Purification and characterization of tannase and tannase gene from Enterobacter sp.

Article abstract of DOI:10.1016/j.procbio.2010.08.016

Tannase of Enterobacter sp. was purified and characterized at molecular level. It was found to be 90 kDa in molecular weight. The purified enzyme showed maximum activity at 40 °C. The enzyme was also found to be active in acidic range of pH. The nucleotide and amino acid sequence of tannase exhibited resemblance with the other reported tannase sequences of bacteria, fungi and plants. Probably, this is the first report of tannase gene in Enterobacter sp. The investigation suggests that the purified enzyme can be useful to synthesize molecules of pharmaceutical interest. In addition to above, the enzyme tannase and the organism itself can also be employed to protect grazing animals and environment against the toxic effects caused by tannins in them.

Full text of DOI:10.1016/j.procbio.2010.08.016

Process Biochemistry 46 (2011) 240–244
Contents lists available at ScienceDirect
Process Biochemistry
journal homepage: www.elsevier.com/locate/procbio
Purification and characterization of tannase and tannase gene
from Enterobacter sp.
Kanti Prakash Sharma , P.J. Johnb
a,∗
a
Department of Biotechnology, Mody Institute of Technology and Science, Lakshmangah, Sikar, Rajasthan 332311, India
Centre for Advanced Studies, Department of Zoology, University of Rajasthan, Jaipur 302004, India
b
a r t i c l e i n f o
a b s t r a c t
Article history:
Tannase of Enterobacter sp. was purified and characterized at molecular level. It was found to be 90 kDa
in molecular weight. The purified enzyme showed maximum activity at 40 C. The enzyme was also
Received 28 December 2009
Received in revised form 3 August 2010
Accepted 24 August 2010
◦
found to be active in acidic range of pH. The nucleotide and amino acid sequence of tannase exhibited
resemblance with the other reported tannase sequences of bacteria, fungi and plants. Probably, this is the
first report of tannase gene in Enterobacter sp. The investigation suggests that the purified enzyme can be
useful to synthesize molecules of pharmaceutical interest. In addition to above, the enzyme tannase and
the organism itself can also be employed to protect grazing animals and environment against the toxic
effects caused by tannins in them.
Keywords:
Tannase
Tannase gene
Enterobacter sp.
SDS PAGE
©
2010 Elsevier Ltd. All rights reserved.
Kinetic constants
1
. Introduction
hydroxyl groups [5]. In spite of the above harmful effects, few small
molecules of tannins such as monomeric, dimeric and trimeric tan-
nins had shown less inhibitory effect on proteins and enzymes.
Moreover, these small molecules were also found to be associated
with valuable pharmacological activities. This dual nature of tan-
nins was recognized by Chung et al. [6] and thus they crowned
tannins as double edged sword in biology. It was further supported
by the fact that herbs containing smaller molecules of tannins pos-
sess anticarcinogenic activity, host-mediated antitumor activity,
antiviral activity, etc. [7]. Therefore biodegradation seems one of
the efficient methods to degrade larger molecule of tannins into
small tannin molecules with valuable biological activities.
Tannins are defined as naturally occurring water soluble
polyphenols of varying molecular weight. They represent the fourth
most abundant group of plant’s secondary metabolites after cel-
lulose, hemicellulose and lignin [1] and second to lignin in plant
phenolics [2]. These molecules are divided into two major groups;
hydrolysable and condensed tannins. Hydrolyzable tannins are
esters of gallic acid and ellagic acid with a sugar core. These are
readily hydrolyzed by the action of either enzymes or acids into
their respective monomeric units. Besides this, the condensed tan-
nins also known as proanthocyanidins, are lacking in sugar core and
are composed of flavanoids (flavan 3-ol or flavan 3,4-diol) units [3].
