Research Article
Production and Catalytic Properties of Amylases from
Lichtheimia ramosa and Thermoascus aurantiacus by
Solid-State Fermentation

Ana Paula Aguero de Oliveira,1 Maria Alice Silvestre,1

Nayara Fernanda Lisboa Garcia,1 Heloíza Ferreira Alves-Prado,2 André Rodrigues,3

Marcelo Fossa da Paz,1 Gustavo Graciano Fonseca,4 and Rodrigo Simões Ribeiro Leite1

1Laboratory of Enzymology and Fermentation Processes, Faculty of Biological and Environmental Sciences,
Federal University of Grande Dourados (FCBA/UFGD), Rodovia Dourados/Itahum, km 12, 79804-970 Dourados, MS, Brazil
2Faculty of Engineering, Department of Phytotechnology, Food Technology and Social Economy,
São Paulo State University (FEIS/UNESP), Avenida Brasil, No. 56, 15385-000 Ilha Solteira, SP, Brazil
3Laboratory of Fungal Ecology and Systematics, Biosciences Institute, Department of Biochemistry and Microbiology,
São Paulo State University (IB/UNESP), Avenida 24A, No. 1515, 13506-900 Rio Claro, SP, Brazil
4Laboratory of Bioengineering, Faculty of Biological and Environmental Sciences,
Federal University of Grande Dourados (FCBA/UFGD), Rodovia Dourados/Itahum, km 12, 79804-970 Dourados, MS, Brazil

Correspondence should be addressed to Rodrigo Simões Ribeiro Leite; simoesbio@yahoo.com.br

Received 25 February 2016; Revised 11 May 2016; Accepted 31 May 2016

Academic Editor: Maurizio Petruccioli

Copyright © 2016 Ana Paula Aguero de Oliveira et al. This is an open access article distributed under the Creative Commons
Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is
properly cited.

The present study compared the production and the catalytic properties of amylolytic enzymes obtained from the fungi Lichtheimia
ramosa (mesophilic) andThermoascus aurantiacus (thermophilic). The highest amylase production in both fungi was observed in
wheat bran supplemented with nutrient solution (pH 4.0) after 96 hours of cultivation, reaching 417.2U/g of dry substrate (or
41.72U/mL) and 144.5U/g of dry substrate (or 14.45U/mL) for L. ramosa and T. aurantiacus, respectively. The enzymes showed
higher catalytic activity at pH 6.0 at 60∘C.The amylases produced by L. ramosa and T. aurantiacuswere stable between pH 3.5–10.5
and pH 4.5–9.5, respectively. The amylase of L. ramosa was stable at 55∘C after 1 hour of incubation, whereas that of T. aurantiacus
maintained 60%of its original activity under the same conditions. Both enzymeswere active in the presence of ethanol.The enzymes
hydrolyzed starch from different sources, with the best results obtained with corn starch. The enzymatic complex produced by L.
ramosa showed dextrinizing and saccharifying potential. The enzymatic extract produced by the fungus T. aurantiacus presented
only saccharifying potential, releasing glucose monomers as the main hydrolysis product.

1. Introduction

Population growth prompts the discovery of new food and
energy sources, which will be possible with the best use of
the polysaccharides that constitute the vegetal biomass [1, 2].
Starch is one of the major vegetable reserve compounds,
composed of glucose units linked by glycosidic bonds. This
polysaccharide has been used as a major energy source in
several trophic levels of the food chain [3].

The saccharification of starch enables the obtainment of
maltose or glucose syrups as sweeteners for the food industry

and for the production of ethanol derived from fermentation
processes [1, 4]. Hydrolysis of starch by enzymatic methods
has advantages compared to that with chemical methods,
such as operating under mild conditions of pH and tem-
perature, preventing equipment corrosion, and subsequent
neutralization steps. Enzymes have substrate specificity, elim-
inating the formation of undesirable by-products, commonly
observed in acid hydrolysis [5, 6].

