ПРИКЛАДНАЯ БИОХИМИЯ И МИКРОБИОЛОГИЯ, 2013, том 49, № 3, с. 249-254
UDC 577.15:576.80
PURIFICATION AND CHARACTERIZATION OF NITROREDUCTASE FROM RED ALKALIPHILIC BACTERIUM Aquiflexum sp. DL6
© 2013 S. A. Misal, V. D. Bajoria, D. P. Lingojwar, and K. R. Gawai
Biochemistry Division, Department of Chemistry, University of Pune, Pune 411 007, India e-mail: krgawai@chem.unipune.ac.in Received March 29, 2012
Nitroaromatic compounds are toxic to living organisms. Most of them exhibit human mutagenic and carcinogenic potential. Biotransformation and bioremediation processes can convert these compounds into non-toxic compounds. Acclimatization of bacterial strain Aquiflexum sp. DL6 with nitro-aromatics resulted in significant induction of nitroreductase (EC 1.5.1.34). The enzyme was purified by the combination of DEAE-cellulose and Sephadex G-100 column chromatography with 80-fold purification and 22% yield. Molecular weight of purified nitroreductase was estimated to be 29 kDa by SDS-PAGE. The enzyme characteristics were explored by varying the pH and temperatures, and the optimum activity was found at pH 9.5 and 40°C. It was revealed that the substrate specificity of nitroreductase of Aquiflexum sp. DL6 was wide for the most of the tested nitro-aro-matic compounds. The kinetic parameters like Michaelis constant and velocity maxima were determined with o-nitrophenol and NADH as substrates.
DOI: 10.7868/S0555109913030124
Nitro-aromatics compounds are found as potential environmental pollutants representing their wide use as explosives, pesticides, dyes, polyurethane foams, pharmaceuticals and plastics, etc. [1—5]. They are stable and persistent in the environment and toxic to living organisms. Its exposure poses many health hazards by affecting reproductive and central nervous systems, heart and liver, many eventually leading to death [69]. Moreover, its reduced intermediates also exhibit mutagenic and carcinogenic potentials in humans [2, 3, 10-11].
Most reports suggested the physico-chemical methods for removal of nitroaromatic compounds from waste water. Use of microorganisms for removal of these compounds is the best and cost effective approach [12]. Microorganisms can transform the nitro aromatic compounds by reducing the aromatic ring by means of the monooxygenase and dioxygenase enzyme. Monooxy-genase and dioxygenase insert a single oxygen atom or two hydroxyl groups into the aromatic ring, respectively, which lead to an elimination of nitro group. In addition, nitroreductase is able to carry out the reduction of the nitro group to the corresponding amino derivatives [13].
In biotransformation, enzymes which catalyze the reduction of nitro aromatic compounds are termed as nitroreductases. The sensitivity of a particular nitrore-ductase towards oxygen has been used to classify these enzymes into oxygen-sensitive and insensitive type. The mechanism underlying the observed oxygen sensitivity of certain nitroreductases was found to involve the re-oxidation of the one-electron reduced nitro-anion radical to the parent compound with the con-
comitant formation of superoxide [14] while, the oxygen-insensitive enzymes catalyze an obligatory two-electron reduction of this substrate [15-17].
More recently, studies have discovered partial reduction of the nitro group to a hydroxyl amino derivative which eventually releases nitrogen as ammonia. The reductive pathway requires one mole of an oxygen and one mole of NADH to convert nitro aromatic compound to important metabolic intermediates and release ammonia [4, 5, 13].
Alkaliphiles are the microorganisms that grow optimally at pH above 9.0 but cannot or slowly grow near neutral pH values. Differences between internal and environmental pH across the plasma membrane of the cell lead to rapid transportation of ions and substances. Accordingly, the cell keeps the intracellular pH in the range between 7.0 and 8.5 in order to thrive in alkaline environments [18-20]. Various enzymes isolated and well characterized from alkaliphiles were azoreductase, pullulanases, alkaline amylases and cel-lulases, xylanases and pectinases, etc. [21-23].
However, a thorough exploration of the biochemical and kinetic properties of nitroreductase purified from alkaliphiles has not been well focused in the literature. The aim of the study was to purify and carry out of preliminary characterization of a nitroreductase from red alkaliphilic bacterium Aquiflexum sp. DL6.
MATERIALS AND METHODS
Chemicals. Nutrient agar, EDTA, DTT, NADH and nitro compounds were obtained from SRL (India). DEAE-cellulose and Sephadex G-100 were pur-
chased from Sigma, (USA ) and Pharmacia Fine Chemicals (Sweden), respectively. All other chemicals were of the highest grade of purity and commercially available.
Microorganism and growth conditions. The Aqui-flexum sp. DL6 strain was collected from alkaline Crater Lake of Lonar (India). Isolation of pure strain of a microorganism was done by serial dilution and plate methods. The Aquiflexum sp. DL6 bacterial strain was identified by 16S rRNA method described earlier [23] (data not shown). The sequence of the 16S rRNA gene of the strain Aquiflexum sp. DL6 is available on NCBI database (GenBank ID: JF812063).
