Advances in Microbiology, 2013, 3, 421-429
http://dx.doi.org/10.4236/aim.2013.35057 Published Online September 2013 (http://www.scirp.org/journal/aim)
Production of Invertases by Anamorphic (Aspergillus
nidulans) and Teleomorphic (Emericela nidulans)
Fungi under Submerged Fermentation Using
Rye Flour as Carbon Source
Juliana Nunes e Oliveira Alves, João Atílio Jorge, Luis Henrique Souza Guimarães*
Department of Biology, Faculty of Phylosophy, Sciences and Letters of Ribeirão Preto,
University of São Paulo, São Paulo, Brazil
Received June 4, 2013; revised July 3, 2013; accepted August 3, 2013
Copyright © 2013 Juliana Nunes e Oliveira Alves et al. This is an open access article distributed under the Creative Commons At-
tribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is prop-
erly cited.
ABSTRACT
The production of invertases by anamorph (A. nidulans) and teleomorph (E. nidulans) was investigated. The best level
of extracellular enzymatic production for anomorph was obtained in Khanna medium containing sucrose as carbon
source, whereas for teleomorph the best production was archived using M5 medium containing inulin as carbon source.
Despite this, rye flour was selected as carbon source. The extracellular enzyme production was higher for teleomorph
than that observed for anomorph for all carbon sources used. The enzyme production was inhibited by the addition of
fructose and glucose in the medium containing rye flour as carbon source. The best conditions to recover the higher
enzymatic activity were temperature of 54˚C - 62˚C and pH of 4.8 - 5.6 for both enzymes determined by experimental
design (CCRD). The stability of the temperatures at 40˚C and 50˚C were similar for both enzymes. The invertases from
the anomorph and teleomorph were activated by Mn2+, but the response of each one towards the presence of this cation
was different with best activation observed for the anomorph enzyme (+80%). The extracellular enzymes were able to
hydrolyze inulin, sucrose and raffinose. However, the affinity was higher for sucrose than inulin. In conclusion, the
carbon source assimilation and the invertase production, as well as the enzymes properties, were different for the ano-
morph and teleomorph mycelia.
Keywords: Invertase; Fructofuranosidase; Aspergillus; Emericela
1. Introduction
Invertases (EC 3.2.1.26) are enzymes which are able to
catalyze the hydrolysis of β 1-2 bonds from sucrose mole-
cule producing an equimolar mixture of D-glucose and
D-fructose named invert sugar. The first study on the in-
vertase activity was in 1828 using yeast cells. Nowadays,
many works showing the production, characterization
and application of these enzymes have been performed.
Among the invertases sources, microorganisms deserve
attention, especially filamentous fungi as Aspergillus ca-
sepitosus [1], Aspergillus niger [2], Paecylomyces va-
riotti [3], Fusarium oxysporum [4] and Rhizopus sp. [5],
among others. Generally, the fungal invertases have at-
tracted the attention of different sectors of the industry
because of their biotechnological potential. The invert
sugar, for example, can be used in food and beverage in-
dustries. Additionally, some invertases with fructosyl-
transferase activity can be used to produce fructooli-
sosaccharides (FOS), as 1-kestose, nystose and fructofu-
ranosyl nystose, which have important functional proper-
ties such as reduction of cholesterol and ammonia in the
blood, and stimulation of Bifidobacterium growth in the
human colon [6]. Despite the great number of filamen-
tous fungi investigated as producers of invertases, a com-
parative study on the enzyme production considering
both asexual phase and sexual phase has not been con-
sidered, especially for invertase. This aspect is important
to understand the differences on the metabolism at dif-
ferent stages of the life cycle of the fungi. A filamentous
fungus can receive two Latin names corresponding to the
*Corresponding author.
C
opyright © 2013 SciRes. AiM

J. N. OLIVEIRA ALVES ET AL.
422
anomorph (asexual stage) and teleomorph (sexual stage)
[7]. The anomorph is characterized by the production of
spores by mitosis while the teleomorph is characterized
by the presence of specific reproductive structure where
meiosis occurs. According to Geiser (2009) [8], the sex-
ual phase for 60% of the Aspergillus species is not ob-
served. The species that show both stages are mentioned
as pleomorphic [7]. In this article, in a comparative way,
the production and characterization of extracellular in-
vertases from anomorph (A. nidulans) and teleomorph (E.
nidulans) filamentous fungi are described, using an agro-
industrial residue as carbon source.
2. Material and Methods
2.1. Microorganisms and Culture Conditions
The strains Aspergillus nidulans (anamorph) and Eme-
ricela nidulans (teleomorph) were obtained from the soil,
identified by the Laboratory of Microbiology from Uni-
versidade Federal de Pernambuco, Pernambuco, Brazil
and deposited in the Culture Collection from Labora-
tory of Microbiology, Faculdade de Filosofia, Ciências e
Letras de Ribeirão Preto, University of São Paulo, Brazil.
Both strains were maintained in PDA (Potato Dextrose
Agar) medium previously autoclaved at 120˚C, 1.5 atm
for 30 minutes. The cultures were maintained at 4˚C for
30 days.
