Study on a novel actinomycetes

Study on a novel actinomycetes, Streptomyces gancidicus (MF464014) and enhancing its tannase
producing ability by optimizing growth conditions in submerged fermentation
* Archana D. Tripathi1, and Lakshmi B2
1, 2 Department of Biotechnology, Kadi Sarva Vishwavidyalaya, Gandhinagar
E-mail: 1*[email protected] and 2*[email protected]
Abstract :
Tannase (Tannin acyl hydrolase E.C. 3.1.1.20) is a ubiquitous enzyme catalyzes the hydrolysis
of ester and depside bonds of hydrolysable tannins, releasing glucose and gallic acid.Gallic acid
is used in the food industry as a substrate for the chemical synthesis of food preservatives such as
gallates and pyrogallol, propyl gallate being a very important food antioxidant, and in the
pharmaceutical industry, for the synthesis of antibacterial drugs.Tannases are extensively used in
food, feed, pharmaceutical, beverage, brewing and chemical industries. Tannase is also utilized
for bioremediation of effluents from tanneries.Number of bacteria and fungus has been reported
to produce tannase, but tannase producing actinomycetes has not reported. So the present study
was undertaken to isolate an actinomycetes with the ability to produce tannase enzyme. Total of
30 microorganisms were isolated by enrichment culture technique using tannin as carbon
source.The tannase producing strain AT 9 was selected based on the zone of tannic acid
hydrolysis. AT 9 was identified as Streptomyces gancidicus on the basis of 16s rRNA.
Streptomyces gancidicus showed maximum tannase production when cultured in mineral
medium for 24 h using 4 plugs of culture were used as inoculum size, 2% tannic acid as carbon
source and ammonium chloride (0.6%)as nitrogen source. The optimum temperature and pH
required for maximum enzyme production was 30 ºC and 6.5 pH respectively.The role of Ca+2
play a significant role in tannase production by Streptomyces gancidicus.
Key words:Tannase, Gallic acid, Tannic Acid, Streptomyces gancidicus, optimization

1. Introduction
Tannase (tannin acyl hydrolase, E.C: 3.1.1.20) is an extracellular, inducible enzyme
produced in the presence of tannic acid by fungi, bacteria and yeast and catalyzes the hydrolysis
of tannic acid or tannin by breaking its ester and depside bonds releasing glucose and gallic acid
(Sabu et. al, 2005).Tannase enzyme hydrolyses tannins as the natural substrate. These(tannins or
tannase) are widely distributed in various parts of plants such as in barks, needles, heartwood,
grasses, seeds and in flowers. Tannins are a group of complex oligomeric chains characterized by
thepresence of polyphenolic compounds. These have a molecular weight higher than 500 and
reaching upto 20,000 kDa. One of the major characteristic of tannins is its ability to form strong
complexes with protein and other macromolecules such as starch, cellulose and minerals (Lekha
and Lonsane, 1997; Aguilar and Gutiérrez- Sánchez, 2001).
Tannase have shown many potential applications in different industrial sectors,like brewing
industry, food industry, leather industry and have been associated with colon cancer allocating
the possibility of bacterial tannase as biomarker for colon cancer (Lekha and Lonsane,1997;
Mohapatra et. al., 2006). Number of bacteria and fungus has been reported to produce tannase,
but tannase producing actinomycetes has not been reported. So the present study was undertaken
to isolate an actinomycetes with the ability to produce tannase enzyme. An attempt was also
carried out tooptimize growth medium and enzyme assay conditions for maximum production of
crude tannase by Streptomyces gancidicus.

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2. Materials and Methods:

