Preparative Biochemistry & Biotechnology, 45:769–784, 2015 Copyright # Taylor & Francis Group, LLC ISSN: 1082-6068 print/1532-2297 online DOI: 10.1080/10826068.2014.952385

Partial Purification and Characterization of Chromate Reductase of a Novel Ochrobactrum sp. Strain Cr-B4 Anuradha Hora and Vidya K. Shetty Department of Chemical Engineering, National Institute of Technology Karnataka, Surathkal, Mangalore, India 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 (Ea) for the chromate reductase was found to be 34.7 kJ=mol up to 40 C and the activation energy for its deactivation (Ed) 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 s1, and for deactivation of chromate reductase it was found to be 265.66  103 s1. 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, Pb2þ, Ni2þ, Zn2þ, and Cd2þ) inhibited the enzyme activity, while metals like Cu2þ and Fe3þ significantly enhanced the reductase activity. The enzyme followed Michaelis– Menten kinetics with Km of 104.29 mM and a Vmax of 4.64 mM=min=mg. Keywords Ochrobactrum sp. strain Cr-B4, chromate reductase, enzyme purification and characterization, hexavalent chromium [Cr(VI)], Michaelis–Menten kinetics

INTRODUCTION Chromate reductases are a group of enzymes that catalyze the reduction of toxic hexavalent chromium [Cr(VI)] to the less toxic trivalent chromium [Cr(III)]. These proteins have recently raised enormous interest because of their central role in mediating chromium toxicity and their potential use in bioremediation.[1] Cr(VI) is widely used in industries such as tanning, corrosion control, plating, pigment manufacture, and nuclear weapons production, and thus wastewaters from these industries contain Cr(VI). Hexavalent chromium compounds are water-soluble, toxic,

Address correspondence to Vidya K. Shetty, Department of Chemical Engineering, National Institute of Technology Karnataka, Surathkal, Mangalore–575 025, India. E-mail: [email protected] Color versions of one or more of the figures in the article can be found online at www.tandfonline.com/lpbb.

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and probably carcinogenic, while Cr(III), is less soluble and less toxic. Thus, reduction of Cr(VI) to Cr(III) represents a potentially useful detoxification process. Environmental cleanup strategies for Cr(VI) removal involve physical, chemical, or biological detoxification. Major limitations of physical and chemical processes such as electrochemical treatments,[2] adsorption,[3] reverse osmosis,[4] precipitation,[5] and so on are the high energy inputs, use of costly chemicals, and generation of waste sludge, with reactive chemical species as secondary wastes. Some of the processes can only transfer the pollutant from one phase to another and thus not provide permanent solution to the environmental pollution problem. These problems can be overcome by biological Cr(VI) detoxification, which is more ecofriendly and an economically feasible technology and categorized under the class of bioremediation strategy.[2,6] The use of microorganisms for reduction of Cr(VI) to Cr(III) is an attractive strategy that is cost-effective, safe, and produces no secondary pollutants. Bioreduction of Cr(VI) can occur directly as a result of microbial metabolism (enzymatic), or indirectly through a bacterial metabolite such as H2S.[7] Although it is an efficient process and cost effective, use of microbes for Cr(VI) reduction also results in the generation of a large amount of biomass, which confines effective bioremediation to a narrow zone. Also the microbial mediated processes require sterile conditions during handling and maintenance of the culture and other purification steps to remove the microbial biomass after the reduction of Cr(VI). Given these labor-intensive procedures, an enzymatic approach toward reduction of Cr(VI) can be seen as a lucrative alternative. Two enzymatic mechanisms are reported for reduction of Cr(VI). In aerobic conditions, most of the chromate reductase reported is soluble in the cytosol and reduces Cr(VI) to Cr(III) inside or outside the plasma membrane.[8,9] Under anaerobic conditions, CrO2 4 is used as a terminal electron acceptor and is reduced in the membrane during anaerobic respiration.[10] The main mechanism used for Cr(VI) reduction involves chromate reductases reportedly produced by gram-negative strains like Escherichia coli,[11] Rhodobacter sphaeroides,[12] Pseudomonas putida,[13] Pseudomonas ambigua,[14] and Enterobacter sp. Du17.[15] Microbial enzyme-mediated reduction of Cr(VI) to Cr(III) can be considered as an additional chromate resistance mechanism that is not usually a plasmid-associated trait.[9] These enzymes are known to protect microorganisms against chromate toxicity, possibly through minimizing the amount of reactive oxygen species formed. Several researchers have proposed that these chromate reductases might be the serendipitous activity of enzymes with other primary physiological functions, since Cr(VI) is mostly of anthropogenic origins and these fortuitous reactions are often carried out by constitutive enzymes.[16–18] However, very few of the chromate reductases[11,13,14,19–21] have been purified and characterized to elucidate their true function. Hence there arrives a need to purify and characterize the enzymes in order to carry out specific reaction and for large-scale implementation of the process. The enzymes may present advantages over traditional technologies, and also over microbial remediation. Also, easy availability of pure enzymes will permit detailed investigations of their kinetic and inhibition properties, leading to the identification of targets for improvement; it will also permit determination of high-resolution structure of these enzymes so that the desired improvements can be effectively attempted. Previous work has shown that a Cr(VI)-reducing strain, Ochrobactrum sp. strain Cr-B4, isolated from aerator liquid of a wastewater treatment facility of a dye- and pigment-based specialty chemical industry contaminated with chromate, is useful for Cr(VI) detoxification in polluted environments.[22] The present work describes the partial purification of chromate reductase of a novel Ochrobactrum sp. strain Cr-B4 and its characterization.

