The influence of amino acid properties on the adsorption of proteins and peptides to stainless steel surfaces.
Thesis DisciplineChemical Engineering
Degree GrantorUniversity of Canterbury
Degree NameDoctor of Philosophy
Stainless steel (SS) is the material of choice in a number of process industries ranging from food and dairy to pharmaceuticals. Adsorption phenomena on SS surfaces are of paramount importance in these industries. For example, protein adsorption constitutes a major issue in process equipment, as the associated surface fouling decreases the efficiency of the overall process and leads to an increase in operational costs because of the need for regular cleaning. In addition, the adsorption of proteins at solid–liquid interfaces is an important research field with relevance in biosensor and biomaterial applications. The primary aim of this thesis was to understand the underlying adsorption properties of selected protein onto SS surfaces and to identify the influence of specific amino acids on bio-fouling. Protein adsorption experiments were carried out on 316 grade SS sensors using a quartz crystal microbalance with dissipation (QCM-D). The proteins consisted of milk proteins (α-lactalbumin, β-lactoglobulin, α-casein, β-casein, κ-casein and bovine serum albumin), blood proteins (cytochrome-c, haemoglobin and myoglobin) and proteins of industrial and medical relevance (α-chymotrypsinogen, human recombinant insulin, lysozyme and papain). The adsorption characteristics of the test proteins were studied and an empirical correlation relating the amount of protein adsorbed to their physical properties was proposed. Adsorption onto a SS surface was followed on the QCM-D in real time and the amounts adsorbed calculated using the Sauerbrey model. In addition, the binding kinetics was modelled using different theoretical models to describe the adsorption mechanism. In all the proteins tested, the conformational change model was found to fit considerably well the adsorption data. Finally, the data collected were used to identify the physical properties of proteins that induce surface binding, with hydrophobic and aromatic amino acids having the most effect on binding. A second aspect investigated in the present work was the determination of hydration water present in the adsorbed layer. In fact, water molecules, solvated ions and other small molecules in the vicinity of the surface all play an important role in protein adsorption and often constitute a large fraction of the total measured adsorbed mass. The fraction of water present on SS surfaces along with adsorbed proteins was determined using fluorescently labelled proteins through a comparative study that included QCM-D experiments as well as fluorescent light intensity measurements. The results were similar for all proteins tested, indicating that 32-45.8% of the total mass adsorbed composed of water. One last aspect considered in this thesis was the influence of the putative adhesive amino acid 3, 4-dihydroxyphenylalanine (DOPA). DOPA residues are present in high levels in the adhesive proteins from marine mussels, hence are thought to facilitate surface attachment. The role of DOPA residues in mediating protein adhesion on SS surfaces was studied using QCM-D. Two repetitive peptide motifs extracted from the sequence of the mussel foot protein mefp-5, KGYKYYGGSS and KGYKYY, were selected for this study. The two peptides contained unmodified tyrosine (Y) residues, which were chemo-enzymatically modified to DOPA using mushroom tyrosinase. Adsorption of the two sequences on SS surfaces was tested before and after modification of tyrosine residues to DOPA. Conversion was linearly related to the incubation time of the peptide fragments with mushroom tyrosinase, Amount of DOPA formed was 70-99% of the tyrosine content in the peptides. QCM-D adsorption experiments on the DOPA-modified sequences revealed four-fold greater adhesion than for unmodified mefp-5 motifs, indicating the paramount role that DOPA has on the adsorption of peptides on 316 grade stainless steel.