Generally tannins are quite resistant to microbial attack and
thus are recalcitrant to biodegradation [4]. These compounds
retard the rate of decomposition of soil organic matter and there-
fore slower down the process of soil formation [1]. Tannins are
also responsible to reduce food digestibility in animals [3]. The
above facts were suggested due to their ability to form com-
plex with proteins, digestive enzymes including amylase, lipase,
protease, ␤-galactosidase, cellulase and to a lesser extent with
other macromolecules like cellulose and pectin by their phenolic
The degradation of tannins is initiated by tannase or tannin acyl
hydrolase enzyme in living organisms. Many fungal species belong-
ing to genera Aspergillus [8], Paecilomyces [9] and Rhizopus [10], etc.
are very well characterized for tannase production. Besides fungi,
bacterial species such as Lactobacillus plantarum [11] and Bacillus
licheniformis [12] have been also identified as tannase producers.
However, little is known about the genes encoding for bacterial
tannases and their properties. In one study Noguchi et al. [13]
investigated the association of tannase producing Staphylococcus
lugdunensis with colon cancer and suggested that it tannase can be
a good marker to identify S. lugdunensis. In this organism a gene
named as tanA, encodes for a polypeptide of 613 amino acids of
T
tannase activity. L. plantarum ATCC 14917 was also isolated from
∗ Corresponding author at: Faculty of Arts Science and Commerce, Mody Institute
of Technology and Science, Lakshmangah, Sikar, Rajasthan 332311, India.
Tel.: +91 9829659294; fax: +91 1573 225044.
E-mail addresses: kantipsharma@gmail.com (K.P. Sharma),
placheriljohn@yahoo.com (P.J. John).
fermented food products and characterized for various biochemical
and molecular properties by Iwamoto et al. [14]. In this bacteria a
regions of 1410 bp of bacterial genome code for a polypeptide con-
sisting of 469 amino acids residues. It was also found 28.8% identical
1
359-5113/$ – see front matter © 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.procbio.2010.08.016

K.P. Sharma, P.J. John / Process Biochemistry 46 (2011) 240–244
2.5. Effect of pH and temperature on enzyme activity
241
to tannase sequence of S. lugdunensis. But tannase of L. plantarum
did not show any similarity in size, pH optima and kinetic con-
stant to that of Aspergillus oryzae. So the present investigation was
designed to characterize tannase at molecular level in Enterobac-
ter sp. not only because of its importance in bioremediation, animal
nutrition and pharmaceuticals but also because of inadequate infor-
mation about tannase gene in literature till date.
Effect of pH on purified enzyme was studied over a pH range of 4.0–6.5. The
buffer systems used were 50 mM citrate buffer for pH 4.0–5.5 and 50 mM phos-
phate buffer for pH 6.0 and 6.5. To study the effect of temperature, the activity
was determined at different temperatures (25–60 C) under standard enzyme assay
conditions.
◦
2
.6. Kinetic constant of Enetrobacter sp. tannase
Kinetic constant of tannase for most commonly used substrate methyl gallate
1–10 mM) was determined. The reactions were conducted under standard assay
2. Materials and methods
(
conditions. Km value was calculated out using the Lineweaver–Burk transformation
of Michaelis–Menten equation.
2.1. Chemicals
All chemicals unless specified otherwise were obtained from Sigma Chemical,
USA and were of certified reagent grade. Rhodanine was purchased from Merck-
Schuchardt, Germany. Sephadex G-100 was supplied by Pharmacia Fine Chemicals,
Sweden and DEAE cellulose was obtained from GE healthcare, USA. Chemicals and
reagents used in gene identification and cloning were purchased from Invitrogen,
USA, and Bangalore Genei, India. Dialysis tubing of 10–12 kDa cut off and bacterio-
logical medium were purchased from Hi-media, Mumbai, India.
2.7. Isolation of genomic DNA
Genomic DNA was isolated from Enterobacter sp. by following the methodology
of Laing et al. [18]. The purity and amount of DNA was calculated spectrophoto-
metrically. Isolated genomic DNA was subjected to PCR along with various sets of
degenerate primers.
