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Purification and partial characterization of azoreductase from Enterobacter agglomerans
ABB Archives of Biochemistry and Biophysics 413 (2003) 139–146 www.elsevier.com/locate/yabbi
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 Blaghena a
Unit of Bio-industry and Molecular Toxicology, Laboratory of Microbiology, Biotechnology and Environment, Faculty of Sciences A€ın Chock, University Hassan II-A€ın Chock, Km 8 route dÕEl Jadida, B.P. 5366 M^ 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 approximately 90-fold with 20% yield by ammonium sulfate precipitation, followed by three steps of column chromatography (gelfiltration, 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 Km values for both NADH and MR were 58.9 and 29.4 lM, respectively. The maximal velocity (Vmax ) was 9.2 lmol of NADH min1 mg1 . The purified enzyme is inhibited by several metal ions including Fe2þ and Cd2þ . 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 (R1 AN@NAR2 ). More than 800,000 tons of dyes are produced annually worldwide [1], of which 60–70% are azo dyes [2]. During manufacturing, an estimated 10–15% is released into the environment [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 interesting approach is to promote the bacterial degradation of these compounds in wastewater treatment systems. In contrast, bacterial degradation of these dyes does not
* Corresponding author. Fax: +212-22-23-06-74. E-mail address: [email protected] (A. Moutaouakkil).
have similar problems. To establish biological wastewater 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 azoreductase, 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 azoreductases [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 monomer and shows substrate specificity [18,21,22]. However,
0003-9861/03/$ - see front matter Ó 2003 Elsevier Science (USA). All rights reserved. doi:10.1016/S0003-9861(03)00096-1
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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)1 by 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). Biochemical analysis according to the standardized micromethod API 20 E (bioMerieux, Inc.) allowed bacterial identification. 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 centrifugation at 9500g for 10 min, washed three times with 50 mM sodium phosphate buffer (pH 7.0), and suspended 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. Cellular debris and unbroken cells were removed by centrifugation 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 ððNH4 Þ2 SO4 Þ, 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 G75 (Pharmacia Fine Chemicals, Uppsala, Sweden) column (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 preparation 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. Elution 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 containing 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 spectrophotometrically at 25 °C, using a Jenway 6405 UV/ Visible spectrophotometer, by monitoring NADH disappearance at 340 nm based on the procedure described by Zimmermann et al. [16]. In general, enzyme preparation 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
1
Abbreviations used: MR, methyl red; BSA, bovine serum albumin; DCM, dichloromethane; DMPD, N ; N 0 -dimethyl-p-phenylenediamine; ABA, 2-aminobenzoic acid.
Protein concentration was measured according to the Bradford [23] procedure, using bovine serum albumin (BSA) as a standard.
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Analytical gel electrophoresis Denaturing polyacrylamide gel electrophoresis. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) was performed as described by Laemmli [24] on one-dimensional 12% polyacrylamide slab gels containing 0.1% SDS. Gels were run on a miniature vertical slab gel unit (Hoefer Scientific Instruments). After electrophoresis, gels were stained with Coomassie brilliant blue R-250 at 0.025% (w/v) in methanol/acetic acid/water (4:1:5, v/v/v) for 30 min at room temperature. Destaining was done in methanol/acetic acid/water (4:1:5, v/v/v). The apparent subunit molecular weight was determined by measuring relative mobilities and comparing with the prestained SDS–PAGE molecular weight standards (Precision Plus Protein Standards, Bio-Rad). Native molecular weight determination. To determine the native molecular weight of purified azoreductase, nondenaturing polyacrylamide gel electrophoresis was carried out according to the method of Hedrick and Smith [25]. The separating gels (6, 8, 10, and 12% polyacrylamide) were buffered with 1.5 M Tris–HCl (pH 8.8). The running buffer was composed of 25 mM Tris and 320 mM glycine (pH 8.6). All experiments were realized at 4 °C. The electrophoresis running conditions, staining, and destaining were as described for SDS– PAGE. The relative molecular weight of the native purified azoreductase was estimated using a commercial rabbit muscle glyceraldehyde-3-phosphate dehydrogenase (137,000), BSA (66,000), ovalbumin (45,000), and trypsin (23,000) (Sigma) as molecular weight markers. By constructing the Ferguson plot [logðRf 100Þ versus the concentration of polyacrylamide gels (%)], the resulting slopes versus the standard native proteins of known molecular weight allows determination of the molecular weight of purified azoreductase. HPLC analysis of methyl red and its metabolites MR and its degradation products were identified by reverse-phase HPLC analysis with UV detection using the method of Wong and Yuen [26] which we modified according to our conditions. Two assay mixtures containing 0.350 mM NADH and 90 lM MR in 10 ml of 50 mM sodium phosphate buffer (pH 7.0) with or without the purified enzyme were prepared. The assay mixture containing the enzyme was then incubated at 20 °C until no azoreductase activity was noted. The two assay mixtures were extracted three times with equal volumes of dichloromethane (DCM). The DCM extracts were pooled and evaporated to 2 ml at 40 °C in a rotary evaporator and then transferred to a test tube. The remaining DCM was removed by evaporation at room temperature (20 0:5 °C) and placed in a hood overnight. The extracted residue was dissolved in 5 ml acetonitrile and filtered through a 0.2-lm nylon filter;
