Production and characterization of a thermostable antifungal chitinase secreted by the filamentous fungus Aspergillus niveus under submerged fermentation

Article abstract of DOI:10.1007/s13205-018-1397-6

The filamentous fungus Aspergillus niveus produced extracellular antifungal chitinase when cultured under submerged fermentation (SbmF) using crab shells as the carbon source. Maximal chitinase production was achieved at 192?h of cultivation using minimal medium containing 1% chitin. The enzyme was purified 1.97-fold with 40% recovery by ammonium sulfate precipitation and Sephadex G-100 gel filtration. The molecular mass was estimated to be 44?kDa by both 12% SDS-PAGE and Sepharose CL-6B gel filtration. Maximal A. niveus chitinase activity was obtained at 65?°C and pH 5.0. The enzyme was fully stable at 60?°C for up to 120?min and the enzymatic activity was increased by Mn²⁺. In the presence of reducing and denaturing compounds, the enzyme activity was not drastically affected. The chitinase was able to hydrolyze colloidal chitin, azure chitin, and 4-nitrophenyl N-acetyl-β-D glucosaminide; for the latter, the K_0.5 and maximal velocity (V_max) were 3.51?mM and 9.68?U/mg of protein, respectively. The A. niveus chitinase presented antifungal activity against Aspergillus niger (MIC = 84?μg/mL), A. fumigatus (MIC = 21?μg/mL), A. flavus (MIC = 24?μg/mL), A. phoenicis (MIC = 24?μg/mL), and Paecilomyces variotii (MIC = 21?μg/mL). The fungus A. niveus was able to produce a thermostable and denaturation-resistant chitinase able to inhibit fungal development, signaling its biotechnological potential.

Full text of DOI:10.1007/s13205-018-1397-6

3
Biotech (2018) 8:369
https://doi.org/10.1007/s13205-018-1397-6
ORIGINAL ARTICLE
Production and characterization of a thermostable antifungal
chitinase secreted by the filamentous fungus Aspergillus niveus
under submerged fermentation
1
1
1
Thaís Barboni Alves · Pedro Henrique de Oliveira Ornela · Arthur Henrique Cavalcanti de Oliveira ·
2
1,2
João Atílio Jorge · Luis Henrique Souza Guimarães
Received: 24 January 2018 / Accepted: 7 August 2018
©
Springer-Verlag GmbH Germany, part of Springer Nature 2018
Abstract
The filamentous fungus Aspergillus niveus produced extracellular antifungal chitinase when cultured under submerged fer-
mentation (SbmF) using crab shells as the carbon source. Maximal chitinase production was achieved at 192 h of cultivation
using minimal medium containing 1% chitin. The enzyme was purified 1.97-fold with 40% recovery by ammonium sulfate
precipitation and Sephadex G-100 gel filtration. The molecular mass was estimated to be 44 kDa by both 12% SDS-PAGE
and Sepharose CL-6B gel filtration. Maximal A. niveus chitinase activity was obtained at 65 °C and pH 5.0. The enzyme
2
+
was fully stable at 60 °C for up to 120 min and the enzymatic activity was increased by Mn . In the presence of reducing
and denaturing compounds, the enzyme activity was not drastically affected. The chitinase was able to hydrolyze colloidal
chitin, azure chitin, and 4-nitrophenyl N-acetyl-β-D glucosaminide; for the latter, the K and maximal velocity (V ) were
0
.5
max
3
.51 mM and 9.68 U/mg of protein, respectively. The A. niveus chitinase presented antifungal activity against Aspergillus
niger (MIC=84 µg/mL), A. fumigatus (MIC=21 µg/mL), A. flavus (MIC=24 µg/mL), A. phoenicis (MIC=24 µg/mL), and
Paecilomyces variotii (MIC=21 µg/mL). The fungus A. niveus was able to produce a thermostable and denaturation-resistant
chitinase able to inhibit fungal development, signaling its biotechnological potential.
Keywords Aspergillus niveus · Antifungal activity · Chitin · Chitinase · Fungal hydrolases
Introduction
the cleavage mechanism of the chitin polymer (Rathore
and Gupta 2015). Endochitinases randomly cleave β-1,4-
glycosidic bonds of chitin, whereas exochitinases cleave the
chitin from the non-reducing end to form diacetyl-chitobiose
Chitin is a homopolymer of β-1,4-linked N-acetyl-d-glu-
cosamine that is widely distributed in nature, being the sec-
ond most abundant polymer after cellulose. It is the major
structural component of fungal cell walls and the exoskel-
eton of arthropods (Seidl 2008). The enzymatic hydrolysis
of chitin occurs through the action of chitinolytic enzymes
that cleave the β-1,4-glycosidic bonds of this polymer.
This group of enzymes can be classified as endochitinases,
exochitinases, or N-acetylglucosaminidases according to
[(GlcNAc) ]. N-acetylglucosaminidases hydrolyze (Glc-
2
NAc) producing GlcNAc. They also can produce GlcNAc
2
from the non-reducing end of chitin and N-acetyl-chitooli-
gosaccharides (Li 2006). These enzymes can be produced
by different organisms, from bacteria to human. Filamen-
tous fungi can contain between 10 and 25 different chitinase
genes and the role of the chitinases in these microorganisms
includes degradation and the use of exogenous chitin for
nutrition, cell wall remodeling during the fungal life cycle,
and competition or defense against other fungi or arthro-
pods. Entomopathogenic and nematode-trapping fungi use
chitinases as “weapons” to attack insects and nematodes,
respectively (Seidl 2008).
