UBC Theses and Dissertations
A mechanism based approach for screening metagenomic libraries for unusual glycosidases Nasseri, Seyed Amirhossein
Unsaturated glucuronidases are among the enzymes that a number of pathogens employ to break down glycosaminoglycans, the main structural polysaccharides of our bodies. In order to find out more about these relatively less studied enzymes and the bacteria that produce them and their relative abundance in the microbial populations of our environment, we set out to screen metagenomic libraries made from environmental samples such as the human gut microbiome for unsaturated glucuronidase activity. In functional metagenomics, proteins from various uncultured organisms are reproduced in labs, by means of transferring the DNA extracted from environmental samples into host bacterial cell lines. This method offers an exciting novel method of enzyme discovery as these expressed proteins can be readily screened for novel enzymatic activity. However, screening for some of these enzymes is not straightforward, as background activity from the host cells and/or other similar unwanted enzymes from metagenomics genes, can mask the desired activity. Unsaturated glucuronidases are one of these masked enzymatic activities. In this study, a novel strategy has been developed for screening metagenomic libraries for unsaturated glucuronidases. This was achieved based on the differences in the mechanisms of unsaturated glucuronidases and β-glucuronidases, the main source of background activity. These differences make β-glucuronidases inefficient in hydrolyzing thioglycoside substrates, while unsaturated glucuronidases cleave them rapidly. Two fluorogenic thioglycoside substrates with two self-immolative thiol linkers were designed and synthesized. A small metagenomic library was then successfully screened with these new substrates and the usefulness of the selective substrates were established. We believe that the same strategy is going to be useful when screening metagenomic libraries for some of the other examples of masked activities.
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