Current Research Journal of Biological Sciences 3(3): 246-253, 2011
ISSN: 2041-0778
© Maxwell Scientific Organization, 2011
Received: March 24, 2011
Accepted: April 20, 2011
Published: May 05, 2011
Production, Purification and Characterization of Tannase from
Rhodococcus NCIM 2891
Naiem H. Nadaf and Jai S. Ghosh
Department of Microbiology, Shivaji University, Vidyanagar, Kolhapur 416004, India
Abstract: The study was done with the objective of tannic acid degradation by tannase from the Rhodococcus
NCIM 2891 which is actually a desulfurizing bacteria. Rhodococcus NCIM 2891cultivated in medium
containing 0.1% Tannic acid, produced tannase which showed maximum activity after 24 h. The enzyme was
purified by DEAE cellulose ion exchange chromatography and was shown to be dimeric in nature it has two
subunits as Tannase I and Tannase II, having specific activities of 0.23 U/mg of protein and 0.295 U/mg of
protein, respectively. Both the enzyme showed optimum activity at pH 6 and 30ºC. The Km of both enzymes
was 0.034 and 0.040 mM respectively and Vmax of both enzymes were found to be 40 and 45 U/mL,
respectively. SDS-PAGE analysis of purified proteins fraction revealed that their molecular weights are 60 and
62 kDa. Tannase I was completely inhibited by 10 mM Hg2+, Cu2+, Fe3+, Co2+ and Tannase I and II both was
completely inhibited by1 mM Hg2+ alone. The DNA damage- protecting activity of tannic acid and product
obtained after tannic acid hydrolysis was assessed bringing about the DNA damage with H2O2 + UV exposure
in absence of the hydrolyzed products of tannic acid. The experiment was carried out by using agarose gel
electrophoresis to analyze DNA damage in presence of H2O2 + UV. However, there was no damage of DNA
by H2O2 + UV exposure in presence of the hydrolysis products.
Key words: Gallic acid, NCIM 2891, pollutants, Rhodococcus, tannase, tannin
Tannin acyl hydrolase (EC 3.1.1.20), commonly
called tannase, is an enzyme that hydrolyzes the ester
bonds of tannic acid, to produce gallic acid and glucose
and galloyl esters (Belur and Mugeraya, 2011). Besides
gallic acid production, the enzyme is extensively used in
the preparation of instant tea, wine, beer, and coffeeflavored soft drinks and also as additive for
detanniffication of food (Lekha and Lonsane, 1997). In
addition, it is also used as a sensitive analytical probe for
determining the structure of naturally occurring gallic acid
ester (Haslam and Stangroom, 1996). Although tannase is
present in the plants, animals and microorganisms, it is
mainly produced by the microorganisms (Ayed and
Hamdi, 2002). Filamentous fungi of the genus
Aspergillus, have been widely used for tannase production
(Banerjee et al., 2001; Beniwal and Chhokar 2010).
Members of the genus Rhodococcus occur widely,
and are aerobic, non-sporulating bacteria, with a high GC
content. These organisms are of environmental and
biotechnological importance because of their broad
catabolic diversity and array of unique enzymatic
capabilities (Bell et al., 1998) and their exceptional ability
INTRODUCTION
Tannins are naturally occurring water-soluble
polyphenols
which are often considered as
recalcitrant, that commonly exist in wastewater from
forestry, paper and leather industries and cause many
problems associated with environmental pollution
(Yagu et al., 2000). These are widespread in the plant
kingdom, occurring mostly in leaves, fruits, bark and
wood and are often considered nutritionally undesirable
(Chung et al., 1998; Murugan and Saleh, 2010). Tannins
inhibit growth of various microorganisms, by
precipitating many enzymes and (Field and
Lettinga, 1992a).
Several conventional physiochemical techniques are
available for tannin-containing wastewater treatment
(Svitelska et al., 2004). However, the major drawbacks of
these processes are high cost, tendency to form secondary
hazardous byproducts, and incomplete degradation. The
biological treatment of tannin-containing wastewater is
now considered to be a better alternative, though these too
have certain limitations (Bhat et al., 1998; Ren, 2004;
Field and Lettinga, 1992b).