The submerged fermentation cultures were obtained
by the inoculums of 1 mL of aqueous spore suspension
(106 spores/mL) in 25 mL of Khanna medium [9] and
M5 [10] in 125 mL Erlenmeyer flasks, pH 6.0, for ana-
morphic and teleomorphic strains, respectively. The me-
dia were added with different carbon sources (1% w/v for
oligosaccharides and complex sources, and 2% w/v for
monosaccharides) and autoclaved at 120˚C, 1.5 atm for
30 minutes. Afterwards, the cultures were maintained for
different periods under agitation (100 rpm) at 30˚C.
2.2. Influence of Different Compounds on
Enzyme Production
The culture media for both strains were added (1% w/v)
with organic (yeast extract and peptone) and inorganic
[(NH4)2SO4; NH4H2PO4 and (NH4)2HPO4] nitrogen sources
or NaH2PO4, Na2HPO4, KH2PO4 and K2HPO4 as phos-
phate sources, separately according to that defined for
each experiment.
The influence of different concentrations (0% - 5%
w/v) of glucose and fructose added to the media on in-
vertase production was also analyzed. The microorgan-
isms were also grown in culture media containing 1%
(w/v) glucose as carbon source at 30˚C, for 24 h under
agitation (100 rpm). Then, the mycelia obtained through
vacuum filtration were washed with distilled water and
transferred to a new media containing 1% (w/v) glucose,
1% (w/v) sucrose or 1% (w/v) rye flour as carbon
sources at 30˚C, for 96 h under agitation (100 rpm).
2.3. Obtainment of the Extract Containing
Invertase
The submerged cultures were harvested under vacuum
filtration using Whatman filter paper No. 1. The free cell
filtrate was dialyzed against distilled water overnight at
4˚C and used as source of extracellular invertase. The
mycelia were washed with distilled water, dried using
filter paper, disrupted using acid clean sand, ressus-
pended using distilled water and centrifuged at 23,000 g
for 15 minutes at 4˚C. The supernatant obtained was dia-
lyzed against distilled water at 4˚C overnight and used as
source of intracellular invertase.
2.4. Partial Purification of the Extracellular
Enzymes
The extracellular crude extracts containing the invertase
were loaded in DEAE-Cellulose chromatographic col-
umn (14.5 × 1.0 cm) previously equilibrated with 10 mM
Tris-HCl buffer pH 7.5. Fractions of 3.0 mL were col-
lected at flow rate of 1.0 mL/min. The fractions contain-
ing invertase activity were eluted using a linear gradient
of NaCl (0 - 1.5 M) in the same buffer cited above. These
fractions were pooled, dialyzed against distilled water
overnight at 4˚C, lyophilized, ressuspended in 50 mM
Tris-HCl buffer pH 7.5 + 50 mM NaCl and loaded in
Sephacryl S-200 chromatographic column (180.0 × 1.0
cm) previously equilibrated using the same buffer. Frac-
tions of 1.2 mL were collected at flow rate of 0.3 mL/min.
The fractions containing invertase were pooled, dialyzed
against distilled water overnight at 4˚C and used for en-
zymatic assays.
2.5. Enzyme Assay and Protein Quantification
The invertase activity was determined using 2% sucrose
as substrate in sodium acetate buffer (100 mM, pH 5.0).
The reducing sugars after hydrolysis were quantified
using DNS according to Miller et al. (1959) [11] at 540
nm. One unit of enzyme activity was defined as the
amount of enzyme necessary to produce 1 μmol of glu-
cose per minute under the assay condition.
The protein was quantified using the Lowry method
[12] using BSA as standard.
2.6. Optimization of Temperature and pH of
Activity and Enzyme Stability
The influence of the temperature and pH (independent
variables) on the extracellular invertase activities (de-
pendent variable) from both strains was performed using
a Rotational Central Composite Design with 11 trials

J. N. OLIVEIRA ALVES ET AL. 423
with 3 repetitions at the central point and 2 axial points.
The software Statistica V8.0 (StatSoft, USA) was used to
perform the analysis of variance (ANOVA) with 95% of
significance level and to generate the surface response
plots.
The thermo stability was determined by the incubation
of the samples of enzymes aqueous solution at different
temperatures (40˚C - 70˚C) for 60 minutes and then as-
sayed for invertase activity as described above. The pH
stability was determined by the incubation of the enzyme
samples in different pH values (sodium acetate buffer pH
3.7 - 5.7; Tris-HCl buffer pH 7.0 - 8.0; CAPS buffer pH
9.0 - 10.0) for 60 minutes and then used for invertase
assay as described above.
2.7. Influence of Different Compounds on
Invertase Activity
The influence of 1 mM of different salts and β-mercap-
toethanol added to the reaction medium on invertase ac-
tivity was analyzed.
2.8. Hydrolysis of Different Substrates and
Kinetic Parameters
The hydrolytic activity from the extracellular invertases
on different substrates was analyzed using 0.5% (w/v) of
inulin, rafinose and sucrose in 100 mM sodium acetate
buffer pH 5.0. Mixtures (1:1 v/v) of inulin plus rafinose,
inulin plus sucrose and sucrose plus rafinose were also
analyzed. The kinetic parameters Km and Vmax were de-
termined for sucrose as substrate (0.1 - 20 mM) using
Lineweaver-Burk plot obtained with Origin Graph soft-
ware (OriginLab Corporation).