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EXPERIMENTAL Microorganism and Growth Conditions A strain of Ochrobactrum sp. strain Cr-B4 with GenBank accession number JF824998,[22] with high capacity of reducing Cr(VI) to Cr(III) and high resistance up to 1000 mg=L of Cr(VI), was used in the present work. Cell suspension of Ochrobactrum sp. strain Cr-B4 was prepared by growing the cells in presterilized LB media at 37 C by shaking at 150 rpm for 24 hr. The 5% (v=v) of fully grown culture was transferred into 250 mL flask containing 100 mL of optimized media with Na2HPO4(6 g=L), KH2PO4(3 g=L), MgSO4  7H2O (0.1 g=L), CaCl2(0.1 g=L), NaCl (0.5 g=L), casein hydrolysate (1.61 g=L), and sucrose (10.05 g=L)[29] along with 100 mg=L of Cr(VI). Stock Cr(VI) solution was prepared by dissolving 2.829 g K2Cr2O7 (294.18=gmol) in 1 L of deionized water. The initial pH of the medium was adjusted to 8.19 (optimum value) by using 0.1 M NaOH or 0.1 M H2SO4 before sterilization. Standard Enzyme Assay The reaction mixture for the enzyme assay contained 100 mM of Cr(VI) in 0.8 mL of 100 mM phosphate buffer (pH 7.0). After 5 min of preincubation at 30 C the reaction was initiated by addition of 0.2 mL of chromate reductase enzyme. Cr(VI) reduction was determined by analysis of Cr(VI) in the reaction mixture by the 1,5-diphenyl carbazide (DPC) method with absorbance measurement at 540 nm[23] after 30 min. One unit of enzyme activity was defined as the amount of enzyme that reduced 1 nM of Cr(VI) per minute. Protein Estimation Total protein content of the crude and partially purified samples was estimated by Bradford’s method of total protein estimation at 595 nm.[24] Purification of Chromate Reductase Cell-free supernatant of Cr-B4 for purification of chromate reductase enzyme was prepared by inoculating 500 mL of optimized media supplemented with 100 mg=L of Cr(VI) with 5% (v=v) inoculum (as described in earlier) and incubated at 37 C, 150 rpm, for 24 hr. All purification steps were carried out at room temperature, and enzyme preparations were stored at 4 C without any loss of activity. Partial purification was done in three steps, ammonium sulfate precipitation, dialysis, and gel filtration chromatography. The cells were separated after 24 hr of incubation by centrifuging the cell culture at 10,000  g, 10 min, 4 C, and the resulting supernatant was used for the first step of purification (ammonium sulfate precipitation). The amount of ammonium sulfate required for 100% saturation had been calculated from the ammonium sulfate calculator (www.encorbio. com/protocols/AM-SO_4.html). The initial amount of ammonium sulfate salt used for precipitation was 383.96 g. The ammonium sulfate was added to the cell-free supernatant pinch by pinch with continuous stirring using magnetic stirrer. Once saturation had been reached, the addition of ammonium sulfate was stopped and remaining amount of ammonium sulfate was calculated to

