Characterization of Aspergillus fumigatus CAS-21 tannase with potential for propyl gallate synthesis and treatment of tannery effluent from leather industry

Article abstract of DOI:10.1007/s13205-018-1294-z

One of the tannase isoforms produced by the fungus Aspergillus fumigatus CAS-21 under submerged fermentation (SbmF) was purified 4.9-fold with a 10.2% recovery. The glycoprotein (39.1% carbohydrate content) showed an estimated molecular mass of 60?kDa. Optimum temperature and pH for its activity were 30–40?°C and 5.0, respectively. It showed a half-life (t₅₀) of 60?min at 45 and 50?°C, and it was stable at pH 5.0 and 6.0 for 3?h. The tannase activity was insensitive to most salts used, but it reduced in the presence of Fe₂(SO₄)₃ and FeCl₃. On contrary, in presence of SDS, Triton-X100, and urea the enzyme activity increased. The K_m value indicated high affinity for propyl gallate (3.61?mmol L^?1) when compared with tannic acid (6.38?mmol L^?1) and methyl gallate (6.28?mmol L^?1), but the best K_cat (362.24?s^?1) and K_cat/K_m (56.78?s^?1 mmol^?1 L) were obtained for tannic acid. The purified tannase reduced 89 and 25% of tannin content of the leather tannery effluent generated by manual and mechanical processing, respectively, after 2-h treatment. The total phenolic content was also reduced. Additionally, the enzyme produced propyl gallate, indicating its ability to do the transesterification reaction. Thus, A. fumigatus CAS-21 tannase presents interesting properties, especially the ability to degrade tannery effluent, highlighting its potential in biotechnological applications.

Full text of DOI:10.1007/s13205-018-1294-z

3 Biotech (2018) 8:270
https://doi.org/10.1007/s13205-018-1294-z
ORIGINAL ARTICLE
Characterization of Aspergillus fumigatus CAS-21 tannase
with potential for propyl gallate synthesis and treatment of tannery
effluent from leather industry
Rayza Morganna Farias Cavalcanti1 · João Atílio Jorge2 · Luis Henrique Souza Guimarães1,2
Received: 29 March 2018 / Accepted: 20 May 2018
© Springer-Verlag GmbH Germany, part of Springer Nature 2018
Abstract
One of the tannase isoforms produced by the fungus Aspergillus fumigatus CAS-21 under submerged fermentation (SbmF)
was purified 4.9-fold with a 10.2% recovery. The glycoprotein (39.1% carbohydrate content) showed an estimated molecular
mass of 60 kDa. Optimum temperature and pH for its activity were 30–40 °C and 5.0, respectively. It showed a half-life (t50)
of 60 min at 45 and 50 °C, and it was stable at pH 5.0 and 6.0 for 3 h. The tannase activity was insensitive to most salts used,
but it reduced in the presence of Fe2(SO4)3 and FeCl3. On contrary, in presence of SDS, Triton-X100, and urea the enzyme
activity increased. The Km value indicated high affinity for propyl gallate (3.61 mmol L−1) when compared with tannic acid
(6.38 mmol L−1) and methyl gallate (6.28 mmol L−1), but the best Kcat (362.24 s−1) and Kcat/Km (56.78 s−1 mmol−1 L) were
obtained for tannic acid. The purified tannase reduced 89 and 25% of tannin content of the leather tannery effluent generated
by manual and mechanical processing, respectively, after 2-h treatment. The total phenolic content was also reduced. Addi-
tionally, the enzyme produced propyl gallate, indicating its ability to do the transesterification reaction. Thus, A. fumigatus
CAS-21 tannase presents interesting properties, especially the ability to degrade tannery effluent, highlighting its potential
in biotechnological applications.
Keywords Aspergillus · Propyl gallate · Submerged fermentation · Tannic acid · Tannin acyl hydrolase · Tannery effluent
Introduction
turbidity of alcohol and coffee, inhibition of the growth of
many microorganisms, resistance to microbial attack, and
recalcitrance to biodegradation (Rodríguez-Durán et al.
2011). Fortunately, various microbial tannases can be
applied in different sectors such as food and feed, bever-
ages, chemicals, and for the tannin-rich effluent treatment
(Yao et al. 2014). The most important application of tannase
is the production of gallic acid. Gallic acid is a substrate for
the chemical or enzymatic synthesis of propyl gallate as well
as an antioxidant used as additive in food, oils, and lipid-rich
products (Fernandez-Lorente et al. 2011).