2
.8. PCR amplification, cloning and sequencing of tannase gene
2.2. Microorganism and growth conditions
The tannase gene sequences from five members of different classes (␣, ␤ and ␥)
A Gram negative bacterium which was identified as Enterobacter sp. on the
of phylum proteobacteria (Klebsiella sp., Pseudomonas sp., Ralstonia sp., Burkholderia
sp. and Roseovarius sp.) were aligned. Four sets of degenerate primers were designed
from various locations of alignment file. Following pairs of primers were used to
basis of morphological, biochemical and 16S ribosomal RNA gene sequence fea-
tures, isolated from normal soil to purify and characterize tannase and tannase gene.
®
ꢀ
Escherichia coli DH5 ␣ and pGEM T Easy (Promega, Madison, USA) were used for
amplify tannase gene sequence in isolated organism; F1: 5 -Cgg HTg Cgg Ygg NYT
ꢀ ꢀ ꢀ ꢀ
gTg Cgg SA-3 ; R1:5 -CCR TgC CAS Agg ATM AAK YTT gCC gCC-3 , F2: 5 -ggY TgC TCN
cloning of tannase gene sequences. Enterobacter culture was maintained on a solid
medium containing 0.05% of each KH2PO4, K2HPO4 and MgSO4, 0.3% NH4NO3, 0.5%
tannic acid and 2% agar. E. coli DH5 ␣ cells were maintained on nutrient agar.
ꢀ
ꢀ
ꢀ
ꢀ
AAY ggN ggN CgC VAR gCg-3 ; R2: 5 -AAg ARS CgC gCR AAN gNC STS NgC-3 , F3:
ꢀ
ꢀ
ꢀ
5 -gAN TTC gAC ggC ATY NTC gCN ggC-3 ; R3: 5 -ATC ATY TTg CCR CCN CgN T-3 , F4:
-CBg AYg ANT TCg ACg gYA TCN TCg NC-3 ; R4: 5 -CCg CCN CgC CAR TgN BDC ATN
ꢀ
ꢀ
ꢀ
5
ꢀ
CCN ggC-3 . The PCR mixture (25 ␮l) consisted of 2.5 ␮l of 10× buffer containing
2
.3. Enzyme assay
10 mM MgCl2, 0.4 ␮l of each dNTP (10 mM) 1 ␮l of each forward and reverse primer
(
10 ␮M), 2 ␮l of template (100 ng) and 0.25 ␮l of Taq DNA polymerase (5 U/ml). PCR
Tannase activity was determined spectrophotometrically by the method given
was carried out in a thermal cycler [Gene Amp PCR system 9700 (Applied Biosys-
◦
by Sharma et al. [15]. In this method tannase reacts with methyl gallate and releases
gallic acid which reacts subsequently with rhodanine and forms a complex. The
formation of above complex (gallic acid and rhodanine complex) was recorded at
tem, USA)] according to a programme encompassing initial denaturation at 94 C for
◦
◦
◦
3 min, followed by 30 cycles of 94 C for 30 s, 58 C for 1 min, 72 C for 2 min and final
◦
extension at 72 C for 5 min. PCR products were electrophoresed on 1% agarose gel
5
20 nm.
in 1× Tris–acetate–EDTA (0.04 M Tris–acetate, 0.001 M EDTA) buffer at 75 mAmp for
TM
1
.5 h. The gel was stained and desired fragment was excised, purified (Gen Elute
Gel Extraction Kit of Sigma) and cloned into pGEM-T Easy cloning vector according
to manufacturer instructions.
2.4. Purification
Transformed E. coli DH5␣ cells carrying the recombinant plasmid were selected
by blue–white selection. The cloned product was sequenced by primer walking
method using a BigDye terminator cycle sequencing kit v3.1 (Applied Biosys-
tems, Foster City, USA) in an ABI Prism 3130 xl Genetic Analyzer (Applied
Biosystems) by following the manufacturer’s instructions. Editing was done
to remove the vector sequence and sequence was characterized with tan-
nase gene sequences already available in GenBank using the BLAST programme
(www.ncbi.nlm.nih.gov/blast/blast.cgi).
