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com

Isolation and Characterization of tannase from isolated

Bacillus subtilis

Sarmad Falih Mohammed1*, R. J. Rdam2, Media F. Ali Jan3,

Mohammed M. Sharba4, Ali Abdulmawjood Mohammed5

1. Department of Medical Laboratory Techniques, Dijlah University College, Baghdad, Iraq.

2. Ministry of health, Republic of Iraq.
3. Department of medical laboratory Techniques, college of medical technology, Farahidi
University, Baghdad, Iraq.
4. Department of Medical Laboratory Techniques, Dijlah University College, Baghdad Iraq.
5. Department of Medical Laboratory Techniques, Dijlah University College, Baghdad, Iraq.

Corresponding Author: *
Sarmad Falih Mohammed
E-mail: sarmad.falih@duc.edu.iq

Abstract:
Isolation of tannase-producin bacteria was produced from enriched soil. The tannase
producing ability of this organism was identified by using tannic acid medium. Organism was
identified as Bacillus Subtilis on the basis of biochemical and API identification system.
Tannase enzyme was cultivated in mineral base medium containing 0.1% Tannic acid. Tannase
showed maximum activity after 24 h.The enzyme was purified by DEAE cellulose ion exchange
chromatography and was shown to having specific activities of 0.22 U/mg of protein. Enzyme
showed maximum activity at pH 6 and 50ºC. The Km of enzyme was 0.029 mM respectively
and Vmax of was found to be 40 and 40U/mL. Protien purification by SDS-PAGE for purified
enzyme fraction revealed enzyme have 60 kDa, molecular weight. Heavy metal effect also
shows this enzyme was activated by some proteins and inhibited by the same its higher
concentration.

Key words: Tannase, Bacillus, industrial, environment

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Introduction:
Tannins are commonly present as secondary metabolites in all types of plant and plant parts,
which stand after abundant components of plant like cellulose, hemicellulose, and lignin
(Serrano et al. 2009; Lekha and Lonsane 1997). This compound is water soluble and recognised
as recalcitrant polyphenolic compound composed of phenolic acids such as gallic acid or
ellagic acid, forming gallotannins and ellagitannins, respectively. Because of their difficulties
in degradation they may cause problem to be remain intact in wastewater from various
industries and provoking environmental problems in terms of water and soil pollution (Yagu
et al., 2000). Based on phenolic compounds monomers and carbohydrate core tannins grouped
in to two types one is condensed tannin and another is condensed tannins. (Mingshu et al.,
2006).
Tannins may cause inhibition of different microorganism by precipitating their different
enzyme. (Field and Lettinga, 1992a). However there are some microorganisms adapted for
tannin and acquired capabilities to utilized tannins and gallotannins as a carbon source. The
enzyme involved in this process commonly known as tannase, the tannin acyl hydrolases (Yao
et al. 2014).
Tannase catalyzes the hydrolysis of the ester bonds in tannic acid and produce gallic acid and
glucose and galloyl esters so it is extensively used for the production of gallic acid. In addition
to that since from the discovery of this enzyme it can be used as a industrial enzyme used in
food, feed, beverage, pharmaceutical, and chemical industries. Tannase also used in the
commercial application in the preparation of instant tea, wine, beer, and coffee flavoured soft
drinks and also as additive for detanniffication of food (Belur and Mugeraya,2011; Lekha and
Lonsane, 1997; Jana et al. 2014). Out of different natural resources like microbial, animal, and
vegetal sources, the preferred source for the tannase production is nothing but microorganisms.
Because of genetic simplicity, biochemical diversity and easiness in handling microorganisms
becomes favourite sources for the production of this industrial enzyme. Among all
microorganisms bacteria are the simplest source for this enzyme (Aguilar et al. 2007 ; Zhong
et al. 2004). As tannase from bacterial sources is working as a better alternative for the other
sources like fungi and animal more imporatance was given for these simplest form of the
microorganism. In these paper attempt was taken for the screening of new tannase-producing
bacterial strains with the desired properties its identification and tannase purication with its
characterization.

