Synthesis and characterization of thermo-responsive poly-N-isopropylacrylamide bioconjugates for application in the formation of galacto-oligosaccharides.

The study demonstrates the properties of conjugation of β-galactosidase with a thermo-responsive polymer, poly-N-isopropylacrylamide (PNIPAAm) in comparison to a non-responsive polymer, poly-acrylamide (PAAm). The maximum formation of bioconjugate (PNIPAAm-β-galactosidase) was 75% (yield) with 50% chemically modified enzyme (using itaconic anhydride). The process of bioconjugation (bioconjugate concentration: 7.4%) decreases lower critical solution temperature from 32.5 °C (with pure PNIPAAm) to 26.5 °C. The effect of temperature on the activities of PNIPAAm-β-galactosidase, PAAm-β-galactosidase and native enzyme was also compared. At 70 °C, the maximum activity was observed for PNIPAAm-β-galactosidase while for others it was at 60 °C. However, the effect of pH was insignificant on activities of both the bioconjugates than the native enzyme. The addition of ethylene glycol (20%, v/v) enhances the activity (by 45%) of PNIPAAm-β-galactosidase with no loss in stability; however; the trend is reversed with the addition of ethanol. Further, employing bioconjugates even up to 24 cycles of precipitation (at 40 °C) followed by re-dissolution (4 °C) around 90% of activity could be retained by PNIPAAm-β-galactosidase. The PNIPAAm-β-galactosidase also showed much-improved thermal and storage stabilities. A lower Michaelis-Menten constant (Km) was estimated with the PNIPAAm-β-galactosidase than the native enzyme as well as PAAm-β-galactosidase. Finally, PNIPAAm-β-galactosidase was tested to synthesize galacto-oligosaccharides from lactose solution.