Application of high hydrostatic pressure for increasing activity and stability of enzymes

Abstract

Elevated hydrostatic pressure has been used to increase catalytic activity and thermal stability of α‐chymotrypsin (CT). For an anilide substrate, characterized by a negative value of the reaction activation volume (ΔV≠), an increase in pressure at 20°C results in an exponential acceleration of the hydrolysis rate catalyzed by CT reaching a 6.5‐fold increase in activity at 4700 atm (4.7 kbar). Due to a strong temperature dependence of ΔV≠, the acceleration effect of high pressure becomes more pronounced at high temperatures. For example, at 50°C, under a pressure of 3.6 kbar, CT shows activity which is more than 30 times higher than the activity at normal conditions (20°C, 1 atm). At pressures of higher than 3.6 kbar, the enzymatic activity is decreased due to a pressure‐induced denaturation. Elevated hydrostatic pressure is also efficient for increasing stability of CT against thermal denaturation. For example, at 55°C, CT is almost instantaneously inactivated at atmospheric pressure, whereas under a pressure of 1.8 kbar CT retains its anilide‐hydrolyzing activity during several dozen minutes. Additional stabilization can be achieved in the presence of glycerol, which is most effective for protection of CT at an intermediate concentration of 40% (v/v). There has been observed an additivity in stabilization effects of high pressure and glycerol: thermal inactivation of pressure‐stabilized CT can be decelerated in a supplementary manner by addition of 40% (v/v) glycerol. The protection effect of glycerol on the catalytic activity and stability of CT becomes especially pronounced when both extreme factors of temperature and pressure reach critical values. For example, at approximately 55°C and 4.7 kbar, enzymatic activity of CT in the presence of 40% (v/v) glycerol is severalfold higher than in aqueous buffer. The results of this study are discussed in terms of the hypotheses which explain the action of external and medium effects on protein structure, such as preferential hydration and osmotic pressure. © 1996 John Wiley & Sons, Inc.

Effect of High Hydrostatic Pressure-Temperature Combinations on the Activity of β-Glucanase from Barley Malt

“…Van der Waals forces are presumably favoured by pressure since they tend to maximize the packing density, producing a reduction in volume of the protein 5,11 . Opposing effects of pressure and temperature on hydrophobic interactions and hydrogen bond formation have been put forward as possible explanations for pressure stabilization of proteins against thermal denaturation 3,35 . Mozhaev et al 35 hypothesized that at the initial step of thermal inactivation, a protein loses a number of essential water molecules, and this loss may give rise to structural rearrangements.…”

“…Mozhaev et al 35 hypothesized that at the initial step of thermal inactivation, a protein loses a number of essential water molecules, and this loss may give rise to structural rearrangements. High pressure may hamper this process owing to its favourable effect on hydration of both charged and nonpolar groups 11,35 .…”

Reduced Thermal Inactivation of Trypsin and Marugoto E by High-pressure Treatment

“…On the other hand, β-galactosidase from Kluyveromyces lactis was inactivated at high hydrostatic pressure and high temperature, and the stability of this enzyme at extreme conditions of pressure could be enhanced by appropriately adjusting ionic strength of the buffer solution used (Cavaille-Lefebvre and Combes, 1998). Mozhaev et al (1996) showed that catalytic activity and thermal stability of α-chymotrypsin increased at the hydrostatic pressure below 360 MPa and by the addition of glycerol. Over this pressure limit, however, pressure-induced denaturation of enzyme molecules was strongly favored.…”

“…However, local changes including the rearrangement of hydrophobic groups after exposure to surrounding medium and the dissolution of hydrophobic groups might also take place after the overall changes in the thermal inactivation at higher temperatures, which was represented by biphasic mode of thermal inactivation (Kim, 1992;Tsuboi et al, 1978). High-pressure treatment was likely to favor simple first-order reaction through retarded local changes due to the reduction in thermal inactivation of enzyme molecules (Katsaros et al, 2009;Mozhaev et al, 1996;Rupley et al, 1983). This is supported by the fact that biphasic mode of thermal inactivation of trypsin only occurred at 60℃ at 300 MPa.…”

“…In light of these reports, hydration of charged groups in trypsin by water molecules seemed to be strengthened by high pressure, which possibly compensated for the loosening in hydration of enzyme molecules at high temperature (Nickerson, 1984). The net effect of the preferential hydration as described above might be reduced thermal inactivation of trypsin (Mozhaev et al, 1996). …”