Tannase (tannin acyl hydrolase, EC 3.1.1.20), an enzyme
with biotechnological importance, catalyzes the hydroly-
sis of ester and depsidic linkage in hydrolysable tannins
such as tannic acid, releasing glucose and gallic acid (Yao
et al. 2014). Tannins are one of the most important groups
of polyphenols in plants. They are characterized as anti-
nutritional agents owing to their property to complex with
proteins, digestive enzymes, polysaccharides, and minerals
(Goel et al. 2005; Santos and Mello 2001). Additional effects
of tannins include the formation of tea cream in tea drink,
Despite their importance, the practical use of microbial
tannases is still limited and advanced studies on their proper-
ties are necessary. Recently, special attention has been given
to the production and characterization of fungal tannases,
especially from the genus Aspergillus and Penicillium (Yao
et al. 2014). Both submerged fermentation and solid-state
fermentation have been used for tannase production and a
majority of these enzymes is extracellular (Aguilar et al.
2007). Different factors influence the production of tannase
by microorganisms. These include carbon and nitrogen
* Luis Henrique Souza Guimarães
lhguimaraes@ffclrp.usp.br
1
Instituto de Química de Araraquara- UNESP, Avenida
Professor Mário Degni s/nº, Quitandinha, Araraquara,
São Paulo 14800-900, Brazil
2
Faculdade de Filosofia, Ciências e Letras de Ribeirão
Preto, USP, Avenida Bandeirantes 3900, Ribeirão Preto,
São Paulo 14040-901, Brazil
Vol.:(0123456789)
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sources, chemical composition of the culture medium, pH,
incubation temperature, agitation, and incubation period
(Yao et al. 2014).
harvested using a vacuum pump and Whatman filter paper
no. 1. The filtrate was used as a source of extracellular tan-
nase for enzyme activity determination and purification.
Fungal tannase properties such as molecular mass
(between 50 and 320 kDa) have been determined (Yao et al.
2014). Some of these enzymes are monomeric, while others
have two or more subunits, such as the enzymes produced
by Aspergillus oryzae (octamer) (Hatamoto et al. 1996),
Emericella nidulans (hexamer) (Gonçalves et al. 2011),
and Arxula adeninivorans (tetramer) (Böer et al. 2011). In
general, tannases are active in a broad temperature range
(20–60 °C) under acidic conditions. Ions, organic solvents,
and surfactants also influence tannase activity. Some authors
have reported tannases as inducible enzymes, while others
demonstrated their constitutive expression (Yao et al. 2014).
Despite the determination of the properties of some fun-
gal tannases and their potential application in the removal
of phenolic compounds from tea, juices, wines, beers, and
soft drinks (Beniwal et al. 2013), their effective use for the
treatment of tannin-rich effluents is still limited, especially
for the tannery effluent from the leather industry. This indus-
try is an important economical field for developing coun-
tries (Lofrano et al. 2013). It is responsible for consuming
large volumes of water and producing significant volumes
of wastewater with high concentration of chemicals such as
vegetable tannins and organic matter, contributing to envi-
ronmental pollution (Romero-Dondiz et al. 2015; Dixit et al.
2015). The methods used for the treatment of tannery waste-
water can be biological, chemical, physical, or a combina-
tion of these (Lofrano et al. 2013; Durai and Rajasimman
2011). In this light, our aim was to characterize the tannase
from A. fumigatus CAS-21 and to analyze its potential for
the treatment of tannery effluent from the leather industry
and for propyl gallate production.
Determination of the enzyme activity
The tannase activity was determined using 0.2% (m/v)
methyl gallate (C8H8O5) (Sigma-Aldrich) as substrate in
100 mmol L−1 sodium acetate buffer (pH 5.0) according
to the methanolic rhodanine method (Sharma et al. 2000).
Briefly, the reaction mixture containing 250 µL of substrate
solution and 250 µL of enzyme sample was incubated at
30 °C for 5 min and added to 300 µL of methanolic rhoda-
nine (0.667%; w/v) at 30 °C. After 5 min, 200 µL of 0.5 N
potassium hydroxide was added and maintained at 30 °C.
Finally, the mixture was diluted with 4 mL of distilled
water and incubated at 30 °C for 10 min. The absorbance
was recorded at 520 nm using a spectrophotometer (UV
Mini-1240, Shimadzu). One unit (U) of enzymatic activity
was defined as the quantity of enzyme necessary to produce
1 µmol of gallic acid per minute under the assay conditions.
Protein and carbohydrate quantification
The protein quantification was performed according to
the Bradford method (Bradford 1976) using bovine serum
albumin (BSA) (Sigma-Aldrich) as standard and expressed
as milligrams of protein per mL of the sample. The carbo-
hydrate content was determined according to Dubois et al.
(1956), using mannose as standard (0-0.1 mg mL−1).
