Enzymatic synthesis of gallic acid from tannic acid with an inducible hydrolase of Enterobacter spp

Article abstract of DOI:10.1080/10242422.2017.1306740

Gallic acid acts as a precursor molecule to synthesize various tannin molecules. These are plant polyphenols and were proved to be good anti-oxidant, anti-cancerous, anti-inflammatory, anti-microbial compounds. In order to fully exploit prominent biological activities of specific tannins and to develop tannin-based new medicines, it is necessary to obtain their pure preparations with an aim of high yield and specificity. In the present study, gallic acid is synthesized by the hydrolysis of tannic acid using a microbial based transformation process. The microorganism was isolated and identified. The ability of the isolated microorganism to covert tannic acid into gallic acid was determined by HPLC and enzyme production.Highlights The present investigation signifies the role of Enterobacter spp. in various processes: ?To synthesize gallic acid (a precursor for food oxidant such as propyl gallate) and a bacteriostatic antibiotic (trimethoprim). ?To protect the environment from tannery’s discharge through the process of biodegradation. ?To reduce the toxicity of tannins in animal feed.

Full text of DOI:10.1080/10242422.2017.1306740

Biocatalysis and Biotransformation
ISSN: 1024-2422 (Print) 1029-2446 (Online) Journal homepage: http://www.tandfonline.com/loi/ibab20
Enzymatic synthesis of gallic acid from tannic acid
with an inducible hydrolase of Enterobacter spp
Kanti Prakash Sharma, P. J. John, Pawas Goswami & Manish Soni
To cite this article: Kanti Prakash Sharma, P. J. John, Pawas Goswami & Manish Soni (2017):
Enzymatic synthesis of gallic acid from tannic acid with an inducible hydrolase of Enterobacter spp,
Biocatalysis and Biotransformation, DOI: 10.1080/10242422.2017.1306740
To link to this article: http://dx.doi.org/10.1080/10242422.2017.1306740
Published online: 31 Mar 2017.
Submit your article to this journal
Article views: 6
View related articles
View Crossmark data
Full Terms & Conditions of access and use can be found at
http://www.tandfonline.com/action/journalInformation?journalCode=ibab20
Download by: [Iowa State University]
Date: 07 April 2017, At: 08:10

BIOCATALYSIS AND BIOTRANSFORMATION, 2017
http://dx.doi.org/10.1080/10242422.2017.1306740
RESEARCH ARTICLE
Enzymatic synthesis of gallic acid from tannic acid with an inducible
hydrolase of Enterobacter spp
a
b
c
d
Kanti Prakash Sharma , P. J. John , Pawas Goswami and Manish Soni
a
b
Department of Biotechnology, Mody University, Laxmangarh, India; Department of Zoology, University of Rajasthan, Jaipur, India;
c
d
Department of Microbiology, Bhaskaracharya College of Applied Sciences, University of Delhi, New Delhi, India; Department of
Zoology, University of Rajasthan, Jaipur, India
ABSTRACT
ARTICLE HISTORY
Gallic acid acts as a precursor molecule to synthesize various tannin molecules. These are plant
polyphenols and were proved to be good anti-oxidant, anti-cancerous, anti-inflammatory, anti-
microbial compounds. In order to fully exploit prominent biological activities of specific tannins
and to develop tannin-based new medicines, it is necessary to obtain their pure preparations
with an aim of high yield and specificity. In the present study, gallic acid is synthesized by the
hydrolysis of tannic acid using a microbial based transformation process. The microorganism was
isolated and identified. The ability of the isolated microorganism to covert tannic acid into gallic
acid was determined by HPLC and enzyme production.
Received 5 October 2015
Revised 1 February 2017
Accepted 7 February 2017
KEYWORDS
Tannic acid; gallic acid;
tannase; biotransformation;
PCR; BLAST; HPLC
HIGHLIGHTS
The present investigation signifies the role of Enterobacter spp. in various processes:
ꢀ
ꢀ
ꢀ
To synthesize gallic acid (a precursor for food oxidant such as propyl gallate) and a
bacteriostatic antibiotic (trimethoprim).
