Stability Properties of Thermoresponsive Poly(N-Isopropylacrylamide)-Trypsin Conjugates

Trypsin was covalently coupled to poly(N-isopropylacrylamide) (pNIPAAm), a thermoresponsive polymer, through reactive groups of N-acryloxysuccinimide (NAS), which were incorporated into the polymer and the enzyme, respectively, prior to immobilization. Higher activity was retained for the enzyme that was coupled to the preformed copolymer of NIPAAm-NAS (conjugate I) as compared to conjugate II prepared by the other method. The conjugated trypsin was found to be more resistant to heat treatment than the free enzyme. Just the physical presence of the polymer had no significant influence on the thermal stability of the free trypsin. The activity loss in the conjugated enzymes during repeated precipitation/dissolution cycles was more due to incomplete recovery than to inactivation by temperature during the precipitation step. Conjugate I exhibited significant retention of activity after storage in acetonitrile and DMF, and was also used for dipeptide synthesis in acetonitrile.

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