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Preparation of Thermo-Responsive Polymer Gels Immobilizing Core-Shell Type Bioconjugates

Atsushi Harada, Kunio Johnin, and Kenji Kono. Department of Applied Chemistry, Graduate School of Engineering, Osaka Prefecture University, 1-1 Gakuen-cho, Naka-ku, Sakai, Osaka, 5998531, Japan

Immobilization of enzymes is interesting research topic from both fundamental and applied aspects, and various kinds of immobilization methods including chemical and physical procedures have been actively investigated, e.g. the chemical conjugation of enzyme with water-soluble polymers and the physical entrapment into polymer gels matrix. The advantages of enzyme-immobilization are improvement of storage and operational stabilities. In such studies, the activity of enzyme often decreased after chemical or physical immobilization, although the stabilities were improved. The decrease in enzymatic activity might be induced by the change in environment around enzyme molecule and/or the change in conformation of enzyme molecule through chemical and physical enzyme-immobilization. If the enzyme-immobilization method in consideration of the change in environment and conformation is used, it was expected that it was possible to prevent the decrease in enzyme activity with immobilization. For the chemical conjugation of enzymes and water-soluble polymers concerning limited combinations, which is one of chemical immobilization method, it has been reported that the storage stability of enzyme may improve without a decrease in enzymatic activity. As one of such bioconjugate system, we have recently succeeded the preparation of core-shell type bioconjugates from the mixture of bovine pancreas trypsin and poly(ethylene glycol)-block-poly(aspartic acid) (PEG-PAA) through the crosslinking between trysin and block ionomer by glutaraldehyde. The enzymatic activity of trypsin in the core of core-shell type bioconjugates was higher than that of native trypsin, and both the storage stability and operational stability was drastically improved. Here, core-shell type bioconjugates incorporating trypsin molecules in the core was physically entrapped in thermo-responsive polymer gels, poly(N-isopropylacrylamide) (PNIPAAm) gels. The water content in the gels prepared at varying cross-linking densities were drastically changed around 32 C, and this phase transition temperature was no change even after the entrapment of core-shell type bioconjugagte. Also, the reaction rates of trypsin in the gels at varying temperature were evaluated by colorimetric assay using L-lysine p-nitroanilide as a substrate. The Arrenius plots of reaction rate showed two linear regions bounded at the phase transition temperature of the gels, although the reaction rate of trypsin was drastically decreased at higher temperature region due to lower diffusion of substrate in the gels. The slopes of two linear lines were almost same, indicating no change in essential reactivity of trypsin. That is, thermo-responsive feature of PNIPAAm gels as well as the reactivity of trypsin was maintained even after the physical entrapment of bioconjugates to the gels.