Purification was done by the methodology used by Zeida et al. [16]. The cells
were grown overnight in nutrient broth medium of pH 7. 10% (v/v) overnight cul-
ture was used to inoculate a litre of defined medium of pH 6.0. (15 g tannic acid,
5
g sucrose, 2 g (NH4)2HPO4, 1 g KH2PO4, 0.5 g MgSO4, and 0.5 g yeast extract). The
◦
inoculated medium was incubated at 35 C for 24 h with constant shaking (200 rpm).
Cells were harvested by centrifugation (Sigma 2 16-PK) at 5000 × g for 10 min at
room temperature. The pellet obtained was washed twice with 50 mM citrate buffer
of pH 5.0 (containing 1 mM DTT and 50 mM Na2S2O3) and suspended in minimum
amount of the same buffer. The resting cells suspension was quantified for tannase
enzyme activity and lysed by ultrasonication for 2 min in an ultrasonicator (Sonics
2
.9. Characterization of tannase sequence
◦
Material Inc., USA) at 4 C by giving pulse of 9 s on and 5 s off. The lysate was cen-
Tannase nucleotides and amino acids sequence of Enterobacter sp. was deposited
◦
trifuged at 4 C for 15 min at 13,000 × g. The supernatant obtained was quantified
in the GenBank database of NCBI. The deduced amino acid sequence was character-
ized in terms of percentage homology with the available tannase sequences through
ClustalW2 (www.ebi.ac.uk).
for tannase activity. The protein concentration was also determined by Bradford
method [17].
The lysate was further used as a source of crude enzyme for purification. The
crude enzyme was fractionated with ammonium sulphate in increasing concen-
tration. Three fractions of ammonium sulphate precipitation (0–30%, 30%, 60% and
3. Results and discussions
◦
6
0–100%) were obtained at 4 C with constant gentle shaking. Each precipitated
samples were suspended in 1 ml of 50 mM citrate buffer of pH 5.0 and dialyzed
overnight against the buffer (50 mM citrate buffer of pH 5.0) containing 1 mM DTT
and 50 mM Na2S2O3 at 4 C in dialysis tubing. The activity and protein content were
3
.1. Purification and characterization
◦
In the present investigation the enzyme tannase was purified
determined as described above in all dialyzed samples.
Proteins precipitated at 30–60% concentration of ammonium sulphate subjected
to dialysis and were loaded onto a pre equilibrated DEAE cellulose column (bed
volume 20 ml) (Hi Prep 16/10 DEAE FF column). 50 mM citrate buffer of pH 5.0
from its native conditions. Osawa et al. [11] and Mondal and Pati
[12] isolated bacterial species for tannase production but none of
them characterized tannase at molecular level. Tannase was finally
purified with a yield of 7.1% by using DEAE cellulose followed by
Sephadex G-100 column. Bhardwaj et al. [8], Mahendran et al. [9]
and Iwamoto et al. [14] also purified tannase from various sources
and obtained the yield of process as 20%, 17% and 4.8%, respectively.
In addition to it, the tannase of Enterobacter sp. was eluted as a
single peak through DEAE cellulose and Sephadex G-100 column
in the present investigation which is different from the observa-
tions made by Bhardwaj et al. [8] during the purification of tannase
TM
containing 1 mM DTT and 50 mM Na2S2O3 was used as pre equilibration buffer.
Elution was done with increasing concentration of NaCl (0–0.5 M) in 50 mM citrate
buffer of pH 5.0 containing 1 mM DTT and 50 mM Na2S2O3 at room temperature with
a flow rate of 5 ml/min. The eluted fractions were checked for tannase activity and
active fractions were pooled. Pooled samples were concentrated by freeze-drying
method and loaded onto G-100 Sephadex column (bed volume 60 ml). The protein
was finally eluted with 50 mM citrate buffer of pH 5.0 containing 1 mM DTT, 50 mM
Na2S2O3 and 50 mM NaCl at a flow rate of 0.3 ml/min. The activity, protein content
and purity were determined and the purified enzyme was characterized in terms of
pH optima, temperature optima and Km.

