Research Journal of Environmental and Earth Sciences 3(5): 608-613, 2011
ISSN: 2041-0492
© Maxwell Scientific Organization, 2011
Received: May 11, 2011
Accepted: June 15, 2011
Published: August 10, 2011
Purification and characterization of catechol 1, 2-dioxygenase from
Rhodococcus sp. NCIM 2891
Naiem H. Nadaf and Jai S. Ghosh
Department of Microbiology, Shivaji University, Vidyanagar, Kolhapur-416004, India
Abstract: The main aim of this study was to purify Catechol 1, 2 dioxygenase from phenol utilizing
Rhodococcus sp. NCIM 2891 which is basically an organism used wide for desulfurization purposes like
desulfurization of diesel. The organism was cultivated in minimal medium supplemented with 500 mg/L phenol,
as a sole carbon source. The temperature of incubation was 30ºC at pH 7.0±0.2. The growth and phenol
degradation studies show that organism was able to degrade the phenol completely within 120 h. The stationary
phase of growth of the organism, however, started after 72 h. This implies that the degradation activities starts
from late log phase and carries on till late stationary phase of growth. The enzyme responsible for phenol
degradation was catechol 1, 2 dioxygenase. The optimal activity of catechol 1, 2 dioxygenase was observed
at 500 mg/L phenol concentration. The enzyme was purified 2.34 fold by ion exchange chromatography. The
Km was found to be 5 :mole with Vmax of 62.5 U/mg of protein. The optimal pH and temperature of the enzyme
was 7.5 and 30ºC respectively. The enzyme activity was completely inhibited by Cu++, Hg++ and Fe+++ metal
ions. The SDS-PAGE analysis reveals that the enzyme has a molecular weight of 30 k Dalton.
Key words: Catechol, dioxygenase, Rhodococcus, muconate, phenol
their biochemical and structural properties. Kim et al.
(1997, 2002, 2003) (Caposio et al., 2002) Cis-cis muconic
acid is the main intermediary metabolite during benzenefree adipic acid synthesis, a process with undeniable
environmental benefit over the conventional chemical
production of nylon (Niu et al., 2002). Catechol 1,2dioxygenases play important roles in the degradation
pathways of various aromatic compounds and are
ubiquitous in microorganisms (Broderick et al., 1991;
Latus et al., 1995; Sauret-Ignazi et a1., 1996).
Rhodococcus are non-sporulating, aerobic bacteria
classified into mycolate-containing nocardioform
actinomycetes (Finnerty, 1992). The genus Rhodococcus
is comprised of genetically and physiologically diverse
bacteria, which have been isolated from various habitats
like soil and sea water. Rhodococcus strains are utilized
as industrial organisms, primarily for biotransformation
and the biodegradation of many organic compounds
(Bell et al., 1998). Rhodococcus can thus be applied in
environmental remediation and in the pharmaceutical and
chemical industries (Van and Dijkhuizen, 2004;
Larkin et al., 2005). Other possible biotechnological
applications of aromatic ring-degrading organisms and
their constituent enzymes, include the use of bioreactor
systems for removal of toxic pollutants, as well as their
incorporation into diagnostic systems and biosensors
(Ali, 1998; Nakajima, 2002).
The present study is regarding single step purification
and characterization of the catechol 1, 2-dioxygenase
from phenol utilizing Rhodococcus sp. NCIM 2891.
INTRODUCTION
Phenols and phenolic compounds are found mostly in
plants and effluents of industries, such as coal conversion,
paper and pulp manufacturing, wood preservation, metal
casting, etc. Phenolic compounds are considered to be
hazardous pollutants (Klibanov et al., 1980). Furthermore,
the breakdown products of phenol may be more harmful
when phenol is incompletely degraded by physical and
chemical methods or by natural oxidation. Therefore, the
complete removal of these substances from industrial and
domestic water bodies is of great importance for
conservation of native biodiversity (Yan et al., 2006).
Aerobic biodegradation of these compounds is
accomplished via pathways in which catechol (1, 2benzenediol) and its derivatives play a key role (Caposio
et al., 2002) as it is a ring cleavage substrate in which an
exceptionally large number of peripheral pathways come
together (Loh and Chua, 2002) to yield metabolites, such
as pyruvic acid, acetic acid, succinic acid, and acetyl-CoA
which are intermediates of the Krebs cycle (Houghton and
Shanley, 1994; Johnson and Stanier, 1971). Catechol 1, 2dioxygenase (Catechol oxygen 1,2-oxidoreductase, EC
1.13.11.1; pyrocatechase) play a central role in the
cleavage of the aromatic ring of catechol to cis,cismuconic acid in presence of 1 mol of oxygen (Stanier and
Ornston, 1973).
