A novel separation process based on the hydrophobic adsorption at the n-hexadecane-water interface was developed for the recovery of Acinetobacter radioresistens lipase from a pre-treated fermentation broth. In a mixture containing water, lipase and n-hexadecane, a water-in-oil emulsion was formed when the n-hexadecane-to-water ratio (o/w ratio) was larger than 3, and a large amount of lipase was found to be adsorbed at the interface. Compared with the oil-in-water emulsion (occurring when o/w ratio < 3), the water-in-oil emulsion generated smaller droplets and larger interfacial area, and was more stable. The harvested emulsion phase could be centrifuged to give an aqueous, concentrated lipase solution. Adsorption of lipase at the interface could be described by the Langmuir isotherm. For lipase concentrations ranging from 8.4 to 87.2 U cm(-3), a single-stage adsorption resulted in a six to four-fold concentration and 16-45% activity recovery, where lipase concentration was the dominant factor. A method using data from a single-stage adsorption to predict multiple-stage operation was described, and the agreement between the experimental and the predicted results was good. To improve the enzyme recovery, a multiple-run adsorption process was proposed. The use of salts enhanced the hydrophobic interaction between lipase and n-hexadecane. Advantages of the proposed process include simple operation, low operational cost, environmentally friendly, no requirement for pre-concentration of the enzyme solution, and negligible enzyme denaturation. (C) 2003 Society of Chemical Industry