Inactivation of enzymes by organic solvents: New technique with well‐defined interfacial area

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Abstract
A liquid-liquid bubble column apparatus allows exposure of enzyme solutions to water-immiscible organic solvents with a known total interfacial area and welldefined time scales and flow. It allows clear distinction of the different classes of inactivation mechanism. With urease as a model enzyme, octan-2-one and butylbenzene act only through the effects of solvent molecules dissolved in the aqueous phase, giving first-order inactivation at 0.34 and 0.21 h−1, respectively. Hexane and tridecane act only through exposure to the interface. The amount of urease inactivated is proportional to the total area of interface exposed, rather than to elapsed time, and may be characterized by a rate of about 0.5 μkat m−2. This is consistent with the formation and (partial) inactivation of a complete adsorbed monolayer of protein. With butan-1-ol, both mechanisms contribute significantly to the observed inactivation. The presence of O2 increases the rate of interfacial inactivation, but not that by dissolved solvent. © 1994 John Wiley & Sons, Inc.