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20 ll was analyzed by a Jasco HPLC system equipped with a Model 875 variable-wavelength detector (detection wavelength was 254 nm) and a reverse-phase C18 column (25 cm 4 mm) packed with 5-lm particles. The mobile phase was composed of 25 mM phosphate buffer (pH 3.0) and acetonitrile (4:6, v/v) with a flow rate of 0.2 ml/min. The standards of methyl red (BDH Chemicals, England), N ; N 0 -dimethyl-p-phenylenediamine (DMPD) (Fluka, Switzerland), and 2-aminobenzoic acid (ABA) (Fluka, Switzerland) were injected for comparison. Determination of optimal pH and temperature of purified azoreductase The influence of pH on the azoreductase activity was studied over a wide range of pH (from 4.0 to 11.0) using a mixture of different buffers that have different pKa (Tris, 4-morpholineethanesulfonic acid, Hepes, sodium phosphate, and sodium acetate) adjusted to the same ionic strength as the standard reaction mixture. Thermal activation experiments were carried out by measuring the activity in 50 mM sodium phosphate buffer (pH 7.0) at temperature range from 5 to 70 °C using a thermostated cuvette holder connected to a refrigerated bath circulator. Thermal denaturation experiments were carried out by enzyme incubation in 50 mM sodium phosphate buffer (pH 7.0) over a temperature range from 5 to 70 °C. After 10 min of incubation, aliquots were cooled in an ice bath and the residual activity was determined at 20 °C as described above. Kinetic studies of purified azoreductase Initial velocities of the enzymatic reaction were performed by varying the concentration of one substrate, MR (from 0.005 to 0.040 mM) or NADH (from 0.037 to 0.300 mM), while the concentration of the other substrate was kept constant (NADH or MR). Values of the Michaelis constants (Km ) and maximal velocity (Vmax ) for the reduction of MR and the oxidation of NADH by the purified azoreductase were determined from Lineweaver–Burk double-reciprocal plots. Effect of cations on azoreductase activity The purified enzyme was incubated in the presence of different concentrations of cations (from 0.025 to 10 mM). For each concentration, residual activity was measured in comparison with control (aliquot without cations). The I50 was then estimated according to the procedure described by Job et al. [27]. Data can be plotted as Vo =Vi versus concentration of inhibitor. After plotting data for several different cation concentrations, a straight line is drawn through the points and the cation concentration that corresponds to Vo =Vi ¼ 2 is the I50 value.
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Results and discussion Purification of azoreductase from Enterobacter agglomerans A total amount of about 590 mg of protein, corresponding to approximately 36 units of azoreductase, was obtained from crude extract of disrupted cells of E. agglomerans (7.1 g wet weight). The purification of the enzyme was performed by ammonium sulfate precipitation, followed by three steps of column chromatography (gel-filtration, anion-exchange, and dye-affinity) which were carried out at 4 °C. After ammonium sulfate precipitation, which eliminated more than 80% of contaminating proteins, the concentrated enzyme solution was applied to Sephadex G-75 and the enzyme was eluted at a flow rate of 10 ml/h. The elution volume of the fraction with maximal activity was approximately 95 ml. The recuperated enzyme solution was loaded onto DEAE– cellulose and the enzyme was eluted with a linear gradient of NaCl (0–500 mM) at a flow rate of 6 ml/h. Following DEAE–cellulose, which allowed the recovery of 15.6 units in 6.3 mg of protein, azoreductase was bound to a Cibacron blue–agarose column which was extensively washed at a flow rate of 20 ml/h and then the enzyme was eluted with 10 mM NADH. The washing step and the low flow rate were essential to remove contaminating proteins. Table 1 summarizes a representative purification protocol. Values of approximately 5.5–5.6 U/mg of protein were obtained for the purified enzyme with a yield of approximately 20% and a purification factor of approximately 90-fold. Molecular weight determination The SDS–PAGE analysis of the different fractions obtained during the purification procedure showed a progressive enrichment in 28-kDa protein (Fig. 1A). Only this protein band was observed in the electrophoretically homogeneous final enzyme preparation. This protein band corresponds to the E. agglomerans azoreductase subunit whose molecular weight could be estimated at 28,000 (1000) Da (Fig. 1B). To determine the molecular weight of the native enzyme, electrophoresis in a nondenaturing system was
Fig. 1. (A) SDS–PAGE pattern showing different purification fractions of azoreductase purified from Enterobacter agglomerans. Lane M, standard proteins; lane 1, crude extract; lane 2, 32–48% ammonium sulfate fraction; lane 3, gel filtration eluate pool; lane 4, anion-exchange chromatography eluate pool; lane 5, affinity chromatography eluate pool (pure protein preparation). (B) Molecular weight of purified azoreductase was estimated by plotting [log (molecular weight of standard proteins) vs. relative mobilities of proteins].