Material and Method:

Isolation of Tannase producing microorganism:
Enrichment of soil for tannase producing microorganism:
For the isolation of organism 1gm soil sample was enriched in Czapek Dox medium having
composition mineral base medium supplemented with 0.1%tannic acid. Overnight incubation
of this medium was done at shaking condition.

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Screening of isolated organism for the production of tannase:

Tannase production by isolate was checked by growing the microorganism on nutrient agar
supplemented with 0.1% tannic acid which was filter sterilized. The addition of tannic acid to
the nutrient agar plate forms a complex of tannic acid and proteins and after cleavage of this
complex, organism produces clear zone around colony.

Isolation and identification of tannase producing microorganisms:

The enriched sample is then serially diluted and streaked on nutrient agar plate with 1% tannic
acid. Plates are then incubated in incubator for overnight. On next day the zone of clearance of
tannic acid degradation was observed on the plate. Pure colonies of isolated microorganisms
were identified using morphological, biochemical tests including API (analytical profile index)
system. (Aruwa, and Olatope, 2015).

Enzyme extraction and purification:

The enzymes tannase was purified from cell free medium. In first step cell free medium was
saturated up to 55% with ammonium sulphate and allowed to precipitate for overnight at 4ºC.
This precipitate was discarded and the supernatant was saturated with ammonium sulphate up
to 70 %, 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-1, using NaCl concentration gradient from 0.1 to 0.5M in 50 mM citrate buffer (pH
= 6.0). The protein content of the fractions was analyzed by taking absorbance at 280 nm and
was tested for tannase activities. The fractions showing tannase activities were concentrated
using sucrose. The fractions were analyzed for tannase activities, and later used for SDS-PAGE
analysis

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.

Effect of temperature and pH on Tannase enzyme

The tannic acid degradation were checked at different pH and temperature pH of medium
adjusted in range of to 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, and 8.0 and temperature assessed are 10ºC,
20ºC, 30ºC, 40ºC, 50ºC, 60ºC and 70ºC. The tannic acid concentration was assessed by
estimation of precipitated residual tannic acid.

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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 was determined by Lineweaver-Burk plots of reciprocal
reaction velocities versus reciprocal substrate 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 1mL. and enzyme activity was monitored under the same conditions that are
mentioned above.

Result and Discussion:

Screening of tannase producing organism from soil:
The preliminary study to check tannase production from isolate included plate assay. The cross
streaked plates of organism taken from enriched sample on tannic acid medium shows a clear
zone after 48 h. incubation. The clear zone was observed around the growth of organism
(Figure 1). From that, it is clear that the microorganism producing tannase enzyme cleave
tannic acid protein complex by hydrolyzing tannic acid and shown clear zone around colony.
This colony was further purified for the production of tannase and identified as Bacillus subtilis
on the basis of Biochemical’s performed and API system Table 1. (Aruwa, and Olatope, 2015).

Isolation of enzyme:
The purification profile of tannase enzyme and the activity of extracted tannase at different
stages of purification is given in Table 2. It has been noted from Table 1, that the precipitate at
70% saturation of ammonium sulfate was giving the best yield of the enzyme. The specific
activities of tannase was measured crude and after ammonium sulfate precipitation and it will
measured about 0.0.27 U/mg of protein and Ion exchange purification it will 0.22 U/mg of
protein using tannic acid as substrate Figure. 2
. The specific activity of tannase was measured about 0.23 U/mg of protein using tannic acid
as substrate. SDS/PAGE analysis revealed that the two enzymes had molecular weights of
63kDa respectively as shown in Figure. 3 the molecular weight of tannase enzyme was found
to be in the range of 40 to 67kDa most of the bacterial tannase showed molecular weight nearby
40 to 60 kDa. (Muñoz et al., 2019)

Characterization of enzyme
pH Effect:
The effect of pH on the enzyme activity is determined by the nature of the amino acids at the
active site, which undergoes protonation and deprotonation, and by the conformational changes
induced by the ionization of the amino acids. Enzymes are very sensitive to changes in pH and
they function best over a very limited range, with a indefinite pH optimum (Sabu et al.