In addition to the production of biofuels from starch
sources, amylases are applied in bread-making, in detergents

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Volume 2016, Article ID 7323875, 10 pages
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2 The Scientific World Journal

for industrial cleaning, and in the degumming processes of
textile fibers. Thus, amylases represent about 25–33% of the
global enzymemarket [7, 8]. However, the production cost of
enzymes on an industrial scale is still elevated. It is estimated
that the formulation of microbial culture medium represents
about 30–40% of the final cost of an enzyme [9].

The need for reducing the production costs of industrial
enzymes encourages the search for low-costmicrobial culture
medium. In this regard, agroindustrial residues have been
used as substrates for microbial-derived enzymes under
solid-state fermentation (SSF) [10, 11] and several published
works have used this process for the production of various
enzymes [6, 12–14].

Solid-state fermentation has some similarities to the
natural environment of the microorganisms, especially for
the filamentous fungi, as a promising alternative for the
cultivation of these organisms. However, some disadvantages
can also be highlighted owing to the low accessibility of the
substrates and low homogeneity of the medium, making it
difficult to control the operating parameters (pH, temper-
ature, moisture, and others). These aspects have stimulated
research to improve the use of SSF for industrial processes
[10, 11].

In this study, we compared the production and the
catalytic properties of amylases produced by the filamentous
fungi Lichtheimia ramosa andThermoascus aurantiacus, cul-
tivated by SSF in agroindustrial residues. These strains were
isolated in the Midwest Brazilian region (less explored for
microbial bioprospecting) and selected for amylase produc-
tion.

2. Materials and Methods

2.1.Microorganisms. Thefilamentous fungi L. ramosa (meso-
philic species) and T. aurantiacus (thermophilic species)
were assessed for amylase production.The microorganism L.
ramosa was isolated from sugarcane bagasse processed in a
sugarcane ethanol plant [15]. The fungus T. aurantiacus was
isolated from leaf litter of Atlantic seasonal forest fragment
located in Dourados, Mato Grosso do Sul State, Brazil.
Working-stock cultures of both fungi were incubated on
Sabouraud dextrose agar medium at 4∘C.

2.2. Inoculum. The fungi were cultivated in 250mL Erlen-
meyer flask containing 40mL of Sabouraud dextrose agar,
incubated for 48 hours at 28∘C and 45∘C for L. ramosa
and T. aurantiacus, respectively.The fungal suspensions were
obtained by gently scraping the surface of the culturemedium
and putting it into 30mL of nutrient solution (0.1% ammo-
nium sulfate, 0.1%magnesium sulfate heptahydrate, and 0.1%
ammonium nitrite, Merheb-Dini et al. [16]). The fungi were
inoculated in the agroindustrial residues by transfer of 5mL
of the microbial suspension.

2.3. Solid-State Fermentation. The fungi were cultivated in
250mL Erlenmeyer flasks containing 5 g of different agroin-
dustrial residues (wheat bran, soy bran, corn cob, corn
straw, rice peel, and sugarcane bagasse). Other fermentative
parameters were also varied in this study such as the nutrient

solution pH (3.0–5.0), the initial moisture (50–90%), and
the cultivation time (24–168 h). The best growth conditions
established in each step were adopted in subsequent assays.
All material was autoclaved for 20min at 121∘C. All assays
were performed in duplicate and the values described repre-
sent the respective averages.

2.4. Enzyme Extraction. Enzymes were obtained from the
fermented residues by adding 50mL of distilled water and
shaking constantly at 100 rpm for 1 h. The samples were
filtered through nylon cloth and centrifuged at 3,000×g for
5min at 5∘C. The supernatant was considered the extracellu-
lar enzymatic extract and used in the subsequent steps.