To protect the environment from tannery’s discharge through the process of
biodegradation.
To reduce the toxicity of tannins in animal feed.
Introduction
proved as strong natural antioxidants (Khan et al. 2000;
Pulido et al. 2000; Zheng and Wang 2001; Metelitza
et al. 2001) and very good anti-carcinogenic (Abu-
Amsha Caccetta et al. 2001; Landrault et al. 2001) mole-
cules. They have also shown potent antifungal (Kubo
et al. 2001; Tao et al. 2009), antibacterial (Kubo et al.
Gallic acid (GA)-3,4,5-trihydroxybenzoic acid, and its
derivatives are biologically active compounds
(
Kahkonen et al. 1999; Lee et al. 2000). These are
widely present in plants for their defensive actions
against herbivores and pathogens and collectively
known as tannins. These are water soluble polyphenols
of varying molecular weight and an important con-
stituent of plant secondary metabolites. These are of
two types: condensed and hydrolysable. Condensed
tannins are composed of flavonoids (flavan 3-ol or fla-
van 3, 4-diol) and commonly known as proanthocyani-
dins (Aguilar et al. 2001). The hydrolysable tannins are
polyesters of gallic acids (gallotannins) or egallic acid
2004; Bravo et al. 2005; Chanwitheesuk et al. 2007) and
antiviral (Hurtado et al. 2008; Kratz et al. 2008; Choi
et al. 2010) activities against a range of microorganisms.
Besides the above GA based compounds were also
found to be involved in prevention of atherosclerosis
(Miura et al. 2001) and of inflammation (El-Saadany
et al. 2008) based diseases. In industrial processes, GA is
often incorporated into chemical syntheses to provide
the trihydroxylated aromatic ring of biologically active
molecules such as the antibiotic trimethoprim, the
muscle relaxant gallamine triethiodide and the insecti-
cide bendiocarb (Kambourakis et al. 2000).
In order to fully exploit prominent biological activ-
ities of specific gallate derivatives and to develop new
pharmaceutical molecules, it is necessary to obtain
(
ellagitannins) with simple sugars. Gallotannins are
cross-linked structure of phenolic groups of GA resi-
dues with depside bond formation, while in ellagitan-
nins, galloyl groups are linked but not through
depside bonds.
Literature reveals that GA molecules and their esters
are very important biological molecules. These are
CONTACT Kanti Prakash Sharma
kantipsharma@gmail.com
Department of Biotechnology, Mody University, Laxmangarh, Sikar, Rajasthan 332311,
India
ß 2017 Informa UK Limited, trading as Taylor & Francis Group

2
K. P. SHARMA ET AL.
pure preparations by isolation from natural sources, by
chemical synthesis or by biotransformation reactions
with an aim of obtaining high yield and specificity.
The natural availability of GA is restricted by its current
isolation from insect carapaces, gall nuts harvested in
China and tara powder derived from the ground seed
pod of a tree found in Peru (Kambourakis et al. 2000).
The chemical approach utilizes acid hydrolysis of tan-
nic acid but it also resulted in low yield. Lekha and
Lonsane (1997) encouraged the use of microorganisms
over plants for tannin transformations owing to stabil-
ity and higher production rate. Werner et al. (1997)
demonstrated synthesis of GA by oxidation of 3-dehy-
droshikimic acid (DHS) to a diketo intermediate fol-
lowed by spontaneous aromatization and by
dehydration of DHS followed by hydroxylation of the
intermediate protocatechuic acid (PCA) using microor-
ganisms. However, the above suggested pathway uti-
lizes glucose to produce GA (Kambourakis et al. 2000).