2.1 Chemicals:
The chemicals used throughout the study were of analytical reagent grade and obtained from
Sigma, Aldrich and Merck.
2.2 Collection of Samples:
Tannery effluent sample was collected from Central Leather Research Institute (CLRI), GIDC
(Gujarat Industrial Development Corporation), Ahmedabad, Gujarat, India in sterile
polypropylene bottles. Microorganisms were isolated from the soil samples contaminated by dye,
catechu and tea waste as well as from the soil near the roots of Amla andJamun trees and tannery
effluent of CLRI.
2.3 Enrichment, isolation and identification of Tannase Producing Microorganisms and
Estimation of tannase activity:
The methods of enrichment, primary and secondary screening for tannase isolates were carried
out according to the method of Archana et al. (2016). The tannase activity was estimated by
modified spectrophotometric method of Sharma et al., 2000. The potential bacterial strain was
characterized by cultural, morphological (Gram Staining and growing on different
media(Glucose Aspergine Agar, Nutrient agar, Czapek dox agar, Bennett’s Agar, Potato
Dextrose agar and Glycerol Aspergine agar)), physiological, biochemical methods. The culture
was subjected to the genenotyping using 16s rRNA sequencing. The sequencing was carried out
at Gujarat State Biotechnology Mission (GSBTM). The partial 16S rRNA sequence (1400 bp) of
the isolate has been submitted to Genbank. (The highest tannase activity was shown by the strain
coded AT 9and it was identified as Streptomyces gancidicus).
2.6. Optimization of Tannase production by AT9 (Streptomyces gancidicus ) under submerged
fermentation:
Various cultural and nutritional parameters that influenced enzyme yield during submerged
fermentation (SmF) were optimized.
a. Study on the effect of Inoculum Size, Temperature and pH on Tannase Production
The activity of an enzyme is affected by its environmental conditions.Changing
these alter the rate of reaction caused by the enzyme. AT 9 cultures were grown on tannic
acid agar medium and incubated for 4 days. The cultures were then transferred to broth by
using sterile corkborer. Culture disc varying from 2,3,4,5 and 6 plugs/discs were inoculated
into 5 different flasks containing tannic acid (TA) broth. Broths were then incubated at 30 °C

temperature for 72-96 hrs. After incubation tannase enzyme activity was measured after
every 24 hours of incubation. To study the effect of temperature, TA broths were inoculated
with 4 plugs of isolate AT 9 and incubated at different temperatures like Room Temperature
(RT), 30 °C, 37 °C, 40 °C and 45 °C and the tannase enzyme activity was measured by using
rhodanine method. To evaluate the effect of pH on enzyme activity, the pH of the production
medium was varied from 5.5, 6.0, 6.5 and 7.0 and was inoculated with 4 plugs of AT 9.The
tannase enzyme activity was measured after every 24 hours of incubation till 96 hours.
b. Optimization of Nutritional parameters for production of Tannase enzyme
Effect of different concentrations of inducers like Tannic Acid, different carbon and
nitrogen sources on Tannase production were carried out. To study the role of inducer tannic acid
in the production of tannase, 4 plugs/culture discs of AT 9 from edge of actively growing 4 days
old colony were inoculated into four different flasks with different concentrations of Tannic acid
(1% to 4%) of 100ml of TA media in 250 ml of flask. Enzyme activity was measured after every
24 hours of incubation.Several Carbohydrates like Glucose, Fructose, Sucrose and Lactose were
incorporated at 0.5% (w/v) concentration into TA broth to study the role of carbon sources.
Tannase enzyme activity was measured after every 24 hours of incubation till 96 hours.
Role of organic and inorganic nitrogen sources in the induction of tannase enzyme
production were also studied. Different Inorganic nitrogen sources like NH4Cl, NaNO3 (0.6%)
and organic nitrogen sources like Peptone, yeast extract and Urea (0.6%) were used in TA broth.
Tannase enzyme activity was measured after every 24 hours of incubation.
Effect of Metal Ions on Tannase Activity
Various metals play a major role in tannase activity. They act as inhibitors or stimulators. Metal
ions like Mg+2, Mn+2, Ca+2, Na+, and K + stimulated the tannase activity, while Cu+2, Fe+3 and
Co+2 acted as inhibitors of the enzyme. Different metal ions like ZnSo4, MnSo4, CuSo4, CaCl2
with 1 mM concentration were added in TA broth and their roles in tannase enzyme produced by
the isolate were studied.

Statistical Analysis
To compare the role of crude tannase enzyme in converting tannic acid to gallic acid in the study
the experimental data were analysed statistically using t-test.

