4-Nitrophenyl-β-D-glucuronide

Recently worldwide food safety authorities indicated the rise of foodborne outbreaks linked to Salmonella: this highlighted the need to intensify monitoring and apply more targeted controls to help manage the spread of the disease. The aim of this study was to assess the prevalence and distribution of Salmonella serotypes in 7 slaughterhouses, located in different areas of Naples province (Regione Campania, Italy). Meat samples collected from the slaughterhouses were submitted for standardized microbiological analysis in 2015. Results of routine testing for Salmonella spp. were analyzed and then compared to biochemical and molecular evaluations. Salmonella spp. were detected in 12% of 320 samples examined (39/320) and the isolation rates ranged from 87% (32 samples) for raw poultry meat to 13% (7 samples) for pork meat. Biochemical serotyping showed that approximately 50% of the isolates belonged to Salmonella enterica serotype Choleraesuis. Rapid detection methods, such as molecular analysis (polymerase chain reaction and gel electrophoresis), able to confirm food matrices contamination, represent a valid support to the fast identification of Salmonella species. A further aspect of the study consisted, indeed, on analyzing isolated strains through molecular evaluations. By amplifying bacterial DNA—using invA primers, selective for Salmonella—it was possible, in less than 3 h, to classify the isolates as Salmonella spp., confirming the results of microbiological outcomes. Results of distribution analysis, supported by rapid molecular approaches, showed the difficulty of reducing Salmonella risk on food chain. This emphasized the importance of periodic surveillance to prevent outbreaks.

Glycoside hydrolases (GHases) of higher performance are immediately needed for efficient degradation of plant biomass into fermentable sugars in industrial processes. The current study represents functional characterization of the enzymatic repertoire involved in crude cotton biomass degradation. Physical contact between cells and substrate is necessary for efficient hydrolysis of cellulose. Cytophagales, which plays a major role in cotton biomass decomposition, was identified as a prevalent community member by 16 S rRNA analysis. From the metagenome data, 2058 GHase homologs were identified, of which sixteen were successfully expressed in E. coli. Four enzymes showed activities on p-nitrophenyl-β-D-xylopyranoside, four showed activities on p-nitrophenyl-β-D-glucopyranoside, two had activities p-nitrophenyl-β-D-glucuronide, one showed activity on laminarin, three had activities against p-nitrophenyl-N-acetyl-β-D-glucosaminide, one had activity towards carboxymethyl cellulose, and one towards p-nitrophenyl-β-D-mannopyranoside. Metagenomics provides a good resource for mining novel biomass degrading enzymes. The sixteen GHases that were cloned may have potential application for biomass conversion and bioproduct production. Functional characterization of the enzymatic repertoire in cotton biomass degradation and analysis of the GHases provide insight into the composition and interaction of enzymes and pathways of plant biomass degradation.

In this work, a self-reporting hydrogel for the rapid in situ detection of bacterial enzymes is reported. To implement the reporting function for the bacterium Escherichia coli into a film-based sensing format, chitosan hydrogel films on solid backing supports were equipped with a reporting function for the enzyme β-glucuronidase (β-GUS), which is secreted by >98% of all known E. coli strains. Covalent coupling of the fluorogenic substrate 4-methylumbelliferyl-β-D-glucuronide or the complementary chromogenic substrate 4-nitrophenyl-β-D-glucuronide via amide bond formation afforded an attachment that is stable for >24 h under physiological conditions. By contrast, in the presence of β-GUS, the reporter dyes were very rapidly cleaved and produced a signal for the presence of the enzyme, which was detectable by bare eye under appropriate illumination. Detailed investigations of the enzymatic reaction for both types of substrates in neat enzyme solution as well as in bacterial supernatant revealed the apparent reaction kinetics and allowed us to determine the concentration of β-GUS in the supernatant. Under optimized conditions, the 4-methylumbelliferyl-β-D-glucuronide-functionalized hydrogel reported the presence of β-GUS within 15 min with a limit of detection of <1 nM. Finally, the function of the generally applicable hydrogel-film-based sensing approach, which is compatible with polymer-film-based applications, including wound dressings and packaging materials, and is also amenable to address noncultivatable pathogenic bacteria by using appropriate fluorogenic or chromogenic substrates, was demonstrated by direct application with bacterial medium.

In the first systematic study of the enzyme β-glucuronidase, Masamune showed that oxkidney preparations did not hydrolyse (-)-menthyl α-D-glucuronide, and this compound was also not hydrolysed by mouse-liver β-glucuronidase and by a snail preparation rich in β-glucuronidase and in other simple glycosidases. Nevertheless, although it is not so ubiquitous as the β-glucuronide residue, there is evidence for a naturally occurring α-conjugated glucuronic acid, for example in wheat straw. It has been inferred that uridine diphosphoglucuronic acid also has the α-configuration.