Corresponding Author: Dr. J.S. Ghosh, Assistant Professor, Department of Microbiology, Shivaji University, Vidyanagar,
Kolhapur 416004, India. Ph: +91 9850515620
246
Curr. Res. J. Biol. Sci., 3(3): 246-253, 2011
Tannase assay: The reaction mixture consisted of 0.3
mL of tannic acid (0.7% (w/v) in 0.2 M citrate buffer (pH
6.0) and 0.1 mL of the enzyme extract. This was
incubated at 30ºC for 20 min. The enzymatic reaction was
paralyzed by the addition of 3 mL of bovine serum
albumin solution - BSA (1 mg/mL), leading to the
precipitation of the remaining tannic acid. This was then
centrifuged at 9000×g for 15 min at 4ºC and the residue
was dissolved in 2 mL of SDS-triethanolamine, followed
by the addition of 1 mL of FeCl3 reagent and holding for
15 min for colour stabilization. The absorbance was
measured at 530 nm. One unit of tannase activity was
defined as the amount of tannic acid hydrolyzed by 1 mL
of enzyme minute of reaction.
to degrade hydrophobic natural compounds and
xenobiotics. However, there are no reports of tannic acid
degradation or production of tannase by Rhodococcus.
This investigation focuses about potential uses of
Rhodococcus in tannin waste recycling and production of
valuable products, applicable for the various industrial
processes.
MATERIALS AND METHODS
The study was conducted between August 2010 and
February 2011. The work was carried out at the
Department of Microbiology, Shivaji University,
Kolhapur, India.
Microorganism: Rhodococcus NCIM 2891 was obtained
from NCIM (National Collection of Industrial
Microorganisms, NCL, Pune, India). The culture was
maintained on GYEME ( Glucose Yeast extract Malt
extract ) agar having the following composition 1%
glucose, 0.3% yeast extract, 0.3% malt extract 0.5%
peptone and 1.5 % agar-agar.
High performance thin layer chromatography
(HPTLC): The gallic acid produced was analyzed by
using High performance thin layer chromatography
(HPTLC) system (CAMAG, Switzerland). Stationary
phase HPTLC silica gel 60 F254 (Merck, Germany),
predeveloped with methanol, dried at 120ºC for 20 min,
cooled to room temperature, and equilibrated with the
relative humidity of the laboratory. The plates were
developed using saturated chamber containing developing
solvent (butanol: acetic acid: water in 4:1:1 ratio). The
chamber was saturated for 20 min prior to plate
development. After development the plate was scanned at
280 nm. The results were analyzed by using HPTLC Win
CATS 1.4.4.6337 software.
Growth of Rhodococcus on tannic acid: The
Rhodococcus NCIM 2891 was grown in medium
containing 0.3% NaNO3, 0.1% K2HPO4, 0.05% MgSO4,
0.05%KCL, 0.0001% FeSO4, and 0.1% tannic acid. The
pH was adjusted to 7.0.
Screening of Rhodococcus for the production of
tannase: Tannase production by Rhodococcus NCIM
2891 was checked by growing the microorganism on
nutrient agar supplemented with 2% tannic acid which
was filter sterilized. Addition of tannic acid to nutrient
agar plate forms a complex of tannic acid and proteins
and after cleavage of this complex, organism produces
clear zone around colony.
Effect of pH and temperature: The effect of pH and
temperature was checked for both the enzymes. The effect
of pH on enzyme activity was studied at varying pH range
from 4.0 to 10.0, using citrate- buffer (pH 3.0-5.0),
sodium- phosphate buffer (pH 6.0-8.0) and glycineNaOH buffer (pH 9.0-10.0). The effect of temperature on
enzyme activity was studied between temperatures 10 to
90ºC. In all cases the preincubation of enzyme was carried
out for 30 min with respective pH and temperatures and
then activity was measured by standard assay as described
above.
Tannic acid degradation:
Enzyme extraction and purification: The enzymes
tannase was purified from cell free medium. In first step
cell free medium was saturated up to 40% with
ammonium sulphate and allowed to precipitate for 6 hrs
at 4ºC. This precipitate was discarded and the supernatant
was saturated with ammonium sulphate up to 80%, which
was kept overnight for residual enzyme precipitation. This
precipitate was dissolved in 20 mL of 1 mM citrate buffer
(pH = 6.0) and was further purified by DEAE - Cellulose
ion exchange column chromatography. The elution was
carried out at a flow rate of 0.5 mL/min, using NaCl
concentration gradient from 0 to 0.2 M in 50 mM citrate
buffer (pH = 6.0). The fractions were analyzed for tannase
activities, and later used for SDS-PAGE analysis.