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determine percentage saturation. The protein precipitate obtained from ammonium sulfate precipitation was collected by centrifugation (12,000 rpm, 15 min, and 4 C) and resuspended in 20 mL of 100 mM phosphate buffer (pH 7.0). After desalting by dialysis (carried out at 25 C) against 0.5 mM phosphate buffer (pH 7.0) (5 changes, 24 hr, 1 L each), the protein sample was concentrated by reverse osmosis by immersing the dialysis bag containing protein sample in polyethylene glycol (PEG-6000). The concentrated protein samples were then resuspended in 5 mL of 100 mM phosphate buffer (pH 7.0) and the partially purified chromate reductase enzyme was applied to a gel filtration column (10 cm, Sephadex G-100 superfine) that had been preequilibrated with 100 mM phosphate buffer (pH 7.0). Then 1.5 mL of partially purified protein sample was applied on the top of 10 mL of packed column. The column elution was carried out using 100 mM potassium phosphate buffer at a constant flow rate of 0.5 mL=min at 20 C. In total, 40 fractions (0.5 mL each) were collected. The concentration of chromate reductase activity and protein was determined after each step of purification by standard assay protocols (already described here) and also subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).[25] SDS-PAGE was carried out using 12% polyacrylamide gel. A broad-range standard protein molecular marker of range 3 kD to 205 kD obtained from Genei was used for comparison. Characterization of Partially Purified Chromate Reductase Effect of pH and Temperature on Chromate Reductase Activity Optimum pH and temperature for chromate reductase activity were determined by incubating the reaction mixture under the standard assay conditions at different pH values ranging from 3.0 to 9.0 at 37 C and at different temperatures ranging from 30 to 90 C at pH 8.0, respectively, for 30 min. The buffers (100 mM each) citrate buffer (pH 3.0–6.0), phosphate buffer (pH 6.0–7.0), and Tris-HCl buffer (pH 7.0–9.0) were used for maintaining the pH. The use of two overlapping pH compensated for the buffer-associated effects. Assays were done in triplicate with blank rates taken at each assayed pH and temperature value. The activation energy (Ea) for chromate reductase from Cr-B4 was also calculated by employing Arrhenius equation as follows: k ¼ A  eEa=RT

ð1Þ

Ea=RT

The exponential term e describes the fraction of molecules with minimum energy for the reaction. R is the ideal gas constant with a value of 8.314 J=K-mol. Equation (1) relates the specific activity (k) and temperature T (Kelvin). A is called the preexponential factor or frequency factor and is the preexponential constant in the Arrhenius equation indicating how many collisions have the correct orientation to lead to product. From the intercept of the Arrhenius plot (ln(k) vs. 1=T), the value of frequency factor can be calculated. The activation energy (Ea) can be found from the slope (–E=2.303R) of Arrhenius plot. Similar equation was used to determine the activation energy and frequency factor for deactivation of the chromate reductase. Effect of pH and Temperature on Chromate Reductase Stability The pH and temperature stability of the enzyme was determined by preincubating the chromate reductase in buffers of different pH ranging from pH 3.0 to 9.0 and different temperature

