Overall, the copolymers are working in the way we hypothesized, however pNIPAM has the highest activity at all temps. and almost all time points. As a chaotropic conjugate, pNIPAM enhances the enzymatic activity of AP and also appears to protect AP from denaturing when subjected to temps. above its LCST (lower critical solution temperature) [3]. As a kosmotropic conjugate, the trehalose glycopolymer stabilized AP and protected enzymatic activity at room temp., however it did not appear to behave in this expected manner when incubated at 37℃ and 55℃ [1]. These unexpected results could be due to uncontrolled pH and salt levels in the incubation mixtures. It could also be that the expected behavior only applies to select enzymes, AP being in an outlying group. The difference in copolymer performance possibly based on structure and temperature is of interest for further investigation.
Use polymers of higher molecular weights (MW) --> previous studies suggest MW should be 50+ kDa for more significant results [1]
Control for pH change from polymers --> use PBS as main solvent instead of water to account for pH and salt changes
Use slightly higher concentration of AP for deeper color change in Quanti-Blue assay
Test polymers with pyridyl disulfide end group CTA to observe if the covalent attachment of the enzyme with the polymers through disulfide exchange could impact enzymatic activity