Purification
The crude filtrate from SbmF was loaded in DEAE-Cellu-
lose (Santa Cruz Biotechnology) chromatographic column
(1×12 cm) equilibrated with 10 mmol L−1 sodium acetate
buffer (pH 5.0, buffer A) and eluted by a linear gradient of
NaCl (0–1.5 mol L−1) in the buffer (A). Three-milliliter frac-
tions were collected at a flow rate of 1.5 mL min−1. Fractions
with tannase activity were pooled, dialyzed against distilled
water for 24 h at 4 °C, lyophilized, suspended in 50 mmol
L−1 Tris–HCl buffer (pH 7.5) containing 100 mmol L−1
KCl (buffer B) and loaded in Sepharose CL-6B (1×70 cm)
(Pharmacia Fine Chemicals) chromatographic column pre-
viously equilibrated and eluted with the buffer (B) One
milliliter fractions were collected at a flow rate of 0.4 mL
min−1. Fractions with tannase activity were pooled, dialyzed
against distilled water for 24 h at 4 °C and used for enzyme
characterization. β-amylase (200 kDa), alcohol dehydroge-
nase (150 kDa), and BSA (66 kDa) were used as molecular
mass markers. The void of 56 mL was estimated using Blue
Dextran 2000.
Materials and methods
Microorganism and culture conditions
The endophytic fungus Aspergillus fumigatus CAS-21
was maintained on slants of PDA (Potato-Dextrose-Agar;
Neogen® Corporation) medium at 4 °C. For obtaining new
cultures, spores from 30-day-old cultures were collected
with a loop, transferred to the PDA medium, incubated at
37 °C for 4 days, and stored at 4 °C.
Submerged cultures (SbmF) were obtained by inoculation
of 1 mL spore suspension (105 spores mL−1) in a mineral
medium (Costa et al. 2008), containing 2% (m/v) tannic
acid as carbon source, previously autoclaved at 120 °C and
1.5 atm for 20 min. The cultures were kept at 37 °C for
24 h under orbital agitation (120 rpm) using a shaker equip-
ment (Tecnal—TE422). After cultivation, the medium was
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3 Biotech (2018) 8:270
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by a separation using a gradient of 5–60% acetonitrile from
3 min to 10 min. Then, a gradient of 60–90% acetonitrile
was applied from 10 min to 15 min, followed by 90% ace-
tonitrile for the next 5 min, and 5% acetonitrile for another
5 min. The flow rate was maintained at 400 µL min−1 and the
compounds present in the samples were monitored by DAD
at 220–600 nm. Tannic acid, gallic acid, and propyl gallate
were used as standards.
Denaturing electrophoresis—SDS–PAGE
The purified enzyme was subjected to 12% SDS-PAGE
according to Laemmli (1970), using a power source (Pow-
erPac™ Bio-Rad®) adjusted to 120 V and 40 mA. After the
run, the gel was stained with Coomassie Blue Silver G-250
(Candiano et al. 2004). The Precision Plus Protein Kaleido-
scope Standards (Bio-Rad®) was used as molecular mass
marker at a range from 10 to 250 kDa.
Treatment of leather effluent using tannase
Influence of different compounds on enzyme
activity
The industrial effluent samples were obtained from the tan-
ning process of he-goat leather in the Arteza cooperative,
Paraíba, Brazil. Effluent samples obtained after mechanical
and manual processing of the leather were treated with crude
filtrate containing tannase (1:1 v/v) for 1 and 2 h at 30 °C,
and the content of tannins and total phenols was determined.
The total phenol content was determined using a modified
Folin–Ciocalteu method from Agostini-Costa et al. (1999).
After the treatment, 50 µL of the effluent samples was added
with 2.5 mL of the Folin–Ciocalteu (10% v/v) (Merck) rea-
gent, 2 mL of sodium carbonate (25% m/v) and maintained
in absence of light for 30 min. Thereafter, the tube content
was analyzed at 760 nm. For both analyses, in the blank tube
(control) the enzyme sample was replaced by distilled water.
The tannin levels of the treated and untreated effluent
samples were determined according to the methodology
described by Hagerman and Butler (1978). The reaction
consisted of 1 mL of the sample and 3 mL of BSA prepared
in 0.2 mol L−1 sodium acetate buffer (pH 5.0) added with
0.17 mol L−1 sodium chloride and maintained for 15 min at
room temperature. The tubes were centrifuged at 5000×g for
10 min (Centrifuge 5415 C—Eppendorf). The supernatant
was discarded and the pellet was dissolved in 1 mL of 1%
(w/v) BSA solution, added with 3 mL of 1% (w/v) SDS
in 5% (v/v) triethanolamine solution and 1 mL of 0.01 mol
L−1 FeCl3. After 15 min at room temperature, the absorb-
ance was recorded at 510 nm using a spectrophotometer (UV
Mini-1240, Shimadzu).
The effect of different salts (1 mmol L−1: AgNO3, BaCl2,
CaCl2, CoCl2, CuCl2, CuSO4, Fe2(SO4)3, FeCl3, HgCl2,
KH2PO4, MgCl2, MgSO4, MnCl2, MnSO4, NaCl, NH4Cl,
Zn(NO3)2, and ZnSO4), denaturing agents (1 mmol L−1
:
β-mercaptoethanol and urea), 1 mmol L−1 EDTA, detergents
(0.01% v/v: SDS, Triton X-100, Tween-20) and organic sol-
vents (1% v/v: methanol, acetonitrile, ethanol, acetone, iso-
propanol, n-butanol) was evaluated. The effect of different
concentrations (0–50 mmol L−1) of glucose and gallic acid
(C7H6O5) (Sigma-Aldrich) on the enzyme activity was also
evaluated.
