Synthetic substrate developped by Megazyme. Assay based on a soluble chromogenic substrate which is combined with two ultra-pure ancillary enzymes: a GH67 alpha-glucuronidase and a GH43 Beta-xylosidase. GEUX3 is a p-nitrophenyl-linked aldotriouronic acid which contains the key 4-O-methyl substitution and features a methyl ester on the carboxylate group of the glucuronic acid residue. Upon hydrolysis of the methyl ester group by a glucuronoyl esterase, the sequential hydrolytic activity by the ancillary enzymes alpha-glucuronidase and beta-xylosidase results in the release of the colourimetric group p-nitrophenol.
In plant cell walls, covalent bonds between polysaccharides and lignin increase recalcitrance to degradation. Ester bonds are known to exist between glucuronic acid moieties on glucuronoxylan and lignin, and these can be cleaved by glucuronoyl esterases (GEs) from carbohydrate esterase family 15 (CE15). GEs are found in both bacteria and fungi, and some microorganisms also encode multiple GEs, although the reason for this is still not fully clear. The fungus Lentithecium fluviatile encodes three CE15 enzymes, of which two have previously been heterologously produced, although neither was active on the tested model substrate. Here, one of these, LfCE15C, has been investigated in detail using a range of model and natural substrates and its structure has been solved using X-ray crystallography. No activity could be verified on any tested substrate, but biophysical assays indicate an ability to bind to complex carbohydrate ligands. The structure further suggests that this enzyme, which possesses an intact catalytic triad, might be able to bind and act on more extensively decorated xylan chains than has been reported for other CE15 members. It is speculated that rare glucuronoxylans decorated at the glucuronic acid moiety may be the true targets of LfCE15C and other CE15 family members with similar sequence characteristics.
Title: beta-Glucuronidase-coupled assays of glucuronoyl esterases Franova L, Puchart V, Biely P Ref: Analytical Biochemistry, 510:114, 2016 : PubMed
Glucuronoyl esterases (GEs) are microbial enzymes with potential to cleave the ester bonds between lignin alcohols and xylan-bound 4-O-methyl-d-glucuronic acid in plant cell walls. This activity renders GEs attractive research targets for biotechnological applications. One of the factors impeding the progress in GE research is the lack of suitable substrates. In this work, we report a facile preparation of methyl esters of chromogenic 4-nitrophenyl and 5-bromo-4-chloro-3-indolyl beta-D-glucuronides for qualitative and quantitative GE assay coupled with beta-glucuronidase as the auxiliary enzyme. The indolyl derivative affording a blue indigo-type product is suitable for rapid and sensitive assay of GE in commercial preparations as well as for high throughput screening of microorganisms and genomic and metagenomic libraries.
