catechol 1 2 dioxygenase from naphthol degrading thermophilic geobacillus sp strain purification and properties

Cent Eur J Biol • 4(1) • 2009 • 68–73 DOI: 10.2478/s11535-008-0049-y Central European Journal of Biology Catechol 1,2-dioxygenase from α-naphthol degrading thermophilic Geobacillus sp strain: purification and properties Research Article Gražina Giedraityte*, Lilija Kalėdienė Department of Plant Physiology and Microbiology, Faculty of Natural Sciences, Vilnius University, LT–03101 Vilnius, Lithuania Received July 2008; Accepted October 2008 Abstract: T he purpose of this study was purification and characterization of catechol 1,2–dioxygenase from Geobacillus sp G27 strain, which degrades α–naphthol by the β–ketoadipate pathway The catechol 1,2–dioxygenase (C1,2O) was purified using four steps of ammonium sulfate precipitation, DEAE–celullose, Sephadex G–150 and hydroxylapatite chromatographies The enzyme was purified about 18–fold with a specific activity of 7.42 U mg of protein-1 The relative molecular mass of the native enzyme estimated on gel chromatography of Sephadex G–150 was 96 kDa The pH and temperature optima for enzyme activity were and 60oC, respectively A half-life of the catechol 1,2–dioxygenase at the optimum temperature was 40 The kinetic parameters of the Geobacillus sp G27 strain catechol 1,2–dioxygenase were determined The enzyme had apparent Km of 29 µM for catechol and the cleavage activities for methylcatechols were much less than for catechol and no activity with gentisate or protocatechuate was detected Keywords: Thermophilic bacteria • α–naphthol • Catechol 1,2–dioxygenase • Purification © Versita Warsaw and Springer-Verlag Berlin Heidelberg Introduction Environmental aromatic pollutants have been reported to be biodegraded by a variety of microorganisms, which contain various dioxygenases capable of cleaving aromatic compounds [1] Many microorganisms use a catabolic sequence for the degradation of these compounds called the β-ketoadipate pathway Catechol 1,2–dioxygenase (C1,2O) is the key enzyme for the β-ketoadipate pathway, catalyzing the cleavage of the aromatic ring of catechol to cis, cis-muconic acid with incorporation of atoms of molecular oxygen into the substrate Enzymes of the various aromatic hydrocarbons biodegradation pathway, including C1,2O, are inducible in microorganisms [2-4] C1,2O have been purified from a variety of organisms comprising Pseudomonas, Alcaligenes, Ralstonia, Rhodococcus, Acinetobacter and Candida albicans [5-10] The search of thermophilic microorganisms that degrade environmental pollutants has recently been extended These microorganisms contain enzymes 68 that function at elevated temperatures Some of these enzymes are stable at elevated temperatures up to 140oC for more than an hour Such enzymes are of great interest for industrial applications [11-13] Only catechol 2,3–dioxygenase from decyclizing dioxygenases have been purified from thermophilic bacteria Bacillus termoleovorans [14], B stearothermophilus [15] and protocatechuate 3,4–dioxygenase from thermophilic Geobacillus strain [16] with half-life of the enzyme activity at the optimum temperature (60oC) - 40 Higher temperatures in the degradation of contaminated wastes have the advantages of increasing the solubility of the aromatic hydrocarbons and lowering the risk of contamination by pathogenic microorganisms We described here the purification, the biochemical properties and the thermal stability of the first isolated enzyme in the α–naphthol degradation pathway, the catechol 1,2–dioxygenase (catechol : oxygen 1,2-oxidoreductase) * E-mail: grazina.giedraityte@gf.vu.lt G Giedraityte, L Kalėdienė Experimental Procedures 2.1 Bacterium growth and extract preparation α-Naphthol using the bacterial strain Geobacillus sp.G 27 [17] was grown in a mineral salt medium The medium contained (g L-1): K2HPO4 – 1.0, NaH2PO4 – 0.5, (NH4)2SO4 – 2.0, MgCl2 –0.2, FeSO4 – 0.008, CaCl2 – 0.01, yeast extract – 0.1, α–naphthol – 0,1 (as the sole carbon source to induce C1,2O), and mL of trace element solution [18] The pH of the medium was adjusted to 7.0 The flask was inoculated with 10% (v/v) of pre-culture grown overnight in a same mineral salt medium at 60oC to an OD590 of 0.7 Batch culture