3. Results and Discussion
3.1. Enzyme Production
The Aspergillus genus comprises many fungal species
recognized as anamorph, which are classified in different
teleomorph genera as, for example, A. nidulans, which is
part of the teleomorph genus Emericela [13]. Aspergillus
spp. has been mentioned as a good producer of different
enzymes that act on polymers to obtain nutrients. In ad-
dition, these enzymes, according to their properties, have
biotechnological potential for many industrial purposes.
Among these enzymes, invertases deserve attention. A.
nidulans was selected because it has been mentioned as a
model for production of different extracellular enzymes
with biotechnological potential [14]. In addition, the
teleomorph E. nidulans was observed as an important
enzyme producer in our laboratory. So far, the compari-
son between invertase production by anamorph and
teleomorph filamentous fungi has not been elucidated.
The growth of filamentous fungi and, consequently,
the enzyme production, are influenced by the nutrients
used, including the carbon source. As it can be observed
in the Table 1, the highest enzymatic production for both
intra and extracellular invertases by E. nidulans was
achieved using inulin as substrate. On the other hand, the
sucrose was the best substrate for extracellular enzyme
production by A. nidulans. The fungi Aspergillus niger
and A. nidulans were mentioned as models for enzyme
secretion, since invertase production is induced by su-
crose [15]. Considering the best conditions obtained for
each one, the enzyme production by E. nidulans was
around 8-fold higher than that observed for intra and ex-
tracellular enzymes from A. nidulans. This fact shows
that the carbon source assimilation by filamentous fungi
is related to the life cycle period. The extracellular en-
zyme production by the teleomorph was higher than that
observed for the anamorph for all carbons sources tested.
The second best carbon source for both E. nidulans and A.
nidulans was rye flour. Considering this agroindustrial
product, the extracellular production was around 10-fold
higher for E. nidulans if compared to the A. nidulans and
similar for the intracellular enzymes. Agroindustrial
residues/products are low-cost substrates that can be used
to produce enzymes with industrial application. In addi-
tion, this type of carbon source is similar to the carbon
sources found in the natural environment of the filamen-
tous fungi, which are able to hydrolyze different poly-
mers.
Table 1. Influence of different carbon sources on produc-
tion of intra and extracellular invertases by A. nidulans
(anamorph) and E. nidulans (telomorph) using Submerged
Fermentation.
Invertase activity (total U)
Teleomorph Anamorph
Carbon
sources
Extra Intra Extra Intra
Without 182.9 ± 32.45.4 ± 1.2 7.8 ± 3.4 1.7 ± 0.7
Corncob 188.1 ± 13.017.5 ± 5.8 0.22 ± 0.4 9.0 ± 1.6
Crushed corn106.4 ± 19.050.1 ± 10.8 1.2 ± 0.2 4.8 ± 1.3
Fructose 64.4 ± 7.021.7 ± 3.6 3.5 ± 1.0 26.0 ± 7.3
Glucose 28.3 ± 8.430.1 ± 2.3 0 12.8 ± 0.7
Inulin 714.4 ± 100.3224.4 ± 6.5 11.4 ± 2.6 23.5 ± 4.5
Oat meal 279.8 ± 24.842.5 ± 2.4 19.0 ± 11.4 16.3 ± 4.5
Rice straw204.3 ± 11.92.5 ± 1.5 6.2 ± 1.5 3.1 ± 0.4
Rye flour 361.9 ± 30.628.3 ± 2.3 33.6 ± 8.3 30.0 ± 5.0
Sucrose 244.3 ± 17.030.0 ± 4.1 93.0 ± 12.3 28.3 ± 10.0
Sugar cane
bagasse 103.8 ± 5.016.6 ± 0.1 13.7 ± 2.0 31.9 ± 7.3
The fungi A. nidulans and E. nidulans were cultured in Khanna and M5
media, respectively, with initial pH 6.0, maintained at 30˚C, under orbital
agitation for 72 h.

J. N. OLIVEIRA ALVES ET AL.
424
The production of the extracellular enzyme by both
anamorph and teleomorph was inhibited when the mono-
saccharides glucose and fructose were used as supple-
ment in the media containing rye flour as the main car-
bon source (Figure 1). However, the influence of fruc-
tose on extracellular enzyme production by E. nidulans
was less pronounced than that observed for glucose.
Considering the production of the intracellular enzyme, it
is possible to see an increment for A. nidulans invertase
production at 0.5% (w/v) and 1.0% (w/v) of glucose,
differing from that observed for E. nidulans, with a re-
duction of enzyme production in all glucose concentra-
tions analyzed. The use of 1.0% (w/v) of fructose pro-
moted an increase in the intracellular enzyme production
by A. nidulans, but not for E. nidulans. On the other hand,
these monosaccharides are related with the regulation of
enzyme secretion at a range of 0.5% - 1.0% for A. nidu-
lans, since there was an increment in the level of intra-
cellular enzyme and reduction in the level of extracellu-
lar enzyme.
High concentrations of glucose and fructose promoted
a reduction in the production of intracellular enzyme.