performed using different separating gels (6, 8, 10, and 12% polyacrylamide). From the Ferguson plot (Fig. 2), a value of 28,000 (1000) Da was estimated for the molecular weight of the native azoreductase. This result, compared with that obtained from SDS–PAGE which shows a single band corresponding to the 28-kDa protein (Fig. 1A, lane 5), suggests that azoreductase purified from E. agglomerans should have a monomeric structure. These results are in accordance with those reported by Zimmermann et al. [16] for the same enzyme from Pseudomonas sp., but they differ from those described for azoreductases from S. dysenteriae type 1 and Escherichia coli K12 [19,20].
Table 1 Purification of azoreductase from Enterobacter agglomerans Fraction
Total protein (mg)
Specific activity (U/mg of protein)
Total activity (U)
Purification factor (fold)
Yield (%)
Crude extract Ammonium sulfate (32–48%) Sephadex G-75 DEAE–cellulose Blue agarose
590.2 101.3 23.6 6.3 1.3
0.062 0.325 1.085 2.490 5.540
36.5 32.9 25.6 15.6 7.2
1.0 5.2 17.5 40.1 89.3
100.0 90.1 70.1 42.7 19.7
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Fig. 4. Reductive cleavage of methyl red (MR) by the purified azoreductase. Products analyzed by HPLC were identified as 2-aminobenzoic acid (ABA) and N ; N 0 -dimethyl-p-phenylenediamine (DMPD). Fig. 2. Determination of the native molecular weight of Enterobacter agglomerans azoreductase by native gel electrophoresis of various concentrations of polyacrylamide (6, 8, 10, and 12%). Molecular weight marker proteins were glyceraldehyde-3-phosphate dehydrogenase (138 kDa), BSA (66 kDa), ovalbumin (45 kDa), and trypsin (23 kDa). Relative mobilities of proteins plotted as log (Rf 100) vs gel concentration are indicated in the inset. A plot of the obtained slopes vs molecular weight was linear and used to determine native azoreductase molecular weight.
HPLC analysis of methyl red and its metabolites The HPLC analysis of the extract of the assay mixture without the enzyme exhibits one peak corresponding to MR at a retention time of 8.05 min (Fig. 3A). The same retention time was obtained when the MR standard was injected. On the other hand, the HPLC chromatogram of the extract of the assay mixture incubated in the presence of the purified enzyme shows the disappearance of the MR peak (8.06 min) and the appearance of two major peaks, X1 and X2 , with retention
times of 2.12 and 4.21 min, respectively (Fig. 3B). Tentative assignment of the by-products (X1 and X2 ) was made on the basis of the retention times of standards, supported by previous knowledge of MR degradation processes [26,28,29]. Therefore, products X1 and X2 were identified as DMPD and ABA, respectively. Similar compounds were reported as metabolites of MR degradation by other bacteria [26,28,29]. Consequently, these results suggest that the purified azoreductase catalyzes the reductive cleavage of the azo bond of MR in the presence of NADH as electron donor and yields DMPD and ABA (Fig. 4). Influence of pH and temperature on the purified azoreductase activity The pH activity profile of purified azoreductase was determined in a pH range from 4.0 to 11.0 using a mixture of different buffers (Fig. 5A). The enzyme had a typical bell-shaped profile covering a broad pH range.
Fig. 3. HPLC chromatograms of the extracts of the assay mixture incubated without (A) and with (B) the purified azoreductase. MR, methyl red; X1 and X2 were identified, in comparison to standards, as DMPD and ABA, respectively.
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Fig. 5. (A) Enzymatic activity of purified azoreductase from Enterobacter agglomerans in the pH range 4.0–11.0 using a mixture of different buffers. (B) Enzymatic activity of purified azoreductase at temperature range from 5 to 70 °C; effects of the assay temperature (activation) and the enzyme preincubating temperature (denaturation).
The maximum enzymatic activity occurred at about pH 7.0. The influence of temperature on enzymatic activities was determined between 5 and 70 °C at pH 7.0 (Fig. 5B). Studies on the effect of assay temperature on enzyme activity (thermal activation) revealed an optimal value of about 35 °C with an activation energy Ea ¼ 2:1 kcal/ mol calculated on the basis of the formulae described by Raison [30]: [Ea ðcal=molÞ ¼ 4:576 p], where p is the slope of the straight line drawn using the Arrhenius plot [log (specific activity) versus 1/T (°K)] (data not shown). Preincubation of E. agglomerans azoreductase for 10 min at temperature range varying from 5 to 35 °C (thermal denaturation) did not affect irreversibly the enzyme activity. Thermal inactivation did, however, occur above 40 °C and resulted in total activity loss at 60 °C.
