Purification and characterization of superoxide dismutase from Phanerochaete chrysosporium

Enzyme and Microbial Technology 25 (1999) 392–399

Purification and characterization of superoxide dismutase from Phanerochaete chrysosporium ¨ ztu¨rka, L. Arzu Bozkayaa, Esin Atavb, Necdet Sag˘lamb, Leman Tarhana,* Raziye O a

Department of Chemistry, Faculty of Education, University of Dokuz Eylu¨l, 35150, Buca-Izmir, Turkey Department of Biology, Faculty of Education, University of Hacettepe, 06532, Beytepe-Ankara, Turkey

b

Received 29 July 1998; received in revised form 8 April 1999; accepted 4 May 1999

Abstract The superoxide dismutase that protects against oxidative stress of superoxide radicals in living cells was isolated and purified from Phanerochaete chrysosporium and partially characterized. Cells cultivated under optimized growth conditions were distrupted by grinding with glass beads in a mixer-mill. Partial protein precipitation in crude extract was affected by using (NH4)2SO4, polyethylene glycol, and methanol methods. Fractionation of superoxide dismutase was performed by O-diethylaminoethyl-cellulose ion-exchange chromatography, followed by Sephadex G-100 gel-filtration chromatography. Purified enzyme has a molecular weight of 44 000 ⫾ 800 and is comprised of two equal sized subunits each having an Mn element. The optimum pH of purified MnSOD was obtained as 8.8. Enzyme remained stable at pH 7.0 – 8.8, 25°C and up to 45°C at pH 7.4 for 1 h incubation. The enzyme was insensitive to cyanide, hydrogen peroxide, ditiothreitol, sodium azide, Triton X-100, and ␤-mercaptoethanol and was sensitive to sodium dodecyl sulfate. Phenylmethylsulfonyl fluoride (2 mM) and iodoacetamide showed only a 15% inhibition effect on the enzyme. Up to 50% activity reduction was observed for 100 mM ionic strength of phosphate and chloride. © 1999 Elsevier Science Inc. All rights reserved. Keywords: Superoxide dismutase; Superoxide anion radical; Phanerochaete chrysosporium

1. Introduction Superoxide dismutases (SODs) catalyze the dismutation of highly reactive superoxide anion radicals (O2 . ) to molecular oxygen and hydrogen peroxide in all oxygen-metabolizing organisms and in some anaerobes. The catalytic mechanism involves sequential oxidation and reduction of the metal ion, which provides electrostatic channeling of superoxide radicals to the active site. Superoxide dismutases that are metalloproteins contain iron or manganese or copper plus zinc as the prosthetic groups. Copper-zinc SODs were isolated from various eukaryotic sources and surprisingly in an increasing although limited number of bacteria [1,2]. Mn-SOD were found in prokaryotes and in mitochondria of eukaryotes [3,4]. Fe-SOD has been found so far only in prokaryotic organisms such as Escherichia coli and algae and most recently in the eucaryote Euglena gracilis [5,6]. SODs play an essential role in allowing organisms to * Corresponding author. Tel.: ⫹90-232-420-4882/420-1317; fax: ⫹90232-420-4895. E-mail address: [email protected] (L. Tarhan)

survive in the presence of O2. This enzyme already has been shown to influence aging, cancer, and some very important diseases, such as artheriosclrosis, cataract, retinal damage, essential hypertension, amyloidosis, ischemia, and age-dependent immune deficiency disease [7]. In the present study, we aimed to isolate and purify MnSOD from Phanerochaete chrysosporium cultivated under optimized conditions, providing higher activity in comparison to most other prokaryotic organisms, and to carry out a comparative study of its enzymatic properties based on the differences in the metal content.

2. Materials and methods The experiments were performed by using 6-hydroxydopamine (2,4,5 trihydroxyphenyl ethylamine), Coomassie brillant blue G and R, phenylmethylsulfonyl fluoride, gel filtration and electrophoresis molecular weight marker kits, O-diethylaminoethyl (DEAE)-cellulose (Sigma, St. Louis, MO, USA), and Sephadex G-100 (Pharmacia, Sweden). These and all other chemicals reagents used were of analytical grade.

