Dechlorination of chlorophenols using extracellular peroxidases produced by streptomyces albus ATCC 3005

Enzyme and Microbial Technology 29 (2001) 62– 69

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Dechlorination of chlorophenols using extracellular peroxidases produced by Streptomyces albus ATCC 3005 Vasileios T. Antonopoulos*, Abdul Rob, Andrew S. Ball, Michael T. Wilson Department of Biological Sciences, John Tabor Laboratories, University of Essex, Wivenhoe Park, Colchester CO4 3SQ, United Kingdom Received 28 March 2000; received in revised form 12 February 2001; accepted 5 March 2001

Abstract Streptomyces albus ATCC 3005 was found to produce higher levels of extracellular peroxidase activity (3.420 U mg⫺1) than previously reported for any other actinomycete. Maximum peroxidase activity was obtained after 72 h of incubation at a temperature of 30°C in a liquid medium (pH 7.6) containing (in w/v) 0.8% to 0.9% oat spelts xylan and 0.6% yeast extract, corresponding to a C:N ratio of around 8.4:1. Characterization of the peroxidases revealed that the optimal temperature for peroxidase activity, using the standard 2,4-dichlorophenol (2,4-DCP) assay was 53°C, when the enzyme reaction was performed at pH 7.2. A study of the effect of temperature on the stability of peroxidase over time, showed that the enzyme was stable at 40°C, with a half-life of 224 min, while at higher temperatures the stability and activity was reduced such that at 50°C and 70°C the half-life of the enzyme was 50 min and 9 min respectively. The optimum pH for the activity of the enzyme occurred between pH 8.1 and 10.4. In terms of substrate specificity, the peroxidase was able to catalyze a broad range of substrates including 2,4-DCP, L-3,4-dihydroxyphenylalanine (L-DOPA), 2,4,5-trichlorophenol and other chlorophenols in the presence of hydrogen peroxide. Ion exchange chromatography was used to confirm that the enzyme was able to release chloride ions from a range of chlorophenols. © 2001 Elsevier Science Inc. All rights reserved. Keywords: Peroxidase; Streptomyces albus ATCC 3005; 2,4-Dichlorophenol; 2,4-DCP; L-DOPA; Chlorophenols; Oat spelts xylan; Yeast extract; C:N ratio

1. Introduction Peroxidases, biological catalysts involved in the oxidation of a range of inorganic and organic compounds in the presence of hydrogen peroxide, have been found widely throughout plants, animals and microorganisms, signifying their important role in biological systems [1–2]. Although peroxidases are primarily found intracellularly [3] in almost all organisms examined [4], extracellular peroxidases are less widespread, with the exception of fungi [5] and bacteria [6 –7] where they are readily produced. Attention has been given to these organisms and their peroxidases due to their involvement in the degradation of the complex lignin polymer [8] as was shown from early experiments with 14C [9], lignin substructure model compounds [10 –12] and APPL [13]. Since then, many reports have been written involving microbial peroxidases in the degradation of a wide-range of compounds including dyes [14 –19], olive * Corresponding author. Tel.: ⫹44-0-1206-873332; fax: ⫹44-0-1206873333. E-mail address: [email protected] (V.T. Antonopoulos).

mill waste waters [20], nitroaromatics [21], dioxins [22], chlorinated [23] and many other compounds as reviewed by [24]. Organochlorine compounds accumulate in large quantities in wastewaters as a result of industrial processes such as the pulp and paper production [25–26]. They are mainly formed during the first chlorination and alkaline extraction stage of pulp processing [27–28]. The effluents from such processes contain high-, medium- and low-molecular-mass (LMM) organochlorine compounds [27,29] of great environmental concern that have to be degraded before they reach their discharge sites. Previous reports have indicated the potential of streptomycetes for the biological treatment of bleach effluents [27], with high-molecular-mass (HMM) organochlorines being efficiently degraded within bio-reactors [29]. However, if the HMM and medium-molecularmass (MMM) organochlorines reach the environment untreated, they slowly decompose to the small LMM compounds (⬍1,000) which are toxic and bio-accumulate in the receiving waters or sediments [29]. These LLM compounds comprise mainly chlorinated phenols and guaiacols [27]. Chlorophenols are very persistent, toxic and health risk-