3. Result and Discussion:
3.1 Primary /Qualitative screening of bacterial isolates for tannase production
A total of 30 microbes were isolated from the tannery waste, by serial dilution and plating on
mineral salt agar medium (supplemented with tannic acid as a sole source of carbon). Of these 30
isolates 15 tannase producers were selected on the basis of tannic acid hydrolysis zone
formation. The zone formed by isolates on Tannic Acid Agar (TAA) plates were not very clear
and was not differentiable between tannase-producing and non-tannase-producing bacteria.
Osawa and Walsh (1993), Kumar et al.,(2010) and Mondal and Pati (2000) reported about
tannase screening methods, but all the methods are time consuming and less sensitive which
have made these limited towards specificity determination and the isolation and screening of
bacteria. To overcome this, the TAA plates were flooded with Gram’s iodine instead of FeCl3 as
reported by Kumar et al 2010. Gram’s iodine formed a dark brown complex with tannic acid but
not with hydrolyzed tannic acid and giving a sharp distinct zone around the tannase producing
microbial colonies even in cases of low levels of tannase production within 3-5 minutes. In the
present technique, gram’s iodine reacted with the tannin–protein complex, yielding a brown to
black colour, providing a dark background allowing clear visibility of zones of tannic acid
hydrolysis (Fig.1b). Actinomycetes isolate AT 9 which showed maximum (19 mm) diameter of
zone of hydrolysis was selected for further studies.
3.2 Effect of incubation period on the production of tannase
Incubation period is one of the most critical parameter in optimization state of affairs.
Tannase enzyme production by AT9 was determined by tannase enzyme assay. Higher tannase
production of the isolate AT 9 was observed after 24 hr of incubation period.The enzyme activity
of AT 9 was 7.73 U/ml/min at 24 hrs. The results are represented in Fig1a.A zig-zag pattern of
enzyme activity was observed.

Figure-1a: Gallic Acid Production by AT 9 at Varying Time Interval
7.7
2.5
4.9 5.1
1.9
6.3
1.5
2.8
0.0
2.0
4.0
6.0
8.0
10.0
24 h48 h72 h96 h120 h144 h168 h192 hEnzyme
Activity
(U/ml/min.)

Time in hours

The enzyme activity was decreased at 48 hrs and increased at 96 hrs in all cases. The reason
behind the decrease of enzyme activity at 48 hrs might be due to decrease in tannic acid and
increased availability of glucose produced as a byproduct of tannic acid hydrolysis. As
mentioned above the large availability of glucose in the medium made the organism to utilize
glucose instead of tannic acid. The sharp increase of enzyme activity at 96 hrs might be due to
depletion of glucose in the medium and the bacterium starts utilizing tannic acid in the medium.
3.3 Identification of Bacterial Isolates:
3.3.1 Morphological characteristics of AT 9:
AT 9 formed a white, powder colony on the TAA plate. Gram’s staining and microscopic view
revealed a gram-positive, filamentous structure.(Figure 1c)

Figure-1. (b) Zone of tannic acid hydrolysis by various isolates on TAA plate (c) Growth of
AT 9 on TAA Plate and Microscopic View of AT 9 after Gram staining (1000X)

3.3.2 Culture Characteristics of AT 9 on Different Media:

AT 9 was grown on different media (Glucose Aspargin Agar,Nutrient Agar, Czapek Dox Agar,
Bennett’s Agar, Potato Dextrose Agar and Glycerol Aspargine Agar) to study their growth
pattern. A comparison of growth of isolates on different media is shown in Figure 2. The colour
of aerial mycelium of AT 9 was found white on Nutrient Agar, Potato Dextrose Agar and
Czapek Dox Agar. In Glucose Aaspargine Agar,AT 9 gave grey mycelium colour. The aerial
mycelium of AT 9 in Glycerol Aspargine Agar was dark brown in colour.

1b
AT 9
1c

Figure-2. Growth of Isolate AT 9 on Different Media
3.3.3 Biochemical Characteristics of isolate AT 9
Biochemical characterization of AT 9 was carried out for identification purpose. AT 9 culture
utilized arabinose and gave good growth in the medium. No growth was observed with sucrose,
mannitol and Trehalose, glucose. Cellulase, Lipase and xylanase production were observed by
the culture. Nitrate reduction, and arginine utilization, were positive, while Citrate utilization and
ONPG utilization,malonate utilization were negative.
3.3.4 Characterization of bacterial isolate by 16s rRNA sequencing.
The isolate AT 9 showing tannase production was identified at GSBTM by sequencing, The
obtained sequences of tannase producing isolate was compared with deposited sequences in
NCBI database, using BLAST as a search tool. In most cases, the high identity values obtained
allowed us to assure that AT 9 isolate belong to Strptomyces spp. with identity values 100 %.
Evolutionary study done by using the 16S rRNA sequence for Actinomycetes reveals that
in the phylogenetic tree the position of AT 9 is near by the Streptomyces gancidicus.The
sequence data of isolate AT 9 was deposited in the Gene bank Database under the accession
number MF464014.The phylogenetic tree was constructed via the neighbor-joining algorithm
using the BLAST package (Figure 3).
Glucose Aspargine Agar Czapek Dox Agar Nutrient Agar
Bennett’s Agar Potato Dextrose Agar Glycerol Aspargine Agar

Figure 3: Unrooted tree showing the phylogenetic relationships of members of the genus
Streptomyces and some related species. The tree constructed by using the neighbor-joining
method
3.4 Optimization Studies
3.4.1 Optimization of physicochemical parameters to maximize the production of Tannase:
by Streptomyces ganncidicus
Effect of Inoculum Size on Tannase Activity
Tannase activity of Streptomyces ganncidicus was maximum when 4 plugs of culture were added
to inoculation medium (TA Broth).The enzyme activity of the bacterium at 24 hrs was 3.9
U/ml/min. The activity decreased after 48 hrs and again increased after 72 hrs. The results are
represented in Figure 4.