Fig. 6. Kinetics of the enzymatic activity of azoreductase purified from Enterobacter agglomerans. Initial velocities were determined by varying the concentration of one substrate, MR (from 0.005 to 0.040 mM (A)) or NADH (from 0.037 to 0.300 mM (B)), while the other substrate concentration was kept constant.
Table 2 Michaelis constants (Km ) and maximal velocities (Vmax ) for azoreductase purified from Enterobacter agglomerans Substrate
Km ðlMÞ
Vmax (U/mg of protein)
NADH MR
58:93 5:43 29:49 3:76
9:20 0:42 9:21 0:36
Values are means SE of three experiments.
family of lines with a common intersection on the left of the 1/V axis. These results indicate that the reaction mechanism was the sequential rather than the ping-pong type. No inhibition by an excess of substrate (MR or NADH) was observed. From secondary plots of 1/V intersections in Figs. 6A and B against the corresponding reciprocals of substrate concentrations, a Vmax value of 9:20 0:42 lmol min1 ðmg of proteinÞ1 and Km values of 58:93 5:43 and 29:49 3:76 lM for NADH and MR, respectively, were obtained (Table 2).
Kinetic studies of purified azoreductase Enzymatic reactions were performed by varying the concentration of one substrate (MR or NADH) and fixing the other substrate concentration. As shown in Figs. 6A and B, double-reciprocal plots of initial reaction velocity against substrate concentration result in a
Relative activity of purified azoreductase with other substrates Purified azoreductase from E. agglomerans was tested with other azo dyes in an assay analogous to that described for MR. Azo dyes tested were disperse yellow,
A. Moutaouakkil et al. / Archives of Biochemistry and Biophysics 413 (2003) 139–146 Table 3 Relative activity of Enterobacter agglomerans azoreductase with different substrates Substrate
Relative activity (%)
Methyl red Disperse yellow Trypan blue Amaranth Orange G
100 85 50 35 30
trypan blue, amaranth, and orange G. The concentration of dyes in the assay mixture was similar to that used for MR (90 lM). Relative activities of purified azoreductase with all tested azo dyes are given in Table 3. Results show that the purified azoreductase utilized all tested azo dyes as substrate but with different activities. MR was the best substrate for the enzyme. Enzyme activities with other azo dyes were significant and followed the order methyl red > disperse yellow > trypan blue > amaranth > orange G. This observed substrate specificity might be due to dye structures: several investigations reported substrate specificity to be related to dye structures [16,18]. Indeed, MR and disperse yellow, whose enzyme relative activities were more than 80%, have similar chemical structures, whereas the chemical structures of amaranth and orange G, which yield a lower enzyme activity, are different from that of MR. Influence of cations on azoreductase activity Results in Table 4 show the effect of cations on enzyme activity. Cations listed were all inhibitors but the degree of inhibition depended upon the nature of the added ion. Thus, the enzymatic activity of purified azoreductase was strongly inhibited by Fe2þ and Cd2þ with I50 s of 111:58 5:85 and 131:38 8:76 lM, respectively. The Mn2þ ions showed a weaker inhibition with I50 of 429:53 13:99 lM. This inhibition could be explained in several ways and the following hypothesis can be put forth: (i) excess ions interact with the same amino acid residues in the active center of azoreductase molecules and in this way diminish the binding of a substrate and/or (ii) these ions Table 4 Effect of cations on Enterobacter agglomerans purified azoreductase activity Cations
I50 ðlMÞ
Fe2þ Cd2þ Hg2þ Zn2þ Mn2þ
111:58 5:85 131:38 8:76 170:75 11:32 283:62 10:65 429:53 13:99
The values of I50 were calculated as described under Materials and methods.
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bind to the amino acid residues far from the active center of the enzyme but cause, in same way, a partial conformational change of the whole azoreductase molecule, altering its catalytic properties which, consequently, results in a decrease of the enzymatic activity. On the other hand, in the present study, we have found that the purified azoreductase did not require any metal ion for its activity. In conclusion, the results indicate that E. agglomerans, isolated from dye-contaminated sludge, produces an azoreductase enzyme catalyzing the reductive cleavage of the azo bond and initiating the azo dye degradation. This azoreductase differed in a number of instances from those described previously from other sources [12,19,20]. The physicochemical properties of the purified azoreductase may open new possibilities for its biotechnological applications and allow the use of E. agglomerans in the treatment of azo dyes in industrial effluents. We could then suggest the immobilization of this bacterium on various carriers to be used in a continuous process for the degradation of azo dyes in industrial effluents.
Acknowledgments This work was supported by the Moroccan CNCPRST and the urban community of Casablanca. The authors thank Dr. Nourrddine Chafik (University Hassan II-A€ın Chock, Casablanca) for helpful corrections of the text.
