Purification and characterization of NADPH-dependent Cr(VI) reductase from Escherichia coli ATCC 33456

A soluble Cr(VI) reductase was purified from the cytoplasm of Escherichia coli ATCC 33456. The molecular mass was estimated to be 84 and 42 kDa by gel filtration and SDS-polyacrylamide gel electrophoresis, respectively, indicating a dimeric structure. The pI was 4.66, and optimal enzyme activity was obtained at pH 6.5 and 37 degrees C. The most stable condition existed at pH 7.0. The purified enzyme used both NADPH and NADH as electron donors for Cr(VI) reduction, while NADPH was the better, conferring 61%; higher activity than NADH. The Km values for NADPH and NADH were determined to be 47.5 and 17.2 micromol, and the Vmax values 322.2 and 130.7 micromol Cr(VI) min(-1)mg(-1) protein, respectively. The activity was strongly inhibited by N-ethylmalemide, Ag2+, Cd2+, Hg2+, and Zn2+. The antibody against the enzyme showed no immunological cross reaction with those of other Cr(VI) reducing strains.     

Evaluation of in vitro Cr(VI) reduction potential in cytosolic extracts of three indigenous Bacillus sp. isolated from Cr(VI) polluted industrial landfill

Three efficient Cr(VI) reducing bacterial strains were isolated from Cr(VI) polluted landfill and characterized for in vitro Cr(VI) reduction. Phylogenetic analysis using 16S rRNA gene sequencing revealed that the newly isolated strains G1DM20, G1DM22 and G1DM64 were closely related to Bacillus cereus, Bacillus fusiformis and Bacillus sphaericus, respectively. The suspended cultures of all Bacillus sp. exhibited more than 85% reduction of 1000 microM Cr(VI) within 30 h. The suspended culture of Bacillus sp. G1DM22 exhibited an ability for continuous reduction of 100 microM Cr(VI) up to seven consecutive inputs. Assays with the permeabilized cells and cell-free extracts from each of Bacillus sp. demonstrated that the hexavalent chromate reductase activity was mainly associated with the soluble fraction of cells and expressed constitutively. The Cr(VI) reduction by the cell-free extracts of Bacillus sp. G1DM20 and G1DM22 was maximum at 30 degrees C and pH 7 whereas, Bacillus sp. G1DM64 exhibited maximum Cr(VI) reduction at pH 6. Addition of 1mM NADH enhanced the Cr(VI) reductase activity in the cell-free extracts of all three isolates. Amongst all three isolates tested, crude cell-free extracts of Bacillus sp. G1DM22 exhibited the fastest Cr(VI) reduction rate with complete reduction of 100 microM Cr(VI) within 100 min. The apparent K(m) and V(max) of the chromate reductase activity in Bacillus sp. G1DM22 were determined to be 200 microM Cr(VI) and 5.5 micromol/min/mg protein, respectively. The Cr(VI) reductase activity in cell-free extracts of all the isolates was stable in presence of different metal ions tested except Hg(2+) and Ag(+).     

A membrane-associated protein with Cr(VI)-reducing activity from Thermus scotoductus SA-01

A membrane-associated chromate reductase from Thermus scotoductus SA-01 has been purified to apparent homogeneity and shown to couple the reduction of Cr(VI) to NAD(P)H oxidation, with a preference towards NADH. The chromate reductase is a homodimer with a monomeric molecular weight of 48 kDa and a noncovalently bound FAD coenzyme. The enzyme is optimally active at a pH of 6.5 and 65 degrees C with a K(m) of 55.5+/-4.2 microM and a V(max) of 2.3+/-0.1 micromol Cr(VI) min(-1) mg(-1) protein. The catalytic efficiency (k(cat)/K(m)) of the enzyme was found to be comparable to that found for quinone reductases but more efficient than the nitroreductases. N-terminal sequencing and subsequent screening of a genomic library of T. scotoductus revealed an ORF of 1386 bp, homologous (84%) to the dihydrolipoamide dehydrogenase gene of Thermus thermophilus HB8. These results extend the knowledge of chromate reductases mediating Cr(VI) reduction via noncovalently bound or free redox-active flavin groups and the activity of dihydrolipoamide dehydrogenases towards physiologically unrelated substrates.     