Aromatic ring-cleaving enzymes such as the catechol
dioxygenases of these catechol 1,2-dioxygenases from
bacteria have been extensively characterized in terms of
Corresponding Author: Dr. J.S. Ghosh, Department of Microbiology, Shivaji University, Vidyanagar, Kolhapur 416004. India.
Ph: +91 9850515620
608
Res. J. Environ. Earth Sci., 3(5): 608-613, 2011
Enzyme assay: Catechol 1, 2 dioxygenase activity was
assayed spectrophotometrically using a Hitachi U-2800
spectrophotometer. The standard assay of enzyme activity
was performed by making an assay mixture containing 5
:L of Catechol 1, 2 dioxygenase, 20 :L of 10 mM
Catechol as a substrate and final volume adjusted to1 mL
with 50 mM sodium phosphate buffer (pH 7.0). The
enzyme activity was monitored by measuring the
formation of cis, cis-muconic acid at 260 nm (, = 16.8
mM). One unit (U) of the enzyme activity was defined as
the amount of the enzyme required to catalyze the
formation of 1 µ mol of product per min at 25°C.
MATERIALS AND METHODS
The study was conducted between June 2010 and
September 2011. The work was carried out at the
Department of Microbiology, Shivaji University,
Kolhapur, India.
Microorganism: Rhodococcus sp. NCIM 2891 was
obtained from NCIM, (National Collection of Industrial
Microorganisms) India. The culture was repeatedly
transferred grown and maintained on YEM (Yeast Extract
Mannitol) agar containing 10 g mannitol, 4 g CaCO3, 0.5
g K2HPO4, 0.4 g Yeast Extract, 0.2 g MgSO4, 0.1 g NaCL
l-1 pH 7.0 at 4ºC during study.
Effect of pH and temperature: The effect of pH and
temperature was checked after incubation of the purified
enzyme at different pH and temperature for 30 min (Tsai
and Li, 2007). The effect of pH on enzyme activity was
studied at pH ranging from 4.0 to 10.0, using citratephosphate buffer (pH 3.0-5.0), sodium- phosphate buffer
(pH 6.0- 8.0), glycine- NaOH buffer (pH 9.0-10.0). The
effect of temperature on enzyme activity was studied
between temperatures 10 to 90ºC.
Growth of bacteria and phenol degradation:
Rhodococcus sp. NCIM 2891 was grown in flask
containing 3.0 g Na2NO3, 1.0 g K2HPO4, 0.5 g MgSO4,
0.5 g KCL, 0.01 g FeSO4, 0.5 g yeast extract and 0.5 g
phenol l-1 pH 7.0 at static condition. The growth of
microorganism was routinely assessed through turbidity
measurement as absorbance at 530 nm and residual
phenol concentrations remaining in broth were assayed
routinely by a colorimetric method using 4aminoantipyrine
and
potassium
ferricyanide
(Ettinger et al., 1951). The growth of Rhodococcus sp.
NCIM 2891 and the residual phenol concentration were
monitored at regular time intervals of 24 h.
Effect of metal ions: To study the effect of metal ions on
enzyme activity, enzyme was pre-incubated in 50 mM
sodium phosphate buffer having pH 7.0 in presence of
metal ions like Ca2+, Hg2+, Fe3+, Co2+, Mg2+, Cu2+ using
their respective water soluble salts in different
concentrations ranging from 1 and 10 mM concentration.
The residual enzyme activity was subsequently measured
by standard assay (Tsai and Li, 2007).
Enzyme extraction and purification: After 72 h of
incubation the cells were harvested for the enzyme
extraction. The harvested cells were washed twice with 30
mL of 50 mM sodium phosphate buffer (pH = 7.0) and
resuspended in the same buffer (pH = 7.0) and then
homogenized by sonication with vibra model VC.130PB
sonicator at 50 amplitude for 5 min. Sonicated sample
was centrifuged for 30 min at 8000×g at 4ºC. After
centrifugation to remove cell debris, the supernatant was
subjected for DEAE - Cellulose chromatography. The
column was eluted with 50 fractions of a linear gradient
of NaCl with concentration of 0.1 to 0.5 M in 50 mM
sodium phosphate buffer (pH 7.0) at a flow rate of 0.5
mL/min. The protein concentration of eluted fractions was
analyzed by taking absorbance at 280 nm. The fractions
having protein were tested for Catechol 1, 2 dioxygenase
activities.