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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 Blaghena a
Unit of Bio-industry and Molecular Toxicology, Laboratory of Microbiology, Biotechnology and Environment, Faculty of Sciences A€ın Chock, University Hassan II-A€ın Chock, Km 8 route dÕEl Jadida, B.P. 5366 M^ 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 approximately 90-fold with 20% yield by ammonium sulfate precipitation, followed by three steps of column chromatography (gelfiltration, 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 Km values for both NADH and MR were 58.9 and 29.4 lM, respectively. The maximal velocity (Vmax ) was 9.2 lmol of NADH min1 mg1 . The purified enzyme is inhibited by several metal ions including Fe2þ and Cd2þ . 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 (R1 AN@NAR2 ). More than 800,000 tons of dyes are produced annually worldwide [1], of which 60–70% are azo dyes [2]. During manufacturing, an estimated 10–15% is released into the environment [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 interesting approach is to promote the bacterial degradation of these compounds in wastewater treatment systems. In contrast, bacterial degradation of these dyes does not
* Corresponding author. Fax: +212-22-23-06-74. E-mail address: [email protected] (A. Moutaouakkil).
have similar problems. To establish biological wastewater 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 azoreductase, 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 azoreductases [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 monomer and shows substrate specificity [18,21,22]. However,
0003-9861/03/$ - see front matter Ó 2003 Elsevier Science (USA). All rights reserved. doi:10.1016/S0003-9861(03)00096-1
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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)1 by 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). Biochemical analysis according to the standardized micromethod API 20 E (bioMerieux, Inc.) allowed bacterial identification. 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 centrifugation at 9500g for 10 min, washed three times with 50 mM sodium phosphate buffer (pH 7.0), and suspended 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. Cellular debris and unbroken cells were removed by centrifugation 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 ððNH4 Þ2 SO4 Þ, 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 G75 (Pharmacia Fine Chemicals, Uppsala, Sweden) column (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 preparation 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. Elution 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 containing 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 spectrophotometrically at 25 °C, using a Jenway 6405 UV/ Visible spectrophotometer, by monitoring NADH disappearance at 340 nm based on the procedure described by Zimmermann et al. [16]. In general, enzyme preparation 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
1
Abbreviations used: MR, methyl red; BSA, bovine serum albumin; DCM, dichloromethane; DMPD, N ; N 0 -dimethyl-p-phenylenediamine; ABA, 2-aminobenzoic acid.
Protein concentration was measured according to the Bradford [23] procedure, using bovine serum albumin (BSA) as a standard.
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Analytical gel electrophoresis Denaturing polyacrylamide gel electrophoresis. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) was performed as described by Laemmli [24] on one-dimensional 12% polyacrylamide slab gels containing 0.1% SDS. Gels were run on a miniature vertical slab gel unit (Hoefer Scientific Instruments). After electrophoresis, gels were stained with Coomassie brilliant blue R-250 at 0.025% (w/v) in methanol/acetic acid/water (4:1:5, v/v/v) for 30 min at room temperature. Destaining was done in methanol/acetic acid/water (4:1:5, v/v/v). The apparent subunit molecular weight was determined by measuring relative mobilities and comparing with the prestained SDS–PAGE molecular weight standards (Precision Plus Protein Standards, Bio-Rad). Native molecular weight determination. To determine the native molecular weight of purified azoreductase, nondenaturing polyacrylamide gel electrophoresis was carried out according to the method of Hedrick and Smith [25]. The separating gels (6, 8, 10, and 12% polyacrylamide) were buffered with 1.5 M Tris–HCl (pH 8.8). The running buffer was composed of 25 mM Tris and 320 mM glycine (pH 8.6). All experiments were realized at 4 °C. The electrophoresis running conditions, staining, and destaining were as described for SDS– PAGE. The relative molecular weight of the native purified azoreductase was estimated using a commercial rabbit muscle glyceraldehyde-3-phosphate dehydrogenase (137,000), BSA (66,000), ovalbumin (45,000), and trypsin (23,000) (Sigma) as molecular weight markers. By constructing the Ferguson plot [logðRf 100Þ versus the concentration of polyacrylamide gels (%)], the resulting slopes versus the standard native proteins of known molecular weight allows determination of the molecular weight of purified azoreductase. HPLC analysis of methyl red and its metabolites MR and its degradation products were identified by reverse-phase HPLC analysis with UV detection using the method of Wong and Yuen [26] which we modified according to our conditions. Two assay mixtures containing 0.350 mM NADH and 90 lM MR in 10 ml of 50 mM sodium phosphate buffer (pH 7.0) with or without the purified enzyme were prepared. The assay mixture containing the enzyme was then incubated at 20 °C until no azoreductase activity was noted. The two assay mixtures were extracted three times with equal volumes of dichloromethane (DCM). The DCM extracts were pooled and evaporated to 2 ml at 40 °C in a rotary evaporator and then transferred to a test tube. The remaining DCM was removed by evaporation at room temperature (20 0:5 °C) and placed in a hood overnight. The extracted residue was dissolved in 5 ml acetonitrile and filtered through a 0.2-lm nylon filter;