2.5. Determination of Amylase Activity. The enzyme activity
was determined by adding 0.1mL of enzymatic extract to
0.9mL of sodium acetate buffer (0.1M, pH 5.0) containing 1%
corn starch. After 10minutes of reaction at 50∘C, the reducing
sugars released were quantified by measuring the absorbance
at 540 nm by the DNSmethod (3,5-dinitrosalicylic acid) [17].
One unit of enzymatic activity was defined as the amount of
enzyme required to release 1 𝜇mol of the product per minute
of reaction.

2.6. Biochemical Characterization of
the Amylases Produced by SSF

2.6.1. Effect of pH and Temperature. The optimum pH was
determined bymeasuring the enzymatic activity at 50∘Cwith
different pH conditions (3.0–8.0) by using McIlvaine buffer
0.1M. The optimum temperature was determined by mea-
suring the enzymatic activity from 30 to 75∘C at the respec-
tive optimal pH of each enzyme. The enzymatic stability
to variations in pHwas assessed by incubating the enzymes at
25∘C for 24 h at different pH range.The following buffers were
used:McIlvaine 0.1M (3.0–8.0), Tris-HCl 0.1M (8.0–8.5), and
Glycine-NaOH 0.1M (8.5–10.5). The thermostability of the
enzymes was assessed by incubating for 1 h at different tem-
peratures (30–75∘C). The residual activity was determined
under optimum conditions of pH and temperature [18].

2.6.2. Effect of Ethanol on Enzymatic Activity. Enzyme activ-
ity was quantified by adding different concentrations of
ethanol (0–30%) to the reaction mixture. The assays were
performed at 50∘C in McIlvaine buffer (0.1M, pH 6.0)
containing 1% corn starch [18].

2.6.3. Enzymatic Hydrolysis of Starch Derived from Several
Vegetal Sources. Enzymatic assays were performed using
potato, corn, wheat, and cassava starch (1%) as substrates.
The enzymatic reactions were performed in McIlvaine buffer
0.1M (pH 6.0). The sugar released was quantified using the
DNS method [17].

2.6.4. Dextrinization Potential of Enzymatic Extracts. Dex-
trinizing activity was assessed using corn starch (1%) as enzy-
matic substrate in McIlvaine buffer 0.1M (pH 6.0) and the



The Scientific World Journal 3

Table 1: Amylase production by solid-state fermentation in several
agroindustrial residues after 120 hours of cultivation with 75%mois-
ture at pH 5.0. Average production with different letters indicates
significant differences (𝑃 < 0.0001) according to Tukey test.

Substrate Lichtheimia ramosa
(U/g dry substrate)

Thermoascus aurantiacus
(U/g dry substrate)

Sugarcane
bagasse — 3.7 ± 0.0c

Corn
straw 8.8 ± 0.5b 6.2 ± 0.0bc

Soy bran 10.5 ± 0.3b 7.6 ± 0.1b

Rice peel 3.9 ± 0.1b 4.7 ± 0.1bc

Corn cob 9.2 ± 0.5b 5.2 ± 0.9bc

Wheat
bran 320.7 ± 6.1a 44.2 ± 1.0a

iodometric methods described by Fuwa [19] and Pongsawadi
and Yagisawa [20]. The reaction mix contained 0.1mL of
enzymatic extract in 0.3mL of buffer solution containing
starch. After 10 minutes at 60∘C, the reaction was stopped by
adding 4mL of 0.2M HCl. Finally, 0.5mL of reactive iodine
and 10mL of distilled water were added. The absorbance was
quantified at 700 nm. One unit of activity was defined as the
amount of enzyme required to reduce the intensity of the
blue color of the starch iodine complex by 10% per minute
of reaction.