Therefore, it is desired to search for new methods
which can produce GA from molecules of natural
abundance. Tannin acyl hydrolase (Tannase) based
hydrolysis of tannic acid can be meaningful in the
above context. Various groups of microorganisms
including bacteria, yeast and fungi were known to
transform tannins by tannase. Literature reveals that
tannase based transformation of tannic acid to GA by
microorganisms has been exploited much less with
reference to biotransformation industry. Among bac-
teria, Lactobacillis plantarum (Osawa et al. 2000) and
Streptococcus spp. (Goel et al. 2005) were mainly
exploited to overcome the toxicity caused by tannins
in ruminant’s diet whereas Citrobacter freundi (Kumar
et al. 1999) and Bacillus licheniformis (Mondal and Pati
Merck (Kenilworth, NJ) and Sigma-Aldrich (St. Louis,
MO). Tannic acid was obtained from Ranbaxy Fine
Chemical Ltd. (India). Gallic acid was obtained from Hi
Media (Mumbai, India). The bacteria were isolated
from a natural sample and identified.
Isolation of microorganism
Various environmental samples including garden soil,
tannery soil and waste water, goat faecal matter, faecal
matter mixed with soil, soil of hilly area, and fer-
mented foods were taken for the isolation process. A
medium containing 0.05% of KH
PO , K HPO , MgSO
2 4 2 4 4
each, 0.3% NH NO and 0.5% tannic acid was used for
4
3
the isolation and cultivation of bacteria. Tannic acid
was filter sterilized through a 0.45 l filter and added
into medium after autoclaving. Agar at a concentration
of 1.8% was used to prepare solid medium. Samples
were serially diluted in saline water and plated onto a
medium for the isolation process. Inoculated Petri
ꢁ
dishes were incubated aerobically 35 C for 48 hours.
Bacterial colonies grown on solid medium were
checked for tannase activity as determined earlier.
Determination of tannase activity
The bacterial colonies were screened to identify tan-
nase producer organisms among them. They were
screened on the basis of their volume activity in broth
medium (0.05% of KH
PO , K HPO and MgSO each,
4 2 4 4
2
0.3% NH NO and 0.5% tannic acid). The culture was
4
3
prepared by inoculating a part of an isolated colony
into nutrient broth medium (5 ml) of pH 7. The culture
ꢁ
was grown for 12 hours at 35 C in an orbital rotatory
2
000) were studied to know their potential in bio-
shaker at 160 rpm and then 2 ml of it was inoculated
into 25 ml of sterilized broth medium. The inoculated
broth was incubated at 35 C under constant shaking
remediation process. However, GA production is well
known by Rhizopus oryzae (Kar et al. 1999; Kar et al.
ꢁ
2
002) but owing to superiority of the action of bacter-
conditions at 160 rpm for 24 hours. The above
obtained culture broth was centrifuged at 5000ꢂg for
five minutes to separate the medium and resting cells.
Resting cells were suspended into minimum volume of
100 mM phosphate buffer of pH 7 to prepare the rest-
ing cell suspension. Both resting cells and culture
supernatant were used to determine tannase activity
in order to determine the nature of enzyme produc-
tion. Tannase activity was determined spectrophoto-
metrically using rhodanine as done earlier by Sharma
and John (2011) and defined as micromole of GA
released per ml of enzyme in one minute. It included
recording the absorbance of GA released against dis-
tilled water at 520 nm from its substrate 0.01 M methyl
gallate (prepared in 0.05 M citrate buffer of pH 5.0).
0.25 ml of substrate solution was added into each of
ial tannase on natural tannins over fungal and yeast
tannase (Deschamps et al. 1980; Deschamps and
Lebeault 1984), there is a need to develop bacterial
based biotransformation process.
The present study demonstrates isolation, identifica-
tion of a microorganism from soil which can synthe-
size GA from tannic acid, a molecule abundantly found
in nature and quantitative estimation of GA produc-
tion by HPLC and enzyme production.
Materials and methods
Chemicals and microorganism
Unless otherwise specified all chemicals were analytical
grade and obtained from Hi Media (Mumbai, India),

BIOCATALYSIS AND BIOTRANSFORMATION
3
three tubes labelled as blank, test, and control tubes.
.25 ml of the buffer and 0.25 ml of the enzyme sam-
molecular weight makers. The desired fragment was
TM
0
excised from the gel, purified (Gen Elute
Gel
ple were added to blank and test, respectively. It was
followed by incubation at 30 C for five minutes
and addition of 0.3 ml of methanolic rhodanine
Extraction Kit of Sigma, St. Louis, MO) and subjected
to sequencing reaction. The sequence obtained was
characterized by BLAST at NCBI to find out homo-
logues with the sequences already available. A phylo-
genetic tree was also constructed to know the genetic
relatedness of the isolate with other bacteria.
