Purification and characterization of PrbA, a new esterase from Enterobacter cloacae hydrolyzing the esters of 4-hydroxybenzoic acid (parabens).
(33/220)
The esterase PrbA from Enterobacter cloacae strain EM has previously been shown to confer additional resistance to the esters of 4-hydroxybenzoic acid (parabens) to two species of Enterobacter. The PrbA protein has been purified from E. cloacae strain EM using a three-step protocol resulting in a 60-fold increase in specific activity. The molecular mass of the mature enzyme was determined to be 54,619 +/- 1 Da by mass spectrometry. It is highly active against a series of parabens with alkyl groups ranging from methyl to butyl, with K(m) and V(max) values ranging from 0.45 to 0.88 mM and 0.031 to 0.15 mM/min, respectively. The K(m) and V(max) values for p-nitrophenyl acetate were 3.7 mM and 0.051 mM/min. PrbA hydrolyzed a variety of structurally analogous compounds, with activities larger than 20% relative to propyl paraben for methyl 3-hydroxybenzoate, methyl 4-aminobenzoate, or methyl vanillate. The enzyme showed optimum activity at 31 degrees C and at pH 7.0. PrbA was able to transesterify parabens with alcohols of increasing chain length from methanol to n-butanol, achieving 64% transesterification of 0.5 mm propyl paraben with 5% methanol within 2 h. PrbA was inhibited by 1-chloro-3-tosylamido-4-phenyl-2-butanone and 1-chloro-3-tosylamido-7-amino-2-heptanone (TLCK), with K(i) values of 0.29 and 0.20 mM, respectively, and was irreversibly inhibited by Diisopropyl fluorophosphate (DFP) or diethyl pyrocarbonate. The stoichiometry of addition of DFP to the enzyme was 1:1 and only 1 TLCK molecule was found in TLCK-modified enzyme, as measured by mass spectrometry. Analysis of the tryptic digest of the DFP-modified PrbA demonstrated that the addition of a DFP molecule occurred at Ser-189, indicating the location of the active serine. (+info)
Improvement of industry-applied rifamycin B-producing strain, Amycolatopsis mediterranei, by rational screening.
(34/220)
An industrially applied rifamycin B-producing strain, Amycolatopsis mediterranei XC 1-02, was used for further screening. A special mutation and screening procedure was adopted to select a strain, which can alleviate the inhibition caused by both aromatic amino acid and p-hydroxybenzoic acid in the pathway of rifamycin B biosynthesis as well as enhance the production of propionate, one of the precursors of rifamycin B biosynthesis. By the above methods, a strain A. mediterranei XC 9-25 was obtained, and its rifamycin B productivity in shaking flask reaches 10 g/L, which is 2.38 times higher than that of the ancestral strain XC 1-02. The productivity of rifamycin B fed-batch fermentation in 60000 L fermentor with A. mediterranei XC 9-25 reached 19.11 g/L. (+info)
Resistance of a strain of Pseudomonas cepacia to esters of p-hydroxybenzoic acid.
(35/220)
Cells of a strain of Pseudomonas cepacia were isolated from an oil-in-water emulsion containing methyl and propyl p-hydroxybenzoates (methylparaben and propylparaben) as preservative additives. This strain demonstrated the ability to destroy these additives, to utilize the propyl ester as sole carbon source, and to hydrolyze the methyl ester. When the isolate was grown on Eugon agar, exposure to the methyl ester killed 99.9% of the inoculum, but the surviving cells grew logarithmically. On the other hand, cells grown on media containing propylparaben were less susceptible when subsequently exposed to emulsions containing methylparaben. These observations demonstrate one mechanism by which microorganisms develop resistance to antimicrobial preservatives. (+info)
Effects of methyl p-hydroxybenzoate (methyl paraben) on Ca2+ concentration and histamine release in rat peritoneal mast cells.
(36/220)
1 Mechanisms of methyl p-hydroxybenzoate (methyl paraben) action in allergic reactions were investigated by measuring the intracellular Ca(2+) concentration ([Ca(2+)](i)) and histamine release in rat peritoneal mast cells (RPMCs). 2 In the presence or absence of extracellular Ca(2+), methyl paraben (0.1-10 mM) increased [Ca(2+)](i), in a concentration-dependent manner. Under both the conditions, methyl paraben alone did not evoke histamine release. 3 In RPMCs pretreated with a protein kinase C (PKC) activator (phorbol 12-myristate 13-acetate (PMA) 3 and 10 nM), methyl paraben (0.3-3 mM) induced histamine release. However, a high concentration (10 mM) of the agent did not increase the histamine release. 4 U73122 (0.1 and 0.5 micro M), an inhibitor of phospholipase C (PLC), significantly inhibited the methyl paraben-induced histamine release in PMA-pretreated RPMCs. U73343 (0.5 micro M), an inactive analogue of U73122, did not inhibit the histamine release caused by methyl paraben. 5 In Ca(2+)-free solution, PLC inhibitors (U73122 0.1 and 0.5 micro M, D609 1-10 micro M) inhibited the methyl paraben-induced increase in [Ca(2+)](i), whereas U73343 (0.5 micro M) did not. 6 Xestospongin C (2-20 micro M) and 2 aminoethoxydiphenyl borate (30 and 100 micro M), blockers of the inositol 1,4,5-trisphosphate (IP(3)) receptor, inhibited the methyl paraben-induced increase in [Ca(2+)](i) in Ca(2+)-free solution. 7 In conclusion, methyl paraben causes an increase in [Ca(2+)](i), which may be due to release of Ca(2+) from storage sites by IP(3) via activation of PLC in RPMCs. In addition, methyl paraben possibly has some inhibitory effects on histamine release via unknown mechanisms. (+info)
CloR, a bifunctional non-heme iron oxygenase involved in clorobiocin biosynthesis.
