Purification and biochemical characterization of a novel thermostable lichenase from Aspergillus niger US368

Article abstract of DOI:10.1016/j.carbpol.2013.07.009

New β-1,3;1,4-glucanase was purified from Aspergillus niger US368. The pure glucanase has a molecular mass of about 32 kDa. The N-terminal sequence of the purified enzyme (A-G-T-N-P-P-I-G-V) was determined. The optimum pH and temperature recorded for enzyme activity were 5 and 60 °C, respectively. It also displayed marked thermostability with a half-life of 30 min at 70 °C. At 37 °C, the enzyme showed 100% stability from pH 3 to 10. The K _m and V_max values exhibited by the enzyme on barley β-glucan were 0.62 mg ml^-1 and 34.46 U ml^-1, respectively. The enzyme is a retaining-one and was only active toward glucan containing β-1,3;1,4-linkages. The production of β-glucanase with barley flour as the sole carbon source was optimized. This is the first report on the purification and characterization of a β- 1,3;1,4-glucanase from A. niger. This lichenase could be considered as a candidate for future application particularly in the animal feed industry.

Full text of DOI:10.1016/j.carbpol.2013.07.009

Carbohydrate Polymers 98 (2013) 967–975
Contents lists available at SciVerse ScienceDirect
Carbohydrate Polymers
journal homepage: www.elsevier.com/locate/carbpol
Purification and biochemical characterization of a novel thermostable
lichenase from Aspergillus niger US368
Fatma Elgharbia, Aïda Hmida-Sayaria,∗, Mouna Sahnouna, Radhouane Kammouna,
Lobna Jlaeilb, Hajer Hassairib, Samir Bejara
a Laboratoire de Microorganismes et de Biomolécules (LMB), Centre de Biotechnologie de Sfax (CBS), Université de Sfax, Route de Sidi Mansour Km 6,
BP “1177” 3018 Sfax, Tunisia
b Laboratoire d’analyse, Centre de Biotechnologie de Sfax (CBS), Université de Sfax, Route de Sidi Mansour Km 6, BP “1177” 3018 Sfax, Tunisia
a r t i c l e i n f o
a b s t r a c t
Article history:
New ␤-1,3;1,4-glucanase was purified from Aspergillus niger US368. The pure glucanase has a molecular
mass of about 32 kDa. The N-terminal sequence of the purified enzyme (A-G-T-N-P-P-I-G-V) was deter-
mined. The optimum pH and temperature recorded for enzyme activity were 5 and 60 ◦C, respectively.
It also displayed marked thermostability with a half-life of 30 min at 70 ◦C. At 37 ◦C, the enzyme showed
100% stability from pH 3 to 10. The Km and Vmax values exhibited by the enzyme on barley ␤-glucan
were 0.62 mg ml−1 and 34.46 U ml−1, respectively. The enzyme is a retaining-one and was only active
toward glucan containing ␤-1,3;1,4-linkages. The production of ␤-glucanase with barley flour as the sole
carbon source was optimized. This is the first report on the purification and characterization of a ␤-
1,3;1,4-glucanase from A. niger. This lichenase could be considered as a candidate for future application
particularly in the animal feed industry.
Received 2 March 2013
Received in revised form 10 June 2013
Accepted 4 July 2013
Available online xxx
Keywords:
Aspergillus niger US368
␤-1,3 ;1,4-Glucanase
Thermostability
Statistical experimental designs
© 2013 Elsevier Ltd. All rights reserved.
