Purification and partial characterization of azoreductase from Enterobacter agglomerans
Adnane Moutaouakkil, a,* Youssef Zeroual, a Fatima Zohra Dzayri, b Mohamed Talbi, b Kangmin Lee, c and Mohamed Blaghen a
a
Unit of Bio-industry and Molecular Toxicology, Laboratory of Microbiology, Biotechnology and Environment, Faculty of Sciences A€ ıın Chock, University Hassan II-A € ıın Chock, Km 8 route dÕEl Jadida, B.P. 5366 M^ a aarif, Casablanca, Morocco
b
Laboratory of Analytical Chemistry, Faculty of Sciences Ben MÕsik, University Hassan II-Mohammedia, Casablanca, Morocco
c
Laboratory of Enzyme Technology, Chonbuk National University, Chonju, Republic of Korea Received 8 January 2003, and in revised form 20 February 2003
Abstract
Azoreductase, an enzyme catalyzing the reductive cleavage of the azo bond of methyl red (MR) and related dyes, was purified to electrophoretic homogeneity from Enterobacter agglomerans. This bacterial strain, isolated from dye-contaminated sludge, has a higher ability to grow, under aerobic conditions, on culture medium containing 100 mg/L of MR. The enzyme was purified ap- proximately 90-fold with 20% yield by ammonium sulfate precipitation, followed by three steps of column chromatography (gel- filtration, anion-exchange, and dye-affinity). The purified enzyme is a monomer with a molecular weight of 28,000 Da. The maximal azoreductase activity was observed at pH 7.0 and at 35 °C. This activity was NADH dependant. The K
mvalues for both NADH and MR were 58.9 and 29.4 lM, respectively. The maximal velocity (V
max) was 9.2 lmol of NADH min
1mg
1. The purified enzyme is inhibited by several metal ions including Fe
2þand Cd
2þ.
Ó 2003 Elsevier Science (USA). All rights reserved.
Keywords: Enterobacter agglomerans; Azo dye; Methyl red; Azoreductase; Purification; Enzyme kinetics
Azo dyes are aromatic compounds characterized by one or more azo bonds (R
1AN@NAR
2). More than 800,000 tons of dyes are produced annually worldwide [1], of which 60–70% are azo dyes [2]. During manu- facturing, an estimated 10–15% is released into the en- vironment [3]. Aside from their negative aesthetic effects, certain azo dyes have been shown to be toxic [4] and, in some cases, these compounds are carcinogenic and mutagenic [5]. Several physical and chemical treatment methods of dye-contaminated wastewaters have been suggested [6–8] but not widely applied because of the high cost and the secondary pollution which can be generated by the excessive use of chemicals. One inter- esting approach is to promote the bacterial degradation of these compounds in wastewater treatment systems. In contrast, bacterial degradation of these dyes does not
have similar problems. To establish biological waste- water treatment of azo dye, it is essential to discover azo dye-degrading microorganisms and to study the enzymes involved in this degradation.
Bacterial degradation of azo dyes is generally feasible only if the azo linkage is first reduced. The reductive cleavage of the azo bond was catalyzed by the azore- ductase, the key enzyme of azo dye degradation. Several species of anaerobic bacteria that have azoreductase activity have previously been isolated and studied [9–14].
Generally, azo dyes are resistant to attack by bacteria under aerobic conditions. In contrast, some specialized strains of aerobic bacteria have developed the ability to reduce the azo group by special oxygen-tolerant azore- ductases [15–17].
Azoreductase, isolated from several bacteria, has been found to be an inducible [18] flavoprotein [19] and to utilize both NADH and NADPH as electron donors [16,20]. Azoreductase from Pseudomonas sp. is a mono- mer and shows substrate specificity [18,21,22]. However,
Archives of Biochemistry and Biophysics 413 (2003) 139–146
www.elsevier.com/locate/yabbi
ABB
*
Corresponding author. Fax: +212-22-23-06-74.
E-mail address: moutaouakkil@hotmail.com (A. Moutaouakkil).
0003-9861/03/$ - see front matter Ó 2003 Elsevier Science (USA). All rights reserved.
doi:10.1016/S0003-9861(03)00096-1
azoreductase produced by Shigella dysenteriae type 1 is a dimer [19].