ꢁ
(
0.667% w/v) to each of the tubes. All test tubes were
ꢁ
again incubated at 30 C for five minutes and 0.2 ml of
0
.5 N potassium hydroxide was added to each tube
and incubation was continued for five minutes at
ꢁ
3
0 C. The reaction mixture volume was made equal
Growth curve and tannase activity
by adding 0.25 ml of enzyme in the control tube only.
Finally each tube was diluted with 4.0 ml distilled
water and incubated at 30 C for 10 minutes. The
enzyme activity was calculated from the change in
absorbance:
The isolate was grown to determine the time period
of tannase production. The time of maximum enzyme
production was determined by estimating the enzyme
activity at regular intervals of three hours after inocula-
tion. The growth was determined by measuring
absorbance of each withdrawal sample at 660 nm and
enzyme activity was defined as micromole of GA
released by per ml of enzyme in one minute as
described earlier.
ꢁ
ΔA520 ¼ ðA test–A blankÞ − ðA control –A blankÞ
Inducible nature of enzyme
To check the inducible nature of enzyme, the isolate
was grown in isolation medium (0.05% of KH PO ,
2
4
K HPO , MgSO each, 0.3% NH NO and 0.5% tannic
2
4
4
4
3
Biotransformation of tannic acid to gallic acid
acid). A medium without tannic acid was served as
control. Additionally a third medium was also used for
enzyme production which contained glucose at a con-
centration similar to tannic acid (0.5%) and activity
was checked as described earlier in both supernatant
and resting cell suspension.
The manuscript also describes synthesis of GA by
using microbial hydrolytic activity. The hydrolysis of
tannic acid was achieved by tannin acyl hydrolase
(tannase) enzyme of isolated bacteria. Tannase hydrol-
yses tannic acid into GA subunits in the presence of
water. The bacterial cells were grown for above deter-
mined time and cells were separated from broth by
centrifuging the growth at 5000ꢂg for five minutes.
The enzyme production was extracellular (determined
above) and thus cultural filtrate was used as a source
of tannase. 50 ll of samples were filtered through a
Identification of microorganism
Preliminary identification was made on the basis of
morphological, biochemical characteristics as described
in Bergey’s Manual of Systematic Bacteriology
(
Grimont and Grimont 2005). Further identification was
0.45 mm PVDF syringe filter and injected onto the
made on the basis of 16S rDNA gene sequence by
reverse-phase C18 Spherisorb ODS2 column
0
PCR with the help of universal forward (5 -
(
4.6 ꢂ 250 mm) of HPLC-DAD system equipped with
0
0
AgAgTTTgATCCTggCTCAg-3 )
and
reverse
(5 -
Empower 3 software (Waters, Milford, MA). The ana-
lysis was performed at 230 nm at 35 C. The mobile
0
CggTTACCTTgTTACgACTT-3 ) primers (Weisburg et al.
ꢁ
1
991). The amplification was carried out in a thermal
phase was prepared by mixing 10 mM KH PO –H PO
4
2
4
3
cycler with 25 ll volume containing 2.5 ll of 10ꢂ buf-
buffer of pH 2.8 and acetonitrile in 99:1 v/v ratio,
respectively. Above prepared solution was used to
elute the metabolites with a flow rate of 1 ml/min. The
metabolites were identified by comparing the reten-
tion time and spectral data of each peak with those of
standards.