*
Luis Henrique Souza Guimarães
lhguimaraes@ffclrp.usp.br
1
Instituto de Química, Universidade Estadual Paulista,
Rua Prof. Francisco Degni, 55, Quitandinha, Araraquara,
SP 14800-900, Brazil
Fungal chitinases show a biotechnological potential
for different applications such as the biocontrol of pests
that attack different plantations causing economic loss
2
Faculdade de Filosofia Ciências e Letras de Ribeirão Preto,
Universidade de São Paulo, Avenida Bandeirantes 3900,
Monte Alegre, Ribeirão Preto, SP 14040-901, Brazil
Vol.:(0
1
123456789)
3

369
Page 2 of 10
3 Biotech (2018) 8:369
worldwide, reducing or eliminating the use of chemical
pesticides, and minimizing the negative impact on the envi-
ronment. In addition, these enzymes can be used for the iso-
lation of protoplasts from fungi and yeasts, for the prepara-
tion of single-cell protein, and for the treatment of chitinous
waste from the fishery industry (Rathore and Gupta 2015).
N-acetyl-d-glucosamine (an important therapeutic agent in
the treatment of osteoarthritis) and bioactive chitooligosac-
charides (which are important as antitumor agents) can also
be obtained using chitinases (Lodhi et al. 2014). Antifun-
gal chitinases have also been reported (Berini et al. 2017;
Swiontek Brzezinska and Jankiweicz 2012). Considering
the biotechnological potential of chitinases associated with
the importance of finding new fungal strains as sources of
enzymes with distinctive characteristics, we describe the
production and characterization of an extracellular chi-
tinase from Aspergillus niveus under submerged fermenta-
tion using crab shells as an inducer. Moreover, the antifungal
potential of the chitinase is also evaluated.
against distilled water at 4 °C for 24 h, and used to determine
enzyme activity and protein concentration.
Determination of chitinase activity and protein
quantitation
The exochitinase activity was evaluated using 1 mM of the
synthetic substrate 4-nitrophenyl N-acetyl-β-d glucosami-
nide (Sigma-Aldrich) in 100 mM sodium acetate buffer,
pH 5.0. The reaction was performed using 200 µL of the
substrate solution and 200 µL of the enzyme sample. After
incubation at 40 °C for 5 min., the reaction was stopped by
adding 1 mL of 1 M NaOH. The p-nitrophenolate released
was quantified at λ= 405 nm in a spectrophotometer. One
unit of enzyme activity (U) was defined as the amount of
enzyme required to hydrolyze 1 µmol of substrate per minute
under the assay conditions.
The chitinase activity was also evaluated using 10% (m/v)
colloidal chitin as a substrate, using 50 mmol/L sodium ace-
tate buffer pH 5.0 according to Rojas-Avelizapa et al. (1999).
The reaction was performed at 50 °C for 1 h and stopped by
adding 1 mL of 1% NaOH solution, followed by boiling for
Materials and methods
5
min. The tubes containing the reaction medium were cen-
Fungal strain and production of chitinase
trifuged at 7000×g for 5 min and the supernatants were used
to determine the reducing sugars using DNS (3,5-dinitrosali-
cylic acid) (Miller 1959) at 490 nm. One unit of chitinase
activity was defined as the amount of enzyme required to
produce 1 µmol of NAG (N-acetyl-d-glucosamine) per hour
under the assay conditions.
The filamentous fungus Aspergillus niveus, deposited in
the culture collection of the Laboratory of Microbiology of
the Faculdade de Filosofia, Ciências e Letras de Ribeirão
Preto, University of São Paulo (USP), was maintained on
PDA (potato dextrose agar) slants previously autoclaved at
The enzymatic assay was also performed using chitin
1
21 °C, 1 atm, for 15 min. The cultures were kept at 30 °C
®
azure (Sigma ) in 50 mmol/L sodium acetate buffer, pH
for 5 days and then stored at 4 °C.
5
.0 as the substrate as described by Gómez Ramírez et al.
The chitinase production by A. niveus was carried out
using Adams (Adams 1990), Czapek (Wiseman 1975),
Khanna (Khanna et al. 1995), minimal (Hill and Kafer
(
2004). The reaction was conducted at 65 °C for 3 h. There-
after, the tubes containing the reaction medium were centri-
fuged at 7000×g for 10 min and the supernatants obtained
measured at 560 nm. One unit of chitinase activity was
defined as the amount of enzyme that produced an increase
of 0.01 in absorbance. All experiments were performed in
triplicate.
2
001), SR (Rizatti et al. 2001), M5 (Peralta et al. 1990),
and YPD (Gun Lee et al. 1999) culture media containing
0
.5% of chitin from crab shells (Sigma-Aldrich). Flasks with
5
0 mL of sterile media were inoculated with 1 mL of a spore
4
suspension (5 × 10 spore/mL) and incubated at 30 °C for
Protein quantitation was performed according to that
described by Bradford (1976) using bovine serum albu-
min (BSA) as a standard. The protein concentration was
expressed as mg of protein per mL of sample.
9
6 h at 100 rpm.
To find the chitin concentration and time of cultivation
that produced the highest enzyme production by A. niveus,
chitinase production was carried out using minimal medium
(
50 mL) containing different chitin concentrations (w/v, 0.5,
1
, 1.5, and 2%) for 48, 96, 144, 192, and 240 h of cultivation
Chitinase purification
at 30 °C at 100 rpm.
The proteins in crude filtrate (200 mL) were precipitated
using ammonium sulfate to 80% saturation, at 4 °C. After
overnight incubation, the solution was centrifuged at
10,000×g for 20 min at 4 °C. The sediment was dissolved in
20 mM sodium acetate buffer (pH 5.0) and dialyzed against
distilled water overnight at 4 °C.
