An α-Glucuronidase Enzyme Activity Assay Adaptable for Solid Phase Screening

Abstract

Glucuronic acid is a common chemical moiety that decorates the xylan polymer of hemicellulose. This chemical substituent impairs both enzymatic and acidic hydrolysis of xylosidic bonds. The alpha-glucuronidase enzyme hydrolyzes the 1,2-linked glucuronic acid from the terminal, non-reducing xylose of xylo-oligosaccharides. There are relatively few alpha-glucuronidase genes in the public databases. We have developed an assay with commercially available reagents that can be used to search DNA libraries for alpha-glucuronidase genes in a high-throughput, solid phase activity screen.

Isolation and characterization of a novel GH67 α-glucuronidase from a mixed culture

“…This library was screened using a high-throughput assay previously developed to detect -glucuronidase activity [14]. One of the genomic clones we isolated and sequenced was a 3.9-kB fragment that encoded a putative -glucuronidase enzyme (Fig.…”

“…Screening the library for -glucuronidase activity was conducted as previously described [14]. BrieXy, the E. coli SOLR cells containing the genomic DNA library were spread onto nylon membranes (Genetix, MA, USA) that had been placed on Luria-Bertani agar containing ampicillin (25 g/ml) and isopropyl--D-thiogalactopyranoside (IPTG) (1 mM).…”

Isolation of α-Glucuronidase Enzyme from a Rumen Metagenomic Library

“…Using a recently developed high-throughput, solid-phase activity assay [15], we have screened a metagenomic DNA library for a-glucuronidase genes for the first time. The ability to screen for genes from uncultured mixed environmental populations of microorganisms will result in the discovery of many more a-glucuronidase enzymes with a wider spectrum of reaction conditions.…”

“…Screening the library for a-glucuronidase activity was conducted using a high-throughput, solid-phase assay as previously described [15]. The E. coli SOLR cells containing the genomic DNA library were spread onto nylon membranes (Genetix, MA, USA) that were overlaid on LuriaBertani agar containing ampicillin (25 lg/ml) and isopropyl-b-D-thiogalactopyranoside (1 mM).…”

“…The library was screened for a-glucuronidase activity using a high-throughput solid phase assay that had been developed earlier [15]. One of the positive clones encoded a 5.5 kB contig (GenBank JN684207).…”