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(4 C and 30–90 C) at pH 8.0, respectively. The residual activity was measured after every hour until 6 hr of incubation and finally after 12 hr and 24 hr. The percentage relative activity at different pH and temperature was calculated with respect to the maximum activity obtained at a particular pH and temperature being taken as 100%. Effect of Metal Ions and Complexing Agents on Enzyme Activity The effects of various divalent metals (Ni2þ, Zn2þ, Cd2þ, Fe3þ, Pb2þ, Cu2þ) as well as the metal chelator EDTA, protein denaturant b-mercaptoethanol, and respiratory inhibitor NaN3 on the activity of the chromate reductase were determined in the presence of 1 mM of each metal or inhibitor into the standard reaction mixture (pH 8.0 and 40 C). Chromate Reductase Enzyme Kinetics The kinetics of Cr(VI) reduction by partially purified chromate reductase enzyme was evaluated under standard reaction conditions at a pH of 8.0 and temperature of 40 C by measurement of initial rates (specific activity) at various concentrations of Cr(VI) (25–600 mM) by conducting standard enzyme assay as explained earlier. The Michaelis–Menten kinetic equation representing saturation kinetics was tested for its validity and the kinetic constants for partially purified chromate reductase activity (maximum velocity (Vmax) and Michaelis–Menten constant (Km)) were determined by using the Lineweaver–Burk plot of (1=rate) versus (1=Cr(VI) concentration).

RESULTS AND DISCUSSION Purification of Soluble Chromate Reductase From Cr-B4 The chromate reductase enzyme of Cr-B4 was purified from the cell-free supernatant with different purification steps including ammonium sulfate precipitation, dialysis, gel filtration chromatography, and electrophoresis. The overall yield of purification was found to be 0.26% with a purification fold of 16.39. The purification parameters are summarized in Table 1. TABLE 1 Purification Parameters for Soluble Chromate Reductase From Cr-B4

Fraction Cell-free supernatant After precipitation After dialysis and reverse osmosis Gel filtration chromatography 

Volume (mL) 500 20 5 1

Protein (mg=mL)

Total protein (mg)

0.91  0.43 454.69 1.31  0.82 26.20 1.18  0.36 5.89 .071  0.045 .071

Enzyme activity (U=mL)

Total enzyme activity (U)

0.79  0.64 393.60 2.31  0.31 46.11 3.43  0.93 17.15 1.01  0.57 1.01

One unit is defined as the amount of enzyme that converts 1 nmol of Cr(VI)=min.

Specific enzyme activity Purification Yield (U=mg) fold (%) 0.87 1.76 2.91 14.26

1 2.02 3.34 16.39

100 11.71 4.36 0.26

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The supernatant obtained from the whole-cell suspension was subjected to ammonium sulfate precipitation. The protein content and enzyme activity was determined for each purification step. The complete precipitation of proteins in supernatant from Cr-B4 took place at 88% of ammonium sulfate salt. The proteins obtained from ammonium sulfate precipitation showed an enzyme activity of 2.31 U=mL with an increase in protein concentration and a purification fold of 2.02. The proteins precipitate obtained from the preceding step was diluted in 20 mL of 100 mM phosphate buffer (pH 7.0) and subjected to dialysis to remove the excess salt. The sample thus obtained was subjected to reverse osmosis to concentrate the protein sample. The enzyme activity of protein sample obtained from this step was found to be 3.43 U=mL with a purification fold of 3.34. The proteins obtained after reverse osmosis were subjected to the final purification step of gel filtration chromatography on a Sephadex G-100 column in order to separate the targeted reductase enzyme from other interfering components. The fractions obtained from gel filtration chromatography were analyzed for protein concentration and chromate reductase activity to determine the fraction containing maximum chromate reductase activity and maximum protein concentration. The enzyme activity and protein concentration of each fraction were plotted against respective volumes collected to give the chromatogram. From Figure 1, which represents the 40-min interval chromatogram, with a flow rate of 1 mL=min, the chromate reductase activity was found between fraction number 15 to 28, with peak activity obtained at fraction numbers 21 and 22. The peak protein concentration was also obtained at fraction number 22 (0.07 mg=mL). As can be observed from Table 1, the specific enzyme activity has increased by around 14 times from crude enzyme to the enzymes obtained after gel filtration chromatography. Thus, the use of purified enzymes can result in an efficient process. As one moves toward the achievement of completely purified enzyme, by incorporating more purification steps after gel chromatography, the cost involved may be very high. Thus, complete purification of enzymes for the purpose of wastewater treatment is not an economical option. The aim of the study was to get partially purified enzyme, as achievement of completely purified enzyme can be a cost-intensive process. A balance between the cost and technology is

