To develop and test support matrices made from waste prawn shells, for the selective sorption of proteins.
A project was set up to assess chitin and chitosan, derived from the waste shell of prawn and crab, as potential chromatographic support materials for use in dye ligand affinity chromatography, with emphasis on their capacities for dye binding and subsequent ability to further bind and separate chosen enzymes or proteins, in particular bovine serum albumin. Throughout the project, assessments were made for the abilities of a range of both commercially and laboratory prepared chitins and chitosans from a variety of sources to:
bind the chosen dye ligand;
subsequently bind chosen enzyme and protein substrates;
compare physical characteristics of chromatography supports, in the form of beads, for a range of chitosans;
assess the mechanical strengths of prepared support beads;
assess bead formation methods;
perform equilibrium studies on chosen supports to compare dye uptake capacities;
determine specific surface areas of selected commercial and laboratory chitosans to help assess dye uptake capacities;
determine mode of action of dye uptake (ie both adsorption and chemical binding). Chitin from all sources proved to be unsuitable for the preparation of chromatographic supports due to the low reactivity of chitin for the chosen dye suitable for dye ligand affinity chromatography. Chitosans, however, did prove to be exemplary materials, in particular those prepared in the laboratory from waste prawn shell. This was satisfying as waste prawn shell is infinitely more accessible and abundant than any other source. An emulsion forming technique for bead production proved superior to any other but unfortunately required chitosan of an extremely high molecular weight and of high percentage deacetylation, not easily achievable. Monotropic gelation was eventually chosen for both simplicity and satisfactory bead formation. This gave superior bead quality and shape but allowed for the introduction of reactive groups beneficial to dye uptake and therefore subsequent enzyme/protein binding. Bead surface area was also enhanced and mechanical strengths greatly improved.
Affinity chromatography has been a major development and has found widespread application, especially in enzyme purificati on. With the further development of new methods for separating biotechnology products which depend on commercially available textile dyes, protein separation and purification was made infinitely easier.
Despite the intense study on known dye ligands and now on novel dyes based on a modification of the well-tried triazine molecules, scant attention has been paid to the nature of the specific adsorbent media or matrices used as chromatography supports.
The use of dyes containing both charged and hydrophobic groups in the area of dye-ligand affinity chromatography immediately raises the question of dye toxicity. With present support matrices dye leakage is a major problem.
Chitin is the second most abundant naturally-occuring polysaccharide after cellulose. It is the major component of the exoskeletons of arthropods,eg, crabs, prawns, lobsters and shrimps. In plants it is found in algae and fungi. Chitin may be regarded as a derivative of cellulose in which the C2 hydroxyl group has been replaced by the acetamide group -NHCOCH3.
The potential applications of chitin and chitosan are extensive but little exploited to date.