fer (including 1.5 mM of MgCl ), 400 lM of each dNTP,
2
0
.1 ll each primer, 0.125 U of Taq DNA polymerase and
1
00 ng of template. Genomic DNA was isolated and
quantified by standard methods using spectrophotom-
eter. The PCR was performed in a Gene Amp PCR sys-
tem 9700 (Applied Biosystem, Foster City, CA) with
following constituents in a programme encompassing
ꢁ
initial denaturation at 94 C for three minutes, followed
Results and discussion
ꢁ
ꢁ
by 30 cycles of 95 C for two minutes, 55 C for
Isolation of microorganism
ꢁ
3
0 seconds, 72 C for four minutes and final extension
at 72 C for 20 minutes. PCR products were electro-
phoresed in 1% agarose gel along with known
ꢁ
Normal garden soil and ruminant faecal matter did not
produce any colonies on isolation medium. This might

4
K. P. SHARMA ET AL.
be due to lack of adaptation on tannins or absence of
tannin utilization ability in the microorganism or low
pH of medium due tannic acid. Tannery soil showed
fungal growth. It has been attributed to fungal cap-
ability to grow well in acidic range of pH. Ruminant
faecal matter mixed with soil produced bacterial colo-
nies on isolation medium. This may be because of
enrichment, given by soil which is considered as the
best habitat for the growth of microorganisms.
production, VP test and Indole production.
Carbohydrate utilization test revealed that the isolate
possesses the ability to use xylose, glycerol, galactose,
mannitol, mannose, fructose and glucose as carbon
sources whereas sucrose and lactose could not be uti-
lized as a carbon source. Further, above characteristics
revealed that the isolate belongs to the
Enterobacteriaceae family of the bacterial kingdom.
PCR revealed an amplification of 16S rDNA gene of
bacterial genome. A band of about 1500 bp was
detected upon electrophoresis. The band upon purifi-
cation and subsequent sequencing reaction produced
1241 nucleotides sequence. The sequence upon BLAST
analysis revealed that the isolated bacterium belongs
to genus Enterobacter of family Enterobacteriaceae,
order Enterobacteriales, class Gammaproteobacteria
and the phylum Proteobacteria. The phylogenetic ana-
lysis was done through the NCBI website by construct-
ing a phylogenetic tree by neighbour joining method
at a sequence distance of 0.05 (Figure 1). The obtained
nucleotide sequence of the 16S rDNA gene was
deposited in the GenBank of NCBI under accession no.
GQ 438735.2. The above finding has been attributed
to the soil sample from which isolation process was
done.
Determination of tannase activity
The bacterial colonies were further subjected to
screening for tannase activity. It is defined as micro-
mole of GA released by per ml of enzyme in one
minute. 0.02 units/ml tannase activity was found only
in culture filtrate suggesting extracellular production
of enzyme. This may be because of the fact that tannic
acid acts as a sole source of carbon in the medium
and the bacterial cell releases tannase into the
medium so that it can obtain energy by the assimila-
tion of metabolites produced by the hydrolysis of tan-
nic acid with tannase.
Inducible nature of enzyme
The nutrient medium containing tannic acid and glu-
cose as a carbon source showed growth of isolated
microorganism but the control medium without car-
bon source did not allow the isolate to grow. This may
be due to the fact that the isolate is heterotrophic and
cannot obtain energy in the absence of any carbon
source. Further the medium containing glucose as a
carbon source instead of tannic acid showed growth
but its culture supernatant as well as resting cell sus-
pension did not show any tannase enzyme activity.
Tannase enzyme activity was observed from the super-
natant only when isolate was grown in the medium
containing tannic acid as a sole source of carbon. This
suggests the inducible nature of enzyme as attributed
by other researchers (Mahendran et al. 2006; Iwamoto
et al. 2008) also.
Growth curve and tannase activity
Absorbance values at 660 nm and enzyme activity
of each sample were plotted against time interval.
The lag phase of isolate continued up to 18 hours
(OD660nm
:
0.07 ± 0.005; Enzyme activity: 0.04 ±
0.002 Units/ml) after inoculation and thereafter bacter-
ial cells enter into log phase where they extensively
utilize substrate (tannic acid) as a sole source of car-
bon by hydrolysing it with the help of tannase
enzyme. The growth curve (Figure 2) showed
maximum enzyme production after 39 hours of incuba-
tion
(OD660nm
:
0.369 ± 0.3;
enzyme
activity:
0.145 ± 0.01 Units/ml). Growth pattern also suggested
that tannase of Enterobacter sp. is a primary metabol-
ite as its production is associated with growth of bac-
teria. The production of tannase ceases when the
microorganism enters in the stationary phase of its
growth. This may be due to toxicity of tannic acid on
microbial growth or feedback inhibition by GA upon it
production by tannase from tannic acid. This is consist-
ent with earlier findings of Doi et al. (1973).