Hexavalent chromate reduction by alkaliphilic Amphibacillus sp. KSUCr3 is mediated by copper-dependent membrane-associated Cr(VI) reductase

The present study was aimed to localize and characterize hexavalent chromate [Cr(VI)] reductase activity of the extreme alkaliphilic Amphibacillus sp. KSUCr3 (optimal growth pH 10.5). The resting cells were able to reduce about 62 % of the toxic heavy metal Cr(VI) at initial concentration of 200 μM within 30 min. Cell permeabilization resulted in decrease of Cr(VI) reduction in comparison to untreated cells. Enzymatic assays of different sub-cellular fractions of Amphibacillus sp. KSUCr3 demonstrated that the Cr(VI) reductase was mainly associated with the membranous fraction and expressed constitutively. In vitro studies of the crude enzyme indicated that copper ion was essential for Cr(VI) reductase activity. In addition, Ca2? and Mn2? slightly stimulated the chromate reductase activity. Glucose was the best external electron donor, showing enhancement of the enzyme activity by about 3.5-fold. The K (m) and V (max) determined for chromate reductase activity in the membranous fraction were 23.8 μM Cr(VI) and 72 μmol/min/mg of protein, respectively. Cr(VI) reductase activity was maximum at 40 °C and pH 7.0 and it was significantly inhibited in the presence of disulfide reducers (2-mercaptoethanol), ion chelating agent (EDTA), and respiratory inhibitors (CN and Azide). Complete reduction of 100 and 200 μM of Cr(VI) by membrane associated enzyme were observed within 40 and 180 min, respectively. However, it should be noted that biochemical characterization has been done with crude enzyme only, and that final conclusion can only be drawn with the purified enzyme.     

Deferoxamine inhibition of Cr(V)-mediated radical generation and deoxyguanine hydroxylation: ESR and HPLC evidence.

Electron spin resonance (ESR) and high-performance liquid chromatography (HPLC) techniques were utilized to investigate the effect of deferoxamine on free radical generation in the reaction of Cr(V) with H2O2 and organic hydroperoxides. ESR measurements demonstrated that deferoxamine can efficiently reduce the concentration of the Cr(V) intermediate as formed in the reduction of Cr(VI) by NAD(P)H or a flavoenzyme glutathione reductase/NADH. ESR spin trapping studies showed that deferoxamine also inhibits Cr(V)-mediated .OH radical generation from H2O2, as well as Cr(V)-mediated alkyl and alkoxy radical formation from t-butyl hydroperoxide and cumene hydroperoxide. HPLC measurements showed that .OH radicals generated by the Cr(VI)/flavoenzyme/NAD(P)H enzymatic system react with 2'-deoxyguanine to form 8-hydroxy-2'-deoxyguanine (8-OHdG), a DNA damage marker. Deferoxamine effectly inhibited the formation of 8-OHdG also.     

Chromium(VI) reductase activity is associated with the cytoplasmic membrane of anaerobically grown Shewanella putrefaciens MR-1

C.R. MYERS, B.P. CARSTENS, W.E. ANTHOLINE and J.M. MYERS.2000. Shewanella putrefaciens MR-1 can reduce a diverse array of compounds under anaerobic conditions, including manganese and iron oxides, fumarate, nitrate, and many other compounds. These reductive processes are apparently linked to a complex electron transport system. Chromium (Cr) is a toxic and mutagenic metal and bacteria could potentially be utilized to immobilize Cr by reducing the soluble and bioavailable state, Cr(VI), to the insoluble and less bioavailable state, Cr(III). Formate-dependent Cr(VI) reductase activity was detected in anaerobically grown cells of S. putrefaciens MR-1, with highest specific activity in the cytoplasmic membrane. Both formate and NADH served as electron donors for Cr(VI) reductase, whereas l -lactate or NADPH did not support any activity. The addition of 10 μmol l⁻¹ FMN markedly stimulated formate-dependent Cr(VI) reductase, and the activity was almost completely inhibited by diphenyliodonium chloride, an inhibitor of flavoproteins. Cr(VI) reductase activity was also inhibited by p -chloromercuriphenylsulphonate, azide, 2-heptyl-4-hydroxyquinolone- N -oxide, and antimycin A, suggesting involvement of a multi-component electron transport chain which could include cytochromes and quinones. Cr(V) was detected by electron paramagnetic resonance (EPR) spectroscopy, suggesting a one-electron reduction as the first step.     

