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The Production of Tannin Acyl Hydrolase from Aspergillus niger
YUNITA ARIAN SANI ANWAR, HASIM, AND I MADE ARTIKA∗
Department of Biochemistry, Institut Pertanian Bogor, Darmaga Campus, Bogor 16680, Indonesia
The aim of this research was to produce tannin acylhydrolase (tannase) from Aspergillus niger isolated from
cacao pod. The first step of the study included determination of optimal pH, temperature, and incubation period
to produce tannase. Optimal conditions obtained for tannase production were pH 5.5, a temperature of 28 oC and
an incubation period of 3 days. Optimization of production medium was conducted. The media tested were solid
and liquid wheat flour media with a concentration of tannic acid as inducer at 0, 3, 5, and 7% (wt/vol). The best
production medium was solid medium with tannic acid concentration of 5% (wt/vol).
Key words: tannase, Aspergillus niger, solid state fermentation, liquid state fermentation, tannic acid inducer
_____________________________________________
_________________
∗
Corresponding author, Phone/Fax: +62-251-423267,
E-mail: imart171@yahoo.com
Tannin acylhydrolase also known as tannase (EC 3.1.1.20)
is a hydrolytic enzyme that catalyses the hydrolysis of
(hydrolysable) tannins releasing glucose and gallic acid.
Tannins are natural compounds which have a number of
phenolic hydroxyl groups and can precipitate protein. The
ability of these compounds to precipitate protein generates
some problems. Tannins are capable of interacting with
protein and crude-fibres and also with digestive enzymes so
that they could interfere with the digestion process that can
inhibit the growth of livestock (Butler and Rogler 1992).
Tannase is extensively used in food and medical
industries. In the food industry, the enzyme is used in the
manufacture of instant tea, as a clarifying agent of wine, fruit
juices, and in reduction antinutritional effects of tannins in
animal feed. In Brazil, tannase has a potential use for reducing
astringency of cashew apple juice. In the medical industry,
tannase is used in the production of gallic acid, a substrate
for the chemical synthesis of propyl gallate and trimethoprim
(Pinto et al. 2001).
A number of microorganisms such as fungi, bacteria, and
yeast are known tannase producers. Species belonging to
the Aspergillus and Penicillium genuses were reported as
the best tannase producers (Pinto et al. 2001). Purnama (2004)
found that Aspergillus niger isolated from cacao pod reduces
tannin levels up to 79.3% (wt/wt). Hatamoto et al. (1996)
isolated and characterized a tannase gene from Aspergillus
oryzae. The gene was found to encode a protein of 588 amino
acids. The tannase gene product was translated as a single
polypeptide and processed by cleavage into two tannase
subunits linked by disulfide bonds. The native tannase was
concluded to consist of four pairs of the two subunits,
forming a hetero-octamer with a molecular weight of about
300,000 (Hatamoto et al. 1996). Sabu et al. (2005) reported
that the optimum temperature for fungal tannase activity
was between 30 and 40 oC. The enzyme showed optimum
activity at pH value between 5.0 and 6.0.
Tannase is an extracellular enzyme that needs an inducer
to increase the enzyme synthesis. Several studies on
optimum production of tannase by moulds have been
conducted. Tannase was found to be induced by tannic acid
and some of its derivatives (Aguilar et al. 2001). Similarly,
Sanchez (2003) reported that tannase production increases
when the culture media contains 3% (wt/vol) of tannic
acid. The tannase activity obtained in these media reached
7.45 U ml-1.
In the present study tannase was produced using A. niger
isolated from local cacao pod. Optimization of production
medium and characterization of the resultant tannase were
carried out. The optimum concentration for induction was
also determined.
MATERIALS AND METHODS
Maintenance of Culture. A strain of Aspergillus niger
was obtained from the stock collection of the Engineering
and Bioprocess Laboratory, Institut Pertanian Bogor. The
strain was isolated from cacao pod collected from a cacao
plantation in Bogor, West Java. Potato Dextrose Agar (PDA)
(Difco) slants were used for maintenance of A. niger with
incubation at 28 oC for 6 days. Fully sporulated slants not in
use were stored at 4 oC. The slants were subcultured routinely
once every three weeks.
Preparation of Spore Inoculum. Fungal spore inoculum
was prepared by adding 10 ml of sterile distilled water
containing 0.1% (vol/vol) Tween 80 to a fully sporulated
culture. The spores were dislodged using a sterile inoculation
loop under strict aseptic condition and then vortexed in a
slanted position. The volume of 1 ml of the prepared spore
suspension was used as inoculum with concentration of 3 x
107 spores.