was incubated in the dark at 60oC until the late exponential growth phase without shaking Bacterial growth was determined by measuring the optical density at 590 nm The cells were harvested with a refrigerated centrifuge at 5 000 x g for 10 at 4oC, washed twice with 20 mL of 50 mM sodium phosphate buffer (pH 7.0) containing 10% (v/v) glycerol and resuspended in the same volume of the buffer The cells were disrupted by ultrasonic treatment for at 22 kHz by using a sonicator The residue was removed by centrifugation at 4oC for 25 at 14 000 x g 2.2 Enzyme assay Enzyme (C1,2O) activity was assayed by monitoring appearance of products with a UV-visible spectrophotometer with a thermojacketed cuvette holder at 60oC The assay system contained 0.3 µmol of catechol in 3.0 mL of 50 mM sodium phosphate buffer, pH 7.0 The reaction was started by addition of a suitable amount of enzyme One unit of enzyme activity is defined as the amount of enzyme that produces µmol of cis, cis–muconic acid at 260 nm per at 60oC The intradiol cleavage activities for 3– and 4–methylcatechols, protocatechuic and gentisic acids were assayed as reported previously by Aoki [7] The catechol 2,3-dioxygenase (extradiol) activity was determined by measuring the formation of 2-hydroxymuconic semialdehide at 390 nm under the same conditions reported for intradiol activity [14] Protein concentrations were determined spectrophotometrically from the absorbance at 280 nm during purification procedure and by the standard Bradford method [19] for a pure enzyme Initial velocities used in determining enzyme kinetic constants were measured with air–saturated 50 mM sodium phosphate buffer at 60oC The kinetic constants were determined graphically from double reciprocal plots 2.3 Enzyme purification The purification procedure was carried out in 50 mM sodium phosphate buffer containing 10% (v/v) glycerol, pH 7.0 at 4oC Ammonium sulfate fractionation The powdered ammonium sulfate was added to 30% of saturation After hours, the precipitate was removed by a 60 centrifugation at 15 000 x g A 30% supernatant was brought into 50% ammonium sulfate and centrifuged as before The pellet was spooned into dialysis tubing and dialyzed for 20 h against 250 mM sodium phosphate buffer DEAE–cellulose fractionation A dialyzed 30–50% ammonium sulfate cut was applied to a DEAE–cellulose column (1x10 cm, Sigma) equilibrated with 50 mM sodium phosphate buffer and enzyme was eluted with a linear gradient from 0.1 to 0.6 M NaCl in 200 mL of the same buffer Fractions of mL were collected at a flow rate mL min-1 Fractions containing C1,2O activity were pooled and were termed DEAE–cellulose eluent Sephadex G–150 fractionation DEAE–cellulose eluent was concentrated with Sephadex G–25 to approximately mL and applied to a Sephadex G–150 column (1 x 30 cm) equilibrated in 50 mM sodium phosphate buffer and eluted at 0.5 mL min-1 with 50 mM phosphate buffer containing 0.15 M NaCl mL fractions were collected Hydroxylapatite fractionation Fractions from Sephadex G–150 column with a specific activity higher than were pooled and applied to a (5.0 x 5.5 cm) Bio– Gel HTP hydroxyapatite column (Bio–Rad) equilibrated with 50 mM (pH 7) sodium phosphate buffer The enzyme was eluted by a 60 mL linear gradient of phosphate buffer (10 mM–0.3 M) at a flow rate 30 mL h-1, and mL fractions were collected 2.4 Electrophoresis and molecular mass determination Crude extract and purified enzyme fraction were monitored for purity by PAGE [20] in a 12.5% gel system Proteins were stained with silver The native enzyme molecular mass was measured by gel filtration on Sephadex G–150 with phosphorylase b (94 kDa), bovine serum albumin (67 kDa), ovalbumin (43 kDa), carbonic anhydrase (30 kDa) and trypsin inhibitor (20 kDa) as standards The column was calibrated by determining the elution volumes of standard proteins and then calculating the elution volume of each protein with respect to the elution volume of Blue Dextran 69 Catechol 1,2-dioxygenase from α-naphthol degrading thermophilic Geobacillus sp strain: purification and properties Step Volume, (ml) Total protein, (mg) Activity, (U) Specific activity, (U/mg) Yield, (%) Purification, (fold) Crude extract 20 53,42 23,20 0,43 100 30-50% saturated ammonium sulfate 