0141-0229/01/$ – see front matter © 2001 Elsevier Science Inc. All rights reserved. PII: S 0 1 4 1 - 0 2 2 9 ( 0 1 ) 0 0 3 5 7 - X

V.T. Antonopoulos et al. / Enzyme and Microbial Technology 29 (2001) 62– 69

related [30 –31] and have been characterized as first priority pollutants by both the EEU (European Economic Union) and US EPA (American Environmental Protection Agency) [32]; this increased concern has led to the development of appropriate bio-sensors for their monitoring [32]. Several studies have described the involvement of peroxidases in the degradation of a range of chlorophenols which are not only produced as by-products of the paper processing but are used in the manufacture of pesticides, herbicides and wood preservatives [30]. For example 2,4-dichlorophenol is used as a precursor for the synthesis of the herbicide 2,4dichlorophenoxyacetic acid and is shown to be readily degraded by both fungi [33] and bacteria [30]. 2,4,5-trichlorophenol, another herbicide precursor, along with 4-chlorophenol, 2,4-dichlorophenol, 2,4,6-trichlorophenol, 2,3,4,6-tetrachlorophenol and pentachlorophenol have been reported to be efficiently dechlorinated from a model wastewater by immobilized horseradish peroxidase [34] and also by fungal peroxidases [23]. In addition, the pesticide and wood preservative pentachlorophenol has also been degraded by fungal peroxidases and a degradation pathway has been proposed [35]. Dechlorination of 2,4-dichlorophenol, 2,4,5-trichlorophenol, 2,4,6-trichlorophenol and pentachlorophenol following incubation with fungal peroxidases showed that peroxidases catalyze a 4-dechlorination to yield a 4-benzoquinone [23,35]. Other reports showed that dehalogenation may not be caused as a result of the peroxidase-mediated reaction and that chloride ions release depend on the oxidative coupling of the free radicals previously generated [36]. In addition, degradation of 2,4-dichlorophenol and pentachlorophenol by fungal peroxidases indicated that all chlorine atoms are removed prior to ring cleavage [33,35]. The finding that peroxidases from fungi and bacteria have a wide range of potential biotechnological applications has caused an increased interest in finding new species that produce beneficial peroxidase activity [5–7]. Although peroxidases are ubiquitous, two main factors limit their exploitation: a) the levels of enzyme production and b) the instability of peroxidase activity under conditions such as high pH and temperatures, conditions generally encountered in industrial processes. This paper reports the effects of environmental conditions on the production of extracellular peroxidase activity by a new species, S. albus ATCC 3005, indicating a number of key properties of the enzyme and investigates its biotechnological potential in the dechlorination of various chlorophenols.

2. Materials and methods 2.1. Microorganism and growth conditions S. albus ATCC 3005 was maintained as a suspension of spores and hyphal fragments in 50.0% (v/v) glycerol at

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⫺20°C and routinely cultured on plates containing M2 medium, at 30°C, for 120 h. The M2 medium contained (in g L⫺1): yeast extract, 5.0; oat spelts xylan, 10.0; MgSO4.7H2O, 0.5; KH2PO4, 2.0; K2HPO4, 4.0; CaCO3, 3.0; bacteriological agar, 20.0. The pH of M2 medium was adjusted to 7.5 with NaOH (1.0 M). Loops of spores taken from these plates were used to inoculate all liquid cultures. 2.2. Liquid culture Both preinoculum and experimental cultures were performed using 100.0 ml conical flasks containing 20.0 ml autoclaved (121°C, 20 min) liquid medium consisting of (in g L⫺1): yeast extract, 6.0; oat spelts xylan, 8.0; (NH4)2SO4, 0.1; NaCl, 0.3; MgSO4.7H2O, 0.1; CaCO3, 0.02. Trace elements solution (1.0 ml L⫺1) was also added in the medium and the pH was finally adjusted at 7.6 with NaOH (1.0 M). The trace elements solution contained (in g L⫺1): FeSO4.7H2O, 1.0; MnSO4.7H2O, 0.2; ZnSO4.7H2O, 0.9. The pH of this solution was adjusted to 7.6 with NaOH (1.0 M). The preinoculum cultures were inoculated with spores from the M2 plates and left to grow for 48 h while experimental cultures were inoculated with 0.5 ml from the preinoculum cultures and left to grow over a period of 168 h. Both culture types were grown at 30°C with shaking at 180 rev min⫺1. Experimental flasks were removed at regular intervals and the peroxidase activity determined using the standard assay described below. 2.3. Enzyme preparation Culture supernatant solutions were produced after centrifuging the contents of each flask at 10,000 g, for 8 min, at 4°C. The cell-free supernatant was stored at ⫺20°C for future analysis. Cell-free supernatant was concentrated by the Amicon ultrafiltration cell model 8400 using 10 kD molecular weight cut-off membranes (Amicon PM10). 2.4. Determination of peroxidase activity Peroxidase activity was assayed using 2,4-DCP as substrate. The final 1.0 ml volume of the reaction mixture contained 0.2 ml of each one of the following: potassium phosphate buffer (100.0 mM, pH 7.2), 2,4-DCP (25.0 mM), 4-aminoantipyrine (16.0 mM), enzyme solution and hydrogen peroxide (50.0 mM). The reaction was initiated by the addition of hydrogen peroxide and the absorbance was monitored at 510 nm, for one min, at 53°C. One unit (U) of enzyme activity is defined as the amount required for an increase in absorbance of one U min⫺1. In addition, the specific activity is defined as the unit of enzyme activity per mg of protein (U mg⫺1). The absorbance value obtained at the end of the one-min incubation was corrected by subtracting a control absorbance value obtained just after the initiation of the reaction by the addition of hydrogen peroxide.