2.6.5. Saccharification Potential of Enzymatic Extracts. The
saccharifying activity of the enzymatic extracts was assessed
by the glucose oxidase/peroxidase method, using 1% corn
starch as enzymatic substrate in McIlvaine buffer 0.1M (pH
6.0) [21]. The reaction mixture contained 0.1mL of the enzy-
matic extract in 0.4mL of buffer solution containing starch.
After 10 minutes at 60∘C, the reaction was stopped in ice
bath.The glucose released was quantified using an enzymatic
colorimetric kit (Glicose-PP, Gold Analisa Diagnóstica Ltda,
Brazil). The absorbance was measured at 505 nm. One unit
of enzyme activity was defined as the amount of enzyme
required to release 1 𝜇mol of glucose per minute of reaction.

2.7. Statistical Analysis. All experiments were performed as
duplicates and the results are presented as the mean of two
independent tests. Statistical analysis of the data included
a one-way ANOVA followed by Tukey’s test with a 5%
significance level.

3. Results and Discussion

3.1. Amylase Production by SSF. Among the tested substrates,
both microorganisms showed higher production of amylase,
on wheat bran, reaching 320.4U/g of dry substrate (or
32.04U/mL) for L. ramosa and 44.2U/g of dry substrate
(or 4.42U/mL) for T. aurantiacus (Table 1). Regarding the
analysis of variance, the results were significant with ANOVA
𝑃 value being < 0.0001, considered extremely significant.

In general, wheat bran has the highest amount of macro-
and micronutrients when compared to other agricultural
residues such as sugarcane bagasse, rice straw, wheat straw,
and rice bran [22]. According to Haque et al. [23], wheat
bran consists of a complex substrate rich in proteins, car-
bohydrates, minerals, lipids, and vitamins favoring microbial
growth and enzyme production. Additionally, Kunamneni et
al. [24] reported that wheat bran was the best substrate for
amylase production by the fungusThermomyces lanuginosus.
Moreira et al. [25] demonstrated a higher amylase production
when different Aspergillus species were cultivated on wheat
bran, increasing the production of the enzyme by up to 10-
fold compared to that obtained by growth on other residues.

Considering the data presented in Table 1, wheat bran
was used in subsequent cultivation steps to evaluate different
fermentative parameters for amylase production, such as
initial moisture, pH of the nutrient solution, and cultivation
time. There are no significant differences between 55 and 65
but it is clear that highest amylase production was obtained
when L. ramosa was cultivated at 60% moisture (w/v),
with a maximum value of 369.8U/g of dry substrate (or
36.98U/mL). The fungus T. aurantiacus increased amylase
production, about 65.1 U/g of dry substrate (or 6.51 U/mL),
when grown on wheat bran containing 65% moisture (w/v)
(Figure 1(a)). 𝑃 value is 0.0004, considered extremely signifi-
cant.

Moisture in the substrate is a fundamental parameter in
SSF. The medium should contain enough moisture to allow
microbial physiological activities but it cannot exceed the
substrate absorption limit, leaving free water among the solid
particles. Excess moisture results in the decrease of porosity,
affects gas exchange, and favors bacterial contamination,
disfavoring the growth and enzyme production [26, 27].

Significant difference was found in the study of nutrient
solution pH (3.0–5.0). The highest amylase production was
obtained in cultures supplemented with nutrient solution
adjusted to pH 4.0 (Figure 1(b)), with enzymatic activity of
407.9U/g of dry substrate (or 40.79U/mL) for L. ramosa and
103.1 U/g of dry substrate (or 10.31 U/mL) for T. aurantiacus.
Omemu et al. [28] reported that pH 4.0 was optimal for
amylase production by Aspergillus niger and the same was
observed by Gomes et al. [29] for Aspergillus flavus.

The last fermentative parameter evaluated in this study
was the cultivation time. All parameters defined as optimum
in the previous assays were employed for this experiment.
Both organisms had statistical significance for the highest
enzymatic production at 96 hours of cultivation, at which
point 417.2U/g of dry substrate (or 41.72U/mL) for L.
ramosa and 144.5U/g of dry substrate (or 14.45U/mL) for T.
aurantiacus were obtained (Figure 1(c)).