0141-0229/99/$ – see front matter © 1999 Elsevier Science Inc. All rights reserved. PII: S 0 1 4 1 - 0 2 2 9 ( 9 9 ) 0 0 0 6 2 - 9

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Optical measurements were achieved with a spectrophotometer (UV-1601, Shimadzu, Tokyo, Japan) at 490 nm. For experimental work, incubator shaker (Physicotherm, New Brunswick Co., NJ, USA), Mixer-mill MM2 (Retch, Haan, Germany), electrophoresis (BIO-RAD, Protean XII, CA, USA), and atomic absorption (Varian, SpectrAA-300 plus, Mulgrave, Victoria, Australia) also were used. 2.1. Media and growth conditions Cultures of P. chrysosporium (American Type Culture Collection (ATCC) 34541) were maintained on supplemented malt agar slants, and spores (conidia) were prepared by a modification of the procedure of Tien and Kirk [8]. Composition of agar for maintenance and spore production (per liter): glucose, 10 g; malt extract, 10 g; pepton, 2.0 g; ferment extract, 2.0 g; KH2PO4, 2.0 g; MgSO4䡠7H2O, 1.0 g; agar, 20 g; thiamin–HCl, 1.0 mg. Spore production in the slants usually requires 2–5 days of growth at 28°C. Spore concentration was determined by measuring absorbance at 650 nm (an absorbance of 1.0 cm⫺1 corresponds to ⬇5 ⫻ 106 spores/ml). Cultures were grown in a synthetic medium containing (per liter): KH2PO4, 2.0 g; CaCl2 䡠 2H2O, 0.114 g; MgSO4 䡠 7H2O, 0.7 g; NH4Cl, 0.12 g; glucose, 2.0 g; FeSO4 䡠 7H2O, 70 ␮g; ZnSO4 䡠 7H2O, 46 ␮g; MnSO4 䡠 2H2O, 35 ␮g; CoCl2 䡠 6H2O, 7 ␮g; thiamin–HCl, 1 mg; and Tween 80, 0.05 g. The culture medium was adjusted to pH 4.5. Cultures were incubated with agitation at 150 rev./min at 28°C in 250-ml shaking flasks containing 100 ml of culture for 12 days. After cultivation, biomass was filtered and kept at ⫺20°C. 2.2. Isolation and purification of SOD Wet P. chrysosporium cells were frozen at ⫺20°C and thawed overnight at 4°C and then suspended in a 20 mM phosphate buffer, pH 7.4, containing polypropylene glycol1 200 in a volume equal to 1.5 times its weight. A 600-␮l cell suspension was ground in 1.5-ml plastic vials with 0.5 g of glass beads (0.5 mm ␾) in a mixer-mill for 3 min. Cell debris was removed by centrifugation at 18 000 rev./min for 15 min. Crude extract was brought to 65% saturation by gradually adding solid (NH4)2SO4 and was stirred for 1 h at 4°C. The precipitate was removed by centrifugation at 18 000 rev./min, 2°C for 15 min and resuspended in 20 mM phosphate buffer, pH 7.4. SOD solution recovered with 94% activity yield was applied to a DEAE-cellulose (16 ⫻ 27 cm) column, which was equilibrated with 0.02 M potassium phosphate buffer, pH 7.4, at 4°C. Elution of the enzyme was achieved with a flow rate of 0.5 ml/min by establishing a linear gradient first with 0.02– 0.1 M phosphate buffer, pH 7.4. and then with 0.2–1.0 M NaCl at 4°C. Fractions containing SOD activity were concentrated by 30 000 nominal molecular weight cut-off (NMWC) ultrafilter and then