Effect of metal ions: To study the effect of metal ions on
enzyme activity, enzyme was pre-incubated in citrate
buffer (pH 6.0) with metal ions Ca2+, Hg2+, Fe3+, Co2+,
Mg2+, Cu2+ using their respective water soluble salts with
1 and 10 mM concentration. The residual enzyme activity
was subsequently measured by standard assay as
described above.
Enzyme kinetics: The Km and Vmax of the Tannase I and
II was determined by Lineweaver-Burk plots of reciprocal
reaction
velocities versus reciprocal substrate
247
Curr. Res. J. Biol. Sci., 3(3): 246-253, 2011
Protein estimation: Protein estimation was done by the
Lowry method using bovine serum albumin as standard
(Lowry et al., 1951).
concentrations. Reactions were performed in citrate buffer
(pH 6.0); with various concentrations of substrate 1%
tannic acid by the addition of 0.1 to 1 mL. and enzyme
activity was monitored under the same conditions that are
mentioned above.
Statistical analysis: Results obtained were the mean of 5
replications. Analysis of the variants was carried out on
all data at p<0.05 using Graph Pad software. (Graph Pad
Instat version 3.00, Graph Pad software, San Diego, CA,
USA).
Protection against DNA damage induced by H2O2 +
UV: DNA damage and DNA protecting activities on
sample DNA according to protocol of Russo et al. (2000)
modified by Skandrani et al. (2009). The test DNA used
in experiment was 8 phage DNA (Linear double stranded
with 48502 base pairs purchased from Genie, Bangalore
India). The 8 phage DNA was oxidized with H2O2 + UV
treatment in presence and in absence of product obtained
after hydrolysis of tannic acid.
The total volume of reaction mixture prepared was17
µL containing 500 ng of 8 phage DNA, 10 µL tannic acid
(0.1% w/v) and 10 µL incubated cell free medium (filter
sterilized). H2O2 was added to this at a final concentration
of 100 mM. The whole system was exposed to UV light
(wavelength of 312 nm) at ambient temperature for 5 min.
After irradiation, the mixture was incubated at room
temperature for 15 min. Untreated 8 phage DNA was
used as a control during the run of gel electrophoresis
along with UV and H2O2 treated sample of DNA.
RESULTS
Growth of Rhodococcus NCIM 2891: It was observed
that the Rhodococcus NCIM 2891 was able to grow on
Tannic acid as a sole source of carbon. The maximum
Tannic acid degradation was observed in exponential
phase.
Screening of Rhodococcus for the production of
tannase: The clear zone was observed around the growth
of Rhodococcus NCIM 2891 after 48 h of incubation
indicates that the microorganism producing tannase
enzyme cleave tannic acid protein complex by
hydrolyzing tannic acid.
Fig. 1: The elution profile of DEAE-Cellulose Ion exchange chromatography of Tannase enzyme. Protein concentration of fractions
(•) and activity of tannase enzymes (#)Results obtained are mean values±SD, n = 5
Table 1: Purification profile of tannase enzymes
Enzyme
Enzyme activity (units)
Proteins (mg)
Sp activity (Units/mg)
Crude
455
3500±0.05*
0.13±0.08*
After ammonium
301
1005±0.06*
0.29±0.05*
sulfate precipitation
After ion exchange chromatography
Fraction 1
60
268±0.03*
0.23±0.02*
(Tannase - I)
Fraction no 2
190
644±0.06*
0.295±0.05*
(Tannase - II)
Results obtained were the standard error of mean of 5 replications; *: p< 0.05
248
Yield (%)
100
66
Purification fold
1
2.23
13.18
1.76
41.57
2.23
Curr. Res. J. Biol. Sci., 3(3): 246-253, 2011
Purification of Tannase: The purification profile of
tannase enzyme is given in Fig. 1 and the activity of
extracted tannase at different stages of purification is
given in Table 1. It has been noted from Table 1, that the
precipitate at 80% saturation of (NH4)2SO4 was giving the
best yield of the enzyme. The same yield did not show
any improvement after DEAE Cellulose ion-exchange
chromatography, except that the 2 fractions seperated
(Fig. 1).
The specific activities of Tannase I and Tannase II
was measured about 0.23 U/mg of protein and 0.295
U/mg of protein using tannic acid as substrate.
SDS/PAGE analysis revealed that the two enzymes had
molecular weights of 60kDa and 62 kDa respectively as
shown in Fig. 2.
HPTLC analysis: The HPTLC analysis of incubated
broth indicated the production of gallic acid due to
hydrolysis of tannic acid by Tannase, as shown in Fig. 3.