Addition of DNA to Cr(VI) and cytochrome b5 containing proteoliposomes leads to generation of DNA strand breaks and Cr(III) complexes

Chromium (Cr) is a cytotoxic metal that can be associated with a variety of types of DNA damage, including Cr-DNA adducts and strand breaks. Prior studies with purified human cytochrome b(5) and NADPH:P450 reductase in reconstituted proteoliposomes (PLs) demonstrated rapid reduction of Cr(VI) (hexavalent chromium, as CrO(4)(2-), and the generation of Cr(V), superoxide (O(2)(*-)), and hydroxyl radical (HO(*)). Studies reported here examined the potential for the species produced by this system to interact with DNA. Strand breaks of purified plasmid DNA increased over time aerobically, but were not observed in the absence of O(2). Cr(V) is formed under both conditions, so the breaks are not mediated directly by Cr(V). The aerobic strand breaks were significantly prevented by catalase and EtOH, but not by the metal chelator diethylenetriaminepentaacetic acid (DTPA), suggesting that they are largely due to HO(*) from Cr-mediated redox cycling. EPR was used to assess the formation of Cr-DNA complexes. Following a 10-min incubation of PLs, CrO(4)(2-), and plasmid DNA, intense EPR signals at g=5.7 and g=5.0 were observed. These signals are attributed to specific Cr(III) complexes with large zero field splitting (ZFS). Without DNA, the signals in the g=5 region were weak. The large ZFS signals were not seen, when Cr(III)Cl(3) was incubated with DNA, suggesting that the Cr(III)-DNA interactions are different when generated by the PLs. After 24 h, a broad signal at g=2 is attributed to Cr(III) complexes with a small ZFS. This g=2 signal was observed without DNA, but it was different from that seen with plasmid. It is concluded that EPR can detect specific Cr(III) complexes that depend on the presence of plasmid DNA and the manner in which the Cr(III) is formed.     

Role of insulin in Cr(VI)-mediated genotoxicity in Neurospora crassa

Aims: Chromium (III) is an insulinomimetic agent whose biological and/or environmental availability is frequently in the form of Cr(VI), which is known to be toxic. Wall‐less mutant of Neurospora crassa (FGSC stock no. 4761) is known to possess insulin receptor in its cell membrane and hence is a good model for Cr toxicity studies. This study explores the toxicity of Cr(VI) and the possible consequences on simultaneous exposure to insulin in N. crassa. Methods and Results: Comet assay of N. crassa cells treated with 100 μmol l^?1 Cr(VI) showed up to 50% reduction in comet tail lengths when incubated simultaneously with 0·4 U insulin. Fluorescence measurement in Cr(VI)‐treated cells using DCFH‐DA showed six‐ to eightfold increase in free radical generation, which was reduced to fourfold by 0·4 U insulin. Annexin‐V/PI Flow cytometry analysis indicated necrotic cell death up to 28·7 ± 3·6% and 68·6 ± 2·5% on Cr(VI) exposure at concentrations 100 and 500 μmol l^?1 which was reduced by 68·3 ± 3·2% and 48·9 ± 3·6%, respectively, upon addition of insulin. Conclusion: Insulin‐mediated protection from DNA damage by Cr(VI) is because of scavenging of free radicals liberated during exposure to Cr(VI). Significance and Impact of the Study: Overall, Cr(VI) toxicity depends upon available insulin, indicating that Cr(VI) toxicity may be a serious issue in insulin‐deficient individuals with diabetes.     

Reduction of chromium(VI) by D-galacturonic acid and formation of stable chromium(V) intermediates

The interaction of dichromate with D-galacturonic acid in aqueous solution, as a function of pH, is described. The reaction involves the reduction of Cr(VI) to Cr(III), but the reaction rate is remarkably dependent on pH. In fact, the reduction of Cr(VI) to Cr(III) proceeds rather quickly in strongly acidic solutions, while it is slow in neutral or moderately acidic media. In all cases, according to the ESR evidence, Cr(V) species are found as intermediates. The stability of the Cr(V) species increases with increasing pH, so that it may be suggested that the overall reaction rate is controlled by the Cr(V) to Cr(III) conversion.     

