Preparation and properties of thermoresponsive bioconjugates of trypsin.
Covalent attachment of enzymes and other proteins to the smart polymer, poly(N-isopropylacrylamide) [poly (NIPAAm)], has been widely used as a method for the preparation of thermosensitive protein conjugates. In the present study, reversible soluble-insoluble polymer-enzyme conjugates were prepared by conjugating a copolymer of NIPAAm with 5-mol % of 6-acrylaminohexanoic acid to trypsin by the carbodiimide-NHS (N-hydroxysuccinimide) coupling method. Four bioconjugates with different units of enzyme coupled to the matrix were prepared. Increased enzymatic activity in terms of high effectiveness factor (in the range of 3-5) was found in the conjugates. Kinetic parameters for the immobilized and free enzyme were determined. The Vmax/Km value of the enzyme significantly increased on immobilization by the factors in the range of 12-28. The immobilized enzyme also showed stability to autolysis at 50 degrees C.
Raghava
Smita
S
Chemistry Department, Indian Institute of Technology, Delhi, Hauz Khas, New Delhi, India.
Mondal
Kalyani
K
Gupta
Munishwar N
MN
Pareek
Pradeep
P
Kuckling
Dirk
D
D000180:Acrylic Resins / Q000737:chemistry / Q000302:isolation & purification; D000818:Animals; D001329:Autolysis; D001704:Biopolymers / Q000737:chemistry / Q000302:isolation & purification*; D002417:Cattle; D004795:Enzyme Stability; D004800:Enzymes, Immobilized / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D007700:Kinetics; D008422:Materials Testing; D013050:Spectrometry, Fluorescence; D014357:Trypsin / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D014361:Trypsin Inhibitors
D000180:Acrylic Resins; D001704:Biopolymers; D004800:Enzymes, Immobilized; D014361:Trypsin Inhibitors; C052970:poly-N-isopropylacrylamide; D014357:Trypsin