Figure-4. Effect of inoculums size on Tannase Activity by Streptomyces ganncidicus
(*Values are mean ± S.D, n=3 There was significant difference between control group and
enzyme treated group i.e at 5% level of significance when added 4 plugs of culture as inoculum
size in the production of gallic acid from tannic acid)

1.02
3.8 3.9* 3.85 3.72
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
2 plugs3 plugs4 plugs5 plugs6 plugs
Tannase Activity ( U/ml/min)

Effect of inoculum size on Tannase activity by Streptomyces ganncidicus
24 hours
48 hours
72 hours
96 hours

Effect of Temperature, pH, Tannic acid concentration, metal ions on Tannase Activity
Temperature is also another critical factor that needs to be controlled and optimized. The
significance of the optimum temperature in the development of fermentation process is such that
it could determine the inhibition of growth, cell viability and death. In general increase in the
temperature of fermenting medium due to respiration. Highest Tannase activity was observed at
30 ?C (3.4 U/ml) after 24 hrs of incubation, inturn indicating maximum production of tannase
enzyme by Streptomyces ganncidicus cultures at 30 ?C.(Figure 5a) The activity decreased as the
temperature increased. The same has been reported by, Bradoo et al. (1997); Sharma et al.
(1999), Aguilar et al. (2001).
Tannase production by microbes and its enzyme activity is affected by the pH. Maximum
enzyme production was observed at pH 6.5 after 72 hrs and the enzyme activity was 2.57
U/ml/min. The activity decreased after at 96 hrs. It was also observed that though acidic pH is
more favorable for tannase production.The optimum initial pH reported for tannase production
was in the acidic pH range of 4.5 to 6.5 (Rajakumar and Nandy, 1983; Barthomeuf et al., 1994;
Bradoo et al., 1997; Ayed and Hamdi, 2002; Lokeshwari and Raju, 2007; Belur et al. 2010;
Darah et al., 2011).The results are represented in Figure 5b.
The experiments were conducted using different concentration of tannic acid (1%, 2%,3
% and 4 % ) in the TA broth. Tannase production increased in 2% tannic acid concentration, it
was 3.41 U/ml/min after 72 hours of incubation and it was further decreased or the concentration
of tannic acid measured. The results are presented in Figure 5c.
Metals plays a major role as cofactors in enzymes. Figure 5d shows the tannase activity
of AT 9 (Streptomyces gancidicus) when different metals at a concentration of 1mM was added
to TA broth. From all the metal ions, addition of CaCl2 in TA broth induced maximum tannase
production and the production was maximum after 72 hrs of incubation(1.87 U/ml/min). The
results thus show that Ca is an inducer for tannase production and Mn2+is acting as inhibitor for
tannase production. CaCl2 to be the most suitable for tannase production by Aspergillus
japonicus as compared to the other used mineral salts (NaCl, KCl, CuSO4, MnSO4, FeSO4,
ZnSO4 and CdSO4) (Arulpandi, et al.,2008) .The inhibitory effect of heavy metal ions is well
documented in the literature (Vallieeb and Ulmer, 1972) . Further, the decreased activity in the
presence of divalent cations could be due to the nonspecific binding or aggregation of the
enzyme (Kar et al., 2003). Tannase from A. oryzae (Iibuchi et al., 1968) and P. chrysogenum
(Rajkumar and Nandy, 1983) were heavily inhibited by Zn2+, Cu2+ and Fe2+.