18,62 13,76 0,74 59 DEAE cellulose 10,60 12,90 1,21 55 Sephadex G-150 4,50 11,70 2,60 50 Hydroxylapatite 1,00 7,42 7,42 31 18 Table Purification of catechol 1,2-dioxygenase from Geobacillus sp G27 strain 2.5 Determination of pH and temperature optima The effect of pH on enzyme activity was measured at various pH values within the range of 4.0 to 11 by using sodium acetate, sodium phosphate and TRIS/HCl buffer systems The pH values were equilibrated at 60oC The temperature dependence of catechol oxidation reaction at pH 7.0 was investigated in the range 30– 90oC by means of thermostated reaction cuvette The enzyme and substrate solutions were pre-incubated for 10 min, mixed, and the enzymatic reaction was then carried out at the same temperature 2.6 Determination of temperature stability The thermal stability of the enzyme was determined by incubating enzymatic reaction mixtures at 60oC during h and measuring activity under standard conditions 2.7 Inhibitors of catechol 1,2-dioxygenase activity AgNO3, CuSO4 · 5H2O, FeSO4· 7H2O, FeCl3, H2O2 and mercaptoethanol (each 0.1 mM), EDTA (1 mM) and o–phenanthroline (0.05 mM) were used The reaction mixture in 50 mM sodium phosphate buffer (pH 7) having a concentration of enzyme of 50 µg, was incubated for 10 at room temperature in the presence of the inhibitor and the reaction started by adding catechol (3 µM) The activity was then measured at 60oC as described above and expressed as a percentage of the activity obtained in the absence of the added compounds Results and Discussion When Geobacillus sp G27 strain was grown on g L-1 of α–naphthol, we observed catechol 1,2–dioxygenase specific activity of 0.43 U mg-1 in its crude extract, but no catechol 2,3–dioxygenase, protocatechuate dioxygenases, gentisate 1,2–dioxygenase activities, indicating that the bacterium catabolized α–naphthol through catechol via the ortho–cleavage pathway 70 The C1,2O is an inducible enzyme of thermophilic Geobacillus sp G27 strain, since no activity of the enzyme was observed when this organism was grown in a medium containing glucose instead of α–naphthol as the major carbon source The results of purification are summarized in Table The pure C1,2O with the specific activity of 7.42 U mg-1 was obtained after a 18-fold enrichment after fractionation with ammonium sulfate, DEAE–cellulose, Sephadex G–150 , hydroxylapatite and an overall recovery of 31% The C1,2O in the present study corresponded to 9% of the total proteins of the Geobacillus sp G27 strain The purification was repeated several times The final enzyme preparation showed one single protein band on PAGE gel (Figure 1) Specific activities, ranging up from 0.15 to 51 U mg-1, were observed from most purified catechol 1,2–dioxygenases of mesophilic Alcaligenes entrophus [6], Ralstonia species [21], Acinetobacter calcoaceticus [22], Pseudomonas aeroginosa [23], A radioresistens [9], Rhodococcus rhodochrous [8], Rhizobium leguminosarum [24] Hydroxylapatite chromatography suggested that strain induced only one type of catechol 1,2-dioxygenase during α–naphthol degradation because the purified enzyme eluates as a symmetrical peak from hydroxylapatite with C1,2O activity (data not shown) It also migrated as a single protein band on PAGE Several bacteria have been reported to possess more than two C1,2O-ases induced by benzoate, aniline or phenol [9,25,26] The molecular mass of the native enzyme measured by gel filtration was about 96±0.5 kDa and was eluted as a single symmetrical peak from Sephadex G–150 Results of the gel filtration procedure are shown in Figure This data is similar to the C1,2O from Pseudomonas arvilla which has a molecular mass of 90 kDa or from Trichosporon cutaneum (106 kDa) and consist of two identical subunits [22] The kinetic constants of the purified enzyme for various substrates are present in Table Catechol 1,2–dioxygenase from thermophilic Geobacillus sp G27 has a high specificity for catechol with Km value of 29 µM 3–Methylcatechol was oxidized at rates 27% of catechol G Giedraityte, L Kalėdienė Substrate Km (µM) k cat (s-1) Relative values to catechol, (%) Vmax Specific activity Catechol 29 9,4 100 100 methylcatechol 286 4,1 44 27 methylcatechol 400 3,2 35 Gentisic acid 0 0 Protocatechuic acid 0 0 Table