2
42
K.P. Sharma, P.J. John / Process Biochemistry 46 (2011) 240–244
Table 1
Purification of tannase from Enetrobacter sp.
Purification step
Total activity (units)
Total protein (mg)
Specific activity (units/mg)
Purification fold
Yield (%)
Crude
1.69
1.13
0.49
0.12
20.1
9.36
0.0432
0.0088
0.084
0.12
11.34
13.63
1
1.4
135
162
100
66.8
28.9
7.1
Precipitated
DEAE cellulose
G-100 Sephadex
Activity represents micromole of gallic acid released in 1 min.
Fig. 3. Lineweaver–Burk plot of tannase of Enterobacter sp. Data points represent
the means of three replicate with standard deviation ± 5%.
Fig. 1. 12% SDS PAGE of purified tannase. Lane 1: molecular mass protein markers;
Lane 2: purified tannase.
from Aspergillus niger MTCC 2425. They observed two major peaks
corresponding to two different proteins during elution of tannase
through DEAE cellulose column (Table 1).
The molecular mass of tannase was found to be 90 kDa on SDS
PAGE (Fig. 1). Literature reveals that the protein tannase is very
diverse in its structural properties, e.g. tannase of A. niger van
Tieghem MTCC 2425 was found to contain two subunits of 102 and
8
3 kDa in size [8]. Coronel et al. [19] purified a tannase from A. niger
and found it to be a dimer of 180 and 90 kDa subunits. In contrast
to the above reports tannase of L. plantarum was found to contain
a single unit of 50 kDa [14]. In yeast, Arxula adeninivorans, it was
found to be a tetramer of 320 kDa [20]. The above variation in the
protein can be attributed to the presence of various isoforms of the
enzyme or it may be also due to the presence signal sequences
which are required to transport the enzyme molecule from the
cytosolic part of cell to the outside [2].
The effect of pH on purified enzyme revealed maximum tan-
nase activity at pH 5.5 (Fig. 2). The optimal pH for most fungal and
bacterial tannases had been reported to be in acidic range which is
also consistent with the present observation [21]. The increase or
Fig. 4. 1% agarose gel electrophoresis of PCR amplified product. Lane 1: 500 bp
marker; Lane 2: primer combination F1 and R1; Lane 3: primer combination F2
and R2; Lane 4: primer combination F3 and R3; Lane 5: primer combination F4 and
R4; Lane 6 contains reaction control (without primer).
decrease in pH from 5.5 was found to decrease the enzyme activity
probably because of alteration in the enzyme structure.
The effect of temperature on enzyme activity showed maxi-
◦
mum enzyme activity at 40 C (Fig. 2). The activity was reduced
◦
when the temperature was increased above 40 C. These findings
Fig. 2. Effect of pH and temperature on tannase activity of Enterobacter sp. Data points represent the means of three replicate with standard deviation ± 5%.

K.P. Sharma, P.J. John / Process Biochemistry 46 (2011) 240–244
243
Fig. 5. Phylogenetic analysis of Enterobacter sp. tannase protein sequence by neighbour joining method at a sequence difference 0.75.
◦
are attributed to the disturbances created in the enzyme struc-
ture upon increasing the temperature. The enzyme also exhibited
lesser enzyme activity when the temperature is below 40 C. This is
because of the fact that at reduced temperature sufficient energy is
not available in order to catalyze the process by an enzyme. Another
significant feature noticed in the present study is that the enzyme
retained 20% of its activity at 60 C which makes enzyme relevant
to industrial applications. The Km and Vmax values of tannase were
found to be 3.7 mM and 0.166 units for methyl gallate, respectively
(Fig. 3). These observations are not similar to the earlier reports
which may be due to variation in the enzyme reaction mechanism
[14].
◦
Fig. 6. Comparison of the deduced amino acid sequence of tannase of Enterobacter sp. with those of tannase Lactobacillus plantarum ATCC 14917T and Staphylococcus
lugdunensis. Highly conserved residues are shown in red colour. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version
of the article.)