This kind of inhibition is recognized as catabolic repres-
sion by carbon source. When the microorganisms were
grown for 24 h using glucose as carbon source and trans-
ferred to new media containing glucose, sucrose or rye
flour, the best induction of enzyme production (intra and
extracellular forms) was achieved with the latter (Figure
2). For anamorph, the extracellular invertase activity was
not detected when the fungus was grown in presence of
sucrose, a natural inducer, but the intracellular form was
observed in presence of both glucose and sucrose. These
saccharides are transported into the cell by sugar trans-
porters and, consequently, the sucrose can be hydrolyzed
by intracellular enzymes. When the anamorph is directly
cultured in presence of sucrose, it needs to hydrolyze this
carbon source in the extracellular medium to obtain nu-
trients for spore germination, growth and the mycelia
formation, what is not necessary when the mycelium
mass was previously obtained in glucose containing me-
dium. For invertase production by the fungus F. ox-
ysporum, the monosaccharides did not have a repressive
effect [4]. It is important to remember that these sugars
are products from the sucrose hydrolysis and can act as
regulators for enzyme production. In some filamentous
fungi, the products of hydrolysis of sucrose can act as
inducers [16]. The expression of SUC2 gene, that en-
codes two enzymes with different cellular localization in
Saccharomyces cerevisiae, is controlled by glucose [17]
and fructose repression [18]. The synthesis of invertase
was not induced by glucose and fructose in A. niger cul-
tures [19] while fructose acted as inducer in Penicillium
glabrum cultures [16].
The influence of organic and inorganic nitrogen
sources, and phosphate sources on invertase production,
Control 0.5 1 2 5
0
100
200
300
400
500
600
700
800
(a)
Extra invertase activity (Total U)
Glucose (%)
Control 0.5 1 2 5
0
50
100
150
200
250
300
350
(b)
Intra invertase activity (Total U)
Glucose (%)
Control 0.51 2 5
0
100
200
300
400
500 (c)
Extra invertase activity (Total U)
Fruc tose (% )
Control 0.51 2 5
0
50
100
150
200
250
300
350
(d)
Intra invertase activity (Total U)
Fructos e (% )
Figure 1. Influence of different concentrations of glucose ((a)
and (b)) and fructose ((c) and (d)) added to the culture me-
dium containing rye flour as the main carbon source on
extracellular ((a) and (c)) and intracellular ((b) and (d))
invertases from A. nidulans (□) and E. nidulans (■).
was also investigated (data not shown). In this case, the
production of extracellular invertase by A. nidulans was
improved 2.6-fold in the presence of peptone and yeast
extract compared to the control while for E. nidulans
extracellular enzyme production was improved 2-fold in

J. N. OLIVEIRA ALVES ET AL. 425
glucosesucroserye flour
0
50
100
150
200
250
300 A
Extra invertase activity (Total U)
Carbon source (1% w/v)
(a)
glucosesucroserye flour
0
5
10
15
20
25
30
35
40 B
Intra invertase activity (Total U)
Carbon source (1% w/v)
(b)
glucosesucroserye flour
0
100
200
300
400
500
600
700 C
Extra invertase activity (Total U)
Carbon source (1% w/v)
(c)
glucosesucroserye flour
0
100
200
300
400
500
600
700
800 D
Intra invertase ac tivity (Total U)
Carbon source (1% w/v)
(d)
Figure 2. Influence of glucose, sucrose and rye flour on ex-
tracellular ((a) and (c)) and intracellular ((b) and (d)) in-
vertases production by A. nidulans ((a) and (b)) and E. ni-
dulans ((c) and (d)) pre-cultured by 24 h in M5 and Khanna
media, respectively, containing 1% (w/v) glucose.
the presence of (NH4)2SO4 as also observed for intracel-
lular enzyme. The invertase production by other fugal
strains in the presence of peptone and yeast extract has
been mentioned [20-22]. Considering the phosphate
sources, the production of intra and extracellular inverta-
ses by E. nidulans was not significantly affected, while
the extracellular enzyme from A. nidulans was reduced.
On the other hand, the production of intracellular form
was not significantly affected by the phosphate sources
used. This fact indicates that the influence of the phos-
phate source is not on enzyme production, but on the
secretion. Production of invertases by A. caespitosus was
influenced positively in the presence of Na2HPO4 and
KH2PO4 [1] as also observed for S. cerevisiae GCB-K5
[23].
Another aspect that should be considered is the myce-
lium morphology. It is recognized that the mycelium is
heterogeneous considering the gene expression and, con-
sequently, affecting the growth and secretion. The het-
erogeneity has been mentioned for peripheral and central
zones of the colony. Under submerged fermentation,
three types of growth can be observed: 1) disperse, 2) as
clumps and 3) as pellets. These possibilities have consid-
erable impact on enzyme production [13]. For both ana-
morph and teleomorph stages, development as pellets
was observed (data not shown), indicating that the dif-
ferential enzyme production is not associated with the
type of growth. The differential expression of SUC genes
for anamorph and teleomorph stages can be hypothesized,
probably related to nutrient obtainment for development
of vegetative hyphae and reproductive structures.
3.2. Influence of Temperature and pH on
Extracellular Invertase Activity
The influence of the independent variables temperature
(X1) and pH (X2) on the extracellular activity (depend-
ent variable) was determined using factorial design (Ta-
ble 2). According to the ANOVA (Table 3) for enzyme
activity from E. nidulans, the quadratic temperature, lin-
ear and quadratic pH effects were significant (α < 0.05)
with R2 value of 0.92. For the enzyme activity from A.
nidulans, the linear temperature and pH, and quadratic
pH were significant (α < 0.05) with R2 value of 0.94. For
both enzymes, the values of Fcalc. were higher than the
value of Ftab, showing that the models are significant and
predictive (data not shown). The equations (Equation (1))
and (Equation (2)) represent the models for E. nidulans
and A. nidulans extracellular invertase activities, respec-
tively.