Hydrolysis of substrates and kinetic parameters
The ability of the enzyme to hydrolyze different substrates
as tannic acid (C76H52O46) (Sigma-Aldrich), methyl gallate
(C8H8O5) (Sigma-Aldrich) and their mixtures (1:1 v/v), as
well as propyl gallate (C10H12O5) (Sigma-Aldrich), was ana-
lyzed as described above. All substrates were prepared in
100 mmol L−1 sodium acetate buffer (pH 5.0). The kinetic
parameters Km and Vmax, Kcat and Kcat/Km were determined
for each substrate using the software SIGRAF (Leone et al.
1992).
Synthesis of propyl gallate
The enzymatic hydrolysis of the effluent tannins was also
analyzed by CLUE-DAD as described above. For this pur-
pose, the reactions containing effluent or tannic acid samples
were conducted for 2 h at 30 °C.
Propyl gallate was produced by transesterification of tannic
acid in the presence of the purified A. fumigatus CAS-21
tannase. The reaction mixture containing 5 mmol L−1 tan-
nic acid in 100 mmol L−1 MES buffer (pH 6.0), 1-propanol
(1:1 v/v), and the tannase was maintained at 30 °C for dif-
ferent periods (48 h and 72 h). Five-microliter samples of
each reaction were loaded in a column Ascentis® Express
C18 (100×4.6 mm, 2.7 µm particle diameter) from Supelco
in the System ACQUITY UPLC H-Class with the diodes
arrangement detector (CLUE-DAD, Waters Corporation).
An aqueous solution of 0.1% formic acid (solvent A) and
acetonitrile containing 0.1% formic acid (solvent B) were
used as mobile phase. The separation was carried out using
isocratic mode with 5% acetonitrile for 0–3 min followed
Results
Purification
Figure 1a depicts the DEAE-Cellulose chromatographic
profile obtained for the A. fumigatus CAS-21 tannase.
Three peaks with tannase activity were obtained. The first
peak (FI) did not interact with the resin, while the peaks
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FII and FIII interacted and they were eluted with 0.25 and
0.50 mol L−1 of NaCl, respectively. Considering the higher
activity obtained for the FII, it was selected for the next
purification step. The FII fractions were pooled, dialyzed,
lyophilized, and loaded in Sepharose CL-6B (Fig. 1b).
A single peak with tannase activity was obtained. Using
these procedures, one tannase isoform was purified 4.9-
fold with a recovery of 10.2% (Table 1). The native
molecular mass for the purified enzyme was estimated as
60.34 0.84 kDa through the Sepharose CL-6B chroma-
tographic column and 60.85 5.80 kDa by SDS-PAGE
(Fig. 1c), confirming that it is a monomeric glycoprotein
with 39.1% carbohydrate content.
Effect of temperature and pH on the tannase
activity
The maximal activity for A. fumigatus CAS-21 tannase
was observed at a range from 30 to 40 °C (Fig. 2a), while
the optimum pH was 5.0 (Fig. 2b). The A. fumigatus CAS-
21 tannase maintained 80% of its initial activity when
incubated at 30 °C for 60 min (Fig. 2c). Reduction of the
enzyme activity to 50% of the initial value was observed
upon incubation for 120 min at this temperature. At 45
and 50 °C, the half-life (t50) for the enzyme was 60 min.
The enzyme was fully stable at pH 5.0 and 6.0 after 3 h of
incubation, while it retained 70% of its initial activity at
these pH values after 24 h incubation (Fig. 2d).
Effect of salts and other compounds
The effect of different salts on tannase activity is pre-
sented in Table 2. The enzyme was slightly activated in the
presence of AgNO3, KH2PO4, and MnSO4, but inhibited
approximately 50% by 1 mmol L−1 Fe2(SO4)3 and FeCl3.
In case of detergents and denaturing agents, tannase activ-
ity was increased in the presence of SDS (+ 15%), Tri-
ton X-100 (+ 18%), methanol (+ 8%), and especially urea
(Table 3). The activity of the A. fumigatus CAS-21 tannase
was increased at urea concentrations from 1 × 10− 3 mol
L−1 to 1 mol L−1, with the highest activity at a concentra-
tion of 4 × 10− 3 mol L−1 (+ 90%). In addition, the enzyme
activity was maintained at 80–90% at high urea concentra-
tions (2 and 3 mol L−1) (Fig. 2e).
Fig. 1 DEAE-Cellulose (a) and Sepharose CL-6B (b) chromato-
graphic profiles for the tannase produced by A. fumigatus CAS-21
and 12% SDS–PAGE results (c) for proteins obtained from DEAE-
Cellulose (I) and Sepharose CL-6B (II) column chromatography.
