Hydroxynitrile lyase at the diisopropyl ether/water interface: Evidence for interfacial enzyme activity

A novel recycle reactor has been designed to determine the interfacial acti vity of hydroxynitrile lyase in a diisopropyl ether (DIPE)/water two-phase system. The reactor provides a known interfacial area. Enzyme activity towa rd mandelonitrile cleavage is continuously measured in the reactor by follo wing benzaldehyde product formation in the DIPE organic phase with an optic al flow cell. For the first time, we establish that this enzymatic reaction is carried out by the hydroxynitrile lyase residing at the organic solvent /water interface and not in the aqueous bulk phase. Hydroxynitrile lyase ad sorbs at the interface and exhibits extraordinary stability. Denaturation d oes not occur over several hours, although the surface pressure increases u nder the same conditions over this time span. Increases in surface pressure indicate enzyme penetration through the interface although no loss of enzy me activity is observed. Adsorption of p-Hnl at the interface is fit by the Langmuir equilibrium adsorption model with an adsorption equilibrium const ant of 0.032 L mg(-1). For the mandelonitrile-cleavage reaction at ambient temperature, p-Hnl follows Michaelis-Menten kinetics at the interface with a Michaelis constant of 14.4 mM and a specific activity close that for the bulk aqueous phase. (C) 1999 John Wiley & Sons, Inc.