耐盐菌Staphylococcus sp. YZ-1和Bacillus cereus CC-1的Cr(VI)脱毒特性与机理

[背景]高盐含铬废水的去除过程中,Cr(Ⅵ)还原菌是研究者关注的重点,但目前对耐盐菌株的Cr(Ⅵ)脱毒特性及机理的分析仍较少。[目的]比较两株耐盐菌株的Cr(Ⅵ)移除特性,并区分Cr(Ⅵ)耐受机制的差异;通过基因组测序分析,从基因层面推测铬耐受相关基因;构建铬还原菌的混菌体系,考察两者对去除污染物的协同作用。[方法]从青海茶卡盐湖分离耐盐菌Staphylococcus sp.YZ-1,与Bacillus cereus CC-1进行基础特性和Cr(Ⅵ)去除性能的比较,并通过全基因组序列的分析验证特性测试的结果。[结果]两株菌都具有铬移除特性,但CC-1的铬移除效率更高,在初始Cr(Ⅵ)浓度为0.1 mmol/L情况下,CC-1能在12h内移除95.3%的Cr(Ⅵ),而YZ-1只能移除40.1%。在进一步实验中发现YZ-1只能对Cr(Ⅵ)进行还原,将其转化为可溶的有机态Cr(Ⅲ),而CC-1能同时对Cr(Ⅵ)进行还原和吸附。全基因组分析发现YZ-1具有编码外排泵蛋白的基因和编码NAD(P)H氧化还原酶的基因,而CC-1具有编码铬转运蛋白ChrA和细胞色素C氧化还原酶的基因。两株菌的混菌体系在处理含Cr(Ⅵ)、Te(Ⅳ)的废水时,菌群能将还原产物聚集成团并沉淀到底部。[结论]菌株YZ-1和CC-1均为耐盐铬还原菌,但YZ-1中的铬还原酶为诱导型酶,CC-1则为组成型酶。基因组数据分析鉴别出两者可能同时存在多种铬耐受机制相关编码基因。混合菌群可以结合YZ-1的自絮凝特性和两者均有的Te(Ⅳ)/Cr(Ⅵ)还原活性,具有潜在的实用价值。     

Environmental and Kinetic Parameters for Cr(VI) Bioreduction by a Bacterial Monoculture Purified from Cr(VI)-Resistant Consortium

Hexavalent chromium, Cr(VI), is toxic to living systems. Widespread contamination of water and soil by Cr(VI) present a serious public health problem. Chromium-resistant bacteria can reduce and detoxify Cr(VI). Twelve bacteria resistant to high concentrations of Cr(VI) were isolated from soil enrichment cultures. Environmental parameters and kinetic parameters of Cr(VI) bioreduction by one monoculture isolate, identified by 16S rRNA gene sequence as Bacillus sp. PB2, were studied. The optimal temperature for growth and Cr(VI) reduction was 35 degrees C. The isolate grew luxuriantly and substantially reduced Cr(VI) at initial pH 7.5 to 9. Maximal Cr(VI) bioreduction occurred at initial pH 8.0. Substantial Cr(VI) bioreduction was observed in salt media, but removal efficiency was inversely related to salt concentration (1-9%). Michaelis-Menten hyperbolic equation and the Lineweaver-Burk double reciprocal plot were comparatively employed to determine the k (m) and V (max) of Cr(VI) bioreduction. A k (m) of 82.5 microg mL(-1) and V (max) of 7.78 microg mL(-1) h(-1) were calculated by nonlinear regression analysis of the hyperbola curve. Linear regression analysis of the double reciprocal plot revealed k (m) and V (max) of 80.9 microg mL(-1) and 10.6 microg mL(-1) h(-1), respectively. Time course studies displayed about 90% reduction of Cr(VI) at an initial concentration of 8,000 microg L(-1) in 8 h, with an estimated t (1/2) of 4 h. Data from time course analysis of the rate of Cr(VI) bioreduction fitted zero-order model, and the kinetic constant k was calculated to be 840 microg L(-1) h(-1). The monoculture isolate, Bacillus sp. PB2, strongly reduces Cr(VI) and could be used for bioremediation of Cr(VI)-contaminated aquatic and terrestrial environments.     

