Directed evolution of biphenyl dioxygenase: emergence of enhanced degradation capacity for benzene, toluene, and alkylbenzenes.
(49/773)
Biphenyl dioxygenase (Bph Dox) catalyzes the initial oxygenation of biphenyl and related compounds. Bph Dox is a multicomponent enzyme in which a large subunit (encoded by the bphA1 gene) is significantly responsible for substrate specificity. By using the process of DNA shuffling of bphA1 of Pseudomonas pseudoalcaligenes KF707 and Burkholderia cepacia LB400, a number of evolved Bph Dox enzymes were created. Among them, an Escherichia coli clone expressing chimeric Bph Dox exhibited extremely enhanced benzene-, toluene-, and alkylbenzene-degrading abilities. In this evolved BphA1, four amino acids (H255Q, V258I, G268A, and F277Y) were changed from the KF707 enzyme to those of the LB400 enzyme. Subsequent site-directed mutagenesis allowed us to determine the amino acids responsible for the degradation of monocyclic aromatic hydrocarbons. (+info)
Genetic and functional analysis of the tbc operons for catabolism of alkyl- and chloroaromatic compounds in Burkholderia sp. strain JS150.
(50/773)
Burkholderia sp. strain JS150 is able to metabolize a wide range of alkyl-and chloroaromatic hydrocarbons through multiple, apparently redundant catabolic pathways. Previous research has shown that strain JS150 is able to synthesize enzymes for multiple upper pathways as well as multiple lower pathways to accommodate variously substituted catechols that result from degradation of complex mixtures of monoaromatic compounds. We report here the genetic organization and functional characterization of a gene cluster, designated tbc (for toluene, benzene, and chlorobenzene utilization), which has been cloned as a 14.3-kb DNA fragment from strain JS150 into vector pRO1727. The cloned DNA fragment expressed in Pseudomonas aeruginosa PAO1c allowed the recombinant to grow on toluene or benzene and to transform chlorobenzene, trichloroethylene, phenol, and cresols. The tbc genes are organized into two divergently transcribed operons, tbc1 and tbc2, each comprised of six open reading frames. Similarity searches of databases revealed that the tbc1 and tbc2 genes showed significant homology to multicomponent cresol and phenol hydroxylases and to toluene and benzene monooxygenases, respectively. Deletion mutagenesis and product analysis were used to demonstrate that tbc2 plays a role in the initial catabolism of the unactivated alkyl- or chloroaromatic substrate and that the tbc1 gene products play a role in the catabolism of the first metabolite that results from transformation of the initial substrate. Phylogenetic analysis was used to compare individual components of these tbc monooxygenases with similar sequences in the databases. These results provide further evidence for the existence of multiple, functionally redundant alkyl- and chloroaromatic monooxygenases in strain JS150. (+info)
Use of expert judgment in exposure assessment. Part I. Characterization of personal exposure to benzene.