345 µg of protein was applied in (I) and 390 µg of protein in (II). In
(III): Molecular Mass Markers (Bio-Rad). Symbols: (open circle) tan-
nase activity; (closed circle) absorbance at 280 nm
Similarly, it was verified that the addition of glucose
in the enzymatic reaction did not affect tannase activity
under all concentrations analyzed (Fig. 2f). On the con-
trary, addition of gallic acid at a concentration higher than
5 mmol L−1 reduced the enzyme activity, and at 10 mmol
L−1 the tannase activity was totally inhibited (Fig. 2f).
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3 Biotech (2018) 8:270
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Table 1 Purification of the extracellular tannase produced by A. fumigatus CAS-21
Step
Volume (mL)
Activity (total U)
Protein (total mg)
SA (U/mg)
Yield (%)
Purifi-
cation
(fold)
Crude filtrate
DEAE – cellulose (peak FII)
Sepharose CL-6B
200
47
16
8669.0
2105.3
881.3
204.7
10.8
4.2
42.3
194.6
209.2
100
24.3
10.2
1
4.6
4.9
SA specific activity
Fig. 2 Influence of temperature (a), pH (b), thermal stability (c), pH
stability (d), and concentration of urea (e), gallic acid and glucose (f)
on tannase activity from A. fumigatus CAS-21. Symbols: in c—(open
circle) 30 °C, (open square) 45 °C, (open upwards triangle) 50 °C and
(open downards triangle) 65 °C; in d: (closed upwards triangle) 3 h
and (closed downwards triangle) 24 h. 100% activity corresponds to
48.74 0.90 U mL−1 (d) and 31.61 0.82 U mL−1 (e)
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Table 2 Influence of different salts on the activity of tannase from A.
fumigatus CAS-21
Salts (1 mmol L−1
)
Relative activity (%)a
Control
AgNO3
BaCl2
CaCl2
CoCl2
100.0
110.7 1.2
78.2 5.7
95.0 4.8
74.7 5.4
100.3 8.3
92.8 5.9
58.2 6.7
49.7 3.2
96.3 0.1
110.3 1.0
75.6 6.2
97.6 3.9
87.1 5.8
112.9 2.1
78.4 2.2
93.2 7.5
100.8 7.0
101.2 0.2
CuCl2
CuSO4
Fe2(SO4)3
FeCl3
HgCl2
KH2PO4
MgCl2
MgSO4
MnCl2
MnSO4
NaCl
NH4Cl
Zn(NO3)2
ZnSO4
Fig. 3 Hydrolysis of the substrates methyl gallate (open circle), tan-
nic acid (closed circle), and methyl gallate+tannic acid (1:1 v/v)
(closed triangle) as a function of time of reaction by the tannase from
A. fumigatus CAS-21
Table 4 Kinetic parameters for the extracellular tannase produced by
A. fumigatus CAS-21
Substrate
Km (mmol
L−1
Vmax (U
mg−1
Kcat (s−1
)
Kcat/Km
(s−1 mmol−1 L)
a100% relative activity corresponds to 31.61 0.82 U mL−1
)
)
Tannic acid 6.38
360.20
152.00
362.24
152.86
56.78
24.34
Methyl gal- 6.28
Table 3 Influence of different compounds on the activity of tannase
from A. fumigatus CAS-21
late
Propyl gal- 3.61
late
111.30
111.93
31.00
Compounds
Relative activity (%)a
Control
100.0
Acetone (1%)
Acetonitrile (1%)
Butanol (1%)
EDTA (1 mmol L−1
Ethanol (1%)
90.0 2.9
69.4 0.5
94.3 2.7
80.6 7.8
88.1 2.4
87.6 6.0
108.8 4.5
115.7 1.7
118.4 4.9
89.2 2.6
154.7 4.0
82.8 7.9
but the best Kcat (362.24 s−1) and Kcat/Km (56.78 s−1
mmol−1 L) were obtained for tannic acid (Table 4).
)
Propyl gallate synthesis by transesterification
Isopropanol (1%)
Methanol (1%)
The A. fumigatus CAS-21 tannase was able to catalyze
transesterification reaction in presence of 1-propanol to pro-
duce propyl gallate, evident from the CLUE-DAD profiles
(Fig. 4). The peaks with retention times of 10.53 min and
10.56 min obtained after 48 h (Fig. 4d) and 72 h (Fig. 4e)
reactions, respectively, were similar to that obtained for the
propyl gallate standard (10.56 min) (Fig. 4c). Moreover,
residual tannic acid could be observed (Fig. 4a), while gal-
lic acid was not detected.