Retrieval of the filtrate containing chitinase
The crude filtrate was separated from the mycelial mass
by vacuum filtration using Whatman filter paper No. 1 in
a Büchner funnel. The resulting crude filtrate was dialyzed
1
3

3
Biotech (2018) 8:369
Page 3 of 10 369
Two milliliters of the concentrated proteins was loaded
Kinetic parameter determination (K0.5 and Vmax)
into Sephadex G-100 (Sigma-Aldrich) gel filtration chro-
matographic column (1×60 cm) equilibrated with sodium
acetate buffer 20 mM, pH 5. The enzyme was eluted using
the same buffer at a flow rate of 0.4 mL/min and fractions
of 1 mL were collected. Both the enzyme activity and the
absorbance at 280 nm for each fraction were analyzed.
Fractions with chitinase activity were pooled, dialyzed
against distilled water for 24 h at 4 °C, and used for enzyme
characterization.
The kinetic parameters K and V for the purified enzyme
0
.5
max
were determined using the synthetic substrate 4-nitrophenyl-
N-Acetyl-β-d-glucosaminide (0.05–1.7 mM) in 100 mM
sodium acetate buffer pH 5. The K and maximal velocity
0
.5
(V ) were calculated using the SigrafW software.
max
Antifungal activity
The antifungal activity from the purified A. niveus chitinase
was evaluated against A. fumigatus CAS21, A. flavus, A.
niger, A. phoenicis, and Paecilomyces variotii according to
Mania et al. (2010) with modification. Briefly, 100 µL of
minimal medium was added to each well in a 96-well micro-
titer plate. Thereafter, purified chitinase and Amphotericin
B (positive control) were added to the wells at different con-
Electrophoresis
1
2% SDS-PAGE was performed according to Laemmli
(
1970). After running at 120 V and 40 mA for 45 min, the
gel was stained using Coomassie blue-silver. The Preci-
®
sion Plus Protein™ (Bio-Rad ) (10–250 kDa) was used as
4
centrations (0.65–84 µg/mL) and 10 spores/mL from each
molecular mass marker.
fungal strain were inoculated. The plates were maintained
The native molecular mass of the A. niveus chitinase was
estimated using a Sepharose CL-6B (1×70 cm) chromato-
graphic column equilibrated with Tris/HCl buffer 50 mM
pH 7.0 containing KCl 100 mM. Fractions of 1 mL were
collected at a flow rate of 0.4 mL/min. The β-amylase pro-
teins (200 kDa), alcohol dehydrogenase (150 kDa), and
bovine serum albumin (66 kDa) were used as standards.
at 37 °C for 72 h. After this period, the indicator resazurin
®
(
7-hydroxy-3H-phenoxazin-3-one-10-oxide; Aldrich ) was
added to the wells at a final concentration of 100 µmol/L and
monitored at 490 nm using a microplate reader (SpectraMax
M2, Molecular Devices, CA, USA). All fungal strains used
are deposited in the culture collection of the Laboratory of
Microbiology of the Faculdade de Filosofia, Ciências e Let-
ras de Ribeirão Preto, University of São Paulo (USP), and
maintained on potato dextrose agar (PDA) slants.
The void volume (V ) was determined to be 56 mL using
o
Blue Dextran.
Influence of temperature and pH on enzyme activity
Results and discussion
The influence of temperature on chitinase activity was ana-
lyzed by testing enzyme activity from 30 to 85 °C. Thermal
stability was assessed throughout the pre-incubation of the
enzymatic samples in aqueous solution at 40, 50, and 60 °C
for different periods of time (5–120 min) followed by the
determination of chitinase activity.
Production of chitinase by A. niveus
The potential of the fungus A. niveus to produce chitinase
under different medium compositions was analyzed.
Among all culture media tested, the best chitinase produc-
tion (6.5 U/mg of protein) was achieved using minimal
medium with added chitin from crab shells, 3.6-fold higher
than that obtained using the Czapeck medium (1.8 U/mg
of protein) in the same condition (96 h, 30 °C, 100 rpm)
The influence of the pH on the enzyme activity was deter-
mined by performing the enzyme assay using 50 mM citric
acid buffer at different pH values (2–6). To determine the pH
stability, enzyme samples were incubated in 50 mM citric
acid buffer at different pH values (2–8) at 4 °C for 2 and
(
Fig. 1). The enzyme production in M5 and YPD media was
greatly reduced. According to Khan et al. (2010), the pres-
ence of MgSO and KH PO in the medium (as observed
2
4 h. Thereafter, the enzymatic reactions were performed
as described above.
4
2
4
for minimal medium, Adams, Czapek, and Khanna media)
can positively affect chitinase production in the presence of
chitin. The chitinase production by A. niveus was induced
by chitin when any another carbon source was present. All
media used, except for minimal medium, contain yeast
extract, providing carbon and nitrogen sources that can be
used promptly, reducing the need for enzyme production.
In minimal medium (in the absence of the yeast extract),
fungal metabolism is directed towards the degradation of
Effect of chemical compounds on chitinase activity
The effect of several chemical compounds [1 mM: MnCl ,
2
MgSO , KI, CuSO , FeSO , CoCl , CaCl , ZnSO , EDTA,
4
4
4
2
2
4
SDS, urea, β-mercaptoethanol, and dithiothreitol (DTT); 1%
(
v/v): HCl and acetone] on chitinase activity was analyzed
under optimal temperature and pH conditions.
1
3

369
Page 4 of 10
3 Biotech (2018) 8:369
Considering the importance of chitin for inducing chi-
tinase production by A. niveus, the influence of different con-
centrations of this inducer on enzyme production as a func-
tion of the cultivation time course was evaluated. As evident,
the highest level of enzyme was obtained when the micro-
organism was cultured in minimal medium with 1% added
chitin from 144 to 240 h (36 U/mg of protein) (Fig. 2a). The
addition of high chitin content (2%) in the culture medium
for long periods of cultivation reduced chitinase production
significantly. A high concentration of chitin in the medium
leads to increased viscosity, reducing the oxygen supply and
leading to reduced fungal growth and enzymatic production.
