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

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 min.. We

Received 4 July 2008; Accepted 6 October 2008

Department of Plant Physiology and Microbiology, Faculty of Natural Sciences, Vilnius University,

LT–03101 Vilnius, Lithuania

degrading thermophilic Geobacillus sp strain:

purification and properties

Gražina Giedraityte*, Lilija Kalėdienė

Abstract: The 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 am-monium 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 7 and 60oC, respectively

A half-life of the catechol 1,2–dioxygenase at the optimum temperature was 40 min 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

© Versita Warsaw and Springer-Verlag Berlin Heidelberg

Keywords:Thermophilic bacteria • α–naphthol • Catechol 1,2–dioxygenase • Purification

* E-mail: grazina.giedraityte@gf.vu.lt

Research Article

1 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 2 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

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 min 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)

2 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 1 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 min 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 3 min at 22 kHz by using a

sonicator The residue was removed by centrifugation at

4oC for 25 min 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 1 µmol of cis,

cis–muconic acid at 260 nm per min 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 2 hours, the precipitate was removed by a 60 min 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 2 mL were collected at a flow rate 1 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

2 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 1 mL fractions were collected

Hydroxylapatite fractionation Fractions from

Sephadex G–150 column with a specific activity higher than 1 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 2 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

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

2 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 min 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

3 Results and Discussion

When Geobacillus sp G27 strain was grown on 1 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

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 1

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],

[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 2

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 2 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

Step Volume, (ml) protein, Total

(mg)

Activity, (U)

Specific activity, (U/mg)

Yield, (%) Purification, (fold)

Table 1 Purification of catechol 1,2-dioxygenase from Geobacillus sp G27 strain.

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 7 (Figure 3A) The enzyme lost its

activity at pH below 5 and retained 45% of its original

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 2 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 min Meta–cleaving oxygenase

(catechol 2,3-dioxygenase) from phenol–degrading

thermophilic B thermoleovorans was less stable, within

a half–life of 3.3 min at 60oC [14] The authors attributed

Table 2 Determination of kinetic constants and substrate specificity of catechol 1,2-dioxygenase from Geobacillus sp G27 strain.

Figure 1 Electrophoresis under nondenaturing conditions Lane 1 –

purified catechol 1,2-dioxygenase, lane 2 – crude extract

of Geobacillus sp G27.

Figure 2 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).

Figure 3 Effects of pH (A) and temperature (B) on activity of

catechol 1,2–dioxygenase.

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

1 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 min [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 microorganisms are used in bioremediation of contaminated sites

Figure 4 Thermal stability of catechol 1,2–dioxygenase from

Geobacillus sp G27 at 60o C.

Table 3 Determination of kinetic constants and substrate specificity of catechol 1,2-dioxygenase from Geobacillus sp G27 strain.

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