Figure 5: Effect of Temperature (5a), pH(5b),Tannic acid concentration (5c) and metal ions
(5d) on Tannase Activity (*Values are mean ± S.D, n=3 There was significant difference
between control group and enzyme treated group i.e at 5% level of significance at a temp. of 30
?C ,pH-6.5, tannic acid concentrations 2% in the production of gallic acid from tannic acid)
Effect of Carbon and Nitrogen Sources on Tannase production
In submerged fermentation, the concentration of tannic acid was found to be a crucial
factor influencing the levels of enzymes (Lekha and Lonsane, 1997; Mondal et al., 2000). It is
evident from the Figure 6a that only tannic acid supported significant higher value of tannase
activity (6.23 U/ml/min).Other carbon sources like lactose,glucose, sucrose and fructose didnot
showed any significant effect on tannase production. Thus, from the results it was evident that
only tannic acid serves as better carbon source for tannase production.
In order to determine the best nitrogen source for tannase production, Sodium nitrate
(0.6% w/v) was replaced with different inorganic and organic nitrogen sources in the production
medium. All the nitrogen sources used had % equivalent nitrogen similar to that of 0.6% (w/v)
sodium nitrate and the results are presented in Figure 6b.The results suggest that only inorganic
nitrogen sources, ie., Ammonium chloride is efficiently utilized when compared to other nitrogen
1.85
3.4*
1.45
2.85
2.45
0
1
2
3
4
RT30 °C37 °C40 °C45 °C
Tannase Activity ( U/ml/min)

Figure 5a: Effect of temperature on tannase
activity by Streptomyces ganncidicus
24hours
48hours
72hours
96hours0
0.33
2.57*
0
0
0.5
1
1.5
2
2.5
3
5.56.26.57.OTannase activity in (U
/ml/min)
Figure 5b :Effect of pH on Tannase activity by
Streptomyces ganncidicus
24 hours
48 hours
72 hours
96 hours
1.27
3.41* 3.13 2.88
0
1
2
3
4
1%2%3%4%
Tannnase Activity ( U/ml/min.)
Figure 5c :Effect of Tannic Acid Concentration
on Tannase Activity by Streptomyces
ganncidicus
24 hours
48 hours
72 hours
96 hours0.97
0.61 0.6
3.04
0
0.5
1
1.5
2
2.5
3
3.5
Zinc SulphateManganeseSulphateCopper SulphateCalciumChloride
Tannase Activity (U/ml/min.)

Figure 5d:Effect of Metal Ions ( 1mM) on Tannase
Activity by Streptomyces gancidicus
24 hours
48 hours
72 hours
96 hours

sources. Ammonium chloride when added in the medium enhanced the growth of the isolate AT
9 (Streptomyces gancidicus) (observed visually) and thereby enhanced tannase production.

Figure-6. Effect of different carbon(6a) and nitrogen Sources (6b) on Tannase Activity by
Streptomyces gancidicus

5. Conclusion
A tannase producing actinomycetes AT 9 was selected from the isolates obtained from soil
samples collected from CLRI located in Naroda ,Ahmedabad , Gujarat , on the basis of zone of
hydrolysis formed on TAA agar plates The AT 9 was identified as Streptomyces gancidicus
,GenBank Accession Number MF464014. Cellulase, lipase and Xylanase were also produced by
Streptomyces gancidicus.Optimization of various nutritional and physical parameters for
maximum tannase production under submerged fermentation by Streptomyces gancidicus.
During optimization, the maximum tannase activity was obtained in 4 plugs of culture as
inoculum size, 2% tannic acid as carbon source and peptone (0.6%) as nitrogen source. The
optimum temperature for maximum enzyme production was at 30 ºC and 6.5 pH
6.23
0.94 1.11 0.88
3.41
0
1
2
3
4
5
6
7
Tannic AcidLactoseGlucoseSucroseFructose
Tannase Activity ( U/ml/min)
Figure 6a Effect of different Carbon sources On Tannase Activity by Streptomyces
ganncidicus
24 hours
48 hours
72 hours
96 hours
1.74
0.72 0.72 1.64
9.61
0
2
4
6
8
10
12
PeptoneSodium NitrateUreaYeast ExtractAmmonium
Chloride
Tannase
Activity ( U/ml/min.)

Figure 6b Effect of different Nitrogen Sources (0.6%) on Tannase Activity by
Streptomyces ganncidicus
24 hours
48 hours
72 hours
96 hours

respectively.Ca+2 was significant for tannase production by Streptomyces gancidicus. There was
significant difference between control group and enzyme treated group i.e 5% level of
significance at a temp of 30 ºC ,pH 6.5, tannic acid concentrations 2% and 4 plugs of culture as
inoculums size .in the production of gallic acid from tannic acid.
6. Acknowledgments
The Authors are thankful to the head of department of biotechnology and Microbiology,
KSV university for providing all the laboratory facilities. The authors are also thankful to
Biogene department of Gujarat State Biotechnology Mission (GSBTM), Gandhinagar, Gujarat,
India for bacterial strain identification.
7. Compliance with ethical standards
Conflict of Interests: There are no conflicts of interest.
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