The influence of chitin concentration on chitinase production
was also evaluated by Farag and Al-Nusarie (2014), who
observed increments in chitinase content when A. terreus
was cultured using shrimp-shell powder chitin from 5 to
Fig. 1 Production of chitinase by Aspergillus niveus under submerged
fermentation using different culture media supplemented with 1% of
chitin from crab shells
2
0 g/L, whereas, at a concentration above 25 g/L, produc-
tion decreased.
Fungal cultivation for 48 h resulted in low chitinase pro-
duction for all chitin concentrations used, as well as reduced
fungal growth. Incubation for short periods may not lead
to maximal enzyme production, as the microorganism is
in the vegetative growth phase. In addition, the growth of
the microorganism over a long period can lead to nutrient
exhaustion and, consequently, to a decline in growth and
enzyme production. Below 1% chitin in the culture medium,
the chitinase production by A. niveus at 192 h of culture
(36 U/mg of protein) was 2.8-fold higher than that observed
at 96 h (12.5 U/mg of protein). Lee et al. (2009) observed
maximal chitinase production by Penicillium sp. LYG 0704
with 3-day-old cultures, while, for A. terreus, maximum
chitin, which is the only available carbon/nitrogen source
in this medium.
Several fungal species of Aspergillus, Penicillium,
Metarhizium, and Trichoderma have been described as chi-
tinase producers (Farag et al. 2016; Swiontek Brzezinska
and Jankiweicz 2012; Lee et al. 2009; Binod et al. 2005;
Rustiguel 2014). The induction of the enzyme production by
these fungi has been performed using chitin from different
sources, such as shrimp-shell powder, used for A. terreus
(
Farag et al. 2016) and A. niger LOCK 62 (Swiontek Brzez-
inska and Jankiweicz 2012), and shrimp shellfish waste used
for Aspergillus sp. S1-13 (Rattanakit et al. 2007).
Fig. 2 Influence of different concentrations of chitin and culture time
on the production of chitinases by A. niveus in minimal medium (a).
Chitin concentrations in the medium: 1% (circle), 0.5% (square),
by A. niveus cultured in minimal medium, containing 1% chitin, for
different time periods (b). Lane 1 48 h; Lane 2 96 h; Lane 3 144 h;
Lane 4 196 h, Lane 5 240 h, and Lane M molecular weight markers.
80 µg of each sample was applied to the gel
1
9
.5% (triangle), and 2% (inverted triangle) at the culture times of 48,
6, 144, 192, and 240 h. SDS-PAGE profile for the proteins secreted
1
3

3
Biotech (2018) 8:369
Page 5 of 10 369
production was obtained with 5-day-old cultures (Farag and
Al-Nusarie 2014). According to Swiontek Brzezinska and
Jankiweicz (2012), the maximal chitinase production by A.
niger LOCK 62 was observed with 6-day-old cultures.
The protein profile for the fungal growth in optimal con-
ditions (minimal medium containing 1% chitin) for different
periods at 30 °C (100 rpm) for the production of high chi-
tinase levels was analyzed using 12% SDS-PAGE (Fig. 2b).
Protein bands with different molecular masses (from 28 to
Table 1 Purification of the extracellular chitinase produced by Asper-
gillus niveus
a
Step
Protein
(total
mg)
Activity SA (U/mg) Yield (%) Purifi-
(total U)
cation
(fold)
Crude
13.3
7.8
88.0
60.0
35.2
6.61
7.69
100
1
filtrate
NH SO
68.18
40.0
1.16
1.97
4
4
(
80%)
Sephadex
2.7
13.03
1
38 kDa) can be observed, especially the band correspond-
G-100
ing to a 44 kDa protein. Increased 44 kDa protein expression
is noted as a function of the increase in culture duration; this
protein being the most prevalent in the cultures.
aSpecific activity
Chitinase purification
ammonium sulfate precipitation and different chromato-
graphic procedures. For example, the chitinases produced by
A. terreus (Farag et al. 2016), A. niger LOCK 62 (Swiontek
Brzezinska and Jankiweicz 2012), and A. fumigatus YJ-407
(Xia et al. 2001), were purified using ammonium sulfate.
Ammonium sulfate is the most commonly used salt in pro-
tein precipitation owing to some of its characteristics such
as high solubility, low cost, and low toxicity (Duong-Ly and
Gabelli 2014).
The A. niveus chitinase was initially precipitated with
ammonium sulfate, followed by Sephadex G-100 gel fil-
tration chromatography. A single peak corresponding with
enzymatic activity was obtained in this last step (Fig. 3a),
recording a 1.97-fold purification with 40% recovery and a
specific activity of 13.03 U/mg of protein (Table 1). In gen-
eral, the purification of most chitinases is inefficient, which
can be explained by the presence of different isoforms with
synergistic actions present in the crude supernatant and, con-
sequently, loss during the purification procedure (Li 2006).
Each step of the purification process was monitored by
Determination of molecular mass
The native molecular mass of A. niveus chitinase was found
to be 44.57 and 44.03 kDa estimated by Sepharose CL-6B
and 12% SDS-PAGE (Fig. 3b), respectively, indicating a
monomeric enzyme. These values were near to the value
1
2% SDS-PAGE (Fig. 3b), and at the end of the purification,
a single protein band was observed. Fungal chitinases have
previously been purified by different methods, including
Fig. 3 Chitinase purifications steps. After precipitation with 80%
ammonium sulfate, the proteins were applied on Sephadex G-100 and
the fractions with enzymatic activity were joined in a single pool as
indicated by arrows in the chromatographic profile (a). Analysis of
chitinase purification steps by 12% SDS-PAGE (b). Lane 1 chitinase
after 80% ammonium sulfate precipitation; Lane 2 purified chitinase;
Lane M molecular weight marker. 50 µg of proteins was applied to
the gel
1
3

369
Page 6 of 10
3 Biotech (2018) 8:369
estimated for the chitinases from produced A. fumiga-
tus YJ-407, with a molecular mass of 46 kDa (Xia et al.