(51/773)
This paper presents the results of the first phase of a study, conducted as an element of the National Human Exposure Assessment Survey (NHEXAS), to demonstrate the use of expert subjective judgment elicitation techniques to characterize the magnitude of and uncertainty in environmental exposure to benzene. In decisions about the value of exposure research or of regulatory controls, the characterization of uncertainty can play an influential role. Classical methods for characterizing uncertainty may be sufficient when adequate amounts of relevant data are available. Frequently, however, data are neither abundant nor directly relevant, making it necessary to rely to varying degrees on subjective judgment. Since the 1950s, methods to elicit and quantify subjective judgments have been explored but have rarely been applied to the field of environmental exposure assessment. In this phase of the project, seven experts in benzene exposure assessment were selected through a peer nomination process, participated in a 2-day workshop, and were interviewed individually to elicit their judgments about the distributions of residential ambient, residential indoor, and personal air benzene concentrations (6-day integrated average) experienced by both the non-smoking, non-occupationally exposed target and study populations of the US EPA Region V pilot study. Specifically, each expert was asked to characterize, in probabilistic form, the arithmetic means and the 90th percentiles of these distributions. This paper presents the experts' judgments about the concentrations of benzene encountered by the target population. The experts' judgments about levels of benzene in personal air were demonstrative of patterns observed in the judgments about the other distributions. They were in closest agreement about their predictions of the mean; with one exception, their best estimates of the mean fell within 7-11 microg/m(3) although they exhibited striking differences in the degree of uncertainty expressed. Their estimates of the 90th percentile were more varied with the best estimates ranging from 12 to 26 microg/m(3) for all but one expert. However, their predictions of the 90th percentile were far more uncertain. The paper demonstrates that coherent subjective judgments can be elicited from exposure assessment scientists and critically examines the challenges and potential benefits of a subjective judgment approach. The results of the second phase of the project, in which measurements from the NHEXAS field study in Region V are used to calibrate the experts' judgments about the benzene exposures in the study population, will be presented in a second paper. (+info)
Effect of benzene, toluene, xylene on the semen quality of exposed workers.
(52/773)
OBJECTIVE: To examine the effects on semen and sperm quality of workers after a short and long term exposure to benzene, toluene, and xylene. METHODS: The semen and blood of 24 married workers exposed to benzene, toluene, and xylene from shoemaking, spray painting, or paint manufacturing factories were collected. The concentration of benzene, toluene, and xylene in the blood and semen was determined by using headspace chromatographic method. Routine sperm test was carried out and acrosin activity detected. RESULTS: The results showed that benzene, toluene, and xylene were found in the blood and semen of some ex-workers in a working environment where the air concentration of benzene, toluene, and xylene exceeded the maximum allowable concentration (MAC). This result was not found in workers of the control group. There were also some effects on the quality of semen in the exposed workers. For example, the percentage of semen with liquefaction time exceeding 30 minutes increased. The sperm vitality, motility and acrosin activity decreased. At the same time, there were a positive correlation between liquefaction time and the level of toluene in semen, and a negative correlation between sperm vitality, sperm activity or acrosin activity and working history. CONCLUSIONS: The results suggested that the mixture could affect the quality of semen and sperm, which might be the main reason of the abnormal pregnancy outcome among the wives of workers exposed to benzene, toluene, and xylene. Further studies are, however, required to confirm these findings. (+info)
Mechanism of ATP-driven electron transfer catalyzed by the benzene ring-reducing enzyme benzoyl-CoA reductase.
(53/773)
Benzoyl-CoA reductase (BCR) from the bacterium Thauera aromatica catalyzes the two-electron reduction of benzoyl-CoA (BCoA) to a nonaromatic cyclic diene. In a process analogous to enzymatic nitrogen reduction, BCR couples the electron transfer to the aromatic ring to a stoichiometric hydrolysis of 2 ATP/2e(-). Reduced but not oxidized BCR hydrolyzes ATP to ADP. In this work, purified BCR was shown to catalyze an isotope exchange from [(14)C]ADP to [(14)C]ATP, which was approximately 15% of the ATPase activity in the presence of equimolar amounts of ADP and ATP. In accordance, BCR (alpha beta gamma delta-composition) autophosphorylated its gamma-subunit when incubated with [gamma-(32)P]ATP. Formation of the enzyme-phosphate was independent of the redox state, whereas only dithionite-reduced BCR catalyzed a dephosphorylation associated with the ATPase activity. This finding suggests that the ATPase- and autophosphatase-partial activities of BCR exhibit identical redox dependencies. BCoA or the nonphysiological electron-accepting substrate hydroxylamine stimulated the redox-dependent effects; the rates of both the overall ATPase and the autophosphatase activities of reduced BCR were increased 6-fold. In contrast, BCoA and hydroxylamine had no effect on oxidized and phosphorylated BCR. The reactivity of the phosphoamino acid fits best with a phosphoamidate or acylphosphate linkage. The results obtained suggest a mechanism of ATP hydrolysis-driven electron transfer, which differs from that of nitrogenase by the transient formation of a phosphorylated enzyme. (+info)
Determination of benzene, toluene, ethylbenzene and xylenes by headspace spectrophotometry with an atomic absorption apparatus.
