Polymer-
enzyme hybrid conjugates modified by a temperature-responsive
polymer,
poly(N-isopropylacrylamide) (
PIPAAm), have been synthesized. We have investigated the molecular architecture of
PIPAAm-
enzyme conjugates by preparing two types of
PIPAAm-
trypsin conjugates, wherein
PIPAAm chains are attached by either single-end or multipoint chemistry. A semitelechelic co-oligomer (IDc) was attached to
trypsin by single-point conjugation (IDc-
trypsin). A copolymer (PIDAAc) consisting of
acrylic acid and IPAAm randomly linked in
polymer chains was attached to
trypsin using multipoint conjugation (PIDAAc-
trypsin). Both conjugates exhibited reversible temperature-responsive phase separation. The IDc-
trypsin conjugate exhibited phase separation at the same temperature as pure IDc, due to the highly mobile free
polymer end group which remains sensitive to small temperature changes. The PIDAAc-
trypsin conjugate precipitated at higher temperatures than pure PIDAAc, whose movement was restricted by multiple binding points. Enzyme stability in
solution was improved after introduction of
PIPAAm chains, which prevented
autolysis attributed to conjugate steric hindrance. Stability under repeated temperature cycling was also dependent on the architecture of conjugates; the IDc-
trypsin conjugate was more stable than the PIDAAc-
trypsin. As a consequence, single-end conjugation of
polymer to
enzyme provides novel bioconjugate with novel functionality attributed to attached
polymer while retaining native
biological function with high stability.