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(+).     

Chromate reduction by cell-free extract of Bacillus firmus KUCr1

Microbial enzymatic reduction of a toxic form of chromium [Cr(VI)] has been considered as an effective method for bioremediation of this metal. This study reports on the in vitro reduction of Cr(VI) using cell-free extracts from a Cr(VI) reducing Bacillus firmus KUCr1 strain. Chromium reductase was found to be constitutive and its activity was observed both in soluble cell fractions (S12 and S150 and membrane cell fraction (P150). The reductase activity of S12 fraction was found to be optimal at 40 microM Cr(VI) with enzyme concentration equivalent to 0.493 mg protein/ml. Enzyme activity was dependent on NADH or NADPH as electron donor; optimal temperature and pH for better enzyme activity were 70 degrees C and 5.6, respectively. The Km value of the reductase was 58.33 microM chromate having a V(max) of 11.42 microM/min/mg protein. The metabolic inhibitor like sodium azide inhibited reductase activity of membrane fraction of the cell-free extract. Metal ions like Cu2+, Co2+, Ni2+ and As3+ stimulated the enzyme but others, such as Ag+, Hg2+, Zn2+, Mn2+, Cd2+ and Pb2+, inhibited Cr(VI) reductase activity.     

Chromate reduction by a pseudomonad isolated from a site contaminated with chromated copper arsenate

A pseudomonad (CRB5) isolated from a decommissioned wood preservation site reduced toxic chromate [Cr(VI)] to an insoluble Cr(III) precipitate under aerobic and anaerobic conditions. CRB5 tolerated up to 520 mg of Cr(VI) liter(-1) and reduced chromate in the presence of copper and arsenate. Under anaerobic conditions it also reduced Co(III) and U(VI), partially internalizing each metal. Metal precipitates were also found on the surface of the outer membrane and (sometimes) on a capsule. The results showed that chromate reduction by CRB5 was mediated by a soluble enzyme that was largely contained in the cytoplasm but also found outside of the cells. The crude reductase activity in the soluble fraction showed a K(m) of 23 mg liter(-1) (437 microM) and a V(max) of 0.98 mg of Cr h(-1) mg of protein(-1) (317 nmol min(-1) mg of protein(-1)). Minor membrane-associated Cr(VI) reduction under anaerobiosis may account for anaerobic reduction of chromate under nongrowth conditions with an organic electron donor present. Chromate reduction under both aerobic and anaerobic conditions may be a detoxification strategy for the bacterium which could be exploited to bioremediate chromate-contaminated or other toxic heavy metal-contaminated environments.     

Purification to Homogeneity and Characterization of a Novel Pseudomonas putida Chromate Reductase

Cr(VI) (chromate) is a widespread environmental contaminant. Bacterial chromate reductases can convert soluble and toxic chromate to the insoluble and less toxic Cr(III). Bioremediation can therefore be effective in removing chromate from the environment, especially if the bacterial propensity for such removal is enhanced by genetic and biochemical engineering. To clone the chromate reductase-encoding gene, we purified to homogeneity (>600-fold purification) and characterized a novel soluble chromate reductase from Pseudomonas putida, using ammonium sulfate precipitation (55 to 70%), anion-exchange chromatography (DEAE Sepharose CL-6B), chromatofocusing (Polybuffer exchanger 94), and gel filtration (Superose 12 HR 10/30). The enzyme activity was dependent on NADH or NADPH; the temperature and pH optima for chromate reduction were 80°C and 5, respectively; and the K_m was 374 μM, with a V_max of 1.72 μmol/min/mg of protein. Sulfate inhibited the enzyme activity noncompetitively. The reductase activity remained virtually unaltered after 30 min of exposure to 50°C; even exposure to higher temperatures did not immediately inactivate the enzyme. X-ray absorption near-edge-structure spectra showed quantitative conversion of chromate to Cr(III) during the enzyme reaction.     

Partial Purification and Characterization of Chromate Reductase of a Novel Ochrobactrum sp. Strain Cr-B4

