A metagenomic search for glycoside phosphorylases using a phosphate dependent 2,4-dinitrophenyl glycoside colorimetric assay

• Main Author: Macdonald, Spence
• Language: English
• Published: University of British Columbia 2014
• Online Access: http://hdl.handle.net/2429/51633

Carbohydrate active enzymes (CAZymes) comprise a large class of enzymes responsible for the assembly and degradation of glycans in biological systems. CAZymes are widely employed enzymes in industry, being used in brewing and food processing, animal feed preparation, industrial pulp and paper applications and increasingly in biofuel development. While the use of CAZymes is cost-effective in glycan degradation, glycan assembly generally requires the use of expensive nucleotide sugar phosphates as a starting material. The high cost of these materials makes an assembly approach towards industrial-scale glycan synthesis difficult and usually non-viable. One class of CAZyme that has received little attention from industry is that of the glycoside phosphorylases (GPases), which offer a potential solution to the high-costs associated with glycan synthesis. These enzymes bypass the need for expensive nucleotide sugar phosphates, and thus, we believe that approaches employing GPases will be of high market value. The bottleneck in this approach to glycan synthesis currently is the very limited range of GPases available, limiting the classes of glycan that can be assembled. To help increase the spectrum of known GPases available, we have turned to metagenomics as means to discover new enzymes belonging to this family. This will involve high-throughput (HT) screening of bacterial genome fragments recovered directly from the environment for expression of novel GPases. Here, we report the development of a novel HT screening methodology that allows the screening of large libraries constructed from metagenomic DNA. A dual approach is described using functional screening and bioinformatic techniques. By using a synthetic substrate analogue that produces a colorimetric response when processed by a GPase we are able to identify functional candidates from libraries containing upwards of 25 000 clones at a time. Likewise, utilising previous sequence data accumulated by our collaborators (Hallam Lab), we were able to identify GPases based on sequence homology. To date, through this screening methodology we have discovered 5 new GPases and a new class of CAZymes: the stereochemistry-retaining β-glycoside phosphorylases. === Medicine, Faculty of === Biochemistry and Molecular Biology, Department of === Graduate