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2005).For the study of optimum pH of tannase producer pH range from 2.0 to pH 8.0 was taken
and Enzyme activity is calculated and the optimum pH of the tannase were (pH= 6) Figure 4.
Ayed and Hamdi 2002 reported Similar type of results was reported from L. plantarum showed
maximum tannase activity at pH 6.0 in other organism shows at pH 5.0 like in B. licheniformis
and B. cereus ( Mondal and Pati 2000).

Temperature Effect:
Temperature affects various metabolic processes such as protein denaturation, enzymatic
inhibition, promotion or inhibition on production of a particular metabolite, cell death, etc. So,
the effect of temperature (10–70oC) on production of tannase was studied and the similar
results were obtained about the optimum temperature of the tannase 50oC Figure 5. However,
no report is available on the temperature stability of bacterial tannase.
The maximum Tannic acid degradation was observed at 50oC. Most of other workers reported
the maximum production of tannase and tannic acid degradation at 37oC (Osawa et al. 2000;
Ayed and Hamdi 2002). (Kumar et al., 1999) reported maximum tannase activity at 30oC by
Citrobacter freundii.

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. Tannase showed increase in activity by 30% above optimum, in presence of 1
mM Cu2+ 10 mM Ca2+, by 19%in presence of Co2+ and up to 8% in presence of Fe3+
Figure.6

Enzyme kinetics:
The Km of tannase is 0.029 mM Vmax 40 U/mL, respectively as shown in figure 7. Different
workers reported Km values for tannic acid of tannases from 0.25 to 1.50 mM (Mukherjee and
Banerjee, 2006; Sharma et al, 1999; Sabu, 2005). Above data suggest isolated organism
produced tannase have higher affinity for tannic acid.

Conclusion:
In recent years tannase grabs attention of all biotechnological research area as it have different
application in food, pharmaceutical, agriculture industries. Its application in various field
increase results in microbial production of tannase. Previously was mostly studied in fungi and
in some extent in other microbial species but due to purification difficulties the preference
would now give to bacterial tannase. However, the following investigation shows promising
results for bacterial tannase as compared to the fungus. Isolated Bacillus species showed high
affinity for tannase and have high productivities at small concentration of tannic acid. The
obtained results not only helpful for the biotechnological application but it can also helps to
maintain microbial biodiversity and also helps to biodegradation of this compound that
otherwise may lead to environmental pollution.

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Acknowledgements:
I cannot express enough thanks to my family and friends for all their unconditional support. I
am also thankful to my university and department for their continued support and
encouragement. Finally, many thanks to all participants that took part in the study and enabled
this research to be possible.

References:
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14. Sabu, A., Shegal Kiran, G. and Pandey, A., 2005. Purification and characterization of tannin
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Table 1. Physiological and Biochemical characteristics of isolated organism

Test Performed Results
Gram Nature +
Morphology Rod Shaped
Spore +
Catalase +
Oxidase +
Methyl Red _
voges proskaue +
Citrate +
Urease _
Starch +
Nitrate +
Urease _
Glucose +
Xylose _

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Lactose +
Sucrose +
Maltose _

Table 2. Activities of tannase at various steps of purifications

Purification step Enzyme activity Proteins (mg) Sp. Activity Purification fold
(units)
Crude 45 350 ± 0.05 0.15 ± 0.03 1
After 33 122 ± 0.02 0.27 ± 0.02 1.80
ammonium
sulfate
precipitation
After ion 20 89 ± 0.03 0.22 ± 0.06 1.46
exchange
chromatography

Figure 1. Isolation of tannase-producing organisms from the soil.

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Figure 2. Ion exchange chromatography purification profile for Tannase enzyme

Figure 3. SDS page analysis of the tannase enzyme

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Figure 4. Effect of pH on Tannase enzyme activity

Figure 5. Graph of optimum Temperature of Tannase enzyme.

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Figure. 6. Effect of heavy metals for tannase enzyme

Figure 7. Enzyme kinetics of tannase enzyme

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