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2.5. Protein estimation Protein estimation was performed spectrophotometrically at 595 nm using the Bio-Rad assay method, which is based on the Bradford method [37]. The total protein concentration of the enzyme solution was estimated from a calibration curve constructed using bovine serum albumin in the range 0.0 ␮g mL⫺1 to 20.0 ␮g mL⫺1. 2.6. The effect of temperature on activity and thermostability of peroxidase The effect of temperature on the activity of peroxidase was monitored by measuring the peroxidase activity of culture supernatant at specific temperatures ranging between 30°C to 70°C using the 2,4-DCP assay. The effect of temperature on the stability of peroxidase over time, was determined by incubating culture supernatant in the absence of substrate over a period of 120 min at various temperatures in the range 40°C to 70°C. Activity of these samples was then measured at 53°C using the standard 2,4-DCP assay. 2.7. Effect of pH on activity The activity of peroxidase over the pH range 3.0 to 13.0 was investigated using culture supernatant and the standard 2,4-DCP assay. However, potassium phosphate buffer was replaced with universal buffer, which contained (in g L⫺1): citric acid, 6.0; boric acid, 1.8; KH2PO4, 3.9; diethylbarbituric acid, 5.3 [38]. The pH was adjusted accordingly with NaOH (1.0 M or 5.0 M). 2.8. Effect of C:N ratio on peroxidase production To examine the effect of the C:N ratio on growth and peroxidase production, cultures were grown as described earlier but the liquid medium was supplemented either with different concentrations (in w/v) of oat spelts xylan ranging between 0.0% to 1.0% or with different yeast extract concentrations in the range 0.0% to 1.2%. All other ingredients were kept fixed at the usual concentrations in both cases. Elemental analysis showed that oat spelts xylan contained (in w/v) 38.8% C and 0.08% N, while yeast extract contained (in w/v) 39.4% C and 10.8% N. 2.9. Substrate specificity The activity of S. albus extracellular peroxidase against various substrates such as L-DOPA, 4-chlorophenol, 2,4DCP, 2,6-dichlorophenol, 2,4,5-trichlorophenol, 2,4,6-trichlorophenol and pentachlorophenol, was studied as described below. In terms of L-DOPA, the reaction mixture (1.0 ml) consisted of 0.25 ml of each one of the following: L-DOPA (16.0 mM), potassium phosphate buffer (100.0 mM, pH 7.2), hydrogen peroxide (50.0 mM) and culture

