°C at 400 MPa was greater (from about 20 to 96% increase) than at low pressure. Activity increased by 110% at low pressure versus a 239% increase at 350 MPa when the temperature was increased from 40 to 80°C. Increasing pressure up to 350 MPa increased lipase activity while pressures greater than 350 MPa maintained or decreased lipase activity. Activation volume (ΔV ^≠) appeared negative between ambient pressure and 200 MPa in contrast to a positive ΔV^≠ between 300 and 600 MPa. Apparent ΔV ^≠ was 14.3 or 15.2 ± 2.2 cm³ mol⁻¹ at 40 or 80°C, respectively, between 300 and 500 MPa.

This study also examines further the negative and positive activation volumes (ΔV^‡) and Michaelis-Menten parameters (K_M and V_max) of immobilized lipase-catalyzed synthesis of isoamyl acetate at HHP in hexane. At 80°C V_max increased (negative ΔV^‡) from 10 to 250 MPa, remained relatively constant between 250 and 350 MPa, then decreased (positive ΔV ^‡) from 350 to 600 MPa. At 40°C V_max increased (negative ΔV^‡) from 1 to 100 MPa, remained relatively constant between 100 to 200 MPa, and then decreased (positive ΔV ^‡) from 200 to 600 MPa. Temperature affected ΔV ^‡ at low pressure but did not at high pressure; indicating that pressure induced activation is affected by temperature while inactivation is not. Pressure significantly increased V_max at both temperatures by approximately 1-log, however pressure only affected K_M at 40°C but not at 80°C. Increased K_M indicates that the enzyme substrate complex formation is being hindered at 40°C but not at 80°C. The V _max was lower at 8°C and low pressure than at 40°C and 200 MPa which may be attributed to lipase inactivation at high temperatures and low pressures.

The application of HHP on the apparent kinetics of immobilized and free lipase (Candida antarctica lipase B) in a biphasic ionic liquid (IL)-alcohol system was investigated. ILs have become an attractive alternative media because they can enhance stability, enantioselectivity, product yield, and reaction rate of enzyme reactions. Although the application of IL and HHP to enzyme catalysis has been previously explored separately, this study is the first to explore the combination of these technologies. Production of isoamyl acetate was up to 10-fold higher with free lipase versus immobilized lipase after 3 h at 300 MPa and 80°C. Rate of catalysis by free lipase also increased up to 15 and 25-fold at 500 MPa versus at 0.1 MPa at 40 or 80°C, respectively. Pressure affected the activation energy (E_a) of immobilized lipase but not free lipase (43.4 ± 3.1 and 55.4 ± 0.1 kJ mol⁻¹ at 0.1 and 300 MPa respectively). Temperature had no effect on activation volume (ΔV^‡) which was −16.1 ± 1.5 and −16.7 ± 1.4 cm³ mol⁻¹ at 40 and 80°C respectively. It was also observed that after treatment at high pressure, the free lipase is temporarily suspended in a semi-solid IL phase.

This study has demonstrated how high pressure can induce activation and stabilization of lipase in a variety of solvents. However, there is a significant lack of data describing the continuous in situ conformational changes induced by combinations of temperature and pressure that may be causing activation and/or stabilization. Fluorescence spectroscopy has been conducted in situ at pressures up to 500 MPa and up to 80°C. Results indicate immediate and profound changes in tryptophan fluorescence as indicated by changes in the intensity at 350 nm. (Abstract shortened by UMI.)