(54/773)
In this study an atomic absorption spectrophotometer equipped with a selenium hollow-cathode lamp was used for analysis of BTEX (benzene, toluene, ethylbenzene and xylenes) in headspace of aqueous solutions. Initially effective factors on headspace such as volume of solution, stirring time, stirring speed, velocity of carrier gas, temperature, number of strippings, addition of salts and salt concentration were investigated and optimum conditions were selected. By addition of salt in different concentrations, different absorbances were obtained for headspace, therefore, binary mixtures of BTEX were analyzed with simultaneous equations. Obtained results agreed with actual amounts and repeatability was very good (RSD% < 3). Correlation coefficients (r) for calibration curves were about 0.999. This proposed method is comparable with absorbance determination of solution with respect to correlation coefficient, linear dynamic range, limit of detection (LOD) and relative standard deviation (RSD), but this method is less susceptible to interferences and more selective. (+info)
Determination of benzene and toluene in blood by means of a syringe-equilibration method using a small amount of blood.
(55/773)
A gas chromatographic determination of benzene and toluene in blood with a small amount of blood sample, 0.02 or 0.1 ml, is described. In the method an aliquot of the blood sample in a sealed hypodermic syringe of 2 ml capacity is equilibrated at 37 degrees C in a thermo-regulated water-bath. After establishing equilibrium 1 ml of overlying air is submitted to gas chromatographic analysis. The value of this method was verified by experiments in which men, rabbits, and rats were exposed to benzene and toluene mixtures of various concentrations. (+info)
The treatment of purified maize oil bodies with organic solvents and exogenous diacylglycerol allows the detection and solubilization of diacylglycerol acyltransferase.
(56/773)
In spite of its importance in the biosynthesis of reserve oils in plants, diacylglycerol acyltransferase (DAGAT, EC 2.3.1.20) has not been purified to homogeneity, and its study has remained incomplete. We found that the microsomal preparations from developing maize embryos contained substantial amounts of endogenous diacylglycerol (DAG). A solubilization procedure for extracting DAGAT from the microsomes (D. Little, R. Weselake, K. Pomeroy, S.T. Furukawa, J. Bagu, Biochem. J. 304 (1994)) was ineffective in eliminating the endogenous DAG, even after gel filtration. DAG removal through the preparation of acetone powders from the embryos led to the loss of DAGAT activity. Labelled triacylglycerol (TAG) was produced in the standard DAGAT assay when labelled DAG was supplied in benzene solution to the freeze-dried microsomes and the sample was dried and resuspended in an aqueous buffer. In contrast, no labelled TAG was produced when a similar sample supplied with non-labelled DAG was assayed with emulsified labelled DAG and acyl-CoA. Repeated washing of the microsomal freeze-dried fraction with benzene resulted in a complete loss of DAGAT activity in the standard assay, but the activity was restored by the addition of DAG plus phosphatidylcholine or Tween 20 in benzene. Although DAGAT has been reported to be confined mainly to the endoplasmic reticulum, we found that DAGAT activity was high in the purified oil bodies from both developing and mature maize embryos and was not removed by repeated washing with 6 M urea. The DAGAT activity was restored from delipidated oil bodies and from microsomes after the preparations had been resuspended in methanol/acetic acid/water (1:1:1, v/v). Although most of the proteins in the suspension were eluted as a single peak at the void volume after gel filtration chromatography, DAGAT activity was found in later fractions. SDS-PAGE of the peak activity fraction revealed no protein bands after silver staining, and the finding suggest that DAGAT protein is of low abundance and has a high k(cat). (+info)