1. Introduction
attractive opportunities in the animal feed industry, particularly
the increase of ␤-glucan digestibility in feed stuffs, the improve-
␤-Glucans are major cell wall components in cereals consisting
of linear ␤-d-glucosyl residues linked through ␤-1,3 and/or ␤-1,4
glycosidic bonds (Tang et al., 2012). Variations have, however, often
been reported in the proportions of ␤-1,3- (25–30%) and ␤-1,4-
linkages as well as the length of mixed-linked segments (Planas,
2000). The degradation of ␤-glucans in nature is catalyzed by ␤-
glucanases, which, based on the type of glycosidic linkage that they
cleave, can be grouped into four main categories, namely ␤-1,3;1,4-
glucanases (lichenases, EC 3.2.1.73), ␤-1,4-glucanases (cellulases,
EC 3.2.1.4), ␤-1,3-glucanases (laminarinases, EC 3.2.1.39), and ␤-
1,3(4)-glucanases (EC 3.2.1.6) (Boyce & Walsh, 2007; McCarthy,
Hanniffy, Savage, & Tuohy, 2003; Yang, Yan, Jiang, Fan, & Wang,
2008).
Due to their suitability for a wide range of industrial appli-
cations, ␤-1,3;1,4-glucanases (lichenases) that belong to the
glycoside hydrolase (GH) family 16 have attracted special atten-
tion in recent research. These enzymes have been reported to
constitute important biotechnological aids in the brewing industry
since they increase extraction yields and lead to high-quality
brewer malt by reducing mash viscosity and turbidity (Celestino,
Cunha, & Felix, 2006). They have also been described to offer
ment of feed conversion efficiency, and the alleviation of sanitary
problems, such as sticky droppings (Beckmann, Simon, & Vahjen,
2006; Mathlouthi, Mallet, Saulnier, Quemener, & Larbier, 2002).
Most of the microbial ␤-1,3;1,4-glucanases so far reported in
the literature have been produced by bacteria, especially the genus
Bacillus (Ekinci, McCrae, & Flint, 1997; Huang et al., 2008; Planas,
2000; Teng et al., 2006). Several extracellular ␤-1,3;1,4-glucanases
have also been purified from various fungal sources, such as Orpino-
myces sp. PC-2 (Chen, Li, & Ljungdahl, 1997), Talaromyces emersonii
(Murray et al., 2001), and Rhizopus microsporus var. microsporus
(Celestino et al., 2006). Overall, most of the ␤-1,3;1,4-glucanases
known to date are, however, optimally active at a temperature
range of 40–60 ◦C and in acidic or neutral pH (Planas, 2000).
Due to their limited properties, the currently marketed commer-
cial ␤-1,3;1,4-glucanases may not be ideally suitable for brewing
and poultry industries. Usually, malting (50–70 ◦C) or feed pelleting
processes (65–90 ◦C) require enzymes with good thermostability.
Therefore, thermostable ␤-1,3-1,4-glucanases will be highly desir-
able owing to their stability at high operation temperatures (Tang
et al., 2012). Accordingly, there are continuing efforts to search
for new sources for the production of glucanases with interesting
properties that can be produced in high yields.
Aspergillus niger, an important microorganism of the genus
Aspergillus, is known to produce a variety of enzymes that attack
different types of ␤-glucosidic linkages (Manners, Stark, Wilson, &
∗
Corresponding author. Tel.: +216 74 440 451; fax: +216 74 440 451.
E-mail address: aidahmida@yahoo.fr (A. Hmida-Sayari).
0144-8617/$ – see front matter © 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.carbpol.2013.07.009

968
F. Elgharbi et al. / Carbohydrate Polymers 98 (2013) 967–975
Brodie, 1976). Although this organism has been extensively used to
produce large quantities of desired enzymes, no previous studies
have investigated its potential with regards to the production of
␤-1,3;1,4-glucanases.
an agitation speed of 150 rpm. Glucanase activity was determined
after 4 days of cultivation.
2.7. Optimization of inoculum size
The present study was undertaken to optimize the produc-
tion conditions for a relatively thermoactive and thermostable
␤-1,3;1,4-glucanase from a new A. niger US368 strain. It also reports
on the purification and physico-chemical characterization of this
interesting ␤-glucanase that could be used in various industrial
applications and processes, particularly in the animal feed industry.