supernatant. The reaction was initiated by the addition of hydrogen peroxide and the formation of the dopachrome pigment from L-DOPA was monitored at 470 nm, for one min, at 53°C. Peroxidase activity of the remaining six substrates was assayed under the conditions described for the 2,4-DCP assay, with substrate concentrations varying as follows: 25.0 mM for 2,4-DCP and 2,6-dichlorophenol, 15.0 mM for 4-chlorophenol, 2,4,5-trichlorophenol and 2,4,6-trichlorophenol and 10.0 mM for pentachlorophenol. The activity of horseradish peroxidase (3.0 ␮g mL⫺1) against these substrates was also assessed under the same conditions. 2.10. Dechlorination of chlorophenols The degree of dechlorination of 4-chlorophenol, 2,4DCP, 2,6-dichlorophenol, 2,4,5-trichlorophenol and 2,4,6trichlorophenol was examined by incubating each chlorophenol individually with crude concentrated (two-fold) peroxidase, for 30 min, at 53°C. The reaction mixture contained equal volumes of potassium phosphate buffer (100.0 mM, pH 7.2), the chlorophenol under examination, enzyme and hydrogen peroxide (50.0 mM). Chlorophenol concentrations were varying as follows: 25.0 mM for 2,4-DCP and 2,6-dichlorophenol, 15.0 mM for 4-chlorophenol and 10.0 mM for 2,4,5-trichlorophenol and 2,4,6-trichlorophenol. Samples were removed at set time points as shown in the relevant figure (Fig. 7) and frozen using dry ice. Immediately after addition of all chemicals, a sample was removed and frozen to serve as control. Defrosted samples were examined for chloride ions using a Dionex ion chromatograph series 2000i/SP (Sunnyvale, California, USA) connected to a conductivity detector. The anion exchange column used was a Dionex AS4A-SC and the samples were eluted with a solution prepared in ultra high purity (UHP) water, containing NaHCO3 (1.7 mM) and Na2CO3 (1.8 mM). The anion exchange column was regenerated using concentrated H2SO4 (0.07% v/v, prepared in UHP water). 3. Results 3.1. Effect of incubation time on peroxidase production Extracellular peroxidase was produced by growing S. albus in a basal mineral liquid medium, in which the main carbon and nitrogen sources were oat spelts xylan and yeast extract respectively. Maximal peroxidase activity (0.650 U mL⫺1) was obtained after 72 h of incubation (Fig. 1). Peroxidase activity was reduced by a total of approximately 50.0% when further incubation (72 h to 168 h) was applied (Fig. 1). 3.2. Effect of C:N ratio on peroxidase production To investigate the effect of substrate concentration on the production of extracellular peroxidase, the effects of C:N

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Fig. 1. Effect of incubation time on peroxidase activity in a basal mineral medium supplemented with 0.8% (w/v) oat spelts xylan and 0.6% (w/v) yeast extract. Peroxidase activity is expressed relative to the maximal value, 0.650 U mL⫺1. The data are presented as means ⫾ SEM for triplicate determinations.

Fig. 3. The effect of temperature on extracellular peroxidase activity produced by S. albus. The reaction was monitored at each one of the specified temperatures using the 2,4-DCP assay. The activity is expressed relatively to the maximal value, 0.580 U mL⫺1. The data are presented as means ⫾ SEM for triplicate determinations.

ratio of the growth medium on enzyme production were investigated. Maximal peroxidase activity (0.650 U mL⫺1) was detected in a minimal salts medium containing 0.9% (w/v) oat spelts xylan and 0.6% (w/v, fixed value) yeast extract corresponding to a C:N ratio of 8.7:1. (Fig. 2). When the effects of C:N ratio of the growth medium on enzyme production were investigated by varying the yeast extract concentration, maximal peroxidase activity (0.600 U mL⫺1)

was detected in a minimal salts medium containing 0.8% (w/v, fixed value) oat spelts xylan and 0.6% (w/v) yeast extract, corresponding to a C:N ratio of 8.1:1 (Fig. 2). Regression analysis for the C:N ratio range 3.5 to 8.7, indicated that there is a linear relationship between peroxidase activity and the C:N ratio (data not shown, R2 ⫽ 0.960). C:N ratios greater than 8.7 reduced peroxidase activity (Fig. 2). 3.3. The effect of incubation temperature on peroxidase activity The effect of temperature on the activity of peroxidase was monitored by measuring the activity using the 2,4-DCP assay at specific temperatures ranging between 30°C and 70°C. Under the conditions applied, maximal peroxidase activity (0.580 U mL⫺1) was obtained when the incubation temperature was set at 55°C (Fig. 3). Non-linear regression analysis of the results indicated that optimal peroxidase activity was obtained when the assay was performed at 53°C (data not shown). At the incubation temperature of 30°C, which was the lowest temperature examined, the activity of the enzyme was 62.0% of the maximal value; at 70°C, the highest temperature examined, activity was found to be 74.0% of the maximal activity (Fig. 3).