YJ-407, with an optimum temperature of 60 °C (Xia et al.
2001). In addition, the A. niveus enzyme was remarkably
thermostable (Fig. 4c); at 40 °C, the enzyme was fully stable.
Interestingly, when maintained at 50 °C, chitinase activity
was increased for all periods analyzed because of structural
changes in the enzyme, which favor a better interaction with
the substrate. At 60 °C, the half-life (t ) of the enzyme was
2
001) and from A. niger LOCK 62, with a molecular mass
of 43 kDa (Swiontek Brzezinska and Jankiweicz 2012).
According to Li (2006), the molecular mass of fungal chi-
tinases can vary from 27 to 190 kDa. The different chitinases
produced by Penicillium aculeatum NRRL 2129 presented
molecular masses of 82.7, 44.6, 28.2, and 26 kDa (Binod
et al. 2005). Rattanakit et al. (2007) purified three chitinases
from Aspergillus sp. S1-13 with molecular masses of 45, 51,
and 73 kDa. For the chitinase from A. terreus, the molecular
mass was estimated as 60 kDa (Farag et al. 2016).
5
0
estimated to be 120 min. The A. fumigatus chitinase retained
40% of its initial activity after heating at 60 °C for 30 min
(Xia et al. 2001), while the A. niger LOCK 62 chitinase
completely lost its activity after incubation at the same tem-
perature for 90 min (Swiontek Brzezinska and Jankiweicz
2
012). The A. terreus chitinase retained 66% and 42% of its
Effect of the temperature and pH on the chitinase
activity
initial activity when maintained at 60 and 70 °C for 60 min,
respectively, and lost 90% of its activity at 90 °C for the
same period of incubation (Farag et al. 2016). According to
Guo et al. (2008), thermostable chitinase had major advan-
tages over industrial catalysis due its high activity at higher
temperatures. For example, the production of chito-oligosac-
carides from chitin waste conducted at high temperature
can minimize the contamination and the viscosity of the
The highest A. niveus chitinase activity was obtained at
6
5 °C (Fig. 4a). This value was higher than that observed
for the enzymes produced by Penicillium aculeatum NRRL
129 and A. terreus, with an optimum temperature of 50 °C
Farag et al. 2016; Binod et al. 2005), and by A. fumigatus
2
(
Fig. 4 Influence of temperature (a) and pH (b) on enzyme activity,
and stability over time at different temperatures (c) at 40 °C (squares),
ric acid buffer (pH 2.0–8.0) for 2 h (black bars) and 24 h (white bars)
for the purified A. niveus chitinase
5
0 °C (circles), and 60 °C (triangles), and to pH (d) using 50 mM cit-
1
3

3
Biotech (2018) 8:369
Page 7 of 10 369
Table 2 Effect of chemical reagents on chitinase activity
medium. Another possible biotechnological application is
the biocontrol agents containing chitinase to combat plant
pathogens (fungi) and insects that should be sprayed on the
plant, exposing them to environmental conditions, such as
high temperatures, for long periods. The preservation of the
enzymatic activity under adverse conditions is important to
achieve positive results in plant protection.
Compounds
Relative activity (%)
None
MnCl2
MgSO4
KI
CuSO4
FeSO4
CoCl2
CaCl2
ZnSO4
EDTA
SDS
Urea
β-Mercaptoethanol
DTT
HCl
Acetone
100a
222.9± 0.6
97.0± 0.3
79.9± 2.6
74.4± 2.1
87.9± 0.1
99.2± 4.2
83.1± 1.4
78.1± 0.1
91.3± 8.6
103.2± 3.5
85.2± 4.4
94.3± 1.4
90.1± 1.7
90.5± 1.7
100.2± 2.4
The optimal pH for A. niveus chitinase was 5.0, retaining
6
2
5–80% of its activity at pH 5.5 and 6.0 (Fig. 4b). At pH
.0–3.5, the enzyme activity was strongly reduced. Most
fungal chitinases have optimum pH of activity from pH 4.0
to 7.0, such as the enzymes produced by A. niger LOCK
6
2 (Swiontek Brzezinska and Jankiweicz 2012), A. fumiga-
tus YJ-407 (Xia et al. 2001), A. terreus (Farag et al. 2016),
and Penicillium aculeatum NRRL 2129 (Binod et al. 2005).
Considering the enzyme stability to pH for 2 h, the activ-
ity of the A. niveus chitinase was slightly increased when
maintained at a pH range from 2 to 5 (Fig. 4d). Progres-
sive reduction in chitinase activity was observed when the
enzyme was incubated at pH 6.0, 7.0, and 8.0. At pH 2.0 and
a100% activity=13.03 U/mg of protein
3
.0 for 24 h, a slight increase in the chitinase activity was
observed. However, at basic pH (8.0), 46% of activity loss
was observed. Rustiguel (2014) reported that the chitinase
from Metarhizium anisopliae IBCB 384 retained 100% of its
activity when incubated at pH 7.0, while the chitinase from
M. anisopliae IBCB 425 maintained 70% of activity in pH
values from 2.5 to 7.0. The chitinase from M. anisopliae
IBCB 167 maintained 60% of its activity at pH 5.5.
of its activity, while, in the presence of HCl, the enzyme
maintained 90% of its activity. The A. fumigatus YJ-407
chitinase maintained its activity when incubated in the pres-
ence of urea and presented a reduction of only 20% of its
activity in the presence of SDS (Xia et al. 2001). In contrast
to the results found in this work, Farag et al. (2016) observed
that a chitinase from A. terreus was strongly inhibited in the
presence of acetone. Resistance to denaturation in organic
solvents, acid, and reducing and denaturing compounds is
favorable characteristics for the industrial application of A.
niveus chitinase.