Chromium(VI) resistance and removal by <Emphasis Type="Italic">Acinetobacter haemolyticus</Emphasis>

The present work highlighted the studies on Cr(VI) reduction by cells of Acinetobacter haemolyticus (A. haemolyticus). The strain tolerated 90 mg Cr(VI) l⁻¹ in LB broth compared to only 30 mg Cr(VI) l⁻¹ in LB agar. From the FTIR analysis, the Cr(III) species formed was also most likely to form complexes with carboxyl, hydroxyl, and amide groups from the bacteria. A TEM study showed the absence of precipitates on the cell wall region of the bacteria. Instead, microprecipitates were observed in the cytoplasmic region of the cells, suggesting the transportation of Cr(VI) into the cells. Intracellular reduction of Cr(VI) was supported by a reductase test using soluble crude cell-free extracts. The specific reductase activity obtained was 0.52 μg Cr(VI) reduced per mg of protein an hour at pH 7.2 and 37°C. Our results indicated that A. haemolyticus can be used as a promising microorganism for Cr(VI) reduction from industrial wastewaters.     

Nonmetabolic Reduction of Cr(VI) by Bacterial Surfaces Under Nutrient-Absent Conditions

We have measured the ability of nonmetabolizing cells of the bacterial species Bacillus subtilis, Sporosarcina ureae , and Shewanella putrefaciens to reduce aqueous Cr(VI) to Cr(III) in the absence of externally supplied electron donors. Each species can remove significant amounts of Cr(VI) from solution, and the Cr(VI) reduction rate is strongly dependent on solution pH. The fastest reduction rates occur under acidic conditions, with decreasing rates with increasing pH. XANES data demonstrate that Cr(VI) reduction to Cr(III) occurs within the experimental systems. Control experiments indicate that the Cr removal is not a purely adsorptive process. Reduction appears to occur at the cell wall, and is not coupled to the oxidation of bacterial organic exudates. Detailed kinetic data suggest that the reduction involves at least a two-stage process, involving an initial rapid removal mechanism followed by a slower process that follows first-order reaction kinetics. Due to the prevalence of nonmetabolizing cells and cell wall fragments in soils and deeper geologic environments, our results suggest that the observed nonmetabolic reduction of Cr(VI) to Cr(III) may significantly affect the environmental distribution of Cr in bacteria-bearing systems.     

Global transcriptional profiling of Shewanella oneidensis MR-1 during Cr(VI) and U(VI) reduction

Whole-genome DNA microarrays were used to examine the gene expression profile of Shewanella oneidensis MR-1 during U(VI) and Cr(VI) reduction. The same control, cells pregrown with nitrate and incubated with no electron acceptor, was used for the two time points considered and for both metals. U(VI)-reducing conditions resulted in the upregulation (> or = 3-fold) of 121 genes, while 83 genes were upregulated under Cr(VI)-reducing conditions. A large fraction of the genes upregulated [34% for U(VI) and 29% for Cr(VI)] encode hypothetical proteins of unknown function. Genes encoding proteins known to reduce alternative electron acceptors [fumarate, dimethyl sulfoxide, Mn(IV), or soluble Fe(III)] were upregulated under both U(VI)- and Cr(VI)-reducing conditions. The involvement of these upregulated genes in the reduction of U(VI) and Cr(VI) was tested using mutants lacking one or several of the gene products. Mutant testing confirmed the involvement of several genes in the reduction of both metals: mtrA, mtrB, mtrC, and menC, all of which are involved in Fe(III) citrate reduction by MR-1. Genes encoding efflux pumps were upregulated under Cr(VI)- but not under U(VI)-reducing conditions. Genes encoding proteins associated with general (e.g., groL and dnaJ) and membrane (e.g., pspBC) stress were also upregulated, particularly under U(VI)-reducing conditions, pointing to membrane damage by the solid-phase reduced U(IV) and Cr(III) and/or the direct effect of the oxidized forms of the metals. This study sheds light on the multifaceted response of MR-1 to U(VI) and Cr(VI) under anaerobic conditions and suggests that the same electron transport pathway can be used for more than one electron acceptor.     