SDS (1 mmol L−1
)
Triton X-100 (0.01%)
Tween-20 (0.01%)
Urea (1 mmol L−1
)
β-Mercaptoethanol (1 mmol L−1
)
a100% relative activity corresponds to 31.61 0.82 U mL−1
Effluent treatment
Hydrolysis of substrates and kinetic parameters
While analyzing the CLUE-DAD profiles for the effluent treat-
ment (Fig. 4f, g), a single peak at 5.3 min that corresponded
to the retention time for the gallic acid was observed, indi-
cating that the A. fumigatus CAS-21 tannase was effective in
hydrolyzing the tannic acid content in effluents generated by
The purified A. fumigatus CAS-21 tannase was able to
hydrolyze tannic acid and methyl gallate, as well as the
mixture of these two substrates (Fig. 3). The Km value
(3.61 mmol L−1) indicated high affinity for propyl gallate,
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3 Biotech (2018) 8:270
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Fig. 4 CLUE-DAD profiles for the propyl gallate synthesis after
48-h (d) and 72-h (e) reactions through esterification of tannic acid
(5 mmol L−1) in 50% of 1-propanol and for the products obtained
from the 2-h treatment of leather effluent generated by manual (f) and
mechanical (g) processes using A. fumigatus CAS-21 tannase. Stand-
ards: tannic acid (a), gallic acid (b), and propyl gallate (c)
mechanical as well as manual processes. The tannase reduced
89 and 25% of the tannin content in manually and mechani-
cally processed samples of the leather effluent, respectively,
after 2 h of treatment (Fig. 5a). In addition, the filtrate contain-
ing tannase from A. fumigatus CAS-21 was also able to reduce
30% of the content of phenolic compounds in both the effluent
samples (Fig. 5b).
Discussion
The fungus A. fumigatus CAS-21 can produce tannase iso-
forms when maintained under SbmF in presence of tannic
acid as carbon source. One of the enzyme isoforms was
effectively purified using DEAE-Cellulose and Sephacryl
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A
B
100
80
60
40
20
0
Control
1 h
2 h
Fig. 5 Treatment of the leather effluent after manual (closed square) and mechanical (open square) procedures, with tannase produced by A.
fumigatus CAS-21, considering the tannin (a) and phenolic (b) content
S-200. Notably, the enzyme recuperation after these pro-
cedures was higher than that reported for the tannases pro-
duced by Aspergillus ochraceus (2.12%) (Gonçalves et al.
2012), A. tamarii (TAH II- 3.84%) (Costa et al. 2012), and
Verticillium sp. (TAH I- 1.6% and TAH II- 0.9%) (Kasiec-
zka-Burnecka et al. 2007).
(Kumar et al. 2015) and A. awamori MTCC9299 (Chhokar
et al. 2010) was 30 °C, while for the isoforms TAH I and
TAH II from A. tamarii tannases, it was 30 °C and 40 °C,
respectively (Costa et al. 2012). For A. fumigatus CAS-
21 tannase, temperatures above 65 °C promoted reduction
in enzymatic activity because under high temperatures,
the weak bonds in the three-dimensional structure of the
protein break, causing its denaturation and loss of activ-
ity (Mukherjee and Banerjee 2006). Considering the fact
that A. fumigatus CAS-21 was isolated from a plant found
in the Caatinga biome, which presents high temperatures
during the day and mild temperatures during the night,
the presence of a tannase able to act at a wide temperature
range is justified. The A. fumigatus CAS-21 tannase was
stable for 1 h at 30 °C, which is also an optimal tempera-
ture for its activity, with a half-life of 30 min at 45 and
50 °C, in agreement with the literature. Interestingly, some
tannases were previously reported to be stable at a tem-
perature range from 10 to 60 °C (Costa et al. 2008; Sharma
et al. 1999; Pinto et al. 2005).
Enzyme purity was confirmed by SDS-PAGE, observing
a single protein band with similar molecular mass obtained
using gel filtration, indicating the monomeric characteris-
tic for this enzyme. Moreover, the analysis of the carbohy-
drate content indicates that the A. fumigatus CAS-21 is a
glycoprotein. In general, tannases have a molecular mass
ranging from 50 to 320 kDa (Yao et al. 2014) and carbo-
hydrate content from 5 to 64% (Kasieczka-Burnecka et al.
2007; Costa et al. 2012). Monomeric tannases were reported
for Aspergillus melleus (Liu et al. 2017), A. niger (Lal and
Gardner 2012), A. ochraceus (Gonçalves et al. 2012), and
Penicillium herquei (Qiu et al. 2011). The TAH I and TAH
II tannases from Verticillium sp. were also characterized as
glycoproteins with 11% and 26% of carbohydrate content,
respectively (Kasieczka-Burnecka et al. 2007), whereas the
enzyme of A. awamori BTMFW032 showed 8.02% carbo-
hydrate content (Beena et al. 2010). Other fungal tannases
were also characterized as glycoproteins such as those from
A. tamarii (Costa et al. 2012), A. phoenicis (Riul et al.
2013), and A. carbonarius (Valera et al. 2015). Remark-
ably, glycosylation is an important characteristic to protect
the tannases from the negative effects of tannin (Lekha and
Lonsane 1997).