Effect of chemical compounds on chitinase activity
The effect of several chemical compounds on chitinase activ-
ity is presented in Table 2. The salt MnCl improved the
2
chitinase activity by around 122% when compared with the
absence of chemical compounds. Chitinase activity from M.
anisopliae (Rustiguel 2014), A. terreus (Farag et al. 2016),
and Penicillium sp. (Lee et al. 2009) were also improved in
Kinetic parameter determination (K0.5 and Vmax
)
The A. niveus chitinase was able to hydrolyze colloidal
chitin (0.05 U/mL), azure chitin (0.02 U), and 4-nitrophe-
nyl N-acetyl-β-d glucosaminide (2.9 U/mL) as substrates.
However, the hydrolysis was more pronounced for the last
substrate, indicating the predominance of the exochitinase
activity. Using 4-nitrophenyl N-acetyl-β-d glucosaminide as
the substrate, the chitinase presented a K of 3.51 mM and
2
+
the presence of Mn . On the other hand, the enzyme activ-
ity from A. niveus was inhibited by 20–26% in the presence
of KI, CuSO , and ZnSO . A slight inhibition (maximum
4
4
1
7%) was observed for the use of the other salts.
Interestingly, the presence of denaturing agents, HCl and
acetone, did not significantly influence A. niveus chitinase
activity. However, in the presence of urea, the enzyme main-
tained 85% of its initial activity. According to other authors,
urea may cause conformational changes in the structure of
the enzymes, promoting denaturation (Pace et al. 2010).
In the presence of β-Mercaptoethanol and DTT, denatur-
ing agents able to attack disulfide bonds between cysteine
residues, the enzyme retained 95 and 90% of its initial activ-
ity, respectively, demonstrating that cysteine residues are
not involved in the formation of the catalytic center. In the
presence of SDS and acetone, the enzyme maintained 100%
0
.5
a V of 9.68 U/mg of protein. The Hill coefficient (n) was
max
estimated as 2.98, indicating a positive cooperativity, which
causes the enzyme to be much more sensitive to substrate
concentration. Likewise, the activity can vary strongly even
at very narrow substrate concentration intervals (Porter and
Miller 2012). Thimoteo et al. (2017) observed that the chi-
tinase MetaChi18A presented an n of 4.12 using colloidal
chitin as a substrate. This phenomenon can occur because
of hysteresis, a delay in the enzyme reaching its fully active
form involving conformational changes. As chitinase is a
1
3

369
Page 8 of 10
3 Biotech (2018) 8:369
Table 3 Antifungal activity
from the A. niveus chitinase
µg/mL
P. variotii
A. phoenicis
A. flavus
A. fumigatus
A. niger
Chi
AmpB
Chi
AmpB
Chi
AmpB
Chi
Amp
Chi
AmpB
8
4
2
1
5
2
1
4
2
1
0.5
.25
.62
.31
−
−
−
+
+
+
+
+
−
−
−
−
+
+
+
+
−
−
+
+
+
+
+
+
−
−
+
+
+
+
+
+
−
−
+
+
+
+
+
+
−
−
+
+
+
+
+
+
−
−
−
+
+
+
+
+
−
−
+
+
+
+
+
+
−
+
+
+
+
+
+
+
−
−
+
+
+
+
+
+
0
.65
Chi chitinase from A. niveus, AmpB amphotericin B, (−) absence of fungal growth, (+) fungal growth. The
assay was performed using minimal medium for 72 h at 37 °C. Resazurin was used as an indicator
multidomain enzyme, the substrate must interact with bind-
ing domains that can organize the conformational change,
facilitating the access of the catalytic domain to the substrate
produced by A. niger LOCK 62 inhibited the growth of
Fusarium culmorum, Fusarium solani, and Rhizoctonia
solani, but not the growth of Botrytis cinerea, Alternaria
alternata, and Fusarium oxysporum (Swiontek Brzezin-
ska and Jankiweicz 2012). The chitinase isolated from A.
griseoaurantiacus KX010988 inhibited the growth of F.
solani (Shehataa et al. 2018).
(
Thimoteo et al. 2017).
Rustiguel (2014) evaluated the kinetic parameters of the
chitinase produced by M. anisopliae IBCB 360 using the
substrate 4-nitrophenyl-N-acetyl-β-d-glucosaminide, and
found a K of 0.57 mM and a V of 3.38 U/mg of protein.
m
max
The A. fumigatus YJ-407 chitinase presented K of 1.12
m
and 1.84 mg/mL when the chitin and chitosan were used as
substrates, respectively (Xia et al. 2001).
Conclusion
Antifungal activity
The chitinase secreted by A. niveus presented very inter-
esting characteristics for possible industrial applications,
such as high temperature of activity and thermostability,
besides the ability to maintain its activity in the presence
of different compounds, including denaturing and reducing
agents. The chitinase secreted by A. niveus may also have
other biotechnological applications such as in the produc-
tion of chitin derivatives by enzymatic hydrolysis and in
the control of mycotoxigenic fungi.