Aerobic Cr(VI) reduction by Thermus scotoductus strain SA-01

AIM: To evaluate Thermus scotoductus SA-01's ability to reduce Cr(VI) aerobically. METHODS AND RESULTS: T. scotoductus SA-01 is able to reduce Cr(VI) aerobically when grown in a complex organic medium containing Cr(VI) concentrations up to 0.5 mmol l(-1). Suspension of T. scotoductus SA-01 cells also reduced Cr(VI) aerobically under nongrowth conditions using a variety of electron donors as well as in the absence of an exogenous electron donor. The optimum temperature and pH for Cr(VI) reduction under nongrowth conditions were found to be 80 degrees C and 7, respectively. It was also found that the Cr(VI) reduction was catalysed by a cytoplasmic, constitutively expressed enzyme. CONCLUSIONS: Apart from SA-01's ability to reduce Cr(VI) through a strictly anaerobic membrane-bound mechanism (unpublished data), it also has a second enzyme localized in the cytoplasm that can reduce Cr(VI) aerobically. As this enzyme is constitutively expressed and not induced by Cr(VI), it remains to be determined whether it has any other physiological functions. SIGNIFICANCE AND IMPACT OF THE Study: This is the first report of a Thermus species able to reduce Cr(VI) aerobically and extends the knowledge of parameters associated with Cr(VI) reduction. Employing thermophiles in bioremediation using industrial bioreactors would cancel the need for expensive cooling systems.     

Reduction of Cr(VI) by a Bacillus sp.

A Bacillus sp. RE was resistant to chromium and reduced Cr(VI) without accumulating chromium inside the cell. When Cr(VI) was 10 and 40 μg ml⁻¹, >95% of the total Cr(VI) was reduced in 24 and 72 h of growth, respectively, whereas at 80 μg Cr(VI) ml⁻¹ only 50% of Cr(VI) was reduced. However growth was not affected; the cell mass was 0.7–0.8 mg ml⁻¹ in all cases. The cell-free extract showed Cr(VI) reducing enzyme activity which was enhanced (>5 fold) by NADH and NADPH. Like whole cells the enzyme also reduced Cr(VI) with decreasing efficiency on increasing Cr(VI) concentration. The enzyme activity was optimal at pH 6.0 and 30 °C. The enzyme was stable up to 30 °C and from pH 5.5 to 8, but from pH 4 to 5 the enzyme was severely destabilized. Its K_m and V_max were 14 μm and 3.8 nmol min⁻¹ mg⁻¹ respectively. The enzyme activity was enhanced by Cu²⁺ and Ni²⁺ and inhibited by Hg²⁺. Received 21 September 2005; Revisions requested 5 October 2005; Revisions received 16 November 2005; Accepted 16 November 2005     

Speciation dependant antioxidative response in roots and leaves of sorghum (Sorghum bicolor (L.) Moench cv CO 27) under Cr(III) and Cr(VI) stress

Growth, lipid peroxidation, H₂O₂ produciton and the response of the antioxidant enzymes and metabolites of the ascorbate glutathione pathway to oxidative stress caused by two concentrations (50 and 100 µM) of Cr(III) and Cr(VI) was studied in 15 day old seedlings of sorghum (Sorghum bicolor (L.) Moench cv CO 27) after 10 days of treatment. Cr accumulation in sorghum plants was concentration and organ dependant. There was no significant growth retardation of plants under 50 µM Cr(III) stress. 100 µM Cr(VI) was most toxic of all the treatments in terms of root and leaf growth and oxidative stress. 50 µM Cr(VI) treated roots exhibited high significant increase in superoxide dismutase (SOD), dehydroascorbate reductase (DHAR) and glutathione reductase (GR) (p < 0.01) and significant increases in catalse (CAT), ascorbate peroxidase (APX) and monodehydroascorbate reductase (MDHAR) (p < 0.05). A high increase in ascorbic acid (AA) level was seen in roots of 50 µM Cr(VI) treated plants in comparison with control. Levels of reduced glutathione (GSH) showed a varied and complex response in all the treatments in both plant parts. GSH/GSSG ratio was not affected by Cr(III) treatment in leaves, in contrast, roots exhibited significant reduction in the ratio. Results indicate that GSH depletion increased sensitivity to oxidative stress (Cr(VI) roots and leaves and Cr(III) 100 µM roots) and AA in tandem with APX compensated for GSH depletion by acting directly on H₂O₂ and the mechanism of defensive response in roots as well as leaves varied in its degree and effectiveness due to the concentration dependant differences observed in translocation of the element itself, reactive oxygen species (ROS) generation and enzyme inhibition based on the oxidation state supplied to the plants.     

