Covalent immobilization of Drosophila acetylcholinesterase for biosensor applications

The synthesized cDNA coding for AChE (acetylcholinesterase) was subcloned in pENTR/D-TOPO plasmid and expressed using baculovirus expression vector and Sf9 insect cells as host. Purified enzyme (specific activity 36374 micromol x min(-1) x mg(-1)) was immobilized on pre-activated perlite (a porous silica matrix) by silanization and glutaraldehyde treatment. The total enzyme immobilized was then measured, and total and specific activity of immobilized AChE was compared with that of soluble enzyme. Using this perlite support not only resulted in a great amount of maintained immobilized enzyme activity (more than 70%, specific activity 26238 micromolx min(-1) x mg(-1)), but also significantly improved stability against temperature (8.7- and 17.7-fold at 50 and 60 degrees C respectively), urea (2.7-fold) and acetonitrile (1.7-fold). Kinetic studies showed that the K(m) value for immobilized enzyme is very similar to the soluble one (0.088 and 0.081 mM respectively). In addition, immobilized enzymes retained 80% of their initial activity after 16 consecutive reactor batch cycles. A comparison of the inhibitory effect of paraoxon on soluble and immobilized AChE showed that immobilization increased the linearity of the inhibition plot particularly in the range 0.1 nM-0.1 microM.