2
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K.P. Sharma, P.J. John / Process Biochemistry 46 (2011) 240–244
3.2. Characterization of tannase gene at nucleotide and amino
Acknowledgements
acid level
The authors are thankful to Indian Academy of Sciences, Ban-
galore to provide summer research fellowship to Kanti Prakash
Sharma for the work. The contribution of Dr. P.S. Ahuja, Director
IHBT, Palampur (CSIR) and Dr. Sanjay Kumar, Scientist, IHBT, Palam-
pur (CSIR) is also duly acknowledged for extending all laboratory
facilities under the fellowship scheme.
PCR revealed a 924 nucleotide fragment of tannase gene from
Enterobacter sp. (Fig. 4). It was submitted in GenBank database
of NCBI under accession no. GQ268323. In the present investiga-
tion protein tannase of Enetrobacter sp. (accession no. ACT35016)
showed resemblance in its amino acid sequence with the earlier
reported tannases of bacteria, fungi (Ascomycetes and Oomycetes)
and plants (Fig. 5). The phylogenetic analysis tree (Fig. 5) suggests
that tannase of Enterobacter sp. is closely related to tannase of
Pantoea sp. It is a matter of fact that both organism share same
family. Besides this, the tree also relates tannase protein sequence
of Enterobacter sp. with other similar protein sequences of various
organism distance wise. In addition to this our study is probably the
first report which identifies a tannase in Enterobacter sp. resembling
in various features with the fungi and plants tannases.
References
[
1] Chowdhury SP, Khanna S, Verma SC, Tripathi AK. Molecular diversity of
tannic acid degrading bacteria isolated from tannery soil. J Appl Microbiol
2004;97:1210–9.
[2] Aguilar CN, Gutierrez-Sanchez G. Review sources, properties, applications and
potential uses of tannin acyl hydrolase. Food Sci Technol Int 2001;7:373–82.
[
3] Bhat TK, Singh B, Sharma OP. Microbial degradation of tannins – a current
perspective. Biodegradation 1998;9:343–57.
[4] Field JA, Lettinga G. The methanogenic toxicity and anaerobic degradability of
a hydrolyzable tannin. Water Res 1987;21:367–74.
It is also evident form the multiple alignment (Fig. 6) that
tannase of Enterobacter sp. is not very much similar to tan-
nase of S. lugdunensis and L. plantarum. The alignment also
revealed the presence of highly conserved amino acid residues
[
5] Scalbert A. Antimicrobial properties of tannins. Phytochemistry
991;30:3875–83.
1
[
6] Chung KT, Wei CI, Johnson MG. Are tannins a double-edged sword in biology
and health. Trends Food Sci Technol 1998;9:168–75.
1
15, 118, 202, 278, 334, 345, 421
[7] Okuda T, Yoshida T, Hatano T. Pharmacological active tannins isolated from
medicinal plants. Basic Life Sci 1992;59:539–69.
among the three enzymes as follows: Gly
,
1
49, 181, 183, 185, 222, 309
161, 225
, Asp215, Gln255, Ser346. The
Ala
, Pro
[
8] Bhardwaj R, Singh B, Bhat TK. Purification and characterization of tannin acyl
hydrolase from Aspergillus niger MTCC 2425. J Basic Microbiol 2003;43:449–61.
9] Mahendran B, Raman N, Kim DJ. Purification and characterization of tannase
from Paecilomyces varioti: hydrolysis of tannic acid using immobilized tannase.
Appl Microbiol Biotechnol 2006;70:444–50.
amino acid sequence alignment revealed that only 10% and 13%
[
amino acid residues of Enterobacter sp. tannase are similar to tan-
T
naseof S. lugdunensis and L. plantarum 14917 tannase, respectively.
The above fact can be attributed to their source of isolation. It is
evident form the literature that S. lugdunensis was isolated from
the colon of cancer patients and L. plantarum was isolated from
fermented food sample.