2
12
2
212
1
Total 267828.8 79.5.3 132.7
271.5 103.8
UX
X
X
X
XX
 

(1)
2
11
22
21
Total 242.832.12.377.7
69.7 2.3
UXX
XXX


2
X
(2)
The surface responses for both extracellular invertase
activities (E. nidulans and A. nidulans) are presented in
Figure 3. The best conditions to recover the higher en-
zymatic activity were temperature of 54˚C - 62˚C and pH

J. N. OLIVEIRA ALVES ET AL.
426
Table 2. Experimental design for influence of the inde-
pendent variables temperature and pH on the extracellular
invertase activities from A. nidulans (anamorph) and E. ni-
dulans (teleomorph).
Encoded (real)
values
Extracellular invertase activity
(total U)
Run
X1 (˚C) X2 (pH) Anamorph Teleomorph
1 1 (60) 1 (6.0) 172.2 ± 13.9 457.7 ± 13.7
2 1 (60) −1 (5.0) 271.6 ± 20.9 927.0 ± 35.3
3 −1 (50) 1 (6.0) 140.6 ± 7.8 461.4 ± 24.1
4 −1 (50) −1 (5.0) 235.4 ± 7.4 723.1 ± 41.3
5 0 (55) 0 (5.5) 227.8 ± 50.0 805.9 ± 60.3
6 0 (55) 0 (5.5) 254.5 ± 69.7 823.0 ± 55.1
7 0 (55) 0 (5.5) 246.1 ± 14.3 857.6 ± 45.6
8 1.41 (62) 0 (5.5) 270.7 ± 2.5 721.9 ± 14.6
9 −1.41 (48) 0 (5.5) 227.8 ± 1.64 639.1 ± 6.9
10 0 (55) 1.41 (6.2) 136.0 ± 23.9 421.8 ± 86.9
11 0 (55) −1.41 (4,8) 218.5 ± 17.2 661.6 ± 21.5
Table 3. ANOVA for the CCRD with temperature and pH
as independent variable for the extracellular invertase ac-
tivities from A. nidulans (anamorph) and E. nidulans (tele-
omorph).
Anamorph
Effects SQ GL QM Fcalc. p value
Temp. (L) 2063.25 1 2063.25 8.13062 0.035764*
Temp. (Q) 7.41 1 7.41 0.02922 0.870981
pH (L) 12081.32 1 12081.32 47.60872 0.000979*
pH (Q) 6856.18 1 6856.18 27.01808 0.003473*
1 × 3 5.38 1 5.38 0.02121 0.889897
Pure error 1268.81 5 253.76
Total 23077.51 10
SQ: Some of Square; GL: Liberty degree; QM: Medium square.
R2: 0.94; *α = 0.05
Teleomorph
Temp. (L) 12587.2 1 12587.2 2.68976 0.161919
Temp.(Q) 24868.6 1 24868.6 5.31419 0.069317*
pH (L) 143165.1 1 143465.1 30.59306 0.002649**
pH (Q) 104066.3 1 104066.3 22.23800 0.005263**
1 × 3 10770.3 1 10770.3 2.30151 0.189697
Pure error 23398.3 5 4679.7
Total 298306.9 10
SQ: Some of Square; GL: Liberty degree; QM: Medium square.
R2: 0.92; *α = 0.1; **α = 0.05
Temp.: temperature; 1 × 3: Temp.(L) × pH(L).
(a) (b)
Figure 3. Surface response for the influence of independent
variables, temperature and pH on extracellular invertase
activities from (a) A. nidulans and (b) E. nidulans.
of 4.8 - 5.6 for both enzymes. The range of temperature
obtained is in agreement with other observations as, for
example, 60˚C for enzymes from A. niger [20] and A.
niveus [24], among others. However, the pH value for A.
niger invertase activity was lower than that observed for
both E. nidulans and A. nidulans enzymes [19].
The extracellular invertases from E. nidulans and A.
nidulans were stable at 40˚C and 50˚C for 1 h, while at
60˚C, 22-minute half-life (T50) was observed for the for-
mer and 30 minutes for the latter (Figure 4). At 70˚C, the
stability was drastically reduced. The thermal stability
observed for both enzymes was higher than that observed
for the invertases from A. flavus [21] and A. caespitosus
[1]. The extracellular enzyme produced by E. nidulans
was fully stable at all pH analyzed for 1 h, differing from
the results observed for extracellular enzyme from A.
nidulans, which was stable only at pH 3.0 and from pH
7.0 to 10.0. The pH stability of invertases produced by
different strains of A. niger has been described. The en-
zyme produced by the strain PSSF21 was stable at a pH
range from 3.5 to 4.5 [20]. For strain IMI 303386, it was
stable from pH 4.0 to 8.0 and for strain AS0023, the en-
zyme was stable from pH 4.0 to 11.0 [25].
3.3. Influence of Different Compounds on
Invertase Activity
The influence of different salts on extracellular invertases
from E. nidulans and A. nidulans was analyzed (Table 4).
