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, respectivel y, prior to immobilization. Higher activity was retained for the enzyme tha t was coupled to the preformed copolymer of NIPAAm-NAS (conjugate I) as com pared 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 t hermal stability of the free trypsin. The activity loss in the conjugated e nzymes during repeated precipitation/dissolution cycles was more due to inc omplete recovery than to inactivation by temperature during the precipitati on step. Conjugate I exhibited significant retention of activity after stor age in acetonitrile and DMF, and was also used for dipeptide synthesis in a cetonitrile.