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20 ll was analyzed by a Jasco HPLC system equipped with a Model 875 variable-wavelength detector (detection wavelength was 254 nm) and a reverse-phase C18 column (25 cm 4 mm) packed with 5-lm particles. The mobile phase was composed of 25 mM phosphate buffer (pH 3.0) and acetonitrile (4:6, v/v) with a flow rate of 0.2 ml/min. The standards of methyl red (BDH Chemicals, England), N ; N 0 -dimethyl-p-phenylenediamine (DMPD) (Fluka, Switzerland), and 2-aminobenzoic acid (ABA) (Fluka, Switzerland) were injected for comparison. Determination of optimal pH and temperature of purified azoreductase The influence of pH on the azoreductase activity was studied over a wide range of pH (from 4.0 to 11.0) using a mixture of different buffers that have different pKa (Tris, 4-morpholineethanesulfonic acid, Hepes, sodium phosphate, and sodium acetate) adjusted to the same ionic strength as the standard reaction mixture. Thermal activation experiments were carried out by measuring the activity in 50 mM sodium phosphate buffer (pH 7.0) at temperature range from 5 to 70 °C using a thermostated cuvette holder connected to a refrigerated bath circulator. Thermal denaturation experiments were carried out by enzyme incubation in 50 mM sodium phosphate buffer (pH 7.0) over a temperature range from 5 to 70 °C. After 10 min of incubation, aliquots were cooled in an ice bath and the residual activity was determined at 20 °C as described above. Kinetic studies of purified azoreductase Initial velocities of the enzymatic reaction were performed by varying the concentration of one substrate, MR (from 0.005 to 0.040 mM) or NADH (from 0.037 to 0.300 mM), while the concentration of the other substrate was kept constant (NADH or MR). Values of the Michaelis constants (Km ) and maximal velocity (Vmax ) for the reduction of MR and the oxidation of NADH by the purified azoreductase were determined from Lineweaver–Burk double-reciprocal plots. Effect of cations on azoreductase activity The purified enzyme was incubated in the presence of different concentrations of cations (from 0.025 to 10 mM). For each concentration, residual activity was measured in comparison with control (aliquot without cations). The I50 was then estimated according to the procedure described by Job et al. [27]. Data can be plotted as Vo =Vi versus concentration of inhibitor. After plotting data for several different cation concentrations, a straight line is drawn through the points and the cation concentration that corresponds to Vo =Vi ¼ 2 is the I50 value.
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Results and discussion Purification of azoreductase from Enterobacter agglomerans A total amount of about 590 mg of protein, corresponding to approximately 36 units of azoreductase, was obtained from crude extract of disrupted cells of E. agglomerans (7.1 g wet weight). The purification of the enzyme was performed by ammonium sulfate precipitation, followed by three steps of column chromatography (gel-filtration, anion-exchange, and dye-affinity) which were carried out at 4 °C. After ammonium sulfate precipitation, which eliminated more than 80% of contaminating proteins, the concentrated enzyme solution was applied to Sephadex G-75 and the enzyme was eluted at a flow rate of 10 ml/h. The elution volume of the fraction with maximal activity was approximately 95 ml. The recuperated enzyme solution was loaded onto DEAE– cellulose and the enzyme was eluted with a linear gradient of NaCl (0–500 mM) at a flow rate of 6 ml/h. Following DEAE–cellulose, which allowed the recovery of 15.6 units in 6.3 mg of protein, azoreductase was bound to a Cibacron blue–agarose column which was extensively washed at a flow rate of 20 ml/h and then the enzyme was eluted with 10 mM NADH. The washing step and the low flow rate were essential to remove contaminating proteins. Table 1 summarizes a representative purification protocol. Values of approximately 5.5–5.6 U/mg of protein were obtained for the purified enzyme with a yield of approximately 20% and a purification factor of approximately 90-fold. Molecular weight determination The SDS–PAGE analysis of the different fractions obtained during the purification procedure showed a progressive enrichment in 28-kDa protein (Fig. 1A). Only this protein band was observed in the electrophoretically homogeneous final enzyme preparation. This protein band corresponds to the E. agglomerans azoreductase subunit whose molecular weight could be estimated at 28,000 (1000) Da (Fig. 1B). To determine the molecular weight of the native enzyme, electrophoresis in a nondenaturing system was
Fig. 1. (A) SDS–PAGE pattern showing different purification fractions of azoreductase purified from Enterobacter agglomerans. Lane M, standard proteins; lane 1, crude extract; lane 2, 32–48% ammonium sulfate fraction; lane 3, gel filtration eluate pool; lane 4, anion-exchange chromatography eluate pool; lane 5, affinity chromatography eluate pool (pure protein preparation). (B) Molecular weight of purified azoreductase was estimated by plotting [log (molecular weight of standard proteins) vs. relative mobilities of proteins].