Hexavalent chromium contamination is a serious problem due to its high toxicity and carcinogenic effects on the biological systems. The enzymatic reduction of toxic Cr(VI) to the less toxic Cr(III) is an efficient technology for detoxification of Cr(VI)-contaminated industrial effluents. In this regard, a chromate reductase enzyme from a novel Ochrobactrum sp. strain Cr-B4, having the ability to detoxify Cr(VI) contaminated sites, has been partially purified and characterized. The molecular mass of this chromate reductase was found to be 31.53 kD, with a specific activity 14.26 U/mg without any addition of electron donors. The temperature and pH optima for chromate reductase activity were 40°C and 8.0, respectively. The activation energy (E_a) for the chromate reductase was found to be 34.7 kJ/mol up to 40°C and the activation energy for its deactivation (E_d) was found to be 79.6 kJ/mol over a temperature range of 50–80°C. The frequency factor for activation of chromate reductase was found to be 566.79 s^?1, and for deactivation of chromate reductase it was found to be 265.66 × 10³ s^?1. The reductase activity of this enzyme was affected by the presence of various heavy metals and complexing agents, some of which (ethylenediamine tetraacetic acid [EDTA], mercaptoethanol, NaN₃, Pb²⁺, Ni²⁺, Zn²⁺, and Cd²⁺) inhibited the enzyme activity, while metals like Cu²⁺ and Fe³⁺ significantly enhanced the reductase activity. The enzyme followed Michaelis–Menten kinetics with K_m of 104.29 µM and a V_max of 4.64 µM/min/mg.     

Proteogenomic and functional analysis of chromate reduction in Acidiphilium cryptum JF-5, an Fe(III)-respiring acidophile

Acidiphilium cryptum JF-5, an acidophilic iron-respiring Alphaproteobacterium, has the ability to reduce chromate under aerobic and anaerobic conditions, making it an intriguing and useful model organism for the study of extremophilic bacteria in bioremediation applications. Genome sequence annotation suggested two potential mechanisms of Cr(VI) reduction, namely, a number of c-type cytochromes, and a predicted NADPH-dependent Cr(VI) reductase. In laboratory studies using pure cultures of JF-5, an NADPH-dependent chromate reductase activity was detected primarily in soluble protein fractions, and a periplasmic c-type cytochrome (ApcA) was also present, representing two potential means of Cr(VI) reduction. Upon further examination, it was determined that the NADPH-dependent activity was not specific for Cr(VI), and the predicted proteins were not detected in Cr(VI)-grown cultures. Proteomic data did show measureable amounts of ApcA in cells grown with Cr(VI). Purified ApcA is reducible by menadiol, and in turn can reduce Cr(VI), suggesting a means to obtain electrons from the respiratory chain and divert them to Cr(VI). Electrochemical measurements confirm that Cr reduction by ApcA is pH dependent, with low pH being favored. Homology modeling of ApcA and comparison to a known Cr(VI)-reducing c-type cytochrome structure revealed basic amino acids which could interact with chromate ion. From these studies, it can be concluded that A. cryptum has the physiologic and genomic capability to reduce Cr(VI) to the less toxic Cr(III). However, the expected chromate reductase mechanism may not be the primary means of Cr(VI) reduction in this organism.     

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.     

Reduction of chromate by cell-free extract of Brucella sp. isolated from Cr(VI) contaminated sites

A locally isolated gram negative strain of Brucella sp., identified by biochemical methods and 16SrRNA analysis, reduced chromate to 100%, 94.1%, 93.2%, 66.9% and 41.6% at concentrations of 50, 100, 150, 200 and 300mgl(-1), respectively at pH 7 and temperature 37 degrees C. Increasing concentrations of Cr(VI) in the medium lowered the growth rate but could not be directly correlated with the amount of Cr(VI) reduced. The strain also exhibited multiple heavy metal (Ni,Zn,Hg,Pb,Co) tolerance and resistance to various antibiotics. Assay with crude cell-free extracts demonstrated that the hexavalent chromium reduction was mainly associated with the soluble fraction of the cell. High Cr(VI) concentration resistance and high Cr(VI) reducing ability of the strain make it a suitable candidate for bioremediation.     

Chromate Reduction by a Pseudomonad Isolated from a Site Contaminated with Chromated Copper Arsenate

A pseudomonad (CRB5) isolated from a decommissioned wood preservation site reduced toxic chromate [Cr(VI)] to an insoluble Cr(III) precipitate under aerobic and anaerobic conditions. CRB5 tolerated up to 520 mg of Cr(VI) liter⁻¹ and reduced chromate in the presence of copper and arsenate. Under anaerobic conditions it also reduced Co(III) and U(VI), partially internalizing each metal. Metal precipitates were also found on the surface of the outer membrane and (sometimes) on a capsule. The results showed that chromate reduction by CRB5 was mediated by a soluble enzyme that was largely contained in the cytoplasm but also found outside of the cells. The crude reductase activity in the soluble fraction showed a K_m of 23 mg liter⁻¹ (437 μM) and a V_max of 0.98 mg of Cr h⁻¹ mg of protein⁻¹ (317 nmol min⁻¹ mg of protein⁻¹). Minor membrane-associated Cr(VI) reduction under anaerobiosis may account for anaerobic reduction of chromate under nongrowth conditions with an organic electron donor present. Chromate reduction under both aerobic and anaerobic conditions may be a detoxification strategy for the bacterium which could be exploited to bioremediate chromate-contaminated or other toxic heavy metal-contaminated environments.     