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loaded at a flow rate of 0.21 ml/min. Sephadex G-100 (16 ⫻ 90 cm) column was equilibrated with 0.02 M potassium phosphate buffer, pH 7.4, at 4°C. 2.3. SOD activity assay SOD activity assay system was based on the inhibitory effect of SOD on the spontaneous autoxidation of 6-hydroxydopamine (6-OHDA) [9]. One unit is the amount of SOD required to inhibit the initial rate of 6-OHDA autoxidation by 50%. 2.4. Protein determination During the purification steps, protein was determined spectrophotometrically according to Bradford method, by using bovine serum albumin (BSA) as the standard [10]. 2.5. Molecular weight determination The apparent molecular weight of the purified SOD was determined by gel exclusion chromatography on a Sephadex G-100 column. BSA (66 000), carbonic anhydrase (29 000), and aprotinin (6500) were used as molecular weight markers, and blue dextran (2 000 000) was used for determining the void volume. 2.6. Electrophoresis Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was performed according to the method of Laemmli by using a vertical slab gel apparatus [11]. The following proteins were used as SDS-PAGE electrophoresis molecular weight standards: BSA (66 000), ovalbumin (45 000), trypsinogen (24 000), ␤-lactoglobulin (18 400), and lysozyme (14 300). 2.7. Determination of metal content Metal content of purified P. chrysosporium SOD was determined by a micro cuvette atomic absorption method after the enzyme was dialyzed extensively against 10 mM phosphate buffer, pH 7.4, containing 1 mM ethylenediaminetetraacetic acid and followed by buffer lacking ethylenediaminetetraacetic acid.

3. Results Small-scale cell-disintegration methods such as mechanical grinding by glass beads, enzymatic lysis, and sonication were investigated to determine the optimized crude extract preparation conditions with the highest SOD activity. Mechanical disintegration of white-rot fungus was performed in mixer-mill. Samples (600 ␮l) having different cell concentrations (30 – 65%) in 20 mM phosphate buffer, pH 7.4, were ground with varying amounts of the glass

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Fig. 1. SOD activity variations depending on glass beads amounts at 30% (—J—), 40% (—⌬—), 65% (—䡺—) w/v concentrations of P. chrysosporium and depending on procedure time for 65% (—F—).

beads (0.5 mm ␾) for 10 min, and the SOD activities were measured. The procedure was repeated to get the best ratio of cell concentration to glass beads versus time (Fig. 1). As can be seen from Fig. 1, the highest SOD activity was obtained as 102 U/ml for 65% (w/v) cell concentrations with 0.5-g glass beads (0.5 mm ␾) and for 10 min. This processing time was decreased to 3 min after the freezing and thawing of cells while giving the same activity. Disintegration of cell membranes also was performed in the presence of 1 mg of lysing enzymes (from Rhizoctonia solani) in 1.5 mM ␤-mercaptoethanol at a 65% cell concentration in a volume of 600 ␮l for 1 h. The SOD activity values were obtained by using enzymatic lysis and sonication methods as 100 and 47.8 U/ml, respectively. The results showed that the mechanical grinding with glass beads was the best method for disintegration of cell membrane. The first purification step of SOD from crude extract was achieved by fractional precipitation of proteins by using (NH4)2SO4, polyethylene glycol, methanol, and (NH4)2SO4 ⫹ methanol methods. The highest recovered activity was obtained as 72.2 U/mg for 65% (NH4)2SO4 in the bottom precipitated phase. According to the results of polyethylene glycol (PEG) precipitation method, maximum SOD activity was obtained with a lower amount of PEG of a higher molecular weight; the best value in upper phase recorded as 70.1 U/mg was achieved in the presence of 14% (w/w) of PEG-5 000. Superoxide dismutase in crude extract was not resistant to methanol. The activity values in both phases were observed to be very low after precipitation with various ratios of methanol. The maximum recovered activity was increased to 35% in 1/4 methanol by adding (NH4)SO4 up to 65% saturation. After the (NH4)2SO4 precipitation process, further purification of SOD was achieved with DEAE-cellulose (16 ⫻

27 cm) column chromatography. In the first step, weakly bound proteins were eluted from the column with a linear gradient of phosphate buffer (20 –100 mM, pH 7.4), followed by strongly bound ones using NaCl solutions (0.2–1.0 M). The SOD activities were observed in fractions 18 –21 (Fig. 2). Sixty-seven percent of SOD activity was recovered in the pooled fraction with a 4.6-fold increase in specific activity. The active fractions were concentrated by 30 000 NMWC ultrafilter and were applied to a Sephadex G-100 (16 ⫻ 90 cm) column for further purification. Elutions containing SOD activity were collected in the fractions 20 –22 (Fig. 3). Enzyme was eluted from the column as a major single symmetrical peak resulting in a 53-fold increase in the specific activity that was determined to be 2250 U/mg. A summary of the purification procedures is presented in Table 1. The apparent molecular weight of the P. chrysosporium SOD was estimated to be 44 000 ⫾ 800 by gel filtration on a Sephadex G-100 column and 22 000 ⫾ 500 by SDS slab gel electrophoresis (Fig. 4). These values indicate that the enzyme is comprised of a dimer of equal-sized subunits. Also, atomic absorption spectroscopy revealed that purified SOD contained 1.02 ⫾ 0.16 atom of manganese per subunit. Consistent with the general reports, no activity loss was determined for P. chrysosporium Mn-SOD in the presence of 2 mM KCN and 2 mM H2O2 in the assay medium. 3.1. Variations of activity with pH Activity profiles of P. chrysosporium SOD were investigated at 25°C and at different pH values in 20 mM phosphate, pH 7.4 – 8.0, and borate, pH 8.7–9.5 buffers. As can be seen from Fig. 5, the optimum pH value was determined to be 8.8.