Identification of microorganism
The morphological characterization revealed the bac-
teria to be Gram negative, nonspore forming rods,
without any capsule. Culture morphology revealed yel-
low, smooth, mucoid colonies on agar plates with halo
formation or zone of clearance around their colonies
on carbon deficient agar plate. The isolate was found
to be positive for catalase activity and MR test.
However, it was found to be negative for H2S
Biotransformation of tannic acid to gallic acid
The chromatogram (Figure 3) of un-inoculated broth
(composition 0.05% of KH PO , K HPO and MgSO4
2
4
2
4

BIOCATALYSIS AND BIOTRANSFORMATION
5
Figure 1. Phylogenetic analysis by neighbour joining method at sequence distance of 0.05.
Figure 2. Growth curve and enzyme production.
ꢁ
Figure 3. Chromatogram of extracts of control medium (without inoculation) after 39 hours of incubation at 35 C.
each, 0.3% NH NO and 0.5% tannic acid) showed two
microorganism and subsequent incubation upon ana-
lysis by HPLC produced five peaks in the chromato-
gram. Two of them are of GA (2.645 minutes) and
tannic acid (14.045 minutes), respectively, as attributed
4
3
major peaks. The elution of standard tannic acid mole-
cules confirmed that the second peak is of tannic acid.
The same broth after inoculation with the isolated

6
K. P. SHARMA ET AL.
Figure 4. Chromatogram of pure gallic acid.
ꢁ
Figure 5. Chromatogram of extracts of Enterobacter spp. growth in liquid medium after 39 hours of incubation at 35 C.
to the passing of standard solutions (Figures 3 and 4)
of them. The chromatogram also showed that peak
area of tannic acid was reduced to 31.9% with respect
to 99.7% of control. This suggested that the isolate
transforms 68% of tannic acid into GA. The efficiency
of biotransformation reached its maximum with-in
the study also demonstrates isolation and identifica-
tion of a Gram negative bacterium which is very
closely related to microorganisms of animal’s intestine.
Therefore, the findings can be utilized in animal nutri-
tion to overcome the nutritional toxicity of forage leg-
ume caused by tannins. In addition to above, the
bacteria degrades tannic acid very efficiently thus
proving its potential role in bioremediation processes
to protect the environment from tannery discharges.
3
9 hours of incubation. The other peaks in Figure 5
suggest the formation of various metabolites. This may
be due to the presence to other enzymes or metabo-
lites produced by the microorganism. Thus in order to
obtain higher GA yield, purification of enzyme would
be required in future studies.
Acknowledgements
The authors acknowledge the help and guidance given by
Dr. R. S. Jayani and Dr. Satyendra Kumar in identifying
and characterizing the organism. Assistance given by
Mr. Priyadarshi Meena, Ph.D. Scholar in the Department of
Zoology, University of Rajasthan is greatly acknowledged.
Conclusions
The present investigation reports a valuable process to
synthesize GA from a naturally abundant substrate
molecule by an inducible hydrolase of Enterobacter
spp. The microorganism is very economic as it requires
very few nutrients to produce GA. Thus the process
can be meaningful for industrial exploitation such as
in food and pharmaceutical industries. Besides above,
Disclosure statement
All others contributed equally in the preparation of manu-
script. We hereby declare that we have no conflict of interest
in the submission of manuscript entitled “Enzymatic
Synthesis of Gallic acid from Tannic acid with an Inducible

BIOCATALYSIS AND BIOTRANSFORMATION
7
Hydrolase of Enterobacter spp.” for the publication in
Biocatalysis and Biotransformation.
product isolation with microbial catalysis. J Am Chem Soc
122:9042–9043.
Kar B, Banerjee R, Bhattacharyya BC. 1999. Microbial produc-
tion of gallic acid by modified solid state fermentation.
J Ind Microbiol Biot 23:173–177.