Similar to our results, tannases from different Aspergillus
species presented optimal activity at pH 5.0 as observed for
A. tamarii (Costa et al. 2008), A. ochraceus (Gonçalves et al.
2012), A. awamori nakazawa (Mahapatra et al. 2005), and A.
oryzae (Mizuno et al. 2014), among others. Moreover, most
tannases are stable at pH 5.0–6.0 (Aguilar et al. 2007), such
as those from A. niger GH1 (Mata-Gomez et al. 2009), A.
awamori (Kumar et al. 2015), A. oryzae (Abdel-Nabey et al.
2016), and A. fumigatus CAS-21. Importantly, pH affects the
ionization of amino acids in the protein, which can modify
ionic interactions that determine the molecule structure,
thereby affecting linkage to the substrate, inactivating the
enzyme or even promoting its instability. In addition, the
abundance of acidic amino acids in the enzyme can explain
the stability of fungal tannases at an acidic pH (Abdel-Nabey
et al. 2016).
The optimal temperature (30–40 °C) for A. fumigatus
CAS-21 tannase activity is in agreement with the data
reported by other authors. For example, Gonçalves et al.
(2012) found optimal activity at 30–45 °C for A. ochraceus
tannase. According to Yao et al. (2014), tannases exhibit
optimal activity between 30 and 60 °C. The optimal tem-
perature of activity for the enzymes from A. awamori
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3 Biotech (2018) 8:270
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Different classes of compounds can positively or nega-
tively affect tannase activity. The most pronounced effect
on the A. fumigatus CAS-21 tannase was observed in pres-
ence of FeCl3 and Fe2(SO4)3, which inhibited enzyme
activity. Similarly, inhibition by FeCl3 was also observed
for the enzymes produced by A. carbonarius (Valera et al.
2015), A. niger ATCC 16620 (Sabu et al. 2005), A. oryzae
(Abdel-Nabey et al. 2016), and A. awamori MTCC9299
(Chhokar et al. 2010). This effect can be justified by the
linkage between iron ions and the thiol groups and/or by
the interaction of ions with the carboxylic group of amino
acids at the catalytic site of the protein (Abdel-Nabey et al.
2016). Furthermore, the presence of Mg2+, Co2+, and Ba2+
inhibited 25% of the enzyme activity. Interestingly, the pres-
ence of sulfate or chloride in the salts is an important factor
regulating the enzyme activity. For example, the negative
effects were mostly accentuated in the presence of MnCl2
and MgCl2 than in the presence of MnSO4 and MgSO4. For
the other salts analyzed, the activity was not significantly
affected. For instance, the tannase produced by Pestalo-
tiopsis guepinii URM 7114 was activated by Ca2+ (Sena
et al. 2014), while the A. fumigatus CAS-21 tannase was not
affected by Ca2+. Consistent with our results, Valera et al.
(2015) also observed activation by silver of the enzyme pro-
duced by A. carbonarius. However, for most proteins, silver
acts as a precipitation agent inhibiting their activities.
Other compounds also affected the activity of the A.
fumigatus CAS-21 tannase. Increase in tannase activity by
urea, as observed by us, was also reported for the enzymes
from A. awamori nakazawa and Verticillium sp. in pres-
ence of 1.5 mol L−1 urea (Kasieczka-Burnecka et al. 2007;
Mahapatra et al. 2005). According to Mahapatra et al.
(2005), tannase is denatured only at high urea concentra-
tions, which can be explained by the conformational changes
in the tertiary structure of the enzyme. On the contrary, in
the presence of acetonitrile, the enzymatic activity was
inhibited by 30%. For other compounds, tannase activity
was slightly reduced, retaining 80–90% of the initial activ-
ity. Thus, the ability to remain stable in presence of organic
solvents, surfactants, and denaturants are favorable charac-
teristics for industrial applications of A. fumigatus CAS-21
tannase.
(1999) and El-Fouly et al. (2010), where it acts as a com-
petitive inhibitor of the enzyme. Consistently, the tannase
produced by A. phoenicis was not regulated by glucose, but
it was drastically inhibited at high concentrations of gallic
acid (Riul et al. 2013).
The A. fumigatus CAS-21 was able to hydrolyze differ-
ent substrates, but the highest values of hydrolysis were
obtained using tannic acid. The values of hydrolysis for the
mixture (tannic acid + methyl gallate) were intermediary
between the values obtained for the separate hydrolysis of
each substrate, indicating that there is a single active site for
hydrolysis of the substrates. If there are two different active
sites, one for each substrate, then the expected values for
the hydrolysis of the mixture of the two substrates should be
approximately similar to the addition of the separate values
obtained. Generally, tannases are isoenzymes with differ-
ent specificity for each substrate according to its structure,
exhibiting both esterase and depsidic activities (Rodríguez-
Durán et al. 2011). Other tannases can also hydrolyze differ-
ent substrates, for instance, the enzymes produced by Asper-
gillus oryzae (Mizuno et al. 2014) and Emericella nidulans
(Gonçalves et al. 2011).
