Tannase production under solid and submerged culture by xerophilic strains of Aspergillus and their genetic relationships

Abstract

La enzima tanasa cataliza la hidrólisis de los enlaces éster del ácido tánico. En el presente estudio, se evalúo la producción de tanasa por siete cepas xerófilas de Aspergillus, utilizando medio de cultivo sólido y sumergido. Seis de las siete cepas fueron aisladas de una región semidesértica mexicana y una cepa fue utilizada como control (Aspergillus niger Aa-20). Las cepas fueron caracterizadas molecularmente por secuenciación de la región 18S del ADNr, con el objetivo de analizar sus relaciones genéticas. La cepa GH1 de Aspergillus niger produjo los niveles más altos de actividad enzimática en ambos sistemas de fermentación. Las especies identificadas fueron A. fumigatus, A. niger, A. ornatus, y A. rugulosus.

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Redalyc
Sistema de Información Científica
Red de Revistas Científicas de América Latina, el Caribe, España y Portugal
Cerda Gómez, A. A. de la; Reyes Valdés, M. H.; Meléndez Rentería, N. P.; Rodríguez
Herrera, R.; Aguilar, C. N.
Tannase production under solid and submerged culture by xerophilic strains of
Aspergillus and their genetic relationships
Micología Aplicada Internacional, vol. 23, núm. 1, enero, 2011, pp. 21-27
Colegio de Postgraduados
Puebla, México
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Tannase producTion by xerophilic Aspergillus
MicologiA AplicAdA internAtionAl, 23(1), 2011, pp. 21-27
© 2011, Berkeley, CA, U.S.A.
www.micaplint.com
Micol. Apl. int., 23(1), 2011, pp. 21-27
Tannase producTion under solid and
submerged culTure by xerophilic sTrains of
Aspergillus and Their geneTic relaTionships
a. a. de la cerda gómez1, m. h. reyes Valdés2, n. p. meléndez renTería1,
r. rodríguez herrera1 and c. n. aguilar1
1 Laboratory of Molecular Biology, Department of Food Science and Technology, School of Chemistry,
Universidad Autónoma de Coahuila, Saltillo, Coahuila, Mexico. Fax: + 52 (844) 415-9534.
E-mail: rrh@mail.uadec.mx
2 Department of Plant Breeding, Universidad Autónoma Agraria “Antonio Narro”, Buenavista 25000,
Saltillo, Coahuila, Mexico.
Accepted for publication December 15, 2010
ABSTRACT
Tannase catalyzes the hydrolysis of ester bonds from tannic acid. In this study, tannase
production by seven xerophilic strains of Aspergillus was evaluated under solid and
submerged culture. Six out of seven strains were isolated from a Mexican semi-desert
region, and one strain was used as control (A. niger Aa-20). Fungal strains were
characterized by sequencing the 18S rDNA region, in order to assess their genetic
relationships. A. niger GH1 strain produced the highest enzyme activity titres in both
fermentation systems. Species identified were A. fumigatus, A. niger, A. ornatus, and A.
rugulosus.
Key words: Aspergillus, 18S rDNA region, solid and submerged state culture, tannase
activity, xerophilic strains.
producción de Tanasa en culTiVo sólido y sumergido por cepas xerofílicas
de Aspergillus y sus relaciones genéTicas

