Purification, Characterization, and Structural Studies of a Sulfatase from .

Sulfatases are ubiquitous enzymes that hydrolyze sulfate from sulfated organic substrates such as carbohydrates, steroids, and flavones. These enzymes can be exploited in the field of biotechnology to analyze sulfated metabolites in humans, such as steroids and drugs of abuse. Because genomic data far outstrip biochemical characterization, the analysis of sulfatases from published sequences can lead to the discovery of new and unique activities advantageous for biotechnological applications.

Factors Compromising Glucuronidase Performance in Urine Drug Testing Potentially Resulting in False Negatives.

Next generation β-glucuronidases can effectively cleave glucuronides in urine at room temperature. However, during the discovery studies, additional challenges were identified for urine drug testing across biologically relevant pH extremes and patient urine specimens. Different enzymes were evaluated across clinical urine specimens and commercially available urine control matrices.

Variants of glycosyl hydrolase family 2 β-glucuronidases have increased activity on recalcitrant substrates.

Glucuronidated drug metabolites can be quantified from urine samples by first hydrolyzing conjugates with β-glucuronidase (β-GUS) and then separating free drug molecules by liquid chromatography and mass spectrometry detection (LC-MS). To improve the activity and specificity of various β-GUS, we designed enzyme chimeras and generated site-saturation variants based on structural analyses, then screened them for improved activity on drug metabolites important to clinical and forensic drug-testing laboratories. Often, an increase of activity on one substrate of interest was countered by loss of activity against another, and there was no strong correlation of activity on standard β-glucuronidase substrates to activity on recalcitrant drug glucuronides.

Parallel sample processing using dispersive INtip micro-purification on programmable multichannel pipettes.

Automation gives researchers the ability to process and screen orders of magnitude higher numbers of samples than manual experimentation. Current biomacromolecule separation methodologies suffer from necessary manual intervention, making their translation to high-throughput automation difficult. Herein, we present the first characterization of biomacromolecule affinity purification via dispersive solid-phase extraction in a pipette tip (INtip).

Survey of Candidate Genes for Maize Resistance to Infection by Aspergillus flavus and/or Aflatoxin Contamination.

Many projects have identified candidate genes for resistance to aflatoxin accumulation or infection and growth in maize using genetic mapping, genomics, transcriptomics and/or proteomics studies. However, only a small percentage of these candidates have been validated in field conditions, and their relative contribution to resistance, if any, is unknown. This study presents a consolidated list of candidate genes identified in past studies or in-house studies, with descriptive data including genetic location, gene annotation, known protein identifiers, and associated pathway information, if known.