Fig. 2. Examination of the effect of C:N ratio on the production of extracellular peroxidase by either varying the oat spelts xylan concentration (-■-) or the yeast extract concentration (-F-) in the liquid medium. Cultures were harvested after 72 h of incubation and the 2,4-DCP assay was used to determine peroxidase activity. Peroxidase activity is expressed relative to the maximal values, 0.650 U mL⫺1 and 0.600 U mL⫺1 respectively. The data are presented as means ⫾ SEM for triplicate determinations.

3.4. Thermostability of peroxidase When the effect of temperature on the stability of peroxidase over time was examined, at 40°C the peroxidase activity showed a half-life of 224 min which was reduced to 50 min at 50°C (Fig. 4). The respective half-life values for 60°C and 70°C were 13 min and 9 min. After 60 min of

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Fig. 4. The effect of temperature on the stability of extracellular peroxidase activity over time. Culture supernatant was incubated at 40°C (-■-), 50°C (-F-), 60°C (-}-) and 70°C (-Œ-) over a period of 120 min. Samples were removed at time intervals and peroxidase activity was determined using the standard 2,4-DCP assay at 53°C. The enzyme activity is expressed relative to the maximal value, 0.600 U mL⫺1. The data are presented as means ⫾ SEM for triplicate determinations.

incubation at 40°C, peroxidase activity was reduced by only 10.0%, while for the same period of time the respective activity reduction at 50°C was 60.0% of the maximal value. At the other two higher incubation temperatures of 60°C and 70°C, after 60 min the enzyme was almost entirely inactivated (Fig. 4). 3.5. The effect of pH on peroxidase activity The activity of peroxidase over the pH range 3.0 to 13.0 showed that maximal peroxidase activity (0.400 U mL⫺1) was found to occur at pH 9.9 (Fig. 5). However, statistical analysis showed no significant difference between the activity values corresponding to pH 8.9 and 9.9. At both pH 8.1 and 10.4, the activity detected was 90.0% of the maximal value, while at neutral pH, the activity was 60.0% and decreased with decreasing pH (Fig. 5). 3.6. Substrate specificity Extracellular peroxidase activity produced by S. albus showed activity against six out of seven substrates examined, namely L-DOPA, 4-chlorophenol, 2,4-dichlorophenol, 2,6-dichlorophenol, 2,4,5-trichlorophenol, 2,4,6-trichlorophenol; no activity was detected against pentachlorophenol (Table 1). Under the substrate concentrations applied, highest activity was obtained against L-DOPA, 2,4-dichlorophenol and 2,4,5-trichlorophenol, while lowest activity was obtained when 4-chlorophenol was examined. The control horseradish peroxidase showed similar behavior against the substrates examined (Table 1).

Fig. 5. The effect of pH on the activity of extracellular peroxidase produced by S. albus. The enzyme activity is expressed relative to the maximal value, 0.400 U mL⫺1. The data are presented as means ⫾ SEM for triplicate determinations.

3.7. Dechlorination of chlorophenols Fig. 7 presents the release of chloride ions from two chlorophenols, 2,4-dichlorophenol and 2,4,5-trichlorophenol, during incubation with crude concentrated peroxidase. In the case of 2,4-DCP, release of chloride ions started immediately after incubation of the reaction mixture while for 2,4,5-trichlorophenol a lag phase was observed for the first 8 min. The concentration of chloride ions after 8 min of incubation for 2,4-DCP was 4.2 ␮M while the corresponding value for 2,4,5-trichlorophenol was approximately 10fold less, 0.4 ␮M (Fig. 7). After 8 min of incubation, the reaction mixture containing 2,4,5-trichlorophenol showed an increase in the chloride ions concentration, reaching a maximal value of 9.2 ␮M after 20 min of incubation. After this time point, until the end of the incubation, the concenTable 1 Substrate specificity of S. albus ATCC 3005 peroxidase and horseradish peroxidase Substrate

Final Monitoring substrate wavelength conc (nm) (mM)