The results showed a considerable decrease in enzymatic
activity, after reaching the production peak (Figure 1(c)).
This reduction is likely due to the prolonged incubation
period, which may have led to (i) nutrient depletion in the
culturemedium, (ii) variations in the pH due to themicrobial
metabolic activity, or (iii) the presence of proteolytic enzymes
[30, 31].

After the optimization of the fermentative process, amy-
lase production by L. ramosa rose from 320.4 to 417.2U/g of



4 The Scientific World Journal

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Figure 1: Amylase production by L. ramosa and T. aurantiacus by solid-state fermentation in wheat bran. (a) Influence of initial substrate
moisture; (b) influence of initial cultivation pH; (c) influence of cultivation time. Average productionwith different letters indicates significant
differences according to Tukey test.



The Scientific World Journal 5

dry substrate, an increase of approximately 30% (Table 1 and
Figure 1(c)). On the other hand, the most notable result was
the increase of amylase production of T. aurantiacus from
44.2 to 144.5U/g of dry substrate, representing a gain of more
than 200%when compared to the initial cultures (Table 1 and
Figure 1(c)).

Considering the several studies on amylase production,
our results are promising. Bhatti et al. [32] reported the
glucoamylase production (about 61.35U/g of dry substrate)
when cultivated in Fusarium solani by SSF using wheat bran
as substrate. Moreira et al. [25] obtained amylases by SSF
using wheat bran as substrate with no additional carbon
sources, achieving approximately 350, 240, and 210U/g of dry
substrate by the fungi Aspergillus flavus, Aspergillus fumiga-
tus, andAspergillus tamari, respectively.The authors reported
a 1.5- to 2-fold increase in the production of amylases inwheat
bran supplemented with various carbon sources. Kunamneni
et al. [24] obtained maximum amylase production (about
534U/g of substrate) after 120 hours in SSF forThermomyces
lanuginosus, in wheat bran supplemented with 1% soluble
starch.

3.2. Biochemical Characterization of
the Amylases Produced by SSF

3.2.1. Effect of pH and Temperature. The amylases produced
by both fungi showed optimum activity at pH 6.0 and at 60∘C
(Figures 2(a) and 2(b)).

The enzymes evaluated in this study showed maximum
activity at temperatures higher than those described by Alva
et al. [30] for amylases produced by Aspergillus in SSF. This
suggests high structural stability of enzymes produced by
L. ramosa and T. aurantiacus. However, our results showed
similarity to previously published articles. Giannesi et al.
[33] reported that amylase obtained from different microbial
sourcesmay have optimumpHbetween 4.5 and 7.0.The same
authors described 60∘C as the optimum temperature for 𝛼-
glucosidase purified from enzymatic extracts of Chaetomium
thermophilum var. coprophilum.

Both enzymes were stable in a wide pH range. The amy-
lase produced by L. ramosamaintained its activity at pH from
3.5 to 10.5 (Figure 2(c)) and the enzymeproduced byT. auran-
tiacus was stable at pH 4.5 to 9.5 (Figure 2(c)). According to
Michelin et al. [34] the maintenance of enzymatic activity in
a wide pH range is an advantage for application in industrial
processes, because it requires lower pH adjustments between
the sequential treatments of liquefaction and saccharification
of starch.

The amylase produced by L. ramosa retained its catalytic
activity after 1 hour at 55∘C and 75% of its original activity
when incubated for the same period at 60∘C (Figure 2(d)).
The amylase produced byT. aurantiacus remained stable after
1 hour at 50∘C; when the temperature was raised to 60∘C, the
enzyme showed only 25% of its initial activity (Figure 2(d)).

