Pressure-induced inactivation of E. coli beta-galactosidase: influence of pH and temperature

In order to assess the feasibility of a high-pressure immunodesorption process using a beta-galactosidase-anti-beta-galactosidase complex as a model, the influence of high hydrostatic pressure on the activation of E. coli beta-galactosidase has been investigated. The irreversible activity loss of beta-galactosidase was studied as a function of pH and temperature for pressures comprised between atmospheric pressure and 500 megapascal (MPa; 1 MPa = 10 bar). This enabled us to establish a practical pressure-temperature diagram of stability for this enzyme. The stability domains determined thus appeared to be strongly dependent on the pH under atmospheric pressure of phosphate buffer employed for pressurisation. Therefore, to interpret meaningfully this result, the influence of pressure on the pH-activity curve of beta-galactosidase was investigated by using a high-pressure stopped-flow device. It appeared that the pH-activity curve of this enzyme was also reversibly affected by pressures lower than 150 MPa. An interpretation of these results in relation to the high-pressure induced changes of ionisation constants is proposed. For our practical purpose, the implications for the elaboration of a high-pressure immunodesorption process using beta-galactosidase as a tag, are discussed.