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Fig. 2. Elution profile of P. chrysosporium SOD from DEAE— cellulose. Absorbance at 280 nm (—J—), SOD activity of fractions (—F—).

3.2. Variations of stability with pH and temperature The pH dependence of the enzyme stability was measured from the residual activity at standard activity assay

conditions after 1 h of preincubation at different pH values in 20 mM acetate, pH 4.7–5.0, phosphate, pH 6.0 – 8.0, and borate, pH 9.0 –11.0, buffers at 25°C. As shown in Fig. 5, the purified SOD remained comparatively stable in the al-

Fig. 3. Gel chromatography on Sephadex G-100 of the collected P. chrysosporium SOD fractions from the DEAE-cellulose column. (a) Absorbance at 280 nm (—J—), SOD activity of fractions (—F—), and molecular weight calibration curve. (b) Protein markers (—J—), purified SOD (—F—).

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Table 1 Purification steps of SOD from Phanerochaete chrysosporium Purification steps

Volume activity (IU/ml)

Protein amount (mg/ml)

Specific activity (IU/mg)

Yield

Purification fold

Crude extract 65% (NH4)2SO4 DEAE–cellulose Sephadex G-100

102 96 68.2 90

2.40 1.33 0.35 0.04

42.5 72.18 194.8 2250

100 94 66.9 88.2

— 1.7 4.6 53

observed compared with Mn-SOD in 20 mM phosphate buffer, pH 7.4, for these incubation periods (Fig. 6). As shown in Fig. 6, under the same incubation conditions, the maximum decrease of the retained activity was observed to be 15% after 15 min for 2 mM phenylmethylsulfonyl fluoride and 2 mM iodoacetamide and to be 85% after 60 min for 2 mM SDS; no further variation was observed in the following 4-h period. 3.4. Effect of ionic strength on the P. chrysosporium SOD activity

kaline region of pH 7.0 – 8.8 but was inactivated rapidly out of this range. The thermostability variations were investigated by preincubating the enzyme in 20 mM phosphate buffer, pH 7.4 at 35, 45, 55, 65, and 75°C for 1 h. The remaining activity values were determined under the standard assay conditions (Fig. 5). The P. chrysosporium SOD was stable up to 45°C but was inactivated rapidly at temperatures above this. No significant retained activity was found after incubation at 75°C for 1 h. 3.3. Effect of some compounds on the SOD activity The purified Mn-SOD was incubated in 20 mM phosphate buffer, pH 7.4, each containing 2 mM ␤-mercaptoethanol, 2 mM dithiothreitol, 2 mM H2O2, 6 mM NaN3, and 1% (w/w) Triton X-100 for 4 h at 25°C. No activity loss was

Activity variations depending on the ionic strength of phosphate and chloride in the 0.02– 0.250 M concentration range were investigated by using the standard activity assay conditions. As can be seen from Fig. 7, similar inhibition values were observed for phosphate pH 7.4, 8.0 and chloride buffered with 20 mM phosphate up to 100 mM ionic strength. After this point, the inhibition effect in phosphate was higher at pH 8.0 than at pH 7.4, and monovalent anion chloride was found to be a more effective inhibitor than divalent anion phosphate at pH 7.4. In phosphate pH 7.4, the retained activity value versus increasing ionic strength decreased up to a concentration of 100 mM and then after a 15% rise remained constant, indicating that the dismutation reaction is electrostatically facilitated in this region. 3.5. Storage stability The purified Mn-SOD was stored in 20 mM phosphate buffer at 4°C. The activity was stable during a period of 1 month. Also, no activity decrease was observed for the enzyme stored at ⫺20°C for 3 months.

