No abstract

The anaerobic fermentation of waste prawn shell (Nephrops norvegicus) using lactic acid bacteria, in combination with especially selected proteolytic enzyme producing bacteria, has been established as an effective method for breaking down shell waste and isolating the valuable components contained within the shell structure. One of these components is the biopolymer chitin, which has numerous industrial applications and has traditionally been produced by a harsh chemical method which is extremely hazardous, energy consuming and ultimately damaging to the environment as it employs high concentrations of mineral acid and alkali. This qualitative study investigates the potential of prawn shell fermentation to provide a multi-product process, thereby eliminating waste and generating revenue. Fermentations were carried out in a bench-top, stirred tank bioreactor (5-litres). Various components of the fermentation product were analysed. The chitinous residues were characterised by CHN and calcium analysis. The composition of the protein mixture in the fermentation liquor was assessed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). Two-dimensional SDS-PAGE followed by mass spectrometry allowed remaining proteins in the liquor to be identified. Carotenoid pigments in the liquor, potentially applicable in animal feed supplements, were investigated for their extractability in various solvents. A fine, white precipitate that is formed in the fermentation broth was also examined. Key results of the study showed that the unique microbial mixture used in the fermentation of prawn shell waste served to achieve the desired objectives i.e. (i) the production of a purified chitin with calcium removal as high as 93.8%; (ii) the production of a pigmented liquor containing peptides, amino acids only a few intact proteins. Sarcoplasmic calcium-binding protein was consistently found as a surviving protein in the fermentation broth. This study demonstrates the use of a biotechnological process as an alternative to the traditional chemical approach used in the manufacture of chitin. Trials have begun to assess the nutritive value of the liquor in lobster farming while biotransformation studies on bioprocessed chitin are ongoing.

A series of novel synthetic dipeptides, containing a C-terminal glyoxal grouping (-COCHO), have been tested as inhibitors against typical members of the serine- and cysteine-proteinase families. For example, the sequences benzyloxycarbonyl (Cbz)-Pro-Phe-CHO (I) and Cbz-Phe-Ala-CHO (II), which fulfil the known primary and secondary specificity requirements of chymotrypsin and cathepsin B respectively, have been found to be potent reversible inhibitors of their respective target proteinase. Thus I was found to inhibit chymotrypsin with a Ki of approximately 0.8 microM, whereas II exhibits a Ki of approximately 80 nm against cathepsin B. These Ki values are some 10-fold and 3-fold lower than those reported for the corresponding peptide-aldehyde inhibitors of chymotrypsin and cathepsin B upon which the peptidyl-glyoxals were fashioned. Unexpectedly, the sequence Cbz-Pro-Ala-CHO, which was designed to inhibit elastase-like proteinases, exhibited no inhibitory activity towards porcine pancreatic elastase, even when used at concentrations as high as 200 microM.

Synovial fluid is a potential source of novel biomarkers for many arthritic disorders involving joint inflammation, including juvenile idiopathic arthritis. We first compared the distinctive protein 'fingerprints' of local inflammation in synovial fluid with systemic profiles within matched plasma samples. The synovial fluid proteome at the time of joint inflammation was then evaluated across clinical subgroups to identify early disease associated proteins. We measured the synovial fluid and plasma proteomes using the two-dimensional fluorescence difference gel electrophoresis approach. Image analysis software was used to highlight the expression levels of joint and subgroup associated proteins across the study cohort (n = 32). A defined subset of 30 proteins had statistically significant differences (p < 0.05) between sample types such that synovial fluid could be differentiated from plasma. Furthermore distinctive synovial proteome expression patterns segregate patient subgroups. Protein expression patterns localized in the chronically inflamed joint therefore have the potential to identify patients more likely to suffer disease which will spread from a single joint to multiple joints. The proteins identified could act as criteria to prevent disease extension by more aggressive therapeutic intervention directed at an earlier stage than is currently possible.

The synovial fluid proteome in juvenile idiopathic arthritis was investigated to isolate joint-specific biomarkers that are expressed in patients displaying recurrent joint inflammation. To identify the synovial specific proteome, matched synovial fluid and plasma samples were subjected to protein separation by 2-dimension electrophoresis (2DE). Forty-three protein spots, overexpressed in the joint, were identified. Synovial fluids from children with single-event knee joint inflammation were then compared with a group with recurrent knee disease. Nine synovial specific proteins were significantly differentially expressed in the recurrent group. Proteolytic fragments of collagen X, fibrin β-chain, and T-cell receptor α-region have been identified among this protein cluster. Putative biomarkers, overexpressed in the joint and differentially expressed in children with recurrent joint inflammation, have been identified. These proteins may play a significant role determining the pathological state within the chronically inflamed joint and influence disease progression in JIA. This is the first study of the synovial proteome in children. Keywords: juvenile idiopathic arthritis • biomarker • inflammation • synovial fluid • plasma