S. albus Horseradish ATCC peroxidase 3005 peroxidase

L-DOPA 4-Chlorophenol 2,4-Dichlorophenol 2,6-Dichlorophenol 2,4,5-Trichlorophenol 2,4,6-Trichlorophenol Pentachlorophenol

4.0 3.0 5.0 5.0 3.0 3.0 2.0

⫹⫹⫹ ⫹ ⫹⫹⫹ ⫹⫹ ⫹⫹⫹ ⫹⫹ ⫺

470 510 510 510 510 510 510

⫹⫹⫹ ⫹ ⫹⫹⫹ ⫹⫹ ⫹⫹⫹ ⫹⫹ ⫺

Positive peroxidase activity is denoted as ⫹ while no significant activity detected is denoted as ⫺. All measurements were carried out at 53°C and pH 7.2. Horseradish peroxidase was used as a control protein. Substrate molecular structures are shown in Figure 6.

V.T. Antonopoulos et al. / Enzyme and Microbial Technology 29 (2001) 62– 69

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Table 2 Extent of dechlorination of various chlorophenols after being treated for 30 min with concentrated crude peroxidase produced by S. albus ATCC 3005

Fig. 6. Chemical structures of substrates: a) L-DOPA, b) 4-chlorophenol, c) 2,4-dichlorophenol, d) 2,6-dichlorophenol, e) 2,4,5-trichlorophenol, f) 2,4,6-trichlorophenol and g) pentachlorophenol.

Chlorophenol

Final chlorophenol concentration (mM)

Released chloride ions rate (␮M h⫺1)

4-Chlorophenol 2,4-Dichlorophenol 2,6-Dichlorophenol 2,4,5-Trichlorophenol 2,4,6-Trichlorophenol

3.8 6.3 6.3 2.5 2.5

13.6 21.0 9.0 16.6 5.6

4. Discussion tration of chloride ions did not increase. The 2,4-DCPcontaining reaction mixture showed maximal concentration of chloride ions after 30 min of incubation, equivalent to 10.5 ␮M (Fig. 7). Non-linear regression analysis of the 2,4-DCP-related data, indicated a good fit when a Michaelis-Menten type regression analysis was performed (R2 ⫽ 0.990). Comparison of all chlorophenols examined, indicated that the highest rate for chloride ions release was observed in the case of the 2,4-DCP-containing reaction mixture (21.0 ␮M h⫺1), with the second highest value obtained for 2,4,5-trichlorophenol (16.6 ␮M h⫺1) (Table 2). The smallest rate of chloride ions release was observed in the case of 2,4,6-trichlorophenol (5.6 ␮M h⫺1), while 4-chlorophenol and 2,6-dichlorophenol showed moderate rate values compared to the rate obtained for 2,4-DCP (13.6 ␮M h⫺1 and 9.0 ␮M h⫺1 respectively) (Table 2).

Fig. 7. Release of chloride ions during incubation of a reaction mixture containing either 2,4-dichlorophenol (-■-) or 2,4,5-trichlorophenol (-F-) with crude concentrated peroxidase. The reaction mixture was incubated for 30 min, at 53°C; samples were removed at [0,4,8,12,16,20,25 and 30] min and examined for chloride ions.

This report investigated the effect of environmental conditions on the production of extracellular peroxidase activity by S. albus ATCC 3005 and the effect of factors such as temperature and pH on the activity of the enzyme. The potential use of peroxidase in the dechlorination of some extensively used chlorophenols was also assessed. The production of extracellular peroxidases is a common feature among actinomycetes. The extracellular peroxidase activity of S. albus was produced in a liquid medium, in which the main carbon and nitrogen sources were oat spelts xylan and yeast extract respectively. Under these experimental conditions and without further concentration of the extracellular culture supernatant, peroxidase activity was detected using 2,4-DCP as the substrate. The incubation time required for maximal peroxidase activity to be obtained was 72 h, this period falling within the range frequently reported for actinomycetes (48 h to 72 h), including Streptomyces thermoviolaceus [39], Thermomonospora fusca BD25 [40 – 42], Streptomyces avermitilis UAH30 [2], Streptomyces viridosporus T7A [42]. When the effect of the concentration of the two basic nutrients (oat spelts xylan and yeast extract) was investigated, maximal peroxidase activity was obtained when 0.9% (w/v) oat spelts xylan and 0.6% (w/v) yeast extract was supplied in the culture medium, corresponding to a C:N ratio of around 8.4:1. A comparable C:N ratio (10:1) has been observed in the case of S. viridosporus T7A peroxidase, where the main carbon source was glucose rather than oat spelts xylan [42]. The maximal specific peroxidase activity reported here (3.420 U mg⫺1) is 11-fold higher than that reported for S. viridosporus, 0.300 U mg⫺1 [12], 28-fold higher than that reported for S. avermitilis UAH30, 0.120 U mg⫺1 [2], and 85-fold higher than that reported for T. fusca BD25 [40] and S. thermoviolaceus [39], both of which showed a maximal activity of 0.040 U mg⫺1. To our knowledge this is the most active peroxidase-producing actinomycete reported. The optimal temperature for the peroxidase assay was estimated to be 53°C when the assay was performed at pH 7.2. In the case of S. viridosporus T7A the optimum temperature was found to be 50°C when the assay was carried