FIGURE 1 Gel filtration chromatogram of partially purified chromate reductase from Cr-B4.

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very important especially in adoption of technology for waste water treatment. Thus, use of partially purified enzyme for the wastewater treatment may prove to be a beneficial option and it may succeed in attracting the stakeholders to adopt the technology. Therefore, enzyme was only partially purified and its characteristics were determined after partial purification. SDS-PAGE Analysis of Soluble Chromate Reductase From Cr-B4 SDS-PAGE analysis after each step of purification was done in order to determine the molecular mass of chromate reductase. From Figure 2, representing the SDS-PAGE analysis, it can be observed that the number of protein bands decreased after each step of purification. In the crude maximum bands were observed, followed by the ammonium sulfate precipitated sample. After dialysis followed by reverse osmosis the number of bands further decreased, with a total of four bands observed after the final purification step of gel filtration, demonstrating the effective partial purification of chromate reductase enzyme. In all the samples a common band was observed with approximate molecular mass of 31.53 kD. However, to determine its precise molecular mass and subsequent sequencing, additional purification steps should be performed to obtain a single and homogeneous band. Most of the chromate reductase enzymes had been shown to be composed of more than one subunit. NADH or NADPH-dependent enzyme with 50 kD molecular mass containing two subunits of 20 kD[13] and 42 kD[11] has also been reported.

FIGURE 2 Estimation of molecular weight of partially purified chromate reductase from Cr-B4 by SDS-PAGE. Lane 1: protein marker. Lane 2: whole cell suspension. Lane 3: ammonium sulfate precipitated fraction. Lane 4: dialyzed fraction. Lane 5: gel filtration chromatography fraction.

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Characterization of Partially Purified Chromate Reductase of Cr-B4 Effect of pH To define the optimum pH of the partially purified chromate reductase enzyme of Cr-B4, the standard enzyme assay was carried over a different pH range of 3.0–9.0 using citrate buffer (pH 3.0–6.0), phosphate buffer (pH 6.0–8.0), and Tris-HCl buffer (pH 7.0–9.0) with overlapping pH range. Figure 3 exhibits the effect of pH on enzyme activity at different pH in the presence of 100 mM of Cr(VI), with maximum activity taken as 100%. As shown in Figure 3, the chromate reductase of Cr-B4 showed significant chromate reductase activity over a wide pH range, 5.0 to 9.0, with maximum activity observed at pH 8.0 (3.60 U=mL). However, the chromate reductase activity of Cr-B4 was found relatively very low at acidic pH of 3.0 and 4.0 (9 and 15%, respectively). Farrell and Ranallo[26] postulated that the pH of the reaction medium affects the degree of ionization of the enzyme and changes the protein conformation. The chromate reductase from other strains have shown the highest activity at acidic and alkaline pH; pH 5.0 for P. putida MK1[13]; pH 6.0 for Bacillus sp.[27]; pH 6.3 for Thermus scotoductus SA-01[21]; pH 6.5 for Halomonas sp. TA-04[28]; and pH 8.6 for Pseudomonas ambigua G-1.[14] The optimum pH for chromate reductase correlates well with optimal growth pH for Cr-B4, which was found to be 8.4.[29] Further, different types of buffer with overlapping pH gave similar results, which clearly indicated that it is the pH, not the chemical composition of the buffer, that influences the enzyme activity. The stability of chromate reductase enzyme of Cr-B4 was also determined over a wide pH range of 3.0–9.0. As shown in Figure 4, the enzyme was most stable at pH 7.0 with retention of 96% of activity after 24 hr of incubation, while at pH 8.0 > 90% of activity was retained until 4 hr, which decreased to 77.58% on incubation for 24 hr at pH 8.0. A drastic decrease in enzyme stability was observed at acidic pH 3.0–5.0, with retention of only 10–20% of enzyme activity, while at near neutral pH a slight decrease in the enzyme activity was observed with retention of approximately 75% of enzyme activity after storage for 24 hr at pH 6. At alkaline pH of 9.0 the reductase activity was found to be > 60% until 5 hr, but it reduced to 24% on incubating for 24 hr. Other authors have reported the enzyme stability between 7.0 and 7.4 for the bacteria