Among all salts used, the MnCl2 promoted the highest
activation for both enzymes from E. nidulans (+36%)
and A. nidulans (+80%). The Zn2+ and K+ were also good
stimulators of A. nidulans enzyme activity, while Ca2+
and Ba2+ were the best stimulators for E. nidulans en-
zyme. The enzyme activity was drastically inhibited by
Hg2+ and Pb2+ considering the anamorph, while NH4+
reduced drastically the enzyme activity from the teleo-
morph. The MgCl2, CuCl2, NH4Cl and Ag2SO4 reduced

J. N. OLIVEIRA ALVES ET AL. 427
Table 4. Effect of different salts and compounds on the ex-
tracellular invertases activities from A. nidulans (anamorph)
and E. nidulans (teleomorph).
Relative invertase activity (%)
Salts and compounds
(1 mM) Anamorph Teleomorph
Without 100 100
Ag2SO4 64.8 ± 14.1 94.3 ± 4.2
AlCl3 99.7 ± 0.1 100.4 ± 20.8
BaCl2 106.7 ± 13.7 122.1 ± 24.4
CaCl2 108.0 ± 12.1 132.8 ± 17.6
CoCl2 110.0 ± 15.2 112.5 ± 21.1
CuCl2 89.4 ± 45.3 80.5 ± 23.7
FeCl3 106.5 ± 47.9 109.3 ± 20.2
HgCl2 2.3 ± 0.7 82.2 ± 1.2
KCl 128.5 ± 31.0 77.0 ± 13.2
MgCl2 92.2 ± 6.4 74.0 ± 13.5
MnCl2 180.0 ± 57.3 136.4 ± 16.4
NaCl 110.1 ± 33.7 109.6 ± 16.6
NH4Cl 85.0 ± 14.4 20.0 ± 1.1
Pb(C2H3O3)2 23.5 ± 8.4 65.0 ± 9.0
ZnCl2 120.2 ± 29.7 102.3 ± 5.5
β-mercaptoethanol 107.0 ± 40.5 123.4 ± 24.5
EDTA 100.5 ± 53.6 111.9 ± 24.8
the activity of both enzymes. The β-mercaptoethanol
promoted an activation of 6% - 23% in the enzyme ac-
tivities. The invertases from both strains showed differ-
ent behaviors in the presence of the salt solutions used,
despite the activation by Mn2+, but at a different propor-
tion. The best enzyme activation by Mn2+ was observed
when we used 25 mM and 10 mM of MnCl2 for inverta-
ses from A. nidulans and E. nidulans, respectively (data
not shown). These data reinforce that the enzymes have
different properties according to the period of life cycle.
Activation of the enzyme activity by Mn2+ was also ob-
served for invertases from Aspergillus niveus [24], As-
pergillus phoenicis [26] and Aspergillus ochraceus [27].
Metallic ions can affect the aminoacid residues through
the induction of charge modifications promoting an in-
crease or decrease in the enzyme activity (sometimes due
to structural distortion).
3.4. Hydrolysis of Substrates and Kinetic
Parameters
The extracellular enzymes from anamorph and teleo-
morph fungi were able to catalyze the hydrolysis of all
substrates analyzed and their combinations (Table 5).
The high hydrolytic activity using only one substrate for
Table 5. Hydrolysis of different substrates by the extracel-
lular invertases from A. nidulans (anamorph) and E. nidu-
lans (teleomorph).
Specific activities (U·mg−1 of protein)
Substrates Anamorph Teleomorph
Inulin 14.5 ± 3.5 7.3 ± 3.6
Raffinose 138.8 ± 0.7 51.0 ± 8.9
Sucrose 106.7 ± 7.8 193.0 ± 12.3
Inulin + sucrose 443.5 ± 7.8 232.3 ± 5.7
Raffinose + sucrose659.5 ± 9.9 256.6 ± 1.1
Inulin + raffinose 432.5 ± 2.1 51.2 ± 1.4
0 102030405060
0
20
40
60
80
100
120 A
Extracellular residual activity (%)
Time
(
mi n
)
(a)
0 102030405060
0
20
40
60
80
100
120 B
Extracellular residual activity (%)
Time (min)
(b)
Figure 4. Thermal stability at 40˚C (-■-), 50˚C (-●-), 60˚C
(-▲-) and 70˚C (-▼-) for extracellular invertases produced
by (a) E. nidulans and (b) A. nidulans.
anamorph was observed with raffinose (138.8 U/mg of
protein), and sucrose (193.0 U/mg of protein) for teleo-
morph. However, the activities obtained with raffinose
plus sucrose were 4.7 and 1.3-fold higher than that re-
ported above, respectively. Additionaly, the S/I value
was 7.3 and 26.5 for extracellular enzymes from A.
nidulans and E. nidulans, respectively. The S/I value is
used as a parameter to help determine the real nature of
the enzymes, i.e., invertase or inulinase. Nevertheless,
this parameter cannot be considered isolated and other
aspects as kinetic parameters and structural characteriza-
tion should be performed. One of the factors that can
influence the S/I value is the inulin source, which can be

J. N. OLIVEIRA ALVES ET AL.
428
obtained from chicory and dahlia tubers among others.