Purification to homogeneity and characterization of a novel Pseudomonas putida chromate reductase

Cr(VI) (chromate) is a widespread environmental contaminant. Bacterial chromate reductases can convert soluble and toxic chromate to the insoluble and less toxic Cr(III). Bioremediation can therefore be effective in removing chromate from the environment, especially if the bacterial propensity for such removal is enhanced by genetic and biochemical engineering. To clone the chromate reductase-encoding gene, we purified to homogeneity (>600-fold purification) and characterized a novel soluble chromate reductase from Pseudomonas putida, using ammonium sulfate precipitation (55 to 70%), anion-exchange chromatography (DEAE Sepharose CL-6B), chromatofocusing (Polybuffer exchanger 94), and gel filtration (Superose 12 HR 10/30). The enzyme activity was dependent on NADH or NADPH; the temperature and pH optima for chromate reduction were 80 degrees C and 5, respectively; and the K(m) was 374 microM, with a V(max) of 1.72 micromol/min/mg of protein. Sulfate inhibited the enzyme activity noncompetitively. The reductase activity remained virtually unaltered after 30 min of exposure to 50 degrees C; even exposure to higher temperatures did not immediately inactivate the enzyme. X-ray absorption near-edge-structure spectra showed quantitative conversion of chromate to Cr(III) during the enzyme reaction.     

Comparison of in vitro Cr(VI) reduction by CFEs of chromate resistant bacteria isolated from chromate contaminated soil

Chromate resistant and reducing strains were isolated from chromium contaminated soil and identified as Bacillus sp. (KCH2 and KCH3), Leucobacter sp. (KCH4) and Exiguobacterium sp. (KCH5). KCH3 and KCH4 showed higher Cr(VI) tolerance (2 mM) and Cr(VI) reduction (1.5 mM) than KCH5 (1.5 mM and 0.75 mM, respectively). Cr(VI) reduction by CFEs of KCH3 and KCH4 showed NAD(P)H dependence, optimum activity at pH 5.5, low K(m) (45-55 microM) and substrate inhibition by Cr(VI) (>75 microM), whereas that of KCH5 showed NADH dependence, pH optimum at 6.0, high K(m) (200 microM) and no inhibition by Cr(VI). Cr(VI) reduction was optimum at 35 degrees C for CFEs of KCH3 and KCH5 and 30 degrees C for that of KCH3. Cr(VI) reduction by CFEs of all the strains were inhibited by Hg(2+) and enhanced by Cu(2+). Activity enhancement by Cu(2+) was more predominant (290%) for KCH4. The characterization of Cr(VI) reduction by CFEs of chromate resistant isolates of different genera is useful for development of Cr(VI) bioremediation.     

In vitro reduction of hexavalent chromium by a cell-free extract of Bacillus sp. ES 29 stimulated by Cu2+

Chromium-resistant bacteria (CRB) isolated from soils can be used to reduce toxic Cr(VI) from contaminated environments. This study assessed in vitro reduction of hexavalent Cr using a cell-free extract (CFE) of CRB isolated from soil contaminated with dichromate. One isolate, ES 29, that substantially reduced Cr(VI) was identified as a Bacillus species by 16S rRNA gene-sequence homology. The isolate reduced Cr(VI) under aerobic conditions, using NADH as an electron donor and produced a soluble Cr(VI)-reducing enzyme stimulated by copper (Cu2+). The CFE of the bacterial isolate reduced 50% of Cr(VI) in 6 h. The Cr(VI)-reduction activity of the CFE had a Km of 7.09 microM and a Vmax of 0.171 micromol min(-1) mg(-1) protein. Mercury inhibited the enzyme, but not competitively, with a Vmax of 0.143 micromol min(-1) mg(-1) protein, a Km of 7.07 microM and a Ki of 1.58 microM. This study characterizes the enzymatic reduction of Cr(VI) by Bacillus sp. ES 29 which can be used for the bioremediation of chromate.     

Reduction of Hexavalent Chromium by Cell-Free Extract of Bacillus sphaericus AND 303 Isolated from Serpentine Soil

Cell-free extracts (CFEs) of chromium-resistant bacterium Bacillus sphaericus AND 303 isolated from serpentine soil of Andaman, India reduced Cr(VI) in in vitro condition, and the reductase activity was solely localized in the soluble cell-fractions (S₁₂, S₃₂, and S₁₅₀). The enzyme was constitutive as the CFEs from cells grown in Cr(VI)-free and Cr(VI)-containing media reduced a more or less equal amount of Cr(VI). Optimum Cr(VI) reductase activity was obtained at an enzyme (S₁₅₀) concentration equivalent to 4.56 mg protein/mL, 300 μM Cr(VI) and pH 6.0 after 30 min incubation at 30°C. The enzyme was heat labile; 80% of its activity was lost when exposed at 70°C for 15 min. Kinetics of Cr(VI) reductase activity fit well with the linearized Lineweaver-Burk plot and showed a V_max of 1.432 μmol Cr(VI)/mg protein/min and K_m of 158.12 μM Cr(VI). The presence of additional electron donors accelerated Cr(VI) reductase activity of CFE, and an increase of 28% activity over control was recorded with 1.0 μM NADH. Heavy metal ions such as Ni(II), Cu(II), and Cd(II) were strong inhibitors of Cr(VI) reductase unlike that of 100 μM Co(II), which retained 93% activity over control.     