Preparation of Production Medium. Tannase production
was carried out using both solid and liquid media. The solid
medium for solid state fermentation (SSF) was prepared as
follows: A mass of 5 g of wheat flour was taken into a 125 ml
Erlenmeyer flask and moistened with 5 ml of Czapeck medium
(NaNO
3
3 g l-1, KCl 0.5 g l-1, MgSO
4
⋅3H
2
O 0.348 g l-1,
FeSO
4
⋅7H
2
O 0.01 g l-1, K
2
HPO
4
⋅3H
2
O 1.301 g l-1, and tannic
acid 30 g l-1). The liquid medium used for tannase production
was the Czapeck medium with 70 g l-1 glucose as carbon
source.
Optimization of Condition for Tannase Production.
Determination of optimal pH, temperature and incubation
period for tannase production was carried out using solid
state fermentation. Variable parameters for the enzyme
MICROBIOLOGY INDONESIA, August 2007, p 91-94 Volume 1, Number 2
ISSN 1978-3477
production were pH (4, 4.5, 5, 5.5, 6), temperature (26, 28, 30,
32 oC), and incubation period (1-5 days).
Extraction of Crude Enzyme. Tannase was extracted from
the fermented substrate by adding 50 ml of distilled water
containing 0.01% (vol/vol) Tween 80. Contents were mixed
well using a magnetic stirrer. Crude enzyme was separated
from fermented matter by centrifugation (Beckman J2-21 rotor)
at 7700 g, 4 oC, for 20 min. The supernatant was separated by
filtration through Whatman no. 1 and the filtrate was collected
in bottles for further studies.
Tannase Assay. Tannase activity was determined by
method of Libuchi et al. (1966). The substrate solution,
containing 1 ml of 0.35% (wt/vol) purified tannic acid in
0.05 M citrate buffer (pH 5) was preincubated at 30 oC for
about 5 min. Enzyme solution (0.25 ml) was added followed
by incubation at 30 oC for 15 min. The blank solution was
prepared by adding citrate buffer in place of the enzyme.
Into the solution, 5 ml of 95% (vol/vol) ethanol was added
followed by mixing in order to stop the reaction. After this,
0.25 ml aliquots of the reaction and blank mixtures were
transferred into respective test tubes. Ethanol solution was
added to all tubes and the tubes were mixed thoroughly.
Absorbance was measured at 310 nm. One unit of enzyme
activity was defined as the amount of enzyme required to
hydrolyze 1 µmol of the ester bond in 1 min.
Optimization of Medium for Tannase Production. In the
present study, the effect of different tannic acid
concentrations on tannase production in solid state
fermentation and liquid surface fermentation was studied.
For these purposed, the tannic acid concentration in the
Czapeck medium used for preparation of solid and liquid
production media was varied from 0, 30, 50, and 70 g l-1. The
culture was incubated at 28 oC on rotary shaker (150 rpm) for
72 h. Tannase assay was performed using method of Libuchi
et al. (1966) and total soluble protein was determined by the
method of Bradford (1976).
RESULTS
Effect of Temperature. Temperature for the growth
studies was 26, 28, 30, and 32 oC. Maximum tannase activity
(0.167 U ml-1) was obtained at growth temperature of 28 oC.
Thereafter a declining trend was observed, as shown in
Fig 1.
Effect of pH. To determine effect of pH of the growth
medium on tannase production, a range of pH of the medium
was used being 4, 4.5, 5, 5.5, and 6. The optimum pH of
medium was found to be 5.5 (Fig 2).
Effect of Incubation Period. Incubation period is the most
important parameter for maximum tannase production. The
optimum incubation period was obtained at 72 h. Up to 72 h
there was a rise in tannase activity, after which a decrease
was observed (Fig 3).
Effect of Media and Tannic Acid Concentration. This
research tested solid and liquid media with an inducer
concentration varied at 0, 3, 5, and 7% (wt/vol). The maximum
enzyme activity was obtained with the solid medium with a
tannic acid (inducer) concentration of 5% (wt/vol). The
optimum activity of tannase obtained from solid and liquid
media was 1.441 and 0.603 U ml-1 respectively (Fig 4).
Total soluble protein in solid media was higher than liquid
media (Fig 5). The maximum protein content obtained at liquid
and solid media when the tannic acid concentration of 5%
(wt/vol) was 0.494 and 0.712 mg ml-1 respectively.