FIGURE 3 Effect of different pH (3.0–9.0) on activity of chromate reductase from Cr-B4.

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FIGURE 4 Effect of different pH (3.0–9.0) on stability of chromate reductase from Cr-B4.

Pseudomonas sp. G1DM21,[30] 6.5 and 7.5 in E. coli CFE,[31] and in the range of 5.0 to 8.0 in Bacillus sp.[27] From the experimental results it can be deduced that chromate reductase enzyme obtained from Cr-B4 can work with maximum efficiency at pH 8.0, while the maximum stability of enzyme was found at pH 7.0. Effect of Temperature The chromate reductase of Cr-B4 was found sensitive to temperature and tends to denature at higher temperature. As shown in Figure 5, the optimum temperature for the chromate reductase of Cr-B4 was 40 C, which correlates well with the optimal temperature for growth (37 C). At 30 C the chromate reductase activity of Cr-B4 was found to be 76.81%, while on increasing the temperature to 50 C a gradual decline in activity was observed with retention of approximately 52% of optimal activity. The chromate reductase activity was further lowered with the increase in temperature above 50 C. Wang et al.[31,32] reported that there was no reduction of chromium at 60 C. The decline in reductase activity at higher temperatures may be attributed either to heat

FIGURE 5 Effect of different temperatures on activity of chromate reductase from Cr-B4.

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FIGURE 6 Effect of different temperatures on stability of chromate reductase from Cr-B4.

denaturation of the enzyme or to change in structural conformation of active site at such high temperatures. The stability of chromate reductase from Cr-B4 was determined by incubating the enzyme in 100 mM phosphate buffer (pH 8.0) at various temperatures for different time intervals (0.5, 1, 2, 3, 4, 5, 6, 12, and 24 hr). The temperature stability studies revealed that the enzyme was relatively stable at temperature below 30 C with retention of >75% reductase activity at 30 C after 24 hr. At 30 C, only 12% activity was lost at after 12 hr. But at 4 C the enzyme was found to be more stable, as 92% of reductase activity was retained on incubating the enzyme for 24 hr. At elevated temperatures (50–90 C), the activity loss was very high after 24 hr due to enzyme denaturation at such high temperatures, indicating the thermolabile nature of the enzyme. The thermolabile nature of chromium-reducing enzymes has also been reported for Bacillus subtilis as it failed to show any Cr(VI) reductase activity after heating at 100 C for 10 min.[33] At 40 C, the enzyme was relatively stable up to 6 hr with retention of more than 60% activity, but on incubating for longer time (24 hr) only 23% of activity was retained. However, chromate reductase activity was preserved at lower temperatures of 4 C, with little activity loss (8%) even after 24 hr (Figure 6). The activation energy (Ea) was calculated from the Arrhenius plot [(log k) (where k represents specific activity) versus (1=T)] and is shown in Figure 7. The activation energy for the chromate

FIGURE 7 Arrhenius plot for calculation of activation energy for chromate reductase from Cr-B4.