References
Kar B, Banerjee R, Bhattacharyya BC. 2002. Optimization of
physicochemical parameters for gallic acid production by
evolutionary operation-factorial design technique. Process
Biochem 37:1395–1401.
Khan NS, Ahmad A, Hadi SM. 2000. Anti-oxidant, pro-oxidant
properties of tannic acid and its binding to DNA. Chem
Biol Interact 125:177–189.
Kratz JM, Andrighetti-Frohner CR, Leal PC, Nunes RJ, Yunes
RA, Trybala E, Bergstr o€ m T, Barardi CR, Sim o~ es CM. 2008.
Evaluation of anti-HSV-2 activity of gallic acid and pentyl
gallate. Biol Pharm Bull 31:903–907.
Kubo I, Fujita K, Nihei K, Nihei AT. 2004. Antibacterial activity
of akyl gallates against Bacillus subtilis. J Agric Food Chem
Abu-Amsha Caccetta R, Burke V, Mori TA, Beilin LJ,
Puddey IB, Croft KD. 2001. Red wine polyphenols, in
the absence of alcohol, reduce lipid peroxidative
stress in smoking subjects. Free Radic Biol Med
30:636–642.
Aguilar CN, Guti ꢀe rrez-Sanchez G. 2001. Review sources,
properties, applications and potential uses of tannin acyl
hydrolase. Food Sci Technol Int 7:373–382.
Aijuan Li A, Chen J, Zhu W, Jiang T, Zhang X, Gu Q. 2007.
Antibacterial activity of gallic acid from the flowers of
Rosa chinensis Jacq. against fish pathogens. Aquacult Res
38:1110–1112.
Bravo HR, Copaja SV, Figueroa-Duarte S, Lamborot M, San
Mart ꢀı n J. 2005. 1,4-benzoxazin-3-one, 2-benzoxazolinone
and gallic acid from Calceolaria thyrsiflora Graham and their
antibacterial activity. Z Naturforsch C J Biosci 60:389–393.
Chanwitheesuk A, Teerawutgulrag A, Kilburn JD,
Rakariyatham N. 2007. Antimicrobial gallic acid from
Caesalpinia mimosoides Lamk. Food Chem 100:1044–1048.
Choi HJ, Song JH, Park KS, Baek SH. 2010. In vitro anti-
enterovirus 71 activity of gallic acid from Woodfordia fruti-
cosa flowers. Lett Appl Microbiol 50:438–440.
Deschamps AM, Lebeault JM. 1984. Production of gallic acid
from tara (Caesalpinia spinosa) tannin by bacterial strains.
Biotechnol Lett 6:237–242.
Deschamps AM, Mohudeau G, Conti M, Lebeault JM. 1980.
Bacteria degrading tannic acid and related compounds.
J Ferment Technol 58:93–97.
Doi S, Shinmyo A, Enatsu T, Terui G. 1973. Growth-associated
production of tannase by a strain of Aspergillus oryzae.
J Ferment Technol 51:768–774.
El-Saadany MA, Rawel HM, Raila J, El-Dashloty MS,
Schweigert FJ. 2008. Antioxidants modulate the IL-6
induced inhibition of negative acute-phase protein secre-
tion in HepG2 cells. Cell Biochem Funct 26:95–100.
Goel G, Puniya AK, Singh K. 2005. Tannic acid resistance in
5
2:1072–1076.
Kubo I, Xiao P, Fujita K. 2001. Antifungal activity of octyl gall-
ate: structural criteria and mode of action. Bioorg Med
Chem Lett 11:347–350.
Kumar RA, Gunasekaran P, Lakshmann M. 1999.
Biodegradation of tannic acid by Citrobacter freundii
isolated from
9:161–168.
a tannery effluent. J Basic Microbiol
3
Landrault N, Poucheret P, Ravel P, Gasc F, Cros G, Teissedre
PL. 2001. Antioxidant capacities and phenolics levels of
French wines from different varieties and vintages. J Agric
Food Chem 49:3341–3348.
Lee MW, Lee YA, Park HM, Toh SH, Lee EJ, Jang HD, Kim YH.