Aquiflexum sp. DL6 bacterial strain was grown in the cultivation medium containing (g/l): yeast extract — 5.0, peptone — 5.0 and sodium chloride — 5.0 with trace elements (mg/l): KH2PO4 - 300, Na2HPO4 - 980 and MgSO4 — 10. The pH was adjusted to 9.0. Flasks containing 100 ml of a medium were inoculated with 5 ml of microorganism suspension and incubated at 37°C. After sufficient growth for 24 h, nitro compounds were added from the stock solutions, with final concentration 1 g/l.
Crude extract preparation. After 24 h incubation, the bacterial cells were harvested by centrifugation at 10,000 x g for 10 min at 4°C, washed with physiological saline (0.85% NaCl) and thrice with 0.1 M sodium phosphate buffer (pH 7.4). The pellet was suspended in 50 ml of the same buffer containing 1 mM EDTA, 1 mM DTT, 1 mM lysozyme and 20% glycerol (v/ v). Cells were disrupted at 4°C by sonication for 30 seconds, 6 times with 70% outputs using a Sartorius labsonic (Germany). Cell debris was removed by centrif-ugation at 10,000 x g for 15 min at 4°C. The supernatant obtained constituted the crude bacterial extract and was used for further studies.
Estimation of protein. Concentration of protein at each step of purification was checked by Lowry's method using BSA as a standard. Protein concentration in the chromatographic fractions was monitored by measuring absorbance at 280 nm.
Assay of nitroreductase activity. The activity of ni-troreductase was determined spectrophotometrically at 37°C using a UV-visible spectrophotometer, Jasco V 630 (Japan) by monitoring a decrease of OD410 based on the procedure described by Bryant et al. with slight modifications [15]. The reaction mixture 3 ml contained 0.1 M sodium phosphate buffer (pH 7.4), 1 mM NADH and 0.2 mM substrate o-nitrophenol (ONP) and 100 ^l of enzyme solution. One unit of enzyme activity was defined as the amount of enzyme required to reduce 1 mM of ONP per min. All experiments were performed at least in triplicates.
Purification of nitroreductase from Aquiflexum sp.
DL6. The bacterial culture of Aquiflexum sp. DL6 was incubated with various nitro compounds for the specific induction of nitroreductase enzyme. After the ac-
climatization for 72 h at 37°C for various nitro compounds, nitroreductase was isolated and purified with the combination of following procedures at 4°C.
Ammonium sulfate precipitation. Solid ammonium sulfate was added with constant stirring to the crude extract over a period of 12 h at 4°C to get 40% of the salt saturation. The resulting precipitate was separated by centrifugation at 4°C and 15,000 x g for 20 min and the supernatant saturated up to 80% with solid ammonium sulfate. The resulting precipitate was collected and dissolved in a minimal volume of 0.1 M phosphate buffer (pH 7.4). The protein solution was dialyzed against the same buffer at 4°C for 24 h.
Ion-exchange chromatography. After dialysis, the solution was loaded on pre-equilibrated DEAE—cellulose column (2 x 30 cm) with equilibrating buffer 0.1 M phosphate buffer, pH 7.4. The protein was eluted with a linear gradient of NaCl (0—500 mM) in the same buffer. Fractions of 5 ml were collected, those fractions showed higher nitroreductase activity were pooled and reverse dialyzed against solid sucrose at 4°C to reduce the volume.
Molecular exclusion chromatography. The reverse dialyzed enzyme preparation was applied to a Sepha-dex G-100 column (2 x 30 cm) equilibrated with 2 bed volumes of equilibrating buffer. The enzyme was eluted by the same buffer at a flow rate of 6 ml/h. Fractions of 3 ml were collected and those that showed higher nitroreductase activity were pooled and reverse dia-lyzed against solid sucrose at 4°C to concentrate the protein.
Characterization of purified nitroreductase from Aquiflexum sp. DL6. SDS-PAGE was made with 4.5% stacking and 10% resolving polyacrylamide gels as described by Laemmli [24]. Electrophoresis was performed with at least 20 ^g of protein samples per well and 20 ^g of protein molecular weight markers in a Bangalore Genei Midi Vertical Unit (India) at constant voltage of 100 V for 1 h. To check the homogeneity of nitroreductase, native PAGE was performed without SDS according to the method ofLaemmli. Protein molecular mass standards (Bio Lit, India) were used. Gels were stained for proteins with silver [25].
Determination of optimal pH and temperature ofpu-rified nitroreductase. The influence of pH on the ni-troreductase activity was studied over a wide range of pH (from 3.6 to 10.7) using a mixture ofdifferent buffers adjusted to the same ionic strength 100 mM (carbonate, sodium phosphate, and sodium acetate). At each pH the enzyme was incubated with buffer for 30 min at 37°C, and residual acti
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