title: Synthesis and characterization of thermo-responsive poly-N-isopropylacrylamide bioconjugates for application in the formation of galacto-oligosaccharides.
abstract: 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.
authors.affiliation: Department of Chemical Engineering, Indian Institute of Technology Kanpur, Kanpur 208016, India.
authors.forename: Tapas
authors.initials: T
authors.lastname: Palai
authors.affiliation: Department of Biological Sciences and Bioengineering, Indian Institute of Technology Kanpur, Kanpur 208016, India. Electronic address: ashokkum@iitk.ac.in.
authors.forename: Ashok
authors.initials: A
authors.lastname: Kumar
authors.affiliation: Department of Chemical Engineering, Indian Institute of Technology Kanpur, Kanpur 208016, India. Electronic address: pkbhatta@iitk.ac.in.
authors.forename: Prashant K
authors.initials: PK
authors.lastname: Bhattacharya
mesh_terms: D000180:Acrylic Resins / Q000737:chemistry*; D001426:Bacterial Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D001704:Biopolymers; D002236:Carbohydrate Conformation; D011232:Chemical Precipitation; D004355:Drug Stability; D004356:Drug Storage; D004800:Enzymes, Immobilized / Q000138:chemical synthesis / Q000302:isolation & purification / Q000378:metabolism*; D000431:Ethanol / Q000494:pharmacology; D019855:Ethylene Glycol / Q000494:pharmacology; D005690:Galactose / Q000378:metabolism*; D007700:Kinetics; D007785:Lactose / Q000378:metabolism; D009844:Oligosaccharides / Q000096:biosynthesis*; D012997:Solvents; D013696:Temperature; D001616:beta-Galactosidase / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: D000180:Acrylic Resins; D001426:Bacterial Proteins; D001704:Biopolymers; D004800:Enzymes, Immobilized; D009844:Oligosaccharides; D012997:Solvents; C052970:poly-N-isopropylacrylamide; D000431:Ethanol; D001616:beta-Galactosidase; D019855:Ethylene Glycol; D007785:Lactose; D005690:Galactose
keywords: Bioconjugate; Enzyme immobilization; Galacto-oligosaccharide; Poly-N-isopropylacrylamide; Responsive polymer