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.     

Hexavalent chromium reduction by an actinomycete, Arthrobacter crystallopoietes ES 32

Environmental contamination by hexavalent chromium, Cr(VI), presents a serious public health problem. This study assessed the reduction of Cr(VI) by intact cells and a cell-free extract (CFE) of an actinomycete, Arthrobacter crystallopoietes (strain ES 32), isolated from soil contaminated with dichromate. Both intact cells and CFE of A. crystallopoietes, displayed substantial reduction of Cr(VI). Intact cells reduced about 90% of the Cr(VI) added within 12 h and Cr(VI) was almost completely reduced after 24 h. The K _M and V _max of Cr(VI) bioreduction by intact cells were 2.61 μM and 0.0142 μmol/min/mg protein, respectively. Cell-free chromate reductase of the A. crystallopoietes (ES 32) reduced hexavalent chromium at a K _M of 1.78 μM and a V _max of 0.096 μmol/min/mg protein. The rate constant (k) of chromate reduction was inversely related to Cr(VI) concentration and the half-life (t _1/2) of Cr(VI) reduction increased with increasing concentration. A. crystallopoietes produced a periplasmic chromate reductase that was stimulated by NADH. Results indicate that A. crystallopoietes ES 32 can be used to detoxify Cr(VI) in polluted sites, particularly in stressed environments.     

Cr(VI) reduction in a chromate-resistant strain of Candida maltosa isolated from the leather industry

A Cr(VI)-resistant yeast was isolated from tanning liquors from a leather factory in Leon, Guanajuato, Mexico. Based on morphological and physiological analyses and the D1/D2 domain sequence of the 26S rDNA, the yeast was identified as Candida maltosa. Resistance of the strain to high Cr(VI) concentrations and its ability to chemically reduce chromium was studied. When compared to the three laboratory yeasts Candida albicans, Saccharomyces cerevisiae and Yarrowia lipolytica, the C. maltosa strain was found to tolerate chromate concentrations as high as 100 micro g/ml. In addition to this phenotypic trait, the C. maltosa strain showed ability to reduce Cr(VI). Chromate reduction occurred both in intact cells (grown in culture medium or in soil containing chromate) as well as in cell-free extracts. NADH-dependent chromate reductase activity was found associated with soluble protein and, to a lesser extent, with the membrane fraction.     

Amperometric cytochrome c3-based biosensor for chromate determination

The chromate reductase activity of cytochrome c(3) (Cyt c(3), M(r) 13000), isolated from the sulfate-reducing bacterium Desulfomicrobium norvegicum, was used to develop an amperometric biosensor to measure chromate (CrO(4)(2-)) bioavailability. The performance of various biosensor configurations for qualitative and quantitative determination of Cr(VI) was studied. Biosensor properties depend on the technique used to immobilize the enzyme on the electrode (glassy carbon electrode). Immobilization of Cyt c(3) by entrapment in poly 3,4-ethylenedioxythiophene films denatured the enzyme, while application of an adsorption technique did not affect enzyme activity but the detection range was limited. The best results were obtained with dialysis membranes, which allowed the determination of Cr(VI) from 0.20 to 6.84 mg l(-1) (3.85-132 microM) with a sensitivity of 35 nA mg(-1) l (1.82 nA microM(-1)). No interference was observed with As(V), As(III) and Fe(III). Only a small amount of Cyt c(3) (372 ng of protein) was needed for this biosensor.     

Hexavalent-chromium reduction by a chromate-resistantBacillus sp. strain

Bacillus strain QC1-2, isolated from a chromium-polluted zone, was selected by its high ability to both tolerate and reduce hexavalent chromium [Cr(VI)] to less-toxic trivalent chromium [Cr(III)]. Cell suspensions of strain QC1-2 rapidly reduced Cr(VI), in both aerobic and anaerobic conditions, to Cr(III) which remained in the supernatant. Cr(VI) reduction was dependent on the addition of glucose but sulfate, an inhibitor of chromate transport, had no effect. Studies with permeabilized cells and cell extracts showed that the Cr(VI) reductase of strain QC1-2 is a soluble NADH-dependent enzyme.     

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。     

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.