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a. a. de la cerda gómez et Al.
Micol. Apl. int., 23(1), 2011, pp. 21-27
INTRODUCTION
Tannase or tannin-acyl-hydrolase (EC.3.1.-
1.20) catalyzes the hydrolysis of ester
bonds from tannic acid, which are a group
of water-soluble polyphenolic compounds
of different molecular weight. This enzyme
plays an important role in the metabolism
of plant complex tannins. It is also relat-
ed to fruit ripening, because the enzyme
hydrolyses some esters produced by con-
densation of a glycoside with gallic and/or
hexahydrophenolic acids3. The hydrolysis
reaction of tannins, catalyzed by the tan-
nase, generates gallic acid and glucose as
products3,4. Tannase has potential in food,
beverage, pharmaceutical and chemical
industries, although a commercial applica-
tion has been proposed for instant tea and
gallic acid production6,8,14. Additional food
application of tannase is the improvement
of raw material utilization and quality10.
The enzyme is obtained from different
animal, plant and microbial sources,
the latter being the most important
due to better enzyme stability6. Many
microorganisms steadily produce high
amounts of enzyme1, and may be easily
used for fermentation processes and
genetic manipulation to increase enzyme
production titres or enzyme activity7.
Because tannase enzyme has potential for
the food industry, it is important to find
novel fungal strains producing tannase
from different habitats15.
Filamentous fungi from the Mexican
semi-desert have been isolated, character-
ized, and shown to produce higher tannase
titres than those strains isolated from tropi-
cal areas9. DNA fingerprinting of these fun-
gal strains has been carried using molecu-
lar markers, such as RAPDs, ITS, IGS, and
RFLPs6. The isolation and characteriza-
tion of tannase genes is important not only
for understanding enzymatic activity, but
also for developing strategies to improve
enzyme yield12. In this study, we assessed
tannase production by seven xerophilic
strains of Aspergillus under solid and sub-
merged state culture, and determined their
RESUMEN
La enzima tanasa cataliza la hidrólisis de los enlaces éster del ácido tánico. En el
presente estudio, se evalúo la producción de tanasa por siete cepas xerófilas de
Aspergillus, utilizando medio de cultivo sólido y sumergido. Seis de las siete cepas
fueron aisladas de una región semidesértica mexicana y una cepa fue utilizada como
control (Aspergillus niger Aa-20). Las cepas fueron caracterizadas molecularmente
por secuenciación de la región 18S del ADNr, con el objetivo de analizar sus relaciones
genéticas. La cepa GH1 de Aspergillus niger produjo los niveles más altos de actividad
enzimática en ambos sistemas de fermentación. Las especies identificadas fueron A.
fumigatus, A. niger, A. ornatus, y A. rugulosus.
Palabras clave: Actividad de tanasa, Aspergillus, cepas xerofílicas, cultivo en estado
sólido y sumergido, región 18S del ADNr.

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Tannase producTion by xerophilic Aspergillus
Micol. Apl. int., 23(1), 2011, pp. 21-27
phylogenetic relationships using the 18S
rDNA region.
MATERIALS AND METHODS
Submerged and solid state fermentation
Microorganisms and inoculum prepara-
tion. Fungal strains were isolated and de-
posited in the culture collection from the
University of Coahuila. Spores were stored
using a cryoconservation system2. Each
strain was subcultured on mycological agar
(g/L: soy peptone 10, glucose 10, agar 16),
and incubated five days at 30 C. Spores
were recovered using Tween 80 (0.1%
v/v), and counted in a Neubauer chamber
for starting each culture system. Fungal
strains tested were: Aspergillus fumiga-
tus Fresen. (GS), A. niger Tiegh. (GH1),
A. niger (PSH), A. ornatus Raper, Fennell
& Tresner (ESH), A. rugulosus Thom &
Raper (NS4), A. rugulosus (NH4), A. ter-
ricola Marchal & É. J. Marchal (PSS), and
A. niger (Aa-20; IRD-UAM-I collection)
as control.
Submerged state culture (SmC). Liquid
culture medium studied was (g/L): KH2PO4
(2.19), (NH4)2SO4 (4.38), MgSO4.7 H2O
(0.44), CaCl2.7 H2O (0.044), MnCl2.6 H2O
(0.009), NaMoO4.2 H2O (0.004), FeSO4.7
H2O (0.06), and tannic acid (12.5; Sigma-
Aldrich, no. 403040). The medium was in-
oculated with spores from each strain using
5 x106 spores per reactor (Erlenmeyer flasks,
250 mL), which was incubated at 30 C for
20 h with constant shaking. To obtain the
crude extract, the culture medium of each
reactor was filtered using Whatman 41 filter
paper. Once the extract was obtained, it was
filtered again using a Millipore® membrane
of 45 μm, and stored until its analysis.
Solid state culture (SSC). This fermen-
tation was performed using polyurethane
foam (PUF) as support, 70% moisture
content. PUF was placed into Erlenmeyer
flasks (250 mL) and sterilized for 20 min
at 121 C. The tannic acid medium was
prepared as described for submerged state
culture, then it was inoculated with 2 x 107
spores per gram of PUF, and added to the
support. Flasks were incubated at 30 C for
20 h. The crude enzymatic extract was re-
covered by mechanic compression with a
sterile syringe, filtrated through Millipore®
membrane of 0.45 μm, and stored in 10 mL
sterile flasks until its analysis.
Tannase activity assay. Tannase activ-
ity was assayed using a spectrophotomet-
ric method, in which a chromophore was
formed between released gallic acid and
rodanine16. Three assay tubes were used
(blank, control and sample). Citrate buf-
fer (0.25 mL) was added to the blank, and
equal volume of enzymatic extract to the
sample tube, which was incubated for 5
min. After that, 0.3 mL of methanolic ro-
danine (0.67 % v/v) and 0.20 mL of KOH
0.5 N were added to each tube, incubated
for 10 min at 30 C, and 4.0 mL of distilled
water were added for reading absorbance
at 520 nm. One unit of tannase activity was
defined as the release of one micromole per
minute under assay conditions.