Here we report the purification and some physical and kinetic properties of azoreductase produced by Enterobacter agglomerans. This bacterial strain, which reduces some azo dyes under aerobic conditions, was isolated from dye-contaminated sludge. The catalytical reduction of the toxic azo dye methyl red (MR)
1by the purified E. agglomerans azoreductase in the presence of NADH as electron donor is briefly discussed.
Materials and methods
Microorganism and growth conditions
The E. agglomerans strain used in this work was isolated from dye-contaminated sludge collected from an industrial area in Casablanca (Morocco). Biochemi- cal analysis according to the standardized micromethod API 20 E (bioM eerieux, Inc.) allowed bacterial identifi- cation. This bacterial strain, which was found to have a higher ability to decolorize and degrade the toxic azo dye methyl red under aerobic conditions, was grown aerobically at 37 °C for 24 h in nutrient broth (Topley House, Bury, England) containing 100 mg/L of MR.
The culture was inoculated with 1% (v/v) of overnight preculture in the same medium without MR.
Crude extract preparation
Cells from 2.5 L of culture were harvested by centri- fugation at 9500g for 10 min, washed three times with 50 mM sodium phosphate buffer (pH 7.0), and sus- pended in 50 ml of the same buffer containing 0.5 mM EDTA and 0.1% (v/v) 2-mercaptoethanol (buffer A).
Cells were disrupted in the cold by sonication (30 s, 70%
output, 16) using a Bandelin Sonopuls sonifier. Cel- lular debris and unbroken cells were removed by cen- trifugation at 15,000g for 45 min at 4 °C using a Sigma 3K15 refrigerated centrifuge. The supernatant obtained constitutes the crude bacterial extract (soluble protein fraction).
Purification procedure
The enzyme was purified by a four-step procedure carried out at 4 °C.
Ammonium sulfate precipitation. Crude extract was brought to 32% (w/v) saturation with solid ammonium sulfate ððNH
4Þ
2SO
4Þ, stirred for 2 h, and then centri-
fuged at 15,000g for 45 min. Afterward, the resulting supernatant was precipitated with ammonium sulfate to a final saturation of 48% (w/v). The final pellet after centrifugation (45 min at 15,000g) was dissolved in a minimal volume of buffer A. The protein solution was dialyzed twice against 1 L of the same buffer overnight.
Molecular exclusion chromatography. The dialyzed enzyme preparation was then applied to a Sephadex G- 75 (Pharmacia Fine Chemicals, Uppsala, Sweden) col- umn (1:6 60 cm) equilibrated with two bed volumes of buffer A. The enzyme was then eluted with equilibrating buffer at a flow rate of 10 ml/h. Fractions of 2 ml were collected and those that showed azoreductase activity were pooled.
Ion-exchange chromatography. The enzyme prepara- tion from above was applied at a flow rate of 6 ml/h to a DEAE–cellulose (Serva, Heidelberg, Germany) column (3 12 cm) that had been previously equilibrated with buffer A. The column was extensively washed at the same flow rate with equilibrating buffer solution. Elu- tion was performed with a linear gradient of sodium chloride (NaCl) (0–500 mM; total volume of 200 ml) in buffer A. Fractions of 2 ml were collected and those which showed azoreductase activity were pooled and dialyzed twice against 1 L of buffer A overnight.
Dye-affinity chromatography. The dialyzed enzyme preparation was then loaded onto a Cibacron blue–
agarose 3GA (Sigma, St. Louis, MO, USA) column (1 10 cm) equilibrated with two bed volumes of buffer A. The column was extensively washed at a flow rate of 20 ml/h and then eluted with equilibrating buffer con- taining 10 mM of NADH at a flow rate of 6 ml/h. The fractions with maximal activity were collected and pooled. Purified enzyme was made up to 50% glycerol and stored at ) 20 °C until use.
Assay of azoreductase activity
The activity of azoreductase was determined spec- trophotometrically at 25 °C, using a Jenway 6405 UV/
Visible spectrophotometer, by monitoring NADH dis- appearance at 340 nm based on the procedure described by Zimmermann et al. [16]. In general, enzyme prepa- ration was added to 50 mM sodium phosphate buffer (pH 7.0) containing 0.350 mM NADH (Sigma) and 90 lM MR; the total volume of the reaction mixture was 1.0 ml. One unit of enzyme activity was defined as the amount of enzyme that catalyzes the oxidation of 1 lmol of NADH/min. All experiments and assays were carried out in triplicate.
Protein concentration
Protein concentration was measured according to the Bradford [23] procedure, using bovine serum albumin (BSA) as a standard.
1