The kinetic parameters for the partially purified ex-
tracellular enzymes from both A. nidulans (purification
of 9.3-fold and yield of 10.9%) and E. nidulans (purifica-
tion of 16.6-fold and yield of 32.1%) were determined
using sucrose as substrate. In this case, the A. nidulans
invertase showed higher affinity (Km = 2.0 mM) for the
substrate than the E. nidulans invertase (Km = 4.8 mM)
despite the Vmax value of 66.7 U/ mg of protein for the
former and 117.6 U/mg of protein for the latter. Both
enzymes showed best affinity for the substrate than the
enzymes produced by A. niveus [24] and A. phoenicis
[27]. The ratio Vmax/K m values were 32.6 U/mg of protein
mM−1 and 24.5 U/mg of protein mM−1 for extracellular
enzymes from A. nidulans and E. nidulans, respectively,
showing that the A. nidulans invertase is more efficient
to hydrolyze the substrate sucrose.
4. Conclusion
The invertase production was distinct from anamorph (A.
nidulans) and teleomorph (E. nidulans) stages of the
filamentous fungus life cycle, indicating that this enzyme
has different participation in the fungal metabolism of
nutrient uptake. This fact is confirmed by some distinct
properties of each enzyme such as substrate affinity and
ion influence. Although the different properties of both
enzymes showed biotechnological potential, future in-
vestigation to elucidate the true relation among invertase
synthesis, metabolism and life cycle of filamentous fungi
is a challenge that only has begun.
5. Acknowledgements
This work was supported by grants from the Fundação de
Amparo à Pesquisa do Estado de São Paulo (FAPESP)
and Conselho de Desenvolvimento Científico e Tecnol-
ógico (CNPq). J. A. J. is a recipient of Research Fellow-
ships of the CNPq. We thank Maurício de Oliveira for
the technical assistance.
REFERENCES
[1] A. C. P. Alegre, M. L. T. M. Polizeli, H. F. Terenzi, J. A.
Jorge and L. H. S. Guimarães, “Production of Thermosta-
ble Invertases by Aspergillus caespitosus under Sub-
merged or Solid State Fermentation Using Agroindustral
Residues as Carbon Source,” Brazilian Journal of Micro-
biology, Vol. 40, No. 3, 2009, pp. 612-622.
doi:10.1590/S1517-83822009000300025
[2] A. K. Balasubramaniem, K. V. Nagarajan and G. Parama-
samy, “Optimization of Media for β-Fructofuranosidase
Production by Aspergillus niger in Submerged and Solid
State Fermentation,” Process Biochemistry, Vol. 36, No.
12, 2001, pp. 1241-1247.
doi:10.1016/S0032-9592(01)00166-2
[3] M. A. Giraldo, T. M. Silva, F. Salvato, H. F. Terenzi, J. A.
Jorge and L. H. S. Guimarães, “Thermostable Invertases
from Paecylomyces variotii Produced under Submerged
and Solid-State Fermentation Using Agroindustrial Resi-
dues,” World Journal of Microbiology and Biotechnology,
Vol. 28, No. 2, 2012, pp. 463-472.
doi:10.1007/s11274-011-0837-9
[4] B. Wolska-Mitaszko, J. Jaroszuk-Scise and K. Pszeniczn,
“Isoforms of Trehalase and Invertase of Fusarium ox-
ysporum,” Mycological Research, Vol. 111, No. 4, 2001,
pp. 456-465. doi:10.1016/j.mycres.2007.01.018
[5] A. J. Goulart, P. R. Adalberto and R. Monti, “Purificação
Parcial de Invertase a Partir de Rhizopus sp. em Fer-
mentação Semi-Sólida,” Alimentação e Nutrição, Vol. 14,
No. 2, 2003, pp. 199-203.
[6] L. Novaki, S. D. M. Hasan, M. K. Kadowaki and D.
Andrade, “Produção de Invertase por Fermentação em
Estado Sólido a Partir de Farelo de Soja,” Engevista, Vol.
12, No. 2, 2010, pp. 131-140.
[7] W. Gams and K. A. Seifert, “Anamorphic Fungi,” In:
Encyclopedia of Life Science (ELS), John Wiley & Sons,
Ltd., Chichester, 2008.
doi:10.1002/9780470015902.a0000351.pub2
[8] D. M. Geiser, “Sexual Structures in Aspergillus: Mor-
phology, Importance and Genomics,” Medical Mycology,
Vol. 47, No. S1, 2009, pp. S21-S26.
doi:10.1080/13693780802139859
[9] P. Khanna, S. S. Sundari and N. J. Kumar, “Production,
Isolation and Partial Purification of Xylanase from As-
pergillus sp.,” World Journal of Microbiology and Bio-
technology, Vol. 11, No. 2, 1995, pp. 242-243.
doi:10.1007/BF00704661
[10] R. M. Peralta, H. F. Terenzi and J. A. Jorge, “β-D Glyco-
sidase Activities of Humicola grisea: Biochemical and
Kinetic Characterization of a Multifunctional Enzyme,”
Biochemica et Biophysica Acta, Vol. 1033, No. 3, 1990,
pp. 243-249. doi:10.1016/0304-4165(90)90127-I
[11] G. L. Miller, “Use of Dinitrosalicylic Acid Reagent for
Determination of Reducing Sugar,” Analytical Chemistry,
Vol. 31, No. 3, 1959, pp. 427-429.
doi:10.1021/ac60147a030
[12] O. H. Lowry, N. J. Rosebrough, A. L. Farr and R. J. Ran-
dall, “Protein Measurement with the Folin Phenol Re-
agent,” Journal of Biological Chemistry, Vol. 193, 1951,
pp. 265-275.