The optimization of inoculum size for ␤-glucanase production
was carried out by assaying various sizes of inocula ranging from
102 to 107 spores/ml of sterile media. The cultures were incubated
for 4 days at 30 ◦C with an agitation speed of 150 rpm.
2.8. Identification of critical culture variables using
Plackett–Burman design
2. Materials and methods
Various medium components and culture parameters were
assayed for screening purposes using a Plackett–Burman (PB) fac-
torial design (Yinghua, Xu, Zhijing, Qingbiao, & Gang, 2007). Each
factor was examined in two levels coded as −1 for low level and
+1 for high level. The screened factors were temperature, pH, agi-
tation, oligoelements, yeast extract, casein peptone, soybean meal
hydrolysate, and MgSO4 (12 trials). All experiments were carried
out in triplicate and the average of glucanase activity was taken
as a response. The effect of each parameter was determined as the
difference between the average of measurements made at the high
(+1) and low (−1) levels using the following equation:
2.1. Materials
The strain of A. niger US368 was already described (Hmida-
Sayari, Taktek, Elgharbi, & Bejar, 2012).
Barley ␤-glucan, laminarin, lichenan, and CM-cellulose were
purchased from Sigma Chemical Co., Ltd.
Broad-range (2–212 kDa) molecular weight marker proteins
were from BioLabs.
2.2. Growth condition
ꢀ
ꢀ
Fermentations were carried out in 1 l flasks that were autoclaved
at 121 ◦C for 20 min and then cooled and inoculated with fungal
spores grown on PDA slants. Inocula were prepared by washing
conidiospores from the surface of PDA slants with 25 ml of sterile
distilled water. Spore densities were determined using a Thoma cell
counting chamber.
Yi+
−
N
Yi−
E(X )
=
i
where E(X ) refers to the factor effect or the contrast coefficient; Yi+
i
and Yi− to the glucanase activities of the trials where the variable
(Xi) measured was present at high and low levels, respectively, and
N to the number of these experiments.
2.3. Basal medium
2.9. L18C Chakravarty design
Before optimization, the strain was cultivated in a medium con-
sisting of 1% (w/v) barley flour, 0.5% (w/v) yeast extract and 0.5%
(w/v) casein peptone.
An L18C Chakravarty experimental design was applied to
identify the optimum conditions for enzyme production. This
experimental design consisted of a matrix of 18 trials (Mintong,
Gunjan, Wendy, Yun, & Larry, 2002) aiming to study the 5 major
variables among the ones screened by the PB design to have the
most significant effects on ␤-glucanase activity. Temperature and
oligoelements were studied at low and high levels coded as −1 and
+1, whereas pH, yeast extract, and casein peptone were studied at
low, middle, and high levels coded as −1, 0, and +1, respectively. The
experiments were performed in triplicate, and data given represent
average values.
2.4. Production medium
At optimized conditions, cultures were performed in 1 l flask
containing 100 ml of barley flour (8%, w/v), yeast extract (1.5%, w/v),
casein peptone (0.5%, w/v), and oligoelements solution (0.2%, v/v)
consisting of ZnSO4 (1.4 g/l), FeSO4·7H2O (5 g/l), MnSO4 (1.6 g/l),
and CaCl2 (2 g/l).
2.5. Enzyme assay
2.10. Software tools
␤-Glucanase activity was determined at 60 ◦C using barley ␤-
glucan (0.2%, w/v) as a substrate in 100 mM sodium acetate buffer
(pH 5). The reaction was interrupted after 30 min of incubation by
adding 3,5-dinitrosalicylic acid, and the reducing sugars released
were then quantified (Miller, 1959).
The experimental data were analyzed using the statistical soft-
ware package “SPSS” (Version 11.0.1 2001, LEAD Technologies, Inc.,
USA). The EXCEL software (Version 2003, Microsoft Office, Inc., USA)
was employed to generate the response surface that allows to find
out the levels of the variables for maximal ␤-glucanase production.
One unit (U) of enzyme activity was defined as the amount of
enzyme capable of releasing 1 ␮mol of reducing sugars/min, under
the defined assay conditions.
