The last fifty years has seen an increase in the production of synthetic or artificial enzyme substrates used to identify and quantify enzymes. These substrates have found applications in a range of biomedical science disciplines. Used in biochemistry and clinical chemistry to identify and measure enzymes, some of these substrates have been adapted for use in microbiology, particularly bacterial diagnosis and, in more recent years, molecular biology. The use of artificial chromogenic and fluorogenic enzyme substrates to identify certain bacteria is now common place in medical laboratories worldwide. Not all bacteria can be identified with existing and commercially available artificial substrates. Some of these can be slow to yield results, imprecise, expensive or require a technical method too complicated to provide a viable laboratory test. Therefore, the search for new, more efficient, biochemical tests has progressed, with novel substrates and inventive applications being developed continually. In this study, core compounds were synthesised by various condensation reactions and their characteristics evaluated with respect to colouration/fluorescence and possible enhancement of these properties by metal chelation. Promising candidates were selected for glycosidation, via modified Koenigs-Knorr reactions, in an attempt to synthesise artificial substrates. Several commercially available core molecules were also subjected to glycosidation. The more successful substrates included glycosides of alizarin, nitrosalicylaldehyde and 3- hydroxyflavone. The galactoside of nitrosalicylaldehyde was evaluated in solid agar media and found to be selective for certain Gram-negative bacteria. When similarly investigated, the 3- hydroxyflavone-?-D-glucoside showed the possibility of being used in a procedure for the isolation of the clinically significant pathogens including Listeria monocytogenes. The enzyme kinetics of ?-glucosidase with this substrate were also determined in a novel fluorescence assay and compared favourably to the well documented 4-methylumbelliferyl-?¬D-glucopyranoside. Alizarin-2-yl-?-D-galactoside and p-naphtholbenzein-?-D-galactoside were successfully utilized for the screening of recombinant and non-recombinant Escherichia coli transformants produced routinely in molecular biology. Aminopeptidase substrates have been shown to be useful for the detection of enzymes which hydrolyse peptides that are specific to certain bacteria. To allow the evaluation of novel aminopeptidase substrates, that were to be subsequently synthesised, a cost effective, large scale source of recombinant leucyl aminopeptidase enzyme was developed via gene cloning techniques. Consequently, the products of this study may serve a beneficial purpose in future enzymatic investigations, medical diagnosis and molecular biology.
|Publication status||Accepted/In press - 23 Mar 2010|