Spectrophotometric assay for online measurement of the activity of lipase immobilised on micro-magnetic particles.
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.
Schultz
Nadja
N
Centre for Microbial Biotechnology, BioCentrum-DTU, Technical University of Denmark, Lyngby, Denmark. ns@biocentrum.dtu.dk
Hobley
Timothy John
TJ
Syldatk
Christoph
C
D020099:Coated Materials, Biocompatible / Q000737:chemistry*; D004800:Enzymes, Immobilized / Q000737:chemistry; D018189:Immunomagnetic Separation / Q000379:methods*; D008049:Lipase / Q000737:chemistry*; D008280:Magnetics; D008863:Microspheres; D009862:Online Systems; D011485:Protein Binding; D012680:Sensitivity and Specificity; D013057:Spectrum Analysis / Q000379:methods*
D020099:Coated Materials, Biocompatible; D004800:Enzymes, Immobilized; D008049:Lipase