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

Abstract

A novel recycle reactor has been designed to determine the interfacial activity of hydroxynitrile lyase in a diisopropyl ether (DIPE)/water two-phase system. The reactor provides a known interfacial area. Enzyme activity toward mandelonitrile cleavage is continuously measured in the reactor by following benzaldehyde product formation in the DIPE organic phase with an optical 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 adsorbs at the interface and exhibits extraordinary stability. Denaturation does not occur over several hours, although the surface pressure increases under the same conditions over this time span. Increases in surface pressure indicate enzyme penetration through the interface although no loss of enzyme activity is observed. Adsorption of p-Hnl at the interface is fit by the Langmuir equilibrium adsorption model with an adsorption equilibrium constant 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.

Development of a process model to describe the synthesis of (R)‐mandelonitrile by Prunus amygdalus hydroxynitrile lyase in an aqueous–organic biphasic reactor

“…Close to the interface, the benzaldehyde concentration will be higher than in the bulk and the reaction rate will be higher. At the conditions of Hickel et al (1999), where the aqueous phase was stagnant due to the absence of mixing in that phase, the benzaldehyde and reaction rate gradients may have been steeper than in our case, where the aqueous phase is mixed.…”

“…As the stirring rate in the aqueous phase was not varied, mass transfer limitation could not be detected by Hickel et al (1999). The correlation they found between the magnitude of the interfacial area and the reaction rate was ascribed to activity of the enzyme at the interface.…”

“…An important question to be addressed for the ecient use of PaHnl is the actual site of the enzymatic reaction. Hickel et al (1999) found that stirring of the organic phase had no eect on the conversion rate. It was concluded that mass transfer had no in¯uence.…”

Rigorous kinetic model considering positional specificity of lipase for enzymatic stepwise hydrolysis of triolein in biphasic oil–water system

“…3. The total concentration of the free enzyme and enzyme complex at the interface (kg m -2 ), q E,tot , is expressed by the Langmuir adsorption isotherm [15,[26][27][28][29] as…”

Role of organic solvents on Pa‐hydroxynitrile lyase interfacial activity and stability

“…We focused on the role of solvent polarity in promoting activity and stability in two-phase systems, specifically for the solvents heptane, dibutyl ether (DBE), diisopropyl ether (DIPE), butylmethyl ether (BME), and methyl tert-butyl ether (MTBE). Enzyme activity towards mandelonitrile cleavage was determined in a recycle reactor with a well-defined interfacial area as described by Hickel, et al 1999. Here the recycle reactor was modified to permit exchange of the aqueous phase.…”