Phylogenetic relationships among fungal
strains
Fungal DNA isolation. Fungal strains
were grown in malt extract broth. Mycelium
was recovered by filtration and washed
three times with sterile distilled water. From
each strain, 0.1 g of mycelium was frozen in
liquid nitrogen, ground to fine powder and
transferred to a 1.5 mL tube. DNA isola-
tion was performed according to a modified
method of Graham et al.11. DNA was evalu-
ated using agarose (1%) gel electrophore-
sis, and quantified spectrophotometrically.

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Micol. Apl. int., 23(1), 2011, pp. 21-27
Amplification of 18S ribosomal DNA
from fungal strains. PCR amplification
from the DNA of each fungal strain was
carried out using the following primers:
18S 3, 5’ CCG TTG GTG AAC CAG CGG
AGG GAT C 3’; and 18S 10, 5’ TCC GCT
TAT TGA TAT GCT TAA G 3’. Each re-
action (23 μL ) contained: 14.5 μL sterile
deionized water, 2.5 µL 10X buffer, 1.0
µL MgCl2, 0.5 µL dNTPs, 2.0 μL of each
primer (10 pM), 0.5 μL of Taq polymerase
(5 U/μL), and 2.0 μL of template DNA (100
ng/μL). The PCR cycling protocol was: de-
naturation temperature of 94 C per 1 min,
annealing temperature of 54 C per 1 min,
and primer extension temperature of 72 C
per 1 min in 35 cycles. Amplified products
were visualized using agarose (1.5 %) gel
electrophoresis. Molecular weight markers
used as reference were 50 bp and 100 bp
DNA ladder (Invitrogen).
Sequencing of 18S ribosomal DNA from
fungal strains. DNA sequencing was done
using a Genetic Analyzer ABI PRISM, mod.
3100 (Applied Biosystems, U.S.A.) with 16
capillary tubes, BigDye terminator version
3.1 sequencing kit. A polymer support
DT3100pop6 (BD) v3 and a capillary tube
(50 cm) were used for mobility. Running
time of sequencing electrophoresis was 2.5
h. Dendrogram construction was carried out
using the MEGA3 software13. Sequences
were transformed to FASTA format,
aligned, and processed by the UPGMA
method with 250 repeats and bootstrap test
for generating a phylogenetic tree.
RESULTS AND DISCUSSION
Submerged and solid state fermentation.
The strain that produced the highest tan-
nase titre (313.11 U/L) under SmC was
Aspergillus niger GH1 (Fig. 1), followed
Fig. 1. Tannase production by seven Aspergillus fungal strains in submerged state culture for 20 h at 30
C, and a tannic acid concentration of 12.5 g/L. NS4: A. rugulosus, Aa-20: A. niger, GS: A. fumigatus,
GH1: A. niger, NH4: A. rugulosus, PSH: A. niger, ESH: A. ornatus.
Tannase activity (U/L)
Strains