(37/220)
The aminocoumarin antibiotics novobiocin and clorobiocin contain a 3-dimethylallyl-4-hydroxybenzoate (3DMA-4HB) moiety. The biosynthesis of this moiety has now been identified by biochemical and molecular biological studies. CloQ from the clorobiocin biosynthetic gene cluster in Streptomyces roseochromogenes DS 12976 has recently been identified as a 4-hydroxyphenylpyruvate-3-dimethylallyltransferase. In the present study, the enzyme CloR was overexpressed in Escherichia coli, purified, and identified as a bifunctional non-heme iron oxygenase, which converts 3-dimethylallyl-4-hydroxyphenylpyruvate (3DMA-4HPP) via 3-dimethylallyl-4-hydroxymandelic acid (3DMA-4HMA) to 3DMA-4HB by two consecutive oxidative decarboxylation steps. In 18O2 labeling experiments we showed that two oxygen atoms are incorporated into the intermediate 3DMA-4HMA in the first reaction step, but only one further oxygen is incorporated into the final product 3DMA-4HB during the second reaction step. CloR does not show sequence similarity to known oxygenases. It apparently presents a novel member of the diverse family of the non-heme iron (II) and alpha-ketoacid-dependent oxygenases, with 3DMA-4HPP functioning both as an alpha-keto acid and as a hydroxylation substrate. The reaction catalyzed by CloR represents a new pathway for the formation of benzoic acids in nature. (+info)
Measurement of diffusion coefficients of parabens and steroids in water and 1-octanol.
(38/220)
Diffusion coefficients (D) of parabens and steroids in water and 1-octanol were determined by using the chromatographic broadening method at 37 degrees C, and the relationships between the D values and the physicochemical properties of the drugs were discussed. The D values in 1-octanol were lower than those in water because of the higher viscosity of 1-octanol. The D values depend on not only the molecular weight (MW), but also the lipophilicity of the drugs in water and on the ability for hydrogen-bonding in 1-octanol. When the lipophilic index (LI), calculated from the retention time using in a reverse-phase column, was used as a parameter of drug lipophilicity, the following equation was obtained for D in water (D(w)); log D(w)=-0.215.log MW-0.077.log LI-4.367. When the hydrogen bond index (HI), the logarithm of the ratio of the partition coefficient of the drugs in 1-octanol and cyclohexane, was used as an index of hydrogen-bonding, the following equation was obtained for D in 1-octanol (D(o)); log D(o)=-0.690.log MW-0.074.log HI-4.085. (+info)
Development of a multidose formulation for a humanized monoclonal antibody using experimental design techniques.
(39/220)
The purpose of this study was to identify optimal preservatives for a multidose formulation of a humanized monoclonal antibody using experimental design techniques. The effect of antimicrobial parenteral preservatives (benzyl alcohol, chlorobutanol, methylparaben, propylparaben, phenol, and m-cresol) on protein stability was assessed using size-exclusion chromatography, differential scanning calorimetry, right-angle light scattering, UV spectroscopy, and potency testing using a cell-based fluorescence-activated cell sorting method. A quick, cost-effective preservative screening test was designed. Combinations of preservatives were examined using an I-optimal experimental design. The protein was most stable in the presence of methylparaben and propylparaben, and was compatible with benzyl alcohol and chlorobutanol at low concentrations. Phenol and m-cresol were not compatible with the protein. The I-optimal experimental design indicated that as an individual preservative, benzyl alcohol was promising. The model also indicated several effective combinations of preservatives that satisfied the antimicrobial efficacy and physical stability constraints. The preservative screening test and the experimental design approach were effective in identifying optimal concentrations of antimicrobial preservatives for a multidose protein formulation; (1) benzyl alcohol, and (2) the combination of methylparaben and chlorobutanol were screened as potential candidates to satisfy the regulatory requirements of various preservative efficacy tests. (+info)
Structural signatures of the complex formed between 3-nitro-4-hydroxybenzoate and the Zn(II)-substituted R(6) insulin hexamer.
(40/220)
3-Nitro-4-hydroxybenzoate (3N4H) is a probe of the structure and dynamics of the metal-centered His B10 assembly sites of the insulin hexamer. Each His B10 site consists of a approximately 12 A-long cavity situated on the threefold symmetry axis. These sites play an important role in the storage and release of insulin in vivo. The allosteric behavior of the insulin hexamer is modulated by ligand binding to the His B10 zinc sites and to the phenolic pockets. Binding to these sites drives transitions among three allosteric states, designated T(6), T(3)R(3), and R(6). Although a wide variety of mono anions bind to the His B10 zinc sites of R(3), X-ray structures of ligands complexed to this site exist only for H(2)O, Cl(-), and SCN(-). This work combines one- and two-dimensional (1)H NMR and UV-Vis absorbance studies of the structure and dynamics of the 3N4H complex, which establish the following: (1). relative to the NMR time scale, 3N4H exchange between free and bound states is slow, while flipping among three equivalent orientations about the site threefold axis is fast; (2). binding of 3N4H perturbs resonances within the His B10 zinc site and generates NOEs between ligand resonances and the insulin C-alpha and side chain resonances of ValB2, AsnB3, LeuB6, and CysB7; and (3).3N4H exchange for other ligands is limited by a protein conformational transition. These results are consistent with coordination of the 3N4H carboxylate to the His B10 zinc ion and van der Waals interactions with Val B2, Asn B3, Leu B6, and Cys A7. (+info)