The A. niveus chitinase was able to inhibit the development
of A. fumigatus CAS21, A. flavus, A. niger, A. phoenicis, and
P. variotii (Table 3). The minimum inhibitory concentration
(
MIC) was 21 µg/mL for P. variotii and A. fumigatus, while
it was 24 µg/mL for A. phoenicis and A. flavus, and 84 µg/
mL for A. niger. According to our results, the antifungal chi-
tinase activity was similar to Amphotericin B for A. phoeni-
cis and A. flavus, but not for that observed for P. variotii and
A. niger. On the other hand, when the assay was conducted
with A. fumigatus, the A. niveus chitinase was more effective
compared with the result obtained with Amphotericin B. A.
flavus is an important aflatoxin-producing fungus responsi-
ble for the contamination of food supplies of humans and
animals, resulting in health hazards and sometimes mortality
Acknowledgements We thank Mauricio de Oliveira for technical assis-
tance. The authors also kindly acknowledge the financial support from
FAPESP (Process no. 2011/50880-1) and the research scholarships
from Coordenação de Aperfeiçoamento de Pessoal de Nível Superior
(CAPES). This manuscript is part of the doctoral thesis by T.B.A.
Author contributions LHSG and TBA designed the study; TBA per-
formed the experiments on chitinase production, purification, and char-
acterization; PHOO performed the experiments on antifungal activity;
LHSG, TBA, AHCO, and JAJ analyzed the results; LHSG and TBA
wrote the manuscript.
(Kumar et al. 2017). Other fungal species, including A. niger
and P. variotii, are also able to produce mycotoxins (Hameed
et al. 2012). Therefore, the use of chitinase from A. niveus
is an attractive alternative method to combat mycotoxigenic
fungi, minimizing food contamination problems. In addition,
the fungus A. fumigatus is an important agent that causes
allergic bronchopulmonary aspergillosis in immunocompro-
mised patients (Singh et al. 2014).
Compliance with ethical standards
Conflict of interest On behalf of all authors, the corresponding author
states that there is no conflict of interest.
Other antifungal chitinases produced by filamentous
fungi have also been reported. For example, the chitinase
1
3

3
Biotech (2018) 8:369
Page 9 of 10 369
Li D-C (2006) Review of fungal chitinases. Mycopathologia
References
1
61:345–360. https://doi.org/10.1007/s11046-006-0024-y
Lodhi G, Kim YS, Hwang JW, Kim SK, Jeon YD, Je JY, Ahn BB,
Moon SH, Jeon BT, Park PJ (2014) Chitooligosaccharide and
its derivatives: preparation and biological applications. Biomed
Res Int 2014:1–13. https://doi.org/10.1155/2014/654913 (Arti-
cle ID 654913).
Adams PR (1990) Mycelial amylase activities of thermophilic spe-
cies of Rhizomucor, Humicola and Papulaspora. Mycopatholo-
gia 112:35–37. https://doi.org/10.1007/BF01795178
Berini F, Presti I, Beltrametti F, Pedroli M, Varum KM, Pellegioni L,
Sjöling S, Marinelli F (2017) Production and characterization of
a novel antifungal chitinase identified by functional screening of
a suppressive-soil metagenome. Microb Cell Fact 16:16. https
Mania D, Hilpert K, Ruden S, Fischer R, Takeshita N (2010) Screen-
ing for antifungal peptides and their modes of action in Asper-
gillus nidulans. Appl Environ Microbiol 76(21):7102–7108
Miller GL (1959) Use of dinitrosalicylic acid reagent for determina-
tion of reducing sugar. Anal Chem 31(3):426–428
:
//doi.org/10.1186/s12934-017-0634-8
Binod P, Pusztaheyi T, Nagy N, Sandhya C, Szakács G, Pócsi I,
Pandey A (2005) Production and purification of extracellular
chitinases from Penicillium aculeatum NRRL 2129 under solid-
state fermentation. Enzyme Microbial Technol 36:880–887.
https://doi.org/10.1016/j.enzmictec.2004.12.031
Pace CN, Huyghues-Despointes BMP, Fu H, Takano K, Scholtz JM,
Grimsley GR (2010) Urea denatured state ensembles contain
extensive secondary structure that is increased in hydropho-
bic proteins. Protein Sci 19:929–943. https://doi.org/10.1002/
pro.370
Bradford MM (1976) A rapid and sensitive method for the quantita-
tion of microgram quantities of protein utilization the principle
of protein-dye binding. Anal Biochem 72:248–254. https://doi.
org/10.1016/0003-2697(76)90527-3
Peralta RM, Terenzi HF, Jorge JA (1990) β-d-glycosidase activi-
ties of Humicola grisea var. thermoidea: biochemical and
kinetic characterization of a multifunctional enzyme. Biochim
Biophys Acta 1033:243–249. https://doi.org/10.1016/0304-
Duong-Ly KC, Gabelli SB (2014) Salting out of proteins using
ammonium sulfate precipitation. Methods Enzymol 541:85–94.
https://doi.org/10.1016/B978-0-12-420119-4.00007-0
4
165(90)90127-l
Porter CM, Miller BG (2012) Cooperativity in monomeric enzymes
with single ligand-binding sites. Bioorg Chem 43:44–50. https
Farag MA, Al-Nusarie ST (2014) Production, optimization, char-
acterization and antifungal activity of chitinase produced by
Aspergillus terreus. Afr J Biotechnol 13:1567–1578. https://
doi.org/10.5897/AJB2014.13628
:
//doi.org/10.1016/j.bioorg.2011.11.001
Gómez Ramírez M, Rojas Avelizapaa LI, Rojas Avelizapab NG,
Cruz Camarillo R (2004) Colloidal chitin stained with Rema-
zol Brilliant Blue RR, a useful substrate to select chitinolytic
microorganisms and to evaluate chitinases. J Microbiol Methods
Farag AM, Abd-Elnabey HM, Ibrahim HAH, El-Shenawy M (2016)
Purification, characterization and antimicrobial activity of chi-
tinase from marine derived Aspergillus terreus. Egypt J Aquat
Res 42:185–192. https://doi.org/10.1016/j.ejar.2016.04.004
Gun Lee D, Shin SY, Maeng CY, Jin ZZ, Kim KL, Hahm K-S (1999)
Isolation and characterization of a novel antifungal peptide from
Aspergillus niger. Biochem Biophys Res Commun 263:646–
5
6:213–219
Rathore AS, Gupta RD (2015) Chitinases from bacteria to human:
properties applications, and perspectives. Enzyme Res 2015:1–
8
. https://doi.org/10.1155/2015/791907 (Article ID 791907)
Rattanakit N, Yano S, Plikomol A, Wakayama M, Tachiki T (2007)
Purification of Aspergillus sp. S1-13 chitinases and their role
in saccharification of chitin in mash of solid-state culture with
shellfish waste. J Biosci Bioeng 103:535–541. https://doi.