2.11. Enzyme purification
A. niger ␤-glucanase was purified from a culture grown in
the production medium for 4 days at 37 ◦C and 150 rpm, all
purification steps were carried out at 4 ◦C. The supernatant was
concentrated 10-fold by rotavaporation and ultrafiltration using a
10 kDa cut-off membrane. Purification to homogeneity was per-
formed by HPLC using a gel filtration column (PL aquagel OH 40
column/300 mm × 250 mm) pre-equilibrated with sodium acetate
buffer pH 5.5 (25 mM). The active fractions pooled from the gel-
filtration step were re-injected into the same column OH 40 to
achieve purification.
2.6. Optimization of carbon source
Based on the literature, and aiming to improve enzyme pro-
duction while minimizing production costs, different economical
and inexpensive carbon sources were assayed for their effects
on the expression of ␤-glucanase. A. niger US368 was cultivated
in the basal medium mentioned above where different carbon
sources (wheat bran, starch, corn steep liquor, glucose, soybean
meal and gruel) were assayed instead of barley flour at 30 ◦C with

F. Elgharbi et al. / Carbohydrate Polymers 98 (2013) 967–975
969
2.12. Electrophoresis and zymogram
were periodically withdrawn, and the reaction was stopped by pla-
cing the samples in boiling water for 5 min. Products of enzymatic
hydrolyses were analyzed qualitatively by thin-layer chromatog-
raphy (TLC) on silica gel 60 F254 plates (Merck 1.05554; 20 cm
by 20 cm) with a butan-1-ol–acetic acid–water (2:1:1, v/v/v) sol-
vent system (Yang et al., 2008). The plates were developed with
one run followed by heating for a few minutes at 130 ◦C in an
oven after spraying the plates with an ethanol–sulfuric acid mix-
ture (95:5, v/v). Glucose and cellobiose were used as the standards.
The end-products hydrolysis of the ␤-glucan were also analyzed
by HPLC on an Agilent 1260 chromatograph (column: Sugar KS-
802, rate 0.8 ml min−1, elution: water) and the mass spectra are
recorded, between 50 and 1500 atomic mass units (m/z), using a
Q-TRAP mass spectrometer (Agilent, USA). In the electrospray ion-
ization mass spectrometry (ESI-MS) experiments, the quadrupole
scan mode was under an electrospray voltage of 3 kV at the tip of a
stainless steel capillary needle, with a source block temperature of
350 ◦C.
SDS–PAGE was performed using a 5% stacking gel and 10%
resolving gel under reducing conditions as described by Laemmli
(1970). Protein concentration was determined using Bio-Rad pro-
tein assay kit, based on the method of Bradford using bovine serum
albumin as the standard (Bradford, 1976).
The molecular mass estimated for the native purified glu-
canase was determined by PAGE under denaturating and
non-denaturating conditions. Protein bands were visualized by
Coomassie brilliant blue R-250 (Bio-Rad) staining. Enzyme activity
was detected in situ in SDS/−polyacrylamide gels containing 0.2%
␤-glucan. After electrophoresis, proteins were re-natured by soak-
ing the gel for 2 h in 50 mM sodium acetate buffer (pH 5) at room
temperature using horizontal shaker. The gel was then incubated
for 1 h at 60 ◦C in the same buffer and stained with 0.1% Congo Red
(Teather & Wood, 1982).
2.13. Amino acid sequencing
2.18. Michaelis–Menten kinetics
To determine the NH2-terminal sequence, protein bands from
SDS gels were blotted onto a polyvinylidene difluoride membrane
(Millipore, Bedford, MA). Automated Edman protein degradation
was performed with a protein sequencer (Applied Biosystems Pro-
tein sequencer ABI Procise 492/610A) equipped with a 140C HPLC
system (Hewick, Hunkapiller, Hood, & Dreyer, 1981).
