Spectrophotometric assay for online measurement of the activity of lipase immobilised on micro-magnetic particles
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A spectrophotometric assay has been adapted to directly measure the activity of enzymes immobilised on insoluble magnetic particles. Three different types of lipases (Candida antarctica lipase A and B and Thermocatenulatus lanuginosus lipase) were immobilised on two types of magnetic beads. The activity of the resulting immobilised lipase preparations was measured directly in the reaction solution by using a modified p-nitrophenol ester assay using a spectrophotometer. Removal of the solid particles was not necessary prior to spectrophotometric measurement, thus allowing reliable kinetic measurements to be made rapidly. The method was effective for a wide range of magnetic bead concentrations (0.01–0.2 mg ml−1). In all cases the assay could determine the bead-related specific enzyme activity. The assay was validated by comparing with a pH-stat method using p-nitrophenol palmitate as the substrate with an excellent correlation between the two methods. The utility of the spectrophotometric assay was demonstrated by applying it to identify the best combination of lipase type, activation chemistry and magnetic particle. Epoxy activation of poly vinyl alcohol-coated magnetic particles prior to immobilisation of commercial C. antarctica lipase A gave the best preparation.
KeywordsCandida antarctica lipase Enzyme Immobilisation Lipolase para-Nitrophenyl palmitate Thermocatenulatus lanuginosus
We thank Chemagen Biopolymer Technologie AG (Baesweiler, Germany) for the gift of M-PVA magnetic beads with different surface activation. Trine Petersen is gratefully acknowledged for preparing laboratory made silanised micro magnetic base particles. Special thanks is given to Assoc. Prof. Xuebing Xu for providing us with CALB and for valuable discussions. PD Dr. Matthias Franzreb is thanked for his valuable comments on this manuscript. Financial support by Deutsche Bundesstiftung Umwelt (DBU) in Osnabrück, Germany and the German Academic Exchange Service (DAAD) in Bonn, Germany are gratefully acknowledged.
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