[13] P. Krijgsheld, R. Bleichrodit, G. J. van Veluw, F. Wang,
W. H. Müller, J. Dijksterhuis and H. A. B. Wösten, “De-
velopment in Aspergillus,” Studies in Mycology, Vol. 74,
No. 1, 2013, pp. 1-29. doi:10.3114/sim0006
[14] A. P. MacCabe, M. Orejas, F. N. Tamayo, A. Villanueva
and D. Ramon, “Improving Extracellular Production of
Food-Use Enzymes from Aspergillus nidulans,” Jounal of
Biotechnology, Vol. 96, No. 1, 2002, pp. 43-54.
doi:10.1016/S0168-1656(02)00036-6
[15] G. L. F. Wallis, F. W. Hemming and J. F. Peberdy, “Se-
cretion of Two β-Fructofuranosidases by Aspergillus ni-
ger Growing in Sucrose,” Archieves of Biochemistry and
Biophysics, Vol. 345, No. 2, 1997, pp. 214-222.
doi:10.1006/abbi.1997.0228
[16] M. C. Rubio, M. C. Maldonado, P. Aznar and A. Navarro,

J. N. OLIVEIRA ALVES ET AL.
429
“Production y Caracterizacion de uma Invertasa Extra-
cellular de Penicillium glabrum,” La Alimentacion Lati-
noamerica, Vol. 247, 2003, pp. 40-45.
[17] R. Trumbly, “Glucose Repression in the Yeast Saccha-
romyces cerevisiae,” Molecular Microbiology, Vol. 6, No.
1, 1992, pp. 15-21.
doi:10.1111/j.1365-2958.1992.tb00832.x
[18] J. Dynesen, H. P. Smits, L. Olsson and J. Nielsen, “Car-
bon Catabolite Repression of Invertase during Batch Cul-
tivations of Saccharomyces cerevisiae: The Role of Glu-
cose, Fructose and Mannose,” Applied Microbiology and
Biotechnology, Vol. 50, No. 5, 1998, pp. 579-582.
doi:10.1007/s002530051338
[19] M. C. Rubio and A. R. Navarro, “Regulation of Invertase
Synthesis in Aspergillus niger,” Enzyme and Microbial
Technology, Vol. 39, No. 4, 2006, pp. 601-606.
doi:10.1016/j.enzmictec.2005.11.011
[20] P. P. Reddy, G. S. Reddy and M. B. Sulochana, “Highly
Termostable β-Fructofuranosidase from Aspergillus niger
PSSF21 and Its Application in the Synthesis of Fructoo-
ligosacharides from Agro Industrial Residue,” Asian Jour-
nal of Biotechnology, Vol. 2, No. 2, 2010, pp. 86-98.
[21] C. Uma, D. Gomathi, C. Muthulakshmi and V. K. Gola-
palakrishnnan, “Production, Purification and Characteri-
zation of Invertases by Aspergillus flavus Using Fruit
Peel Was as Substrate,” Advances in Biological Research,
Vol. 4, No. 1, 2010, pp. 31-36.
[22] H. N. Bhatti, A. Muhammad, A. Abbas, R. Nawaz and M.
A. Sheikh, “Studies on Kinetics and Thermostability of
Novel Acid Invertase from Fusarium solani,” Journal of
Agriculture and Food Chemistry, Vol. 54, No. 13, 2006,
pp. 4617-4623. doi:10.1021/jf053194g
[23] K. Shafiq, S. Ali and I. Ul-Hag, “Effect of Different Min-
eral Nutrients on Invertase Production by Saccharomyces
cerevisiae GCB-K5,” Biotechnology, Vol. 1, 2002, pp.
40-44. doi:10.3923/biotech.2002.40.44
[24] L. H. S. Guimarães, A. F. Somera, H. F. Terenzi, M. L. T.
M. Polizeli and J. A. Jorge, “Production of β-Fructofu-
ranosidases by Aspergillus niveus Using Agroindustrial
Residues as Carbon Sources: Characterization of an In-
tracellular Enzyme Accumulated in the Presence of Glu-
cose,” Process Biochemistry, Vol. 44, No. 2, 2009, pp.
237-241. doi:10.1016/j.procbio.2008.10.011
[25] Q. D. Nguyen, J. M. Rezessy-Szabó, M. K. Bhat and Á.
Hoschke, “Purification and Some Properties of β-Fructo-
furanosidase from Aspergillus niger IMI 303386,” Proc-
ess Biochemistry, Vol. 40, No. 7, 2005, pp. 2461-2466.
doi:10.1016/j.procbio.2004.09.012
[26] C. B. Rustiguel, A. H. C. Oliveira, H. F. Terenzi, J. A.
Jorge and L. H. S. Guimarães, “Biochemical Properties of
an Extracellular β-D-Fructofuranosidase II Produced by
Aspergillus phoenicis under Solid-Sate Fermentation Us-
ing Soy Bran as Substrate,” Electronic Journal of Bio-
technology, Vol. 14, No. 2, 2011.
[27] L. H. S. Guimarães, H. F. Terenzi, M. L. T. M. Polizeli
and J. A. Jorge, “Production and Characterization of a
Thermostable Extracellular β-D-Fructofuranosidase Pro-
duced by Aspergillus ochraceus with Agroindustrial Resi-
dues as Carbon Sources,” Enzyme and Microbial Tech-
nology, Vol. 42, No. 1, 2007, pp. 52-57.
doi:10.1016/j.enzmictec.2007.07.021