The Rf value of standard gallic acid was 0.90 where as the
product of hydrolysis gave Rf values 0.89 and 0.88. these
Rf values linearly equals each other, indicating that
tannase produced by the microorganism was able to
degraded tannic acid in to its monomer, gallic acid.
Fig. 2: SDS-PAGE of purified Tannase I (a) Tannase II (b),
from Rhodococcus NCIM 2891 Size of the standard
markers is at left. Markers (da) (m): 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)
d
1000.0
[AU]
800.0
700.0
1000.0
[AU]
600.0
800.0
500.0
700.0
400.0
300.0
600.0
200.0
500.0
400.0
100.0
0.0
50.0
[AU]
40.0
35.0
30.0
25.0
20.0
15.0
300.0
200.0
100.0
0.0
0.20
0.00
0.20
0.40
0.00
10.0
0.00
5.0
[AU]
1.20
0.0
Fig. 3: HPTLC analysis of standard gallic acid and incubated broth after 24 h. and 48 h,(a) standard gallic acid; (b) broth after 24
h. incubation; (c) Broth after 48h. Incubation; (d) 3D peak profile of gallic acid; (e) developed HPTLC plate in that
249
Residual activity of Tannase II (%)
Curr. Res. J. Biol. Sci., 3(3): 246-253, 2011
Residual activity (%)
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
pH
Fig. 4: Effect of pH on enzyme activity tannase I (•) and
Tannase II (#).Results obtained are mean values±SD,
n=5
160
1 mM
10 mM
140
120
100
80
60
40
20
0
Control Hg
Mg
Mn
Ca
Metal ions
Cu
Fe
Co
Fig. 7: Effect of heavy metals on Tannase II with 1mM and 10
mM concentration. Results obtained are mean
values±SD, n = 5
250
100
80
200
60
40
150
20
0
1/V
Recidual activity (%)
120
10
20
30
40
50
60
Temperature °C
70
80
90
100
Residual activity of Tannase I (%)
Fig. 5: Effect of Temperature on enzyme activity Tannase I (•)
and Tannase II (#).Results obtained are mean
values±SD, n = 5
120
50
0
1 mM
10 mM
-50
0
50
100
150
200
1/S
100
Fig. 8: Km of Tannase I with Tannic acid as a substrate results
obtained are mean values±SD, n = 5
80
60
400
40
350
20
0
Control Hg
Mg
Mn
Ca
Metal ions
Cu
Fe
300
Co
250
1/V
Fig. 6: Effect of heavy metals on tannase I with 1mM and 10
mM concentration. Results obtained are mean
values±SD, n = 5
200
150
pH and temperature effect: The optimum pH of the
Tannase I and Tannase II were 6 whereas temperature
optimum for Tannase I and Tannase II were 30ºC as
shown in Fig. 4 and 5.
100
50
-50
Effect of heavy metals: The Tannase I showed complete
inhibition by Hg+2 in 1mM concentration and by 10 mM
Fe3+, Cu2+, Co2+ whereas the activity of tannase was
retained 100 %, by 1 mM Cu2+ as shown in Fig. 6.
0
0
50
1/S
100
150
200
Fig. 9: Km of Tannase II with Tannic acid as a substrate
Results obtained are mean values±SD, n = 5
250
Curr. Res. J. Biol. Sci., 3(3): 246-253, 2011
a
b
c
Monika et al. (2007) reported the production of two
extracellular tannase but cold adapted from Verticillum
sp. 9 each consisting of approximately 40 and 60 kDa sub
units Rhodococcus NCIM 2891 also produces two
enzyme namely Tannase I and Tannase II having
approximately molecular weight around 60 and 62 kDa.
The other known fungal tannase are multimeric proteins
and molecular weight varying from 168 to over 310 kDa
(Sabu et al., 2005; Hatamoto et al., 1996).
Both tannase were optimally active at pH 6 however
the optimum pH was 5-5.5 in case of fungal tannase
(Sabu et al., 2005). The optimum temperature of the
Tannase I and and II is 30oC which is same as reported for
the Bacillus licheniformis KBR 6 (Mondal and
Patil, 2000) as compared many reports regarding
temperature variability among tannase produced by
different type of fungi (Sabu et al. 2005).