performed using different separating gels (6, 8, 10, and 12% polyacrylamide). From the Ferguson plot (Fig. 2), a value of 28,000 (1000) Da was estimated for the molecular weight of the native azoreductase. This result, compared with that obtained from SDS–PAGE which shows a single band corresponding to the 28-kDa protein (Fig. 1A, lane 5), suggests that azoreductase purified from E. agglomerans should have a monomeric structure. These results are in accordance with those reported by Zimmermann et al. [16] for the same enzyme from Pseudomonas sp., but they differ from those described for azoreductases from S. dysenteriae type 1 and Escherichia coli K12 [19,20].
Table 1 Purification of azoreductase from Enterobacter agglomerans Fraction
Total protein (mg)
Specific activity (U/mg of protein)
Total activity (U)
Purification factor (fold)
Yield (%)
Crude extract Ammonium sulfate (32–48%) Sephadex G-75 DEAE–cellulose Blue agarose
590.2 101.3 23.6 6.3 1.3
0.062 0.325 1.085 2.490 5.540
36.5 32.9 25.6 15.6 7.2
1.0 5.2 17.5 40.1 89.3
100.0 90.1 70.1 42.7 19.7
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Fig. 4. Reductive cleavage of methyl red (MR) by the purified azoreductase. Products analyzed by HPLC were identified as 2-aminobenzoic acid (ABA) and N ; N 0 -dimethyl-p-phenylenediamine (DMPD). Fig. 2. Determination of the native molecular weight of Enterobacter agglomerans azoreductase by native gel electrophoresis of various concentrations of polyacrylamide (6, 8, 10, and 12%). Molecular weight marker proteins were glyceraldehyde-3-phosphate dehydrogenase (138 kDa), BSA (66 kDa), ovalbumin (45 kDa), and trypsin (23 kDa). Relative mobilities of proteins plotted as log (Rf 100) vs gel concentration are indicated in the inset. A plot of the obtained slopes vs molecular weight was linear and used to determine native azoreductase molecular weight.
HPLC analysis of methyl red and its metabolites The HPLC analysis of the extract of the assay mixture without the enzyme exhibits one peak corresponding to MR at a retention time of 8.05 min (Fig. 3A). The same retention time was obtained when the MR standard was injected. On the other hand, the HPLC chromatogram of the extract of the assay mixture incubated in the presence of the purified enzyme shows the disappearance of the MR peak (8.06 min) and the appearance of two major peaks, X1 and X2 , with retention
times of 2.12 and 4.21 min, respectively (Fig. 3B). Tentative assignment of the by-products (X1 and X2 ) was made on the basis of the retention times of standards, supported by previous knowledge of MR degradation processes [26,28,29]. Therefore, products X1 and X2 were identified as DMPD and ABA, respectively. Similar compounds were reported as metabolites of MR degradation by other bacteria [26,28,29]. Consequently, these results suggest that the purified azoreductase catalyzes the reductive cleavage of the azo bond of MR in the presence of NADH as electron donor and yields DMPD and ABA (Fig. 4). Influence of pH and temperature on the purified azoreductase activity The pH activity profile of purified azoreductase was determined in a pH range from 4.0 to 11.0 using a mixture of different buffers (Fig. 5A). The enzyme had a typical bell-shaped profile covering a broad pH range.
Fig. 3. HPLC chromatograms of the extracts of the assay mixture incubated without (A) and with (B) the purified azoreductase. MR, methyl red; X1 and X2 were identified, in comparison to standards, as DMPD and ABA, respectively.
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Fig. 5. (A) Enzymatic activity of purified azoreductase from Enterobacter agglomerans in the pH range 4.0–11.0 using a mixture of different buffers. (B) Enzymatic activity of purified azoreductase at temperature range from 5 to 70 °C; effects of the assay temperature (activation) and the enzyme preincubating temperature (denaturation).
The maximum enzymatic activity occurred at about pH 7.0. The influence of temperature on enzymatic activities was determined between 5 and 70 °C at pH 7.0 (Fig. 5B). Studies on the effect of assay temperature on enzyme activity (thermal activation) revealed an optimal value of about 35 °C with an activation energy Ea ¼ 2:1 kcal/ mol calculated on the basis of the formulae described by Raison [30]: [Ea ðcal=molÞ ¼ 4:576 p], where p is the slope of the straight line drawn using the Arrhenius plot [log (specific activity) versus 1/T (°K)] (data not shown). Preincubation of E. agglomerans azoreductase for 10 min at temperature range varying from 5 to 35 °C (thermal denaturation) did not affect irreversibly the enzyme activity. Thermal inactivation did, however, occur above 40 °C and resulted in total activity loss at 60 °C.
