Immobilization of trypsin on polyester fleece via different spacers

The immobilization of trypsin directly on polyester fleece FO 2413 as well as via PEG-diamine, aldehyde dextran, amino dextran and bovine serum albumine (BSA) spacers was investigated. It is well established that longer spacers are required for efficient immobilization of biospecific molecules to solid support materials. These minimize steric hinderance and environmental effects imposed by the surface properties of the matrices. The hydrophobic surface of the polyester fleece causes inactivation of the immobilized enzyme. Therefore, the above-mentioned spacers were employed not only to achieve a high amount of bound trypsin but also to immobilize trypsin in its active conformation. The trypsin activity was tested with the N-benzoyl-DL-arginine-p-nitroanilide hydrochlorid (BAPNA) substrate as well as with the azocasein substrate, whereas the amount of fixed trypsin was determined by BCA method. To characterize the properties of the different linked trypsin molecules, the thermal stability, pH stability and the behaviour in ethanol/water mixtures was investigated. Due to the highest activity values obtained with the BSA spacer, this system was characterized more thoroughly by testing the activity behaviour after repetitive use and various storage conditions. Immobilized trypsin shows a high thermal and pH stability. Furthermore it can be stored for several months without loosing its activity.