Cr(VI) reduction by Enterobacter sp. DU17 isolated from the tannery waste dump site and characterization of the bacterium and the Cr(VI) reductase

Out of nineteen bacteria screened from the tannery waste dump site, the most effective isolate, strain DU17 was selected for Cr(VI) reduction process among the non-pathogenic once. Based on 16S rRNA gene sequence analysis, the bacterium was identified as Enterobacter sp. DU17. Its amplified Cr(VI) reductase gene showed maximum homology with flavoprotein of Enterobacter cloacae. Enterobacter sp. DU17 reduced Cr(VI) maximally at 37 °C and pH 7.0. Various co-metals, electron (e⁻) donors and inhibitors were tested to study their effect on Cr(VI) reduction. In presence (0.2% each) of glucose and fructose, Enterobacter sp. DU17 reduced Cr(VI) completely after 16 and 20 h, respectively. Since the concentration of total Cr was invariable after remediation as detected through AAS analysis, this experiment disclosed that responsible operation was associated with extracellular Cr(VI) reduction process rather than uptake mechanism. Multiple antibiotic resistance index of 0.08 for this bacterium was very low as compared to standard risk assessment value of 0.20. With high Cr(VI) reducing capability, non-pathogenicity and antibiotic sensitivity, Enterobacter sp. DU17 is found to be very efficient in removing Cr(VI) toxicity from the environment.     

Chromium (V) and hydroxyl radical formation during the glutathione reductase-catalyzed reduction of chromium (VI)

Electron spin resonance measurements provide evidence for the formation of long-lived Cr(V) intermediates in the reduction of Cr(VI) by glutathione reductase in the presence of NADPH and for the hydroxyl radical formation during the glutathione reductase catalyzed reduction of Cr(VI). Hydrogen peroxide suppresses Cr(V) and enhances the formation of hydroxyl radicals. Thus Cr(V) intermediates catalyze generation of hydroxyl radicals from hydrogen peroxide through a Fenton-like reaction. Thus the mechanism of Cr(VI) toxicity might involve the interaction between macromolecules and the hydroxyl radicals.     

A novel chromate reductase from Thermus scotoductus SA-01 related to old yellow enzyme

Bacteria can reduce toxic and carcinogenic Cr(VI) to insoluble and less toxic Cr(III). Thermus scotoductus SA-01, a South African gold mine isolate, has been shown to be able to reduce a variety of metals, including Cr(VI). Here we report the purification to homogeneity and characterization of a novel chromate reductase. The oxidoreductase is a homodimeric protein, with a monomer molecular mass of approximately 36 kDa, containing a noncovalently bound flavin mononucleotide cofactor. The chromate reductase is optimally active at a pH of 6.3 and at 65 degrees C and requires Ca(2+) or Mg(2+) for activity. Enzyme activity was also dependent on NADH or NADPH, with a preference for NADPH, coupling the oxidation of approximately 2 and 1.5 mol NAD(P)H to the reduction of 1 mol Cr(VI) under aerobic and anaerobic conditions, respectively. The K(m) values for Cr(VI) reduction were 3.5 and 8.4 microM for utilizing NADH and NADPH as electron donors, respectively, with corresponding V(max) values of 6.2 and 16.0 micromol min(-1) mg(-1). The catalytic efficiency (k(cat)/K(m)) of chromate reduction was 1.14 x 10(6) M(-1) s(-1), which was >50-fold more efficient than that of the quinone reductases and >180-fold more efficient than that of the nitroreductases able to reduce Cr(VI). The chromate reductase was identified to be encoded by an open reading frame of 1,050 bp, encoding a single protein of 38 kDa under the regulation of an Escherichia coli sigma(70)-like promoter. Sequence analysis shows the chromate reductase to be related to the old yellow enzyme family, in particular the xenobiotic reductases involved in the oxidative stress response.