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Tannase producTion by xerophilic Aspergillus
Micol. Apl. int., 23(1), 2011, pp. 21-27
by A. rugulosus NS4 with 228.13 U/L. By
contrast, A. ornatus (ESH) was the strain
that produced the lowest tannase titre. The
strain GH1 of Aspergillus niger showed
lower tannase activity than that previously
reported9, although the initial concentra-
tion of tannic acid (50 g/L) was different
and the fermentation period was longer
than this study.
The strain showing the highest tannase
production titre (437.94 U/L) under SSC
was A. niger GH1 (Fig. 2), followed by the
A. niger PSH, A. rugulosus NS4, and A. ni-
ger Aa-20 strains. It has previously been
reported a tannase production titre of 2,291
U/L by Aspergillus niger GH1, in solid
state culture having an initial tannic acid
concentration of 50 g/L and a fermenta-
tion period of 30 h at 30 C9. The difference
between previous data and this study may
be attributed to the initial concentration of
tannic acid and the fermentation period.
The fact that most fungal strains tested
produced higher tannase titres in SSC than
SmC is important. In recent years, biotech-
nology has been focused on enzyme pro-
duction using solid state fermentation as
fungi produce more extracellular enzyme
in SSC than SmC, and the cost for enzyme
recovery is reduced17. Tannase produc-
tion in SmC has been studied with satis-
factory results. However, higher tannase
yields and substrate degradation in SSC
have been reported using a discontinuous
matrix and shorter fermentation periods
(12 h, data not shown). These results have
been attributed to the support that favored
a rapid adaptation of the microorganism, as
Fig. 2. Tannase production by seven Aspergillus strains in solid state culture at 30 C, an initial concen-
tration of 12.5 g/L of tannic acid, and 20 h of fermentation period. NS4: A. rugulosus, Aa-20: A. niger,
GS: A. fumigatus, GH1: A. niger, NH4: A. rugulosus, PSH: A. niger, ESH: A. ornatus.
Tannase activity (U/L)
Strains

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a. a. de la cerda gómez et Al.
Micol. Apl. int., 23(1), 2011, pp. 21-27
well as substrate degradation14. Strains of
A. niger (GH1, PSH, Aa-20) can degrade
high concentrations of tannic acid, and can
be considered efficient tannase producers9.
Other authors have mentioned that tannase
is induced after fungal growth in the pres-
ence of tannic acid, and that the products
released by tannic acid hydrolysis are gal-
lic acid and glucose3.
Phylogenetic relationships among fungal
strains. A 550 bp product of the 18S rDNA
gene was amplified from seven fungal
strains shown to be tannase-producers (Fig.
3). Variation in DNA sequences between
fungal species is due to mutation and recom-
bination processes. The analysis of strain
sequences allowed a dendrogram construc-
tion using the UPGMA method (Fig. 4).
Results showed the presence of four differ-
ent species, namely: A. fumigatus, A. niger,
A. ornatus, and A. rugulosus. Aspergillus
fumigatus (GS) and Aspergillus rugulosus
Fig. 3. Amplification of a 550 bp product of the 18S rDNA gene from seven fungal strains that produce
tannase. Lanes 1 and 3: Aspergillus niger, Aa-20. Lane 2: Molecular marker ladder. Lane 4: A. terricola,
PSS. Lane 5: A. ornatus, ESH. Lane 6: A. niger, GH1. Lane 7: Molecular marker ladder. Lane 8: A.
rugulosus, NH4. Lane 9: A. niger, PSH. Lane 10: A. niger, Aa-20.
420 bp
123456789 10
Fig. 4. Dendrogram of genetic similarity generated by UPGMA analysis of sequences from the 18S
rDNA region of seven tannase-producing strains of Aspergillus. The bootstrap test considered 1,000
re-samplings.
A. fumigatus (GS)
A. rugulosus (NS4)
A. niger (GH1)
A. niger (PSH)
A. niger (Aa-20)
A. rugulosus (NH4)
A. ornatus (ESH)

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Tannase producTion by xerophilic Aspergillus
Micol. Apl. int., 23(1), 2011, pp. 21-27
(NS4) were closely related strains. Stability
of each node in the dendrogram was deter-
mined using a bootstrap test with 1,000 re-
samplings, which showed that nodes were
very stable. Molecular analyses showed ge-
netic relationships of fungal strains, which
produced tannase, in a similar way to their
morphological classification. The dendro-
gram showed genetic diversity among the
seven tannase producing strains.
ACKNOWLEDGMENTS
This research was supported by a project from the Mexican
Council of Science and Technology, through the funding
program: Fondo Sectorial SEP-CONACYT-2005-24348.
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