4. Discussion

Fig. 4. SDS-polyacrylamide gel electrophoresis of manganese-containing SOD isolated from P. chrysosporium. (a) Protein standards. (b) Purified Mn-SOD.

The crude extract from P. chrysosporium was prepared by using a small-scale disruption method. The best activity yield was obtained by mechanical grinding with glass beads, following a freeze and thaw cycle. The enzymatic lysis process, although providing similar activity values, was not favored because of the possibility of additional contamination brought in by the lysing enzymes and the ␤-mercaptoethanol added to the medium. The specific activity in crude extract from P. chrysosporium is higher than the enzyme level reported for Trichinella spiralis, Escherichia coli, Mycobacterium avium, Moniezia expansa, Hymenolepis diminuta [12–15]. Fractional precipitation of the proteins was performed at 65% saturation of (NH)2SO4 for maximum activity yield of SOD. In this precipitation step, it is an advantage that SOD is present in the bottom phase so no salt removal process is required. Precipitation with PEG was unacceptable because it required an additional purification step because of the presence of SOD in the upper

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Fig. 5. PH-dependent activity variation at 25°C (—⌬—); pH-stability variations for 1 h incubation at 25°C (—J—); temperature-stability variations for 1-h incubation at pH 7.4 (—F—) of P. chrysosporium.

phase together with PEG, although an activity comparable to (NH)2SO4 precipitation was recovered. But, the activity yields were decreased dramatically by using the methanol precipitation method. Reported studies indicate that MnSOD is more susceptible to denaturation by organic solvents than the Cu,Zn-SOD [16].

In general, Mn-SODs are characterized from different sources that contain two or four subunits [15,17]. The cyanideinsensitive P. chrysosporium SOD was a homodimer with an identical molecular weight of 22 000 ⫾ 500 and contained one atom of Mn per subunit. It is remarkably similar to comparable enzymes isolated from a wide range of bacteria [18].

Fig. 6. The effects of some compounds on the stability of P. chrysosporium Mn-SOD: 2 mM H2O2 (—J—), 1% (w/w) Triton X-100 (—F—), 2 mM dithiothreitol (—j—), 6 mM NaN3 (—⫻—), 2 mM ␤-mercaptoethanol (—f—), 2 mM phenylmethylsulfonyl fluoride (—⌬—), 2 mM iodoacetamide (—Œ—), and 2 mM SDS (—多—) at 25°C.

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Fig. 7. The effects of ionic strength on the Mn-SOD activity: phosphate, pH 7.4 (—J—), pH 8.0 (—F—), and chloride, pH 7.4 (—⌬—) at 25°C.

The optimum pH value for P. chrysosporium SOD was 8.8; this is similar to those obtained for the fungus Pleurotus olearius and for Pisum sativum [19,20]. However, the dismutation rate for copper- and zinc-containing enzyme from bovine erythrocytes was essentially independent of pH in the wide range of 6.0 –10.2. It can be said that a positive charge area in the active site region of the enzyme is important for the electrostatic facilitation of the catalyzed dismutation reaction [21,22]. The positive charge loss on this area above pH 9.0 prevents the guidance of O2 . radicals in to the active site channel. This effect also is demonstrated by the decrease in Mn-SOD activity with increasing ionic strength. Thermal, pH, and storage stability of the purified P. chrysosporium SOD is better than that obtained from Ascaris suum [3]. The purified Mn-SOD was not stable even in a medium containing 2 mM SDS, which had no effect on Cu,Zn-SOD up to 70 mM SDS [21]. The interaction of common positive charges and the hydrophobic zones on Mn-SOD with the negative head groups and the alkyl chains of SDS leads to strongly steric strains and stability loss. ␤-Mercaptoethanol and dithiothreitol had no effects on the stability of the enzyme indicating that -SH groups in the Mn-SOD structure do not play an important role for activity [23]. The manganese-containing SOD, purified from P. chrysosporium, belongs to a class of SOD enzymes having remarkably similar properties and is distinguishable by only minor differences.

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