Effect of surface hydrophobicity on activity of immobilized Taq DNA polymerase, its reusability and storage.

Abstract

Method of oriented and activity-preserved immobilization of biologically active proteins based on concepts of active-site masking and kinetic control was previously reported. We extended our study and found that the surface hydrophobicity had a noticeable effect on the activity of active-site protected immobilized (PIM) Taq DNA polymerase on mixed self-assembled monolayer (SAM) manufactured on Au surface. Hydrophobic SAM was created by using 12-mercaptododecanoic acid and 1-heptanethiol. The resulting Taq DNA polymerase activity was measured by performing PCR amplification and compared with previously reported values acquired with hydrophilic SAM. The maximum activity of immobilized Taq DNA polymerase was achieved at 17.5% of 12-mercaptododecanoic acid and within 90 min of reaction time which are higher than those acquired with hydrophilic SAM; maximum activity at 5% of 12-mercaptododecanoic acid and at 10 min. In order to be applicable to commercial level, immobilized enzyme need to be stable, reusable and storable. PIM Taq DNA polymerase was stably attached to the surface and could be used for as many as 10 PCR runs which were comparable to solution-phase enzyme. Even after 56 days of storage at 4 °C, immobilized enzyme pertained 70% of the initial PIM enzyme activity. These data suggest that PIM Taq DNA polymerase can be used for various commercial applications.