2000. Antioxidative phenolic compounds from the roots of
Rhodiola sachalinensis A. Bor. Arch Pharm Res 23:455–458.
Lekha PK, Lonsane BK. 1997. Production and application of
tannin acyl hydrolase: state of the art. Adv Appl Microbiol
44:215–260.
Mahendran B, Raman N, Kim DJ. 2006. Purification and char-
acterization of tannase from Paecilomyces variotii: hydroly-
sis of tannic acid using immobilized tannase. Appl
Microbiol Biotechnol 70:444–450.
Metelitza DI, Eryomin AN, Sviridov DO, Kamyshnikov VS.
2001. Initiation and inhibition of free radical processes
ruminal streptococcal isolates.
5:243–245.
J
Basic Microbiol
0
in H
2
O
2
-metmyoglobin (methemoglobin)-2,2 -azino-bis-(3-
4
ethylbenzthiazoline-6-sulfonic acid) systems. Biochemistry
Mosc) 66:505–514.
Grimont PAD, Grimont F. 2005. The genus Enterobacter. In:
Brenner DJ, Krieg NR, Staley JT, editors. Bergey’s manual
of systematic bacteriology. New York: Springer Inc.
p. 661–669.
Hurtado C, Bustos MJ, Sabina P, Nogal ML, Granja AG,
Gonz ꢀa lez ME, G oꢀ nzalez-Porqu ꢀe P, Revilla Y, Carrascosa AL.
(
Miura Y, Chiba T, Tomita I, Koizumi H, Miura S, Umegaki K,
Hara Y, Ikeda M, Tomita T. 2001. Tea catechins
prevent the development of atherosclerosis in apoprotein
E-deficient mice. J Nutr 131:27–32.
Mondal KC, Pati BR. 2000. Studies on the extracellular tan-
nase from newly isolated Bacillus licheniformis KBR 6.
J Basic Microbiol 40:223–232.
2008. Antiviral activity of lauryl gallate against animal
viruses. Antivir Ther (Lond) 13:909–917.
Iwamoto K, Tsuruta H, Nishitaini Y, Osawa R. 2008.
Identification and cloning of a gene encoding tannase
Osawa R, Kuroiso K, Goto S, Shimizu A. 2000. Isolation of
tannin-degrading Lactobacilli from humans and fermented
foods. Appl Environ Microbiol 66:3093–3097.
Pulido R, Bravo L, Saura-Calixto F. 2000. Antioxidant activity
of dietary polyphenols as determined by a modified ferric
reducing/antioxidant power assay. J Agric Food Chem
48:3396–3402.
Sharma KP, John PJ. 2011. Purification and characterization
of tannase and tannase gene from Enterobacter spp.
Process Biochem 46:240–244.
(
1
tannin acylhydrolase) from Lactobacillus plantarum ATCC
4917(T). Syst Appl Microbiol 31:269–277.
Kahkonen MP, Hopia AI, Vuorela HJ, Rauha JP, Pihlaja K,
Kujala TS, Heinonen M. 1999. Antioxidant activity of plant
extracts containing phenolic compounds. J Agric Food
Chem 47:3954–3962.
Kambourakis S, Draths KM, Frost JW. 2000. Synthesis of gallic
acid and pyrogallol from glucose: replacing natural

8
K. P. SHARMA ET AL.
Tao S, Zhang S, Tsao R, Charles MT, Yang R, Khanizadeh S.
009. In-vitro antifungal activity and mode of action of
Werner I, Bacher A, Eisenreich W. 1997. Retrobiosynthetic
NMR studies with 13C-labeled glucose. Formation of
2
selected antioxidants on Botrytis cinerea. Arch Phytopathol
Plant Prot 43:1564–1574.
gallic acid in plants and fungi.
272:25474–25482.
J
Biol Chem
Weisburg WG, Barns SM, Pelletier DA, Lane DJ. 1991. 16S
ribosomal DNA amplification for phylogenetic study.
J Bacteriol 173:697–703.
Zheng W, Wang SY. 2001. Antioxidant activity and phenolic
compounds in selected herbs.
J Agric Food Chem
49:5165–5170.