title: Influence of protein bulk properties on membrane surface coverage during immobilization.
abstract: Biomolecules immobilization is a key factor for many biotechnological applications. For this purpose, the covalent immobilization of bovine serum albumin (BSA), lipase from Candida rugosa and protein G on differently functionalized regenerated cellulose membranes was investigated. Dynamic light scattering and electrophoresis measurements carried out on biomolecules in solution indicated the presence of monomers, dimers and trimers for both BSA and protein G, while large aggregates were observed for lipase. The immobilization rate and the surface coverage on functionalized regenerated cellulose membranes were studied as a function of biomolecule concentration. Results indicated that the saturation coverage of BSA and protein G was concentration independent (immobilized protein amount of 2.40±0.03mg/g and 2.65±0.07mg/g, respectively). Otherwise, a different immobilization kinetics trend was obtained for lipase, for which the immobilized amount increases as a function of time without reaching a saturation value. Atomic force microscopy (AFM) micrographs showed the formation of monolayers for both BSA and protein G on the membrane surface, while a multilayer structure is found for lipase, in agreement with the trends observed in the related immobilization kinetics. As a result, the morphology of the proteins layer on the membrane surface seems to be strictly dependent on the proteins behavior in solution. Besides, the surface coverage has been described for BSA and protein G by the pseudo second order models, the results indicating the surface reaction as the controlling step of immobilization kinetics. Finally, enzyme activity and binding capacity studies indicated the preservation of the biomolecule functional properties.
authors.affiliation: Institute on Membrane Technology, ITM-CNR, c/o University of Calabria via Pietro Bucci, 17/C, 87036 Rende (CS), Italy.
authors.forename: Francesca
authors.initials: F
authors.lastname: Militano
authors.affiliation: Institute on Membrane Technology, ITM-CNR, c/o University of Calabria via Pietro Bucci, 17/C, 87036 Rende (CS), Italy. Electronic address: t.poerio@itm.cnr.it.
authors.forename: Teresa
authors.initials: T
authors.lastname: Poerio
authors.affiliation: Institute on Membrane Technology, ITM-CNR, c/o University of Calabria via Pietro Bucci, 17/C, 87036 Rende (CS), Italy. Electronic address: r.mazzei@itm.cnr.it.
authors.forename: Rosalinda
authors.initials: R
authors.lastname: Mazzei
authors.affiliation: Institute on Membrane Technology, ITM-CNR, c/o University of Calabria via Pietro Bucci, 17/C, 87036 Rende (CS), Italy.
authors.forename: Emma
authors.initials: E
authors.lastname: Piacentini
authors.affiliation: Institute on Membrane Technology, ITM-CNR, c/o University of Calabria via Pietro Bucci, 17/C, 87036 Rende (CS), Italy.
authors.forename: Annarosa
authors.initials: A
authors.lastname: Gugliuzza
authors.affiliation: Institute on Membrane Technology, ITM-CNR, c/o University of Calabria via Pietro Bucci, 17/C, 87036 Rende (CS), Italy.
authors.forename: Lidietta
authors.initials: L
authors.lastname: Giorno
mesh_terms: D000327:Adsorption; D000818:Animals; D002175:Candida / Q000378:metabolism; D002417:Cattle; D002482:Cellulose / Q000737:chemistry*; D005029:Ethylenediamines / Q000737:chemistry; D005656:Fungal Proteins / Q000737:chemistry; D005976:Glutaral / Q000737:chemistry; D006801:Humans; D055767:Immobilized Proteins / Q000737:chemistry*; D007074:Immunoglobulin G / Q000737:chemistry; D007700:Kinetics; D008049:Lipase / Q000737:chemistry; D008567:Membranes, Artificial; D018625:Microscopy, Atomic Force; D010084:Oxidation-Reduction; D011506:Proteins / Q000737:chemistry*; D012710:Serum Albumin, Bovine / Q000737:chemistry; D013499:Surface Properties
chemical_list: D005029:Ethylenediamines; D005656:Fungal Proteins; D055767:Immobilized Proteins; D007074:Immunoglobulin G; D008567:Membranes, Artificial; D011506:Proteins; D012710:Serum Albumin, Bovine; D002482:Cellulose; D008049:Lipase; D005976:Glutaral
keywords: AFM topography; Biomolecules covalent immobilization; Immobilization rate; Regenerated cellulose