␤-Glucanase assays were performed using barley ␤-glucan at
various concentrations ranging from 0.1% to 1% (w/v). Assays were
performed at 60 ◦C using a 50 ␮g ml−1 enzyme concentration in the
assay mixture. The reducing sugars generated were determined at
regular time intervals and expressed in equivalent micromoles of
glucose per ml of purified enzyme used for the reaction. The Vmax
and Km values were calculated from a Lineweaver and Burk plot
using the hyper-32 program.
2.14. Effects of temperature and pH
The effects of pH and temperature on the activity of ␤-glucanase
were examined using barley ␤-glucan (0.2%) as a substrate. To
determine the pH profile, reactions were performed at 65 ◦C in
100 mM glycine–HCl buffer (pH 3–4), sodium acetate buffer (pH
4–6), Na-phosphate buffer (pH 6–7) and Tris–HCl buffer (pH 7–9).
The effect of temperature on the enzyme was investigated at tem-
perature values ranging from 40 to 80 ◦C and at pH 5 (in 100 mM
sodium acetate buffer).
2.19. Metal ion requirement
Several metallic ions (Co2+, Zn2+, Mg2+, Ca2+, Cu2+, Fe2+, Mn2+
,
and Mg2+) were assayed at 5 mM for their effects on ␤-glucanase
activity under optimal conditions.
2.20. Configuration of the anomeric carbon of the released glucose
2.15. pH and temperature stabilities
The method described in Genta, Dumont, Marana, Terra, and
Ferreira (2007) and Saibi and Gargouri (2011) was used to identify
the anomeric configuration of the glucose liberated following the
action of the purified ␤-glucanase. At the end of the reaction, the
liberated glucose can have ␣ or ␤ form depending on the action
mechanism of the enzyme, qualified respectively as a retaining or
inverting mode. The ␤-glucose might be converted into ␣ form by a
simple heating at 100 ◦C for 10 min. Moreover, the glucose oxidase
can act only on the ␤-glucose. Consequently, the method described
by Genta and his collaborators (2007) and adapted to ␤-glucanase
assessment is based on the comparison of the amount of glucose
liberated by the ␤-glucanase, determined by GOD (glucose oxidase
kit) either directly or after a heat treatment and considering that
glucose oxidase reacts exclusively with ␤-glucose form. In addi-
tion, the amount of ␤-glucan hydrolysis (for 4 h) is also measured
spectrophotometrically (Miller, 1959), in order to compare it to
the GODs determined amount of glucose, without heating. Hence,
the enzyme was incubated with barley ␤-glucan (0.2%, w/v) as a
substrate in 100 mM sodium acetate buffer (pH 5) for 4 h at 60 ◦C.
The reaction was stopped by the addition of MnSO4 (5 mM) which
completely inhibits the ␤-glucanase activity. Two equivalent sam-
ples were removed, the first before and the second after boiling the
reaction tube for 10 min. Heating insures the displacement of the
equilibrium between the two anomeric forms of glucose toward
the ␣-form. The GOD kit was used to measure the liberated glucose
using two controls consisting in the fresh and boiled solution of
glucose.
The thermal stability of the enzyme was assayed at 50, 60, 65,
70, and 80 ◦C. Incubation was performed in 100 mM sodium acetate
buffer pH 5. Aliquots were drawn at regular time intervals and
immediately cooled in ice-cold water. Residual activities, deter-
mined at 60 ◦C under the standard assay conditions, were expressed
as percentage activity of zero-time control of untreated enzyme.
The stability of ␤-glucanase was investigated over a pH range
of 3–10 in 100 mM buffers. Buffers used were glycine–HCl buffer
(pH 3–4), sodium acetate buffer (pH 4–6), phosphate buffer (pH
6–7.5), Tris–HCl buffer (pH 7.5–9), and glycine–NaOH buffer (9–10).
Aliquots were taken after 1 h of incubation at 37 ◦C and immediately
assayed for residual ␤-glucanase activity.