DISCUSSION
The present study clearly showed that various factors
affect tannase production by A. niger isolated from
Indonesian cacao pod. Tannase activity at a growth
temperature of 28 oC was higher than that of growth
temperature of 26 and 32 oC but was not different from that of
24 26 28 30 32 34 36
0.20
0.16
0.12
0.08
Temperature (oC)
T
a
n
n
a
s
e
a
c
ti
v
it
y
(
U
m
l-
1
)
Fig 1 Effect of temperature on tannase activity at pH 5.5 and 3
days incubation period.
T
a
n
n
a
s
e
a
c
ti
v
it
y
(
U
m
l-
1
)
0.17
0.16
0.15
0.14
4.0 4.5 5.0 5.5 6.0 6.5
p H
Fig 2 Effect of pH on tannase activity at 28 oC and 3 days
incubation period.
T
a
n
n
a
s
e
a
c
ti
v
it
y
(
U
m
l-
1
)
0.18
0.16
0.14
0.12
0.10
0.08
0 24 48 72 96 120 144
Incubation period (h)
Fig 3 Effect of incubation period on tannase activity at 28 oC and
pH 5.5.
92 ANWAR ET AL. Microbiol Indones
30 oC. Similar results were reported for tannase from A. niger
(Lekha and Lonsane 1997; Sanchez 2003). Slightly different
results were reported by Banerjee et al. (2005) who found an
optimum growth temperature of 30 oC. This was mainly due
to the difference in the strain used in the study. They used
Rhizopus oryzae and Aspergillus foetidus as tannase
producers.
The lower activity of tannase observed at 26 oC could be
due to a lower enzymatic reaction rate leading to lower
tannase activity compared to the tannase activity obtained
at optimal growth temperature (28 oC). On the other hand, at
high temperature, proteins are denatured because of the
disruption of their tertiary and quaternary structures, and
enzymatic activities decline. In addition, at temperature above
the maximal growth temperature, excretion of protease
proceeds rapidly so that tannase activity decreases
(Mackenzie et al. 1994).
Apart from growth temperature, the pH value of growth
medium also affects tannase activity. Tannase activity was
higher in the culture with growth medium with pH of 5.5
compared to the tannase activity from growth medium with
other pH values. Similar results were reported for tannase
from A. niger (Ramirez-Coronel et al. 2003; Sabu et al. 2005).
Apparently different results were reported by Sanchez (2003)
who found that optimum pH value for tannase production
was 4. It is important, however, to note that in these
experiments the pH values tested were limited to pH 4 and 7,
while the pH value of 5.5 was not tested (Sanchez 2003).
Optimization of incubation period was carried in order to
determine the best time for harvesting tannase from
production culture. Incubation period also affects the
assayable tannase level. The optimum incubation period was
3 days which was similar to that found by Kar et al. (1999)
and Pinto et al. (2001). Different optimum incubation periods,
however, were reported by Alberts (2002) and Rana and Bhat
(2005). Alberts (2002) found that optimum incubation period
was 24 h while Rana and Bhat (2005) reported maximum
tannase production with incubation period of 96 h. The varied
optimal incubation period found by different workers might
be due to differences in fungal strain and medium
composition used for tannase production.
Solid medium was found to be better than liquid medium.
This could be due to effect of catabolite repression is less
significant in solid medium. The present study showed that
solid state fermentation can increase tannase activity about
1.5 folds. In addition, tannase production was affected by
concentration of inducer in the medium. The highest tannase
activity was found in solid state fermentation with tannic
acid concentration of 5% (wt/vol). Tannase activity was lower
at lower (3%, wt/vol) tannic acid concentration. Similarly, at
higher (7%, wt/vol) tannic acid concentration the tannase
activity decreased indicating that the optimum tannic acid
concentration was 5% (wt/vol). Excessive tannic acid was
reported to act as repressor and prevents synthesis mRNA.
In addition, the increased of tannic acid causes an increased
of heat build up and reduced aeration which in turn decreased
productivity of tannase (Banerjee et al. 2005). Similar results
were reported previously by Lekha and Lonsane (1994) and
Aguilar (2001).
Increased productivity of hydrolytic enzyme in solid
media is due rapid oxygen uptake rate which allows fungus
to form abundant aerial mycelium. Aerial mycelium gives a
strong increase in enzyme production (Rahardjo et al. 2002).
In addition, solid media generate higher product stability
and lower catabolic repression compared to liquid media
(Holker et al. 2004). Moreover, protease activity in solid state
fermentation was found to be lower than protease activity in
liquid surface fermentation resulting in higher tannase
productivity (Aguilar et al. 2002).
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T
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a
c
ti
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it
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(
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