Identification of a novel zinc metalloprotease through a global analysis of clostridium difficile extracellular proteins

Clostridium difficile is a major cause of infectious diarrhea worldwide. Although the cell surface proteins are recognized to be important in clostridial pathogenesis, biological functions of only a few are known. Also, apart from the toxins, proteins exported by C. difficile into the extracellular milieu have been poorly studied. In order to identify novel extracellular factors of C. difficile, we analyzed bacterial culture supernatants prepared from clinical isolates, 630 and R20291, using liquid chromatography-tandem mass spectrometry. The majority of the proteins identified were non-canonical extracellular proteins. These could be largely classified into proteins associated to the cell wall (including CWPs and extracellular hydrolases), transporters and flagellar proteins. Seven unknown hypothetical proteins were also identified. One of these proteins, CD630_28300, shared sequence similarity with the anthrax lethal factor, a known zinc metallopeptidase. We demonstrated that CD630_28300 (named Zmp1) binds zinc and is able to cleave fibronectin and fibrinogen in vitro in a zinc-dependent manner. Using site-directed mutagenesis, we identified residues important in zinc binding and enzymatic activity. Furthermore, we demonstrated that Zmp1 destabilizes the fibronectin network produced by human fibroblasts. Thus, by analyzing the exoproteome of C. difficile, we identified a novel extracellular metalloprotease that may be important in key steps of clostridial pathogenesis

Purification of commercial 2,3-dimethyl phenol using supercritical fluid extraction

The commercial value of phenols is often reduced due to the presence of colored impurities. Several conventional techniques have been used for the purification of phenols. However, conventional purification techniques are tedious and make use of hazardous and expensive organic solvents. In this study, we present a new method for purification of an aged-discolored (orange) commercial 2,3-dimethyl phenol (2,3-DMP) reagent (~97%) using supercritical fluid CO2 (SCF CO2), as an extraction solvent. A supercritical fluid extraction (SFE)/purification apparatus was constructed and purification of the reagent under different extraction conditions was investigated. Based on the differential solubility of the 2,3-DMP and the impurities in SCF CO2, the commercial reagent was successfully purified by SFE; the purified 2,3-DMP was a white solid of high purity (\u3e 99.5%). The SFE method was also applied to purify a recently purchased batch of 2,3-DMP reagent. We found that the reagent purified by SFE was of a higher quality than a commercially available analytical standard. © Taylor & Francis Group, LLC

Polar mixed-solid solute systems in supercritical carbon dioxide: Entrainer effect and its influence on solubility and selectivity

The equilibrium solubilities of benzoic acid (BA), salicylic acid (SAL), and acetylsalicylic acid (ASA) were determined in binary (solute + CO 2), ternary (two solutes + CO2), and quaternary systems (three solutes + CO2) at temperatures ranging from (308 to 328) K and pressures ranging from (10.1 to 28.0) MPa. Solubility data were obtained using a dynamic approach with a simple and reliable apparatus. Polar mixed-solid solute systems demonstrated solubility enhancements, which were consistent with the entrainer effect. In all the polar ternary systems studied, at least one component exhibited solubility enhancements. In the polar quaternary system studied, the solubility of each component increased in comparison to each binary system; the solubility of ASA, SAL, and BA was enhanced up to 484 %, 248 %, and 43 %, respectively. The high solubility enhancements observed in our study indicate that solute-solute interactions are significant in the supercritical fluid (SCF) phase. The solubility enhancements observed in the polar mixed-solid solute systems studied resulted in a decrease in selectivity of SCF CO 2. However, in a quaternary system consisting of BA, SAL, and fluoranthene (FLU), the selectivity of SCF CO2 for SAL versus FLU increased by a factor of 2.7 due to specific solute-solute interactions. This study showed that solute-solute interactions in mixed solid solute systems can result in an increase in the solubility of solutes and also the selectivity of SCF CO2. © 2008 American Chemical Society

Solubilities of substituted phenols in supercritical carbon dioxide

The binary (solute + CO 2) equilibrium solubilities of six substituted phenols (2,5-dimethyl phenol, 2,3-dimethyl phenol, 2,4,6-trimethyl phenol, 2,3,5-trimethyl phenol, 4-phenyl phenol, and 4-tert-butyl phenol) were determined at a temperature of 308 K in the pressure range of 10.1 to 28.0 MPa. Solubility data were obtained using a dynamic approach with a simple and reliable apparatus. The mole fraction solubilities of substituted phenols ranged from 5.14 × 10 -5 to 2.56 × 10 -2. Solubilities of three ternary (two solutes + CO 2) systems were investigated at a temperature of 308 K in the pressure range of 10.1 to 28.0 MPa. In the 4-phenyl phenol + 2,3,5-trimethyl phenol and 4-phenyl phenol + 2,4,6-trimethyl phenol systems, the solubility of 4-phenyl phenol was enhanced relative to its binary solubility by 22.9 % and 217 %, respectively. The 2,3,5-trimethyl phenol and 2,4,6-trimethyl phenol did not exhibit any solubility enhancements in the two ternary systems. Accurate solubilities could not be measured for the 2,5-dimethyl phenol + 4-tert-butyl phenol ternary system due to the existence of a liquid phase under the conditions studied. © 2006 American Chemical Society