Covalent binding between chitin and protein
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Chitin is one of the most abundant biopolymers on Earth. The polysaccharide is a linear polymer of β 1,4 linked N-acetyl-D-glucosamine residues. As a structural biopolymer, chitin is mainly occurring in animals with an outer skeleton and the main industrial source for commercial chitin is crab and shrimp shell waste.The three main components of shrimp shells are minerals (mainly CaCO3), proteins and chitin. In order to obtain pure chitin, all other components are extracted from the shells. For high-value biomedical and pharmaceutical applications pure chitin is used as raw material for high-quality and ultrapure chitosan. In order to produce pure chitin without protein, the knowledge about the detailed chemical linkage(s) between chitin and protein is necessary, which from this point is not yet available although different covalent bonds between chitin and protein have been proposed.In this research an attempt has been made to isolate chitin-protein fragments from shrimps shells of Pandalus borealis using the following strategy. The fresh shrimps were peeled and washed. A mild acid treatment was first performed to extract the minerals from the shells in order to prepare a chitin with low ash (mineral) content. In the next step proteins were solubilized by the use of protease. Enzymatic treatments with chitinase B and chitobiase were carried out in order to degrade the chitin to water-soluble low molecular weight chitin oligosaccharides. Thereafter the water-soluble fraction was prepared by centrifugation. High-resolution Size-Exclusion Chromatography was used to isolate the water-soluble fractions that contained possible fragments of chitin-oligosaccharides covalently linked to peptides (CHOS-PEP). At last, characterization of the CHOS-PEP was performed by high-resolution Mass Spectroscopy (MS) and/or Nuclear Magnetic Resonance-spectroscopy (NMR).Structure elucidation with high resolution MS and NMR pointed out that the water-soluble fraction did not contain chitin-protein fragments. Therefore, the treatment was reproduced to obtain more water-soluble CHOS-PEP fragments. However, the enzymatic degradation was performed with another enzyme with different substrate specificity, namely lysozyme.Further characterization of CHOS-PEP is necessary to obtain the knowledge about the chemical linkage(s) between chitin and protein.