Protein estimation: Protein estimation was done by the
Lowry method using Bovine Serum Albumin as a
standard (Lowry et al., 1951).
Enzyme kinetics: The Km and Vmax of the catechol 1,2dioxygenase was determined by Lineweaver-Burk plots of
reciprocal reaction velocities versus reciprocal substrate
concentrations. Reactions were performed in 50 mM
sodium phosphate buffer pH 7.0, with various
concentrations of substrate (10 mM catechol varying
between 1 to 50 :L. and enzyme activity was monitored
by methods mentioned before.
RESULTS
SDS/PAGE analysis of enzyme: The molecular weights
of enzyme were determined by the SDS/PAGE (Laemmli,
1970). The analysis of molecular weight of purified
enzyme were carried out by using protein molecular
weight markers that are phosphorylase b (97.4 kDa),
Bovine Serum albumin (66 kDa), ovalbumin (43 kDa),
carbonic anhydrase (29 kDa), trypsin inhibitor (20.1 kDa),
and lactabumin (14.3 kDa).
Growth of bacteria and phenol degradation: It was
observed that the Rhodococcus sp. NCIM 2891 was able
to grow on phenol as a sole source of carbon. The
maximum phenol degradation was observed after 72 h.
i.e., in log phase. The residual phenol concentration was
240 mg/L at 92 h, that is nearly half of initial phenol
concentration (500 mg/L). In Fig. 1 it was observed that
there was complete degradation of phenol within 120 h.
609
Res. J. Environ. Earth Sci., 3(5): 608-613, 2011
Fig. 1: Growth of Rhodococcus NCIM 2891 and residual phenol concentration. Residual phenol concentration (•) and growth of
Rhodococcus NCIM 2891(#) Results obtained are mean values±SD, n = 5
Fig. 2: Elution profile of catechol 1, 2 dioxygenase from DEAE-cellulose column chromatography. Fractions from the DEAEcellulose anion exchange chromatography (#) and fractions showing catechol 1, 2 dioxygenase activity (•)
Table 1:Purification of catechol 1, 2 dioxygenase Results obtained are mean values±SD, n = 5
Volume (mL)
Protein (mg)
Specific activity (U/mg)
Crude Extract
20
2240±3.77
0.78±0.0262
Ion. exchange
15
510±1.55
1.83±0.06
chromatography
Yield (%)
100
45
Purification fold
1
2.34
The SDS-PAGE analysis reveals that the purified
enzyme has near about 30 KD molecular weight as shown
in (Fig. 3).
Purification of catechol 1, 2 dioxygenase: A single-step
protocol was used to purify catechol 1, 2 dioxygenase.
The purified catechol 1, 2 dioxygenase was obtained from
8 g of wet cell mass the purification profiles is given in
(Fig. 2) and the activity of purified catechol 1, 2
dioxygenases at different stages of purification is given in
Table 1.
Enzyme stability:
Effect of temperature and pH on Catechol 1, 2
dioxygenase: The optimal temperature and pH of
610
Recidual enzyme activity (%)
Res. J. Environ. Earth Sci., 3(5): 608-613, 2011
120
100
80
60
40
20
0
4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5
pH
Fig. 5: Effect of pH on catechol 1, 2 dioxygenase activity.
Residual catechol 1, 2 dioxygenase activity of purified
catechol 1, 2 dioxygenase (#) Results obtained are mean
values±SD, n = 5
Residual activity (%)
120
Fig. 3: SDS-PAGE of purified catechol 1, 2-dioxygenase (C)
crude sample (P) purified sample by ion exchange
chromatography from Rhodococcus NCIM 2891 Size of
the standard markers is at left. Markers (M):
phosphorylase b (97.4 kDa), Bovine Serum albumin (66
kDa), ovalbumin (43kDa), carbonic anhydrase (29 kDa),
trypsin inhibitor (20.1 kDa) and lactabumin (14.3 kDa)
100
80
60
40
20
0
None
Cu 2+
Ca 2+ Mg2+ Hg2+
Heavy matel
Co2+
Fe3+
Fig. 6: Effect of heavy metals on 1, 2 dioxygenase with 1mM
and 10 Mm Concentration Results obtained are mean
values±SD, n = 5
120
100
160
80
140
60
120
40
100
20
1/v
Relative activity (%)
1 mM
10 mM
0
0
10
20
30
40
50
60
70
80
90
100
80
60
Temprature °C
40
Fig. 4: Effect of temperature on catechol 1, 2 dioxygenase
activity. Residual catechol 1, 2 dioxygenase activity of
purified catechol 1, 2 dioxygenase (#) Results obtained
are mean values±SD, n = 5
20
-0.2
0
0.0
0.2
0.4
0.6
1/s
Catechol 1, 2 dioxygenase was 30ºC and pH 7.5,
respectively. Results are shown in Fig. 4 and 5.