Fig. 6. Kinetics of the enzymatic activity of azoreductase purified from Enterobacter agglomerans. Initial velocities were determined by varying the concentration of one substrate, MR (from 0.005 to 0.040 mM (A)) or NADH (from 0.037 to 0.300 mM (B)), while the other substrate concentration was kept constant.
Table 2 Michaelis constants (Km ) and maximal velocities (Vmax ) for azoreductase purified from Enterobacter agglomerans Substrate
Km ðlMÞ
Vmax (U/mg of protein)
NADH MR
58:93 5:43 29:49 3:76
9:20 0:42 9:21 0:36
Values are means SE of three experiments.
family of lines with a common intersection on the left of the 1/V axis. These results indicate that the reaction mechanism was the sequential rather than the ping-pong type. No inhibition by an excess of substrate (MR or NADH) was observed. From secondary plots of 1/V intersections in Figs. 6A and B against the corresponding reciprocals of substrate concentrations, a Vmax value of 9:20 0:42 lmol min1 ðmg of proteinÞ1 and Km values of 58:93 5:43 and 29:49 3:76 lM for NADH and MR, respectively, were obtained (Table 2).
Kinetic studies of purified azoreductase Enzymatic reactions were performed by varying the concentration of one substrate (MR or NADH) and fixing the other substrate concentration. As shown in Figs. 6A and B, double-reciprocal plots of initial reaction velocity against substrate concentration result in a
Relative activity of purified azoreductase with other substrates Purified azoreductase from E. agglomerans was tested with other azo dyes in an assay analogous to that described for MR. Azo dyes tested were disperse yellow,
A. Moutaouakkil et al. / Archives of Biochemistry and Biophysics 413 (2003) 139–146 Table 3 Relative activity of Enterobacter agglomerans azoreductase with different substrates Substrate
Relative activity (%)
Methyl red Disperse yellow Trypan blue Amaranth Orange G
100 85 50 35 30
trypan blue, amaranth, and orange G. The concentration of dyes in the assay mixture was similar to that used for MR (90 lM). Relative activities of purified azoreductase with all tested azo dyes are given in Table 3. Results show that the purified azoreductase utilized all tested azo dyes as substrate but with different activities. MR was the best substrate for the enzyme. Enzyme activities with other azo dyes were significant and followed the order methyl red > disperse yellow > trypan blue > amaranth > orange G. This observed substrate specificity might be due to dye structures: several investigations reported substrate specificity to be related to dye structures [16,18]. Indeed, MR and disperse yellow, whose enzyme relative activities were more than 80%, have similar chemical structures, whereas the chemical structures of amaranth and orange G, which yield a lower enzyme activity, are different from that of MR. Influence of cations on azoreductase activity Results in Table 4 show the effect of cations on enzyme activity. Cations listed were all inhibitors but the degree of inhibition depended upon the nature of the added ion. Thus, the enzymatic activity of purified azoreductase was strongly inhibited by Fe2þ and Cd2þ with I50 s of 111:58 5:85 and 131:38 8:76 lM, respectively. The Mn2þ ions showed a weaker inhibition with I50 of 429:53 13:99 lM. This inhibition could be explained in several ways and the following hypothesis can be put forth: (i) excess ions interact with the same amino acid residues in the active center of azoreductase molecules and in this way diminish the binding of a substrate and/or (ii) these ions Table 4 Effect of cations on Enterobacter agglomerans purified azoreductase activity Cations
I50 ðlMÞ
Fe2þ Cd2þ Hg2þ Zn2þ Mn2þ
111:58 5:85 131:38 8:76 170:75 11:32 283:62 10:65 429:53 13:99
The values of I50 were calculated as described under Materials and methods.
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bind to the amino acid residues far from the active center of the enzyme but cause, in same way, a partial conformational change of the whole azoreductase molecule, altering its catalytic properties which, consequently, results in a decrease of the enzymatic activity. On the other hand, in the present study, we have found that the purified azoreductase did not require any metal ion for its activity. In conclusion, the results indicate that E. agglomerans, isolated from dye-contaminated sludge, produces an azoreductase enzyme catalyzing the reductive cleavage of the azo bond and initiating the azo dye degradation. This azoreductase differed in a number of instances from those described previously from other sources [12,19,20]. The physicochemical properties of the purified azoreductase may open new possibilities for its biotechnological applications and allow the use of E. agglomerans in the treatment of azo dyes in industrial effluents. We could then suggest the immobilization of this bacterium on various carriers to be used in a continuous process for the degradation of azo dyes in industrial effluents.
Acknowledgments This work was supported by the Moroccan CNCPRST and the urban community of Casablanca. The authors thank Dr. Nourrddine Chafik (University Hassan II-A€ın Chock, Casablanca) for helpful corrections of the text.
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