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FIGURE 8 Arrhenius plot for calculation of deactivation energy for chromate reductase from Cr-B4.

reductase of Cr-B4 was found to be 34.7 kJ=mol up to 40 C. The increasing activity up to 40 C suggests the unchanged functional conformation of this enzyme up to 40 C. The deactivation energy (Ed) for chromate reductase of Cr-B4 was found to be 79.6 kJ=mol over a temperature range of 50–90 C (Figure 8). The frequency factor A for activation of chromate reductase was found to be 566.79 s1, and for deactivation of chromate reductase, it was found to be 265.66  103 s1. Effect of Metals and Other Complexing Agents The wastewater containing Cr(VI) may also contain other heavy metals and its treatment may also involve chelation of metals by chelating agents such as EDTA. Moreover, some industries use b-mercaptoethanol, a protein denaturant, as corrosion inhibitor and=or as a solvent. NaN3, a respiratory inhibitor, is used as preservatives and biocides in many industries. The presence of these complexing agents in the industrial effluents may affect the activity of chromate reductase enzyme; therefore, the influence of these complexing agents on Cr(VI) reductase activity was determined in their presence at 1 mM concentration in the reaction mixture (Figure 9). The percentage relative activity for other heavy metals and complexing agents was calculated with respect to activity obtained with only Cr(VI) in the reaction mixture being taken as 100%. All the metals ions except iron and copper were found to inhibit the enzyme activity. Cu2þ ion significantly stimulated chromate reductase activity by 30.16%. Camargo et al.[34] and Elangovan et al.[27] have also reported the stimulation of Cr(VI) reduction in Bacillus sp. on addition of 1 mM of Cu2þ. The role of Cu2þ in stimulation of chromate redustase could be related to its main function as a protective agent for electron transport, as a single electron redox center, and as a shuttle for electrons between protein subunits.[35,36] The stimulation of enzyme activity by Cu2þ might be due to its nature as a prosthetic group of many reductase enzymes.[37] As shown in Figure 9A, addition of Fe3þ into the reaction mixture also stimulated the activity of the chromate reductase of Cr-B4 by 73%. Though both Cu2þ and Fe3þ stimulated the chromate reductase activity, the enhancement of chromate reductase activity in the presence of Fe3þ was higher as compared to that with Cu2þ.

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FIGURE 9 (A) Effect of different metals on activity of chromate reductase from Cr-B4. (B) Effect of different complexing agents on activity of chromate reductase from Cr-B4.

Other divalent cations such as Pb2þ, Ni2þ, Zn2þ, and Cd2þ were found to inhibit the chromate reductase activity to a variable degree. These variations may be due to different functional nature of the reductase enzyme and its interaction with the different metals. The order in which these metals inhibit chromate reductase activity is: Zn2þ > Ni2þ > Cd2þ > Pb2þ. In the presence of Pb2þ, the reductase activity was inhibited by 17.46%, while Zn2þ was found to decrease chromate reductase activity by approximately 76%, followed by Cd2þ, which was found to inhibit 55.55% of enzyme activity, and Ni2þ, which inhibited 49.21% of chromate reductase activity. The results of inhibition of Cr(VI) reduction by Ni2þ have been reported by Pal et al.[37] and the inhibition of Cr(VI) reduction by the addition of Zn2þ has been reported by Elangovan et al.[27] The metals ions are known to form metal–enzyme complexes and may lead to inactivation of chromate reductase enzymes or sites responsible for Cr reduction.[38,39] As exhibited in Fig.9 Figure 9B, the presence of other complexing agents such as the metal chelator EDTA, protein denaturant b-mercaptoethanol, and respiratory inhibitor NaN3 significantly inhibited the chromate reductase activity of Cr-B4 by 44, 73, and 20%, respectively. EDTA, being a chelating agent, may form a complex with the chromate ions and thus prevent binding of the substrate to its enzyme, leading to decreased enzyme activity. The inhibitory effect of b-mercaptoethanol may be attributed to its action as a disulfide reducer, causing denaturation of the chromate reductase enzyme. The inhibitory effect of azide has also been observed in purified chromate reductase of E. coli ATCC 33456 19[11] and aerobic chromate reduction by Bacillus subtilis[33] and Arthrobacter sp. SUK 1201.[40] Respiratory inhibitors act on de novo protein synthesis or affect the respiratory chain intermediates responsible for Cr(VI) reduction, wherein Cr(VI) serves as a terminal electron acceptor.[13] Effect of Cr(VI) Concentration and Kinetics of Chromate Reductase The enzyme kinetics was evaluated by determining the effect of different concentration of Cr(VI) (25–600 mM) on chromate reductase activity. The specific activity of chromate reductase of Cr-B4 was found to increase with the increase in initial Cr(VI) concentration up to 350 mM of

