Retention and reusability of trypsin activity by covalent immobilization onto grafted polyethylene plates

摘要

This study investigated the activity of trypsin that had been covalently immobilized onto acrylic acid (AA)- and methacrylic acid (MAA)-grafted polyethylene (PE) plates-PE-g-PAA and PE-g-PMAA-using a water-soluble carbodiimide as a coupling agent, as a function of the immobilized amount, the grafted amount, the pH value on immobilization, and the pH value and temperature at the activity measurement. The activity of trypsin immobilized on the PE-g-PAA plates at pH 6.0 decreased with an increase in the immobilized amount because of the crowding of trypsin molecules in the vicinity of the surfaces of the grafted PAA layers. The increase in the grafted amount resulted in a decrease in the activity of immobilized trypsin because of a decrease in the diffusivity of BANA molecules caused by the formation of dense grafted PAA layers for the PE-g-PAA plates and led to the increased activity because of the increase in the hydrophilicity of the whole grafted layers for the PE-g-PMAA plates. The activity of trypsin immobilized on the PE-g-PAA and PE-g-PMAA plates at pH 6 increased with an increase in the pH value, probably because of the expansion of trypsin-carrying grafted PAA and PMAA chains and the increased diffusivity of Nalpha-benzoyl-DL-arginine-nitroanilide hydrochloride molecules in the grafted layers. The optimum temperature of the activity of immobilized trypsin shifted to 50degreesC from 30degreesC for native trypsin. Immobilized trypsin was reusable without any denaturation and isolation at temperatures ranging from 20degreesC to 60degreesC and pH values ranging from 6 to 10. Trypsin immobilized on a PE-g-PAA plate had 95% of the remaining activity in relation to native trypsin at 30degreesC after preservation in a pH 7.8 buffer at 4degreesC over 6 months. These results made clear that alkaline and thermal stability, reusability, and storage stability can be much improved by the covalent coupling of trypsin on PE-g-PAA and PE-g-PMAA plates. (C) 2003 Wiley Periodicals, Inc. [References: 48]