On the other hand, the best affinity was observed for pro-
pyl gallate, which is also observed for the tannases produced
by A. phoenicis (Riul et al. 2013) and Penicillium variable
(Sharma et al. 2008). The affinity of the A. fumigatus CAS-
21 tannase for the tannic acid and methyl gallate substrates
was similar to that of the A. ochraceus enzyme (Gonçalves
et al. 2012). The Kcat for the A. fumigatus CAS-21 tannase
was lower than that obtained for the enzyme from A. ory-
zae (Abdel-Nabey et al. 2016), indicating that the catalytic
cycle performed by the CAS-21 tannase is faster than that
performed by the A. oryzae enzyme.
The potential of A. fumigatus CAS-21 tannase for pro-
pyl gallate synthesis was analyzed, considering its favora-
ble characteristics such as the stability over a wide range
of pH and temperature, tolerance to surfactants, organic
solvents and denaturing agents, and a high activity in the
presence of glucose. The propyl gallate production continu-
ously increased during the 72-h reaction, indicating that the
enzyme can be applied for the production of this important
antioxidant in food industry (Fernandez-Lorente et al. 2011).
Tannases produced by A. phoenicis (Riul et al. 2013), A.
awamori BTMFW032 (Beena et al. 2010), A. niger, and
Penicillium variable (Sharma and Saxena 2012) were also
described as potential enzymes to produce propyl gallate.
Another important application of tannases is the treat-
ment of tannin-rich waste generated by leather industry. As
tannin is an important recalcitrant agent found in the tannery
effluent, special attention is required for its degradation prior
to elimination in the environment. In Brazil, many leather
industries use tannic acid to treat the leather. The use of A.
fumigatus CAS-21 tannase to treat the leather effluent was
Glucose and gallic acid are the products of tannic acid
hydrolysis under tannase action. The products obtained from
the substrate hydrolysis inhibit many enzymes. The hydroly-
sis of substrates containing different molecules such lignin,
saccharides, and tannins, can be achieved by application
of enzymatic cocktails (Riul et al. 2013). Considering the
resistance of the A. fumigatus CAS-21 tannase to the inhibi-
tion by glucose, it can be exploited for the formulation of
enzymatic cocktails to hydrolyze tannin substrates at a high
saccharide concentration. The A. fumigatus CAS-21 tannase
was inhibited by gallic acid, as also reported by Kar et al.
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3 Biotech (2018) 8:270
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effective and a single gallic acid peak was observed after
2-h treatment of both mechanically and manually processed
effluents. No tannic acid signal was detected, indicating the
potential of A. fumigatus CAS-21 tannase for an eco-friendly
treatment of tannin-rich effluents, especially in the leather
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To date, no tannase application has been reported for the
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ment of tannery effluent allowed 45 and 36% reduction in
the tannin content, respectively (Sharma and Saxena 2012).
Additionally, Salgado et al. (2016) demonstrated that the
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In conclusion, the fungus A. fumigatus CAS-21, isolated
from the Caatinga biome, was found to be a good source
of tannases. One of these tannases was characterized and
it presented tremendous biotechnological potential for the
treatment of tannery effluent and propyl gallate production
using a transesterification reaction. In addition, the enzyme
presented interesting properties such as thermal and pH
stability, insensitivity to glucose, tolerance to solvents and
detergents, thereby amplifying the possibilities of its bio-
technological application.
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Acknowledgements The authors gratefully acknowledge the finan-
cial support from Fundação de Amparo à Pesquisa do Estado de
São Paulo—FAPESP (2016/11311-5) and the research scholarships
from Coordenação de Aperfeiçoamento de Pessoal de Nível Superior
(CAPES). This manuscript is a part of the Master Degree dissertation
from R.M.F.C. We also thank Maurício de Oliveira and Dr. Eduardo
J. Crevelin for the technical assistance, and Carlos Ambrosio from
ARTEZA, who provided the effluent samples.
Fernandez-Lorente G, Bolivar JM, Rocha-Martin J, Curiel JA, Muñoz
R, Rivas B, Carrascos AV, Guisan JM (2011) Synthesis of propyl
gallate by transesterification of tannic acid in aqueous media by
derivatives of tannase from. Lactobacillus plantarum Food Chem
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Extracellular tannase from Emericella nidulans showing hypertol-
erance to temperature and organic solvents. J Mol Catal B Enzym
71:29–35
Compliance with ethical standards
Gonçalves HB, Riul AJ, Quiapin AC, Jorge JA, Guimarães LHS (2012)
Characterization of a thermostable extracellular tannase produced
under submerged fermentation by Aspergillus ochraceus. Electron
J Biotechnol 15:1–12
Conflict of interest The authors declare that there is no conflict of in-
terest.
Hagerman AE, Butler LG (1978) Protein precipitation method for
the quantitative determination of tannins. J Agric Food Chem
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