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out at pH 5.5 [43]. Moreover, both S. albus and S. viridosporus exhibit high peroxidase activity at 35°C, corresponding to 74.0% and 72.0% respectively of the maximal peroxidase activity. At an assay temperature of 70°C, the peroxidase from S. albus remained active, indicating that the enzyme is capable of efficient activity (⬎70.0%) over a wide range of temperatures (35°C to 70°C). Given the wide range of industrial conditions encountered, this ability would be an important consideration for biotechnological applications. In the case of T. fusca BD25 and S. avermitilis UAH30, the optimal incubation temperature for performing the 2,4-DCP assay was found to be 60°C, justifying the thermophilic nature of the organisms [2,40]. The stability of the enzyme against time over a range of temperatures indicated that at mesophilic temperatures the enzyme was stable, while at higher temperatures, the stability of the enzyme was reduced, with the enzyme having a half-life of 13 min at 60°C. This value is comparable to the thermostability of peroxidase produced by S. avermitilis UAH30, that shows a half-life of 20 min at 60°C [2]. Optimum pH for peroxidase activity was found to occur over a broad pH range (8.1 to 10.4). This is significantly higher than the optimum pH range observed for S. viridosporus (5.5 to 7.5) [43], T. fusca BD25 (6.0 to 8.0) [40], S. thermoviolaceus (6.5 to 7.0) [39] and S. avermitilis UAH30 (6.0 to 8.5) [2], suggesting that this enzyme may be useful for the treatment of alkaline effluents. Assessment of the activity of the enzyme against a range of chlorophenol substrates showed that the enzyme was able to oxidize successfully all chlorophenols except pentachlorophenol, with 2,4-dichlorophenol and 2,4,5-trichlorophenol being the most reactive substrates (Table 1). The catalysis for all reacting chlorophenols proceeded with the formation of a pink/red color due to coupling with the color-forming reagent 4-aminoantipyrine. This observation is in accordance with the suggestion of Spiker et al., that the oxidation of phenols containing one or no carbon in the para position results in coupling with 4-aminoantipyrine to form a pink/red color [44]. The suggestion was also true for L-DOPA. In addition to colorimetric examination, chlorophenols were subjected to direct measurement of released chloride ions after incubation with S. albus peroxidase. Results indicated that the enzyme was capable of dechlorinating mono-, di- and tri-chlorinated substrates, with maximal dechlorination rate detected with 2,4-dichlorophenol, followed by that of 2,4,5-trichlorophenol (Table 2 and Fig. 7). This paper has identified S. albus ATCC 3005 as a high peroxidase producing actinomycete. Production of the extracellular enzyme has been investigated through changes in incubation time and C:N ratio. In terms of intrinsic properties, S. albus peroxidase was shown to be functional under high temperature and pH. Results indicated the involvement of S. albus peroxidase in the dechlorination of a range of chlorophenols, suggesting the use of the enzyme in the treatment of effluents from chemically bleached pulp. Most

importantly, S. albus peroxidase is suitable for the treatment of alkaline pH effluents (e.g. from kraft pulping process [28], textile industry [45]) where known peroxidases from other species would not be applicable.

Acknowledgments The authors acknowledge the studentship awarded to Vasileios T. Antonopoulos by University of Essex (Colchester, United Kingdom) and also Dr. W. Kurtzakowski (Institute of Public Health and Hygiene, Warsaw, Poland) for providing S. albus ATCC 3005.

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