org/10.1263/jbb.103.535
6
51. https://doi.org/10.1006/bbrc.1999.1428
Guo R-F, Shi B-S, Li D-C, Ma W, Wei Q (2008) Purification and
characterization of a novel thermostable chitinase from Ther-
momyces lanuginosus SY2 and cloning of its encoding gene.
Agric Sci China 7(12):1458–1465
Rizatti AC, Jorge JA, Terenzi HF, Rechia CG, Polizeli ML (2001)
Purification and properties of a thermostable extracellular β-d-
xylosidase produced by a thermotolerant Aspergillus phoenicis.
J Ind Microbiol Biotechnol 3:156–160. https://doi.org/10.1038/
sj/jim/7000107
Hameed AAA, Ayesh AM, Mohamed MAR, Mawla HFA (2012)
Fungi and some mycotoxins producing species in the air of soy-
bean and cotton mills: a case study. Atmos Pollut Res 3:126–131
Hill TW, Kafer E (2001) Improved protocols for Aspergillus
minimal medium: trace element and minimal medium salt
stock solutions. Fungal Genet Newsl 48:20–21. https://doi.
org/10.4148/1941-4765.1173
Rojas-Avelizapa LI, Cruz-Camarillo R, Guerrero MI, Rodríguez-
Vázquez R, Ibarra JE (1999) Selection and characterization
of a proteo-chitinolytic strain of Bacillus thuringiensis, able
to grow in shrimp waste media. World J Microbiol Biotechnol
Khan MA, Hamid R, Ahmad M, Abdin MZ, Javed S (2010) Optimi-
zation of culture media for enhanced chitinase production from
a novel strain of Stenotrophomonas maltophilia using response
surface methodology. J Microbiol Biotechnol 20(11):1597–
1
5(2):299–308
Rustiguel CB (2014) Comparison of the biochemical properties of
the chitinases produced by different isolates of Metarhizium
anisopliae. Thesis, University of São Paulo, Brazil
1
602. https://doi.org/10.4014/jmb.0909.09040
Khanna P, Sundari SS, Kumar NJ (1995) Production, isolation and
partial purification of xylanase from Aspergillus sp. World J
Microbiol Biotechnol 11:242–243. https://doi.org/10.1007/
BF00704661
Seidl V (2008) Chitinases of filamentous fungi: a large group of
diverse proteins with multiple physiological functions. Fungal
Biol Rev 22:36–42. https://doi.org/10.1016/j.fbr.2008.03.002
Shehataa AN, Abd El Aty AA, Darwishc DA, Wahabb WAA,
Mostafa FA (2018) Purification, physicochemical and ther-
modynamic studies of antifungal chitinase with production of
bioactive chitosan-oligosaccharide from newly isolated Asper-
gillus griseoaurantiacus KX010988. Int J Biol Macromol
Kumar P, Mahato DK, Kamle M, Mohanta TK, Kang SG (2017)
Aflatoxins: a global concern for food safety, human health
and their management. Front Microbiol 7:2170. https://doi.
org/10.3389/fmicb.2016.02170
Laemmli UK (1970) Cleavage of structural proteins during the
assembly of the head of bacteriophage T4. Nature 227:680–685.
https://doi.org/10.1038/227680a0
1
07:990–999
Singh B, Singh S, Asif AR, Oellerich M, Sharma GL (2014) Allergic
aspergillosis and the antigens of Aspergillus fumigatus. Curr
Protein Pept Sci 15(5):403–423. https://doi.org/10.2174/13892
Lee YG, Chung KC, Wi SG, Lee JC, Bae HJ (2009) Purification
and properties of a chitinase from Penicillium sp. LYG 0704.
Protein Expr Purif 65:244–250. https://doi.org/10.1016/j.
pep.2008.12.004
0
3715666140512120605
Swiontek Brzezinska M, Jankiweicz U (2012) Production of chi-
tinase by Aspergillus niger LOCK 62 and Its potential role in
1
3

369
Page 10 of 10
3 Biotech (2018) 8:369
the biological control. Curr Microbiol 65:666–672. https://doi.
org/10.1007/s00284-012-0208-2
Xia G, Jin CZ, Hou JY, Ang SZ, Hang S, Jin C (2001) A novel chi-
tinase having a unique model of action from Aspergillus fumigatus
YJ-407. Eur J Biochem 208:4079–4085. https://doi.org/10.104
6/j.1432-1327.2001.02323.x
Thimoteo SS, Glogauer A, Faoro H, Souza EM, Huergo LF, Moer-
schbacher BM, Pedrosa FO (2017) A broad pH range and pro-
cessive chitinase from metagenome library. Braz J Med Biol
Res 50(1):e5658. https://doi.org/10.0.6.54/1414-431x20165658
Wiseman A (1975) Industrial practice with enzymes. In: Wiseman
A (ed) Handbook of enzyme biotechnology, 1rd edn. Horwood,
Chichester, pp 243–272
1
3