The results presented in Figure 2 indicate that the amylase
produced by the mesophilic fungus L. ramosa had a higher
structural stability compared to the enzyme produced by
the thermophilic fungus T. aurantiacus. This characteristic is
very appreciable in industrial applications, considering that

industrial environment differs significantly from laboratory
conditions, in regard to the control of pH and temperature.
The structural stability of the enzymes is indispensable to
withstand variations in these parameters during different
processes. According to Bruins et al. [35], there is no complex
structural system that distinguishes a stable protein from
another with less stability. Small molecular alterations as
the number of hydrogen and disulfide bonds, folding, and
hydrophobicity degree of the molecule and the amount of
ionic linkages can produce large modifications in the stability
of a protein.

Although it is not usual to observe high thermostability
in enzymes produced by mesophilic microorganisms, results
from previous studies support this possibility [18]. Gomes
et al. [29] reported thermostability ranging from 10 to 60∘C
for amylases produced by Aspergillus flavus (mesophilic) and
from 10 to 40∘C for amylases of Thermomyces lanuginosus
(thermophilic).

3.2.2. Effect of Ethanol on Enzymatic Activity. Ethanol inhi-
bition is a strong trend in the study of certain enzymes,
since they can be exposed to substantial concentrations of
alcohol for various industrial applications [6]. The results
demonstrate that amylase produced by L. ramosa showed
residual activity around 65% and T. aurantiacus amylase
showed greater than 90% residual activity when incubated at
concentrations of 10% ethanol (Figure 3).

This data reveals that both enzymes have potential for
use in alcoholic fermentation processes derived from starch
sources. In conventional alcoholic production processes, con-
centrations higher than 10% ethanol are extremely harmful
to the fermenters organisms [36]. The increase of catalytic
potential by ethanol may be associated with transferase
activity, with ethanol being used as an intermediate acceptor
by the enzyme; thus, resulting in increased reaction rate
[18, 37].

3.2.3. Enzymatic Hydrolysis of Starch Derived from Several
Vegetal Sources. The action of amylolytic enzymes on starch
from different vegetal sources was evaluated. Enzymatic
extracts showed the highest catalytic potential on corn starch,
obtaining 34.94U/mL and 12.26U/mL for amylases produ-
ced by L. ramosa and T. aurantiacus, respectively (Figure 4).

Differences in the action of amylolytic enzymes may
be related to the composition of the starch molecules, in
particular, the amylose content and the extent of their chains.
The lipid, protein, and mineral levels may also influence the
enzyme activity. Another factor that may be related to the
susceptibility of starch granules to enzymatic attack is the
pore-size present on their surface [38].

The content of amylose and amylopectin varies according
to the botanical source, providing specific characteristics to
starch, reflecting in the granule architecture and its textural
properties [39]. The corn starch has a higher amount of
amylose, compared to other starches and consequently lower
amylopectin content, favoring enzymatic degradation. The
amylopectin exhibits highly ramified structure with high
molecular weight, disfavoring the catalytic performance of
amylolytic enzymes [40].



6 The Scientific World Journal

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Figure 2: Continued.



The Scientific World Journal 7

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L. ramosa T. aurantiacus

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Figure 2: Effect of pH and temperature on amylase activity of L. ramosa and T. aurantiacus. (a) Amylase activity at different pHs and (b)
at different temperatures; (c) amylase residual activity after 24 h at different pHs and (d) after one hour at different temperatures (each data
point was the average of two replicate determinations, and the error bars show the data ranges).

0 5 10 15 20 25 30

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Figure 3: Effect of ethanol on amylase activity. (a) L. ramosa; (b) T. aurantiacus (each data point was the average of two replicate
determinations, and the error bars show the data ranges).

3.2.4. Dextrinization and Saccharification Potential of Enzy-
matic Extracts. Comparing the action of enzymatic extracts
on the starch molecule by different colorimetric methods,
we observed that the enzymatic extract produced by L.
ramosa showed high depolymerizing potential (dextriniz-
ing activity), resulting in high amount of reducing chain
ends (Figures 5(a) and 5(b)), some of which being glucose
monomers, measured by glucose oxidasemethod, specific for
determining glucose (Figure 5(c)).