It is well known that tannins are the main constituent of plant
defence system and degradation of these molecules seems to affect
plant health [22]. The isolated bacteria Enterobacter sp. is similar in
its genetic makeup with Pantoea and Erwinia sp. which are estab-
lished plant pathogens. Since tannase of Enterobacter sp. shares
some of its properties with the tannases of plants and Pantoea sp.,
the present investigation can be helpful in determining the role of
tannase in plant defence system.
[10] Banerjee R, Mukherjee G, Patra KC. Microbial transformation of tannin
rich substrate to gallic acid through co-culture method. Bioresour Technol
2005;96:949–53.
[
[
[
11] Osawa R, Kuroiso K, Goto S, Shimizu A. Isolation of tannin degrading lactobacilli
from humans and fermented foods. Appl Environ Microbiol 2000:3093–7.
12] Mondal KC, Pati BR. Studies on the extracellular tannase from newly isolated
Bacillus licheniformis KBR 6. J Basic Microbiol 2000;40:223–32.
13] Noguchi N, Ohashi T, Shiratori T, Narui K, Hagiwara T, Ko M, et al. Associa-
tion of tannase-producing Staphylococcus lugdunensis with colon cancer and
characterization of a novel tannase gene. J Gastroenterol 2007;42:346–51.
14] Iwamoto K, Tsuruta H, Nishitaini Y, Osawa R. Identification and cloning of a gene
encoding tannase (tannin acylhydrolase) from Lactobacillus plantarum ATCC
[
T
1
4917 . Syst Appl Microbiol 2008;31:269–77.
[15] Sharma S, Bhat TK, Dwara RK. A spectrophotometric method for assay of tan-
nase using rhodanine. Anal Biochem 2000;279:85–9.
[
16] Zeida M, Wieser M, Yoshida T, Sugio T, Nagasawa T. Purification and characteri-
zation of gallic acid decarboxylase from Pantoea agglomerans T 71. Appl Environ
Microbiol 1998;64:4743–7.
4
. Conclusions
[
17] Bradford MM. A rapid and sensitive method for quantitation of microgram
quantities of protein utilizing the principle of protein–dye binding. Anal
Biochem 1976;72:248–54.
The present investigation characterizes tannase in Enterobacter
sp. at molecular level. The findings reveal that tannase of Enterobac-
ter sp. can be useful in the bioremediation aspect and can also be
utilized in biocatalysis and biotransformation process to synthe-
size molecules like gallic acid, trimethoprim and other molecules
of pharmaceutical interest. In addition to these, tannase of Enter-
obacter sp. is closely related to tannases of other members of
Enterobacteriaceae family and thus both protein and organism
itself can be useful in manipulation of gut microflora to combat
the harmful effects of tannins in the digestive tract of ruminants.
Besides this, the study also sets up a platform to understand the
role of tannase in plant defence system.
[18] Laing C, Pegg C, Yawney D, Ziebell K, Steele M, Johnson R, et al. Rapid
determination of Escherichia coli O157:H7 lineage types and molecular sub-
types by using comparative genomic fingerprinting. Appl Environ Microbiol
2008;74:6606–15.
[19] Coronel RMA, Gonzalez GV, Darvill A, Augur CA.
A
novel tannase from
Aspergillus niger with ␤-glucosidase activity. Microbiology 2003;149:2941–6.
20] Boer E, Bode R, Mock HP, Piontek M, Kunze G. Atan lp – an extracellular tannase
from the dimorphic yeast Arxula adeninivorans: molecular cloning of the ATANI
gene and characterization of the recombinant enzyme. Yeast 2009;26:323–37.
21] Mingshu L, Kai Y, Qiang H, Dongying J. Biodegradation of gallotannins and
ellagitannins. J Basic Microbiol 2006;46:68–84.
22] Madhavakrishna W, Bose S, Nayudamma Y. Estimation of tannase and cer-
tain oxidising enzymes in Indian vegetables tanstuffs. Bull Cent Leath Res Inst
1960;7:1–11.
[
[
[