The Km of Tannase I and II are 0.034mM and 0.040
mM which is very low as compared to the Km value for a
similar enzyme produced by Cryphonectria parasitica,
using tannic acid as substrate, have been found to be 0.94
mM (Farias et al., 1994). The Km values for tannases from
Selenomonas ruminantium and Cryphonectria parasatica
using methyl gallate as substrate have been found to be
1.6 mM (Skene and Brooker, 1995) and 7.49 mM
(Farias et al., 1994). The Km values for tannic acid of
tannases produced by other fungi ranged from 0.20 to
1.03
mM
(Mukherjee
and
Banerjee 2006;
Sharma et al., 1999; Sabu et al., 2005).
The enzyme was completely inhibited by Hg2+, Cu2+,
2+
Fe , Co2+ ions the effect of metal ions are similar to those
reported previously but the only difference was that
activity of other tannases are increased in presence of
Mg2+ (Kar et al., 2003).
There are reports which states important application
of tannase that is the production of gallic acid and
propylgallate, fine-chemicals employed as antioxidants in
foods, cosmetics, hair products, adhesives, and lubricant
industry (Kar et al., 2002; Yu et al., 2004; Yu and
Li, 2006). Min-Jer and Chinshuh, (2008) studied
enzymatic modification by tannase which increased the
antioxidant activity of green tea. The antioxidant
properties of tannic acid degradation, was checked by
using DNA damage assay. The oxidative DNA damage
induced by tea catechins especially epigallocatechin
gallate (EGCG) in presence of metals complexes such as
Cu2+ and Fe3+ has been reported to decrease due to EGCG
hydrolysis (Furukawa et al., 2003). These are similar with
our results but the difference was only that there were no
metals complexes.
d
Fig. 10: Profile of agarose gel electrophoresis lane (a) DNA +
H2O2, lane (b) Control DNA, lane (c) DNA+ H2O2+
incubated cell free medium and lane (d) DNA+ H2O2+
Tannic acid
The Tannase II showed complete inhibition by Hg+2
at 1 mM concentration and 25% of the activity was
inhibited by 1 mM Mn+2, and 10mM Mg2+, Cu2+, Co2+.
Tannase I showed increase in activity by 37% above
optimum, in presence of 1 mM Cu2+, 10 mM Ca2+, by 25%
in presence of Co2+ and up to 12% in presence of Fe3+
(Fig. 7).
Enzyme kinetics: The Km of Tannase I is 0.034 mM
whereas the Km Tannase II 0.040mM. The Vmax of both
enzymes were found to be 40 and 45 U/mL, respectively
as shown in (Fig. 8 and 9).
Protection against DNA damage induced by H2O2 +
UV by obtained product after hydrolysis of tannic
acid: Figure 10 shows that the DNA damage was
observed in first sample (a) where the DNA was treated
with 100 mM H2O2. Sample (b) which was unexposed
control, shows no damage of DNA. Sample(c), which was
treated with 100 mM H2O2 and 10 :L incubated cell free
medium containing gallic acid shows similar band as in
control. Sample (d) which had tannic acid, showed some
damage of the DNA.
DISCUSSION
This is a first report of tannase production from
Rhodococcus NCIM 2891 where this microorganism
produces 2 extracellular tannase namely Tannase I and
Tannase II. The study regarding the production of
Tannase by other microorganisms reveals that the fungi
are predominant tannase producers and extensively
studied (Bradoo et al., 1996). Deschamps et al. (1983)
reported that the optimum enzyme production by Bacillus
pumilus, Bacillus polymyxa, Cornybacterium sp. and
Klebsiella pneumoniae in presence of chestnut bark as
sole carbon source. The preliminary study to check
tannase production from Rhodococcus NCIM 2891
included plate assay. The production of gallic acid during
incubation was checked by HPTLC method.
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CONCLUSION
The tannase production by microorganism is
extensively studied with respect to the food and
pharmaceutical industries. However, it was mostly studied
in fungi and in some extent in other microbial species.
The results of this investigation clearly indicate that
tannase produced by Rhodococcus NCIM 2891 has higher
affinity for tannic acid as compared to those produced by
other bacterial species. Tannic acid is water soluble
metabolite of plant showing antagonistic activity against
bacteria and its high concentration in environment can
significantly affect the biodiversity. The findings of this
study clearly indicates that tannase from Rhodococcus
NCIM 2891 can be used as a potent bioremediation tool
for industrial waste containing tannins or their derivatives.
The antioxidant properties of tannic acid degradation
products was another significant, novel finding which
could be used to remove tannins from various substances
like fodder for sheep and goats and thus improve the
health of these animals.
ACKNOWLEDGMENT
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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