These results suggest that the enzymatic extract obtained
by L. ramosa under the conditions described above shows
synergic activity of dextrinizing and saccharifying enzymes
(endo and exoamylases production). The synergic action of

amylases produced by a single microorganism is not com-
monly found. However, previous studies confirm this possi-
bility. Silva et al. [41] reported the production of dextriniz-
ing and saccharifying enzymes by the filamentous fungus
Rhizomucor pusillus. Ezeji and Bahl [42] also reported the
potential for producing 𝛼-amylase and glucoamylase by the
bacterium Geobacillus thermodenitrificans.

The enzymatic extract produced by T. aurantiacus pre-
sented low depolymerizing potential (Figure 5(a)). Similar
amounts of total reducing sugar (quantified by DNS) and free
glucose (glucose oxidase quantified) suggest a predominantly
exoamylolytic activity (Figures 5(b) and 5(c)) with glucose
monomers as the main product, a typical catalytic property



8 The Scientific World Journal

0

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Cassava Potato Corn Wheat 

L. ramosa
T. aurantiacus

A
m

yl
as

e a
ct

iv
ity

 (U
/m

L)

Substrate sources

Figure 4: Enzymatic hydrolysis of starch derived from several vegetal sources (DNSmethod) (each data point was the average of two replicate
determinations, and the error bars show the data ranges).

L. ramosa T. aurantiacus
0

10

20

30

40

50

60

70

80

A
m

yl
as

e a
ct

iv
ity

 (U
/m

L)

Microorganisms

(a)

L. ramosa T. aurantiacus
Microorganisms

0

5

10

15

20

25

30

35

40
A

m
yl

as
e a

ct
iv

ity
 (U

/m
L)

(b)

L. ramosa T. aurantiacus
Microorganisms

0

5

10

15

20

25

A
m

yl
as

e a
ct

iv
ity

 (U
/m

L)

(c)
Figure 5: Enzymatic modifications of the corn starch molecule. (a) Quantification of dextrinizing activity using the iodometric method
(reduction in the starch polymerization degree); (b) quantification of sugars and reducing ends using the DNS method; (c) quantification of
glucose using the glucose/oxidase method (each data point was the average of two replicate determinations, and the error bars show the data
ranges).



The Scientific World Journal 9

of glucoamylase (EC 3.2.1.3) or 𝛼-glucosidase (EC 3.2.1.20).
Previous studies confirm these results. Carvalho et al. [43]
reported 𝛼-glucosidase production by submerged fermenta-
tion of the fungus T. aurantiacus.

4. Conclusions

The two examined strains showed potential for amylase pro-
duction by SSF, using wheat bran as substrate. However, the
production of amylase by L. ramosa was considerably higher,
compared to that of T. aurantiacus. The enzyme produced
by L. ramosa also showed greater stability at different pHs
and temperatures, characteristics that are very appreciable
for industrial application. Enzymes of both microorganisms
retained their catalytic activities in alcoholic conditions, so
they can be applied to processes for obtaining ethanol from
starch sources. Another interesting characteristic observed
in the enzymatic extract produced by L. ramosa is the
synergic potential of liquefaction and saccharification of
starch, indicating the presence of endo and exoamylases in its
composition. The properties described for amylase obtained
from the fungus L. ramosa highlight the importance of this
work, considering that this fungal species is still less explored
for amylase production.

Competing Interests

The authors declare that there is no conflict of interests.

Acknowledgments

The authors acknowledge Conselho Nacional de Desenvolvi-
mento Cient́ıfico e Tecnológico (CNPq), Fundação de Apoio ao
Desenvolvimento do Ensino, Ciência e Tecnologia do Estado
de Mato Grosso do Sul (FUNDECT), and Coordenação de
Aperfeiçoamento Pessoal deNı́vel Superior (CAPES) for finan-
cial support.

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