Fig. 7: Km of Catechol 1, 2 dioxygenase with Catechol as
substrate
Effect of metal ions on enzyme activity: The effects of
metal ions on the activity of this enzyme were
investigated. The results are shown in Fig. 6. The activity
of this enzyme towards catechol was minimally affected
by Co2+ (20%), and Mg2+ (20%) at concentrations of up to
1 mM, whereas the enzymatic reaction inhibited
completely with addition of 1 mM Fe3+, Cu2+, and Hg+2 For
10 mM, used Ca2+, Cu2+, Fe3+ shows 100% inhibition
whereas Mg2+ and Co2+ shows inhibition up to (60 %).
Enzyme kinetics: Km and Vmax values of catechol 1, 2
dioxygenase are found to be 5 :mole/mL and 62.5 U/mg
of protein respectively as shown in Fig. 7.
DISCUSSION
The study of phenol and other phenolic compound
degradation by bacteria, fungi and other microorganisms
were screened and it was found that there are number of
611
Res. J. Environ. Earth Sci., 3(5): 608-613, 2011
ACKNOWLEDGMENT
bacteria which have exceptional capability of phenol
degradation. This organisms include Pesudomonas,
Agrobacterium, Burkholderia, Acinetobacter, Ralstonia,
Klebsiella, Bacillus, Rhodococcus and Rhizobium
(Koutny et al., 2003). In this study the phenol degradation
ability of Rhodococcus sp. NCIM 2891 was demonstrated
with purification of the four phenol oxidases. The
organism was able to use phenol as a sole source of
carbon and energy at a concentration of 500 mg/L under
specified condition.
There are many reports of production of catechol 1,
2 dioxygenase from number of phenol degrading
microorganisms like Ralstonia sp., Pseudomonas
aeruginosa, Rhodococcus sp. AN-22, R. rhodochrous
NCIMB 13259 strains (Wang et al., 2001; Chuan et al.,
2006; Mastumura et al., 2004; Chuan et al., 2006;
Strachan, 1998) with phenol and different substrate. The
strain Acinetobacter sp. was capable of removing 500
mg/L phenol concentration in liquid minimal medium
(Wang et al., 2007).
This is the first report on the purification and
characterization of catechol 1, 2 dioxygenase from
Rhodococcus sp. NCIM 2891 with single step purification
procedure. Though the enzyme activity of catechol 1, 2
dioxygenase shows similarity with that from
Acinetobacter and other Rhodococcus sp., the SDS-PAGE
analysis shows that the molecular weight of the catechol
1, 2 dioxygenase from Rhodococcus sp. NCIM 2891 was
near about 30 KD which was observed in catechol 1, 2
dioxygenase of Ralstonia sp. Ba-0323 (30 KD)
(Wang et al., 2001).
The optimal pH of catechol 1, 2 dioxygenase enzyme
is comparatively lower with that of Catechol 1, 2
dioxygenase that have been previously isolated from other
microorganisms. The enzyme activity remains up to
>80% for 30 min between pH range of 7.0-9.0, whereas
the stability of enzyme greatly decreased in the pH
condition out of this range. The thermal stability of
catechol 1, 2 dioxygenase was found to be similar with
Pseudomonas aeruginosa (Chuan et al., 2006) enzyme
was stable when it was kept at temperature lower than
40ºC.the optimal temperature of catechol 1, 2
dioxygenase was 30ºC.
It was observed that the catechol 1, 2 dioxygenase of
this organism was sensitive to Fe, Cu, Mg, Co, ions that
were also observed in Aniline-Assimilating Bacterium
Rhodococcus sp. AN-22 (Mastumura et al., 2004).
The Km of this enzyme is 5 :mole/mL with Vmax 62.5
U mg-1of protein. The Km value of enzyme indicates that
it has lower affinity for substrate (catechol) that is formed
during phenol assimilation in broth. This Km value
was found to be parallel with Km of P. aeruginosa
(Chuan et al., 2006) and higher than R. rhodochrous
NCIMB 13259 strains (Strachan, 1998).
The authors are very grateful to the Department of
Microbiology, Shivaji University, Kolhapur, for extending
the laboratory facilities to complete the investigation.
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