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FIGURE 10 Lineweaver–Burk plot for kinetics of chromate reductase from Cr-B4.

Cr(VI). The specific chromate reductase activity increased from 0.89 U=mg at 25 mM of Cr(VI) to 2.89 U=mg at 200 mM of Cr(VI), and the specific activity further increased to 4 U=mg at 350 mM of Cr(VI); thereafter the specific activity of chromate reductase was marginally decreased to a value of 3.78 U=mg at a Cr(VI) concentration of 450 mM. Though at concentrations above 350 mM of Cr(VI) very slight inhibition by Cr(VI) was observed, for all practical purposes this marginal decrease in the enzyme activity on increasing the concentration of Cr(VI) may be considered as almost constant, indicating the saturation of enzyme with Cr(VI). The kinetics of Cr(VI) reduction by the chromate reductase of Cr-B4 was found to obey the Michaelis–Menten kinetics. As shown in Figure 10, from the Lineweaver–Burk plot for chromate reductase kinetics using Cr(VI) as substrate, the values of Km and Vmax were found to be 104.29 mM and 4.64 mM= min=mg, respectively. The R2 value for the linear fit of the data on the Lineweaver–Burk plot was found to be 0.9874 with the sum of squared errors (SSE) as 0.446, indicating the good fit of the kinetic data on Michaelis–Menten kinetic model. The kinetics parameters Km and Vmax for this chromate reductase are found to be better than for some other chromate reductase reported earlier,[13,41] which makes it a better enzyme for Cr(VI) removal with respect to its high substrate affinity and Cr(VI) reduction rates without need of any external electron donor.

CONCLUSION The present work reveals the partial purification and characterization of chromate reductase enzyme of a novel Ochrobactrum sp. strain Cr-B4. This chromate reductase enzyme was purified from cell-free supernatant obtained from culture of Cr-B4 and was found to have a molecular mass of 31.53 kD. The enzyme was found to be highly active without any addition of electron donors with a specific activity of 14.26 U=mg. The pH and temperature optimum for this chromate reductase were found to be 8.0 and 40 C, respectively. The activation energy for the chromate reductase of Cr-B4 was found to be 34.7 kJ=mol up to 40 C and the deactivation energy (Ed) 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 s1, and

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for deactivation of chromate reductase, it was found to be 265.66  103 s1. The presence of heavy metals like Cu2þ and Fe3þ stimulated the Cr(VI) reduction by chromate reductase enzyme, while metals like Ni2þ, Zn2þ, Pb2þ, and Cd2þ inhibited the chromate reductase activity to a variable extent. Other complexing agents like EDTA, b-mercaptoethanol, and NaN3 significantly inhibited the chromate reductase activity. The Cr(VI) reduction by partially purified chromate reductase followed Michaelis–Menten kinetics. The kinetic parameters Km and Vmax were found to be 104.29 mM and 4.64 mM=min=mg, respectively. The relatively low Km value in comparison to some other chromate reductases represents the high substrate affinity of this chromate reductase enzyme. The partial purification and characterization of chromate reductase of Cr-B4 helped in investigating the kinetic and inhibition properties of this enzyme, which can help in improving its catalytic performance and its use toward solving Cr(VI) contamination problems.

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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 redu...
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