Determination of kinetic constants and substrate specificity of catechol 1,2-dioxygenase from Geobacillus sp G27 strain Figure Electrophoresis under nondenaturing conditions Lane – purified catechol 1,2-dioxygenase, lane – crude extract of Geobacillus sp G27 Figure Figure A semilogarithmic plot of molecular mass as a function of the distribution coefficient, Kp (from left to right – trypsin inhibitor, carbonic anhydrase, ovalbumin, bovine serum albumin, phosphorylase b, catechol 1,2–dioxygenase) Gentisic and protocatechuic acids were not oxidized by C1,2O The activities with substituted catechols such as 3–methyl and 4–methylcatechols show that the specificity of the Geobacillus sp G27 catechol 1,2–dioxygenase is similar to catechol 1,2–dioxygenases from R rhodochrous and R erythropolis [7] The effects of pH and temperature on enzyme activity were examined The optimal pH for enzyme activity was found to be about (Figure 3A) The enzyme lost its activity at pH below and retained 45% of its original Effects of pH (A) and temperature (B) on activity of catechol 1,2–dioxygenase specific activity over a broad pH range (8–10,5) The optimum temperature for enzyme activity was 60oC (Figure 3B) The enzyme lost only 3% activity at 50 to 70oC and 22% at 80oC, the activity rapidly decreases outside this range The temperature optimum of 60oC for the enzyme activity was the same as the growth optimum of our thermophilic bacterium The purified enzyme retained 100% activity after month storage at 4oC The thermal stability of the enzyme was analyzed at 60oC (Figure 4) The half-life of the purified enzyme at this temperature was 40 Meta–cleaving oxygenase (catechol 2,3-dioxygenase) from phenol–degrading thermophilic B thermoleovorans was less stable, within a half–life of 3.3 at 60oC [14] The authors attributed 71 Catechol 1,2-dioxygenase from α-naphthol degrading thermophilic Geobacillus sp strain: purification and properties Compound Concentration, (mM) Inhibition, ( %) 0,1 100 CuSO4 x 5H2O 0,1 100 FeSO4 x 7H2O 0,1 12 FeCl3 0,1 EDTA 1,0 20 Mercaptoethanol 0,1 98 H2O2 0,1 98 Streptomycin sulfate 5% 80 o-Phenanthroline 0,05 None AgNO3 Table Determination of kinetic constants and substrate specificity of catechol 1,2-dioxygenase from Geobacillus sp G27 strain Figure Thermal stability of catechol 1,2–dioxygenase from Geobacillus sp G27 at 60oC this instability to the oxidation of iron from ferrous to the ferric state Zhang et al [15] purified a catechol 2,3–dioxygenase from B stearothermophilus that had unaltered catalytic activity after heating at 65oC for over hour The temperature stability of the enzyme from Geobacillus sp was higher than that of the mesophilic enzymes from A radioresistens [9], Rhodococcus rhodochrous [8] and P arvilla [25] The C1,2O from Ralstonia, Frateuria, Arthrobacter sp lost its activity at 45oC for 10 min, and the most of mesophilic enzymes lost 20% of their activity at 45oC for 10 [21] The effects of metal ions, sulfhydryl and reducing agents as inhibitors of enzyme activity were determined (Table 3) Among metal ions tested, the enzyme was completely inhibited by AgNO3 and CuSO4 The sulfhydryl compound mercaptoethanol and reducing agent H2O2 were effective inhibitors of C1,2O activity Enzyme activity was inhibited 80% by streptomycin sulfate, which used for nucleic acids elimination According to the literature, the only other C1,2O that was sensitive to streptomycin sulfate was isolated from Rhodococcus erythropolis strain [27] The enzyme purified in this study is the first catechol 1,2–dioxygenase purified from thermophilic microorganisms The bacterium can be used in bioremediation of polluted soil contaminated with various aromatic hydrocarbons ranging from monocyclic to polycyclic, because Geobacillus sp G27 was able to utilize anthracene, napthalene, biphenyl, benzenediols, naphthols, phenol, benzene, cresols [17] Furthermore, due to the important implications of aromatic ring– cleaving dioxygenases in bioremediation processes, C1,2O could be immobilized and used to remove catechols from waste waters The enzyme properties such as substrate specificity, temperature – pH optimum and stability are important determinants to consider when 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