Covalent attachment of trypsin on plasma polymerized allylamine.
This paper focuses on the immobilization of a proteolytic enzyme, trypsin, on plasma polymerized allylamine (ppAA) films. The later have been deposited onto silicon substrate by means of radiofrequency glow discharge. The covalent attachment of the enzyme was achieved in three steps: (i) activation of the polymer surface with glutaraldehyde (GA) as a linker, (ii) immobilization of trypsin and (iii) imino groups reduction treatment. The effects and efficiency of each step were investigated by X-ray photoelectron spectroscopy (XPS) and atomic force microscopy (AFM). Fluorescent spectroscopy was used to evaluate the change of the biological activity following the immobilization steps. The results showed that enzyme immobilization on GA-modified substrate increases the enzyme activity by 50% comparing to adsorbed enzymes, while the imino reduction treatment improves the enzyme retention by about 30% comparing to untreated samples. In agreement with XPS and AFM data, UV-vis absorption spectroscopy, used to quantify the amount of immobilized enzyme, showed that allylamine plasma polymer presents a high adsorption yield of trypsin. Although the adsorbed enzymes exhibit a lower activity than that measured for enzymes grafted through GA linkers, the highest catalytic activity obtained was for the enzymes that underwent the three steps of the immobilization process.
Abbas
Abdennour
A
Laboratory of Biological Processes, Enzymatic and Microbial Engineering (EA 1026), Lille1 University, F59655 Villeneuve d'Ascq, France.
Vercaigne-Marko
Dominique
D
Supiot
Philippe
P
Bocquet
Bertrand
B
Vivien
Céline
C
Guillochon
Didier
D
D000499:Allylamine / Q000378:metabolism*; D000818:Animals; D002417:Cattle; D005470:Fluorometry; D055767:Immobilized Proteins / Q000378:metabolism / Q000648:ultrastructure; D018625:Microscopy, Atomic Force; D011108:Polymers / Q000378:metabolism*; D015203:Reproducibility of Results; D012996:Solutions; D017550:Spectroscopy, Fourier Transform Infrared; D013499:Surface Properties; D014357:Trypsin / Q000378:metabolism* / Q000648:ultrastructure
D055767:Immobilized Proteins; D011108:Polymers; D012996:Solutions; D000499:Allylamine; D014357:Trypsin