2.16. Enzyme specificity
A. niger ␤-glucanase activity was determined under optimal
conditions (60 ◦C, sodium acetate buffer pH 5), on barley ␤-
glucan (0.2%, w/v), lichenan (0.2%, w/v), laminarin (0.2%, w/v), and
CM–cellulose (0.2%, w/v) substrates prepared in Milli-Q water.
2.17. Hydrolysis properties of purified ˇ-glucanase
For the analysis of barley ␤-glucan degradation products, 10 U
of purified ␤-glucanase were incubated with 10 mg of substrate
in 1 ml of 100 mM sodium acetate buffer (pH 5) at 50 ◦C. Aliquots

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F. Elgharbi et al. / Carbohydrate Polymers 98 (2013) 967–975
3. Results
3.1. Optimization of enzyme production
The traditional “one factor at a time” method was used to study
the effects of various carbon sources and inoculum size on ␤-
glucanase production by A. niger US368. Accordingly, ␤-glucanase
production was assayed with different carbon sources, namely
wheat bran, starch, barley flour, corn steep liquor, glucose, soy-
bean meal, and gruel (a by-product of wheat milling; Kammoun,
Naili, & Bejar, 2008), in the basal liquid media at 1%, 30 ◦C, pH 5.5,
and 150 rpm (data not shown). The findings revealed that barley
flour was the most appropriate carbon source for glucanase pro-
duction, with maximum production of 0.6 U ml−1 within 4 days of
growth. Fungal ␤-d-glucanase enzymes production is influenced
by a number of factors including the type of strain used, the cul-
ture conditions and mainly the substrate type (Harman, Howell,
Viterbo, Chet, & Lorito, 2004; Howell, 2003; Martin, McDougall,
McIlroy, Chen, & Seviour, 2007; Pitson, Martin, & McDougall, 1993).
Zymogram activity staining of the enzymatic extraction in the basal
medium with 1% barley flour showed a single active band suggest-
ing that our strain produces only one type of enzyme according
to the culture conditions used. The substitution of barley flour in
the culture medium by soybean meal, gave us different protein pro-
files (Fig. 1). The production of ␤-glucanase is dependent on carbon
sources used (Vázquez-Garciduen˜as, Carlos, Morales, & Estrella,
1998). We were interested on the glucanase produced in medium
containing 1% barley flour since the activity was 0.6 U ml−1 against
0.4 U ml−1 in medium based on 1% soybean meal.
Accordingly, barley flour was further assayed at various con-
centrations ranging from 1 to 12% (w/v) to determine the optimal
concentration required for maximal ␤-glucanase production. The
Fig. 2. (a) Optimization of barley flour concentration. Barley flour was assayed
at various concentrations ranging from 1 to 12% (w/v) to determine the optimal
concentration required for maximal ␤-glucanase production. (b) Optimization of
inoculum size. ␤-Glucanase production activity was studied by assaying various
sizes of inocula ranging from 102 to 107 spores/ml of sterile media.
results indicated that a maximal activity of 1.1 U ml−1 (1.6 U mg−1
)
was obtained with barley flour used at a concentration of 8%
(Fig. 2a).
The size of the inoculums is also reported to have significant
effects on the growth of microorganisms and enzyme production
(Pandey, 1992; Pandey, Soccol, & Mitchell, 2000). ␤-Glucanase pro-
duction activity was, therefore, studied by assaying various sizes
of inocula. The results revealed that optimal glucanase production
was achieved at an initial inoculum size of 106 spores/ml of culture
media. An important decrease in enzyme production was noted
when the size of the inoculums increased (Fig. 2b).
To enhance glucanase production from A. niger US368, different
culture conditions and nutrients were investigated in terms of their
effects on enzyme production. Eight parameters were evaluated
using a Plackett–Burman experimental design. The effects of the
selected variables, namely temperature, oligoelements, pH, yeast
extract, and casein peptone, were further investigated via an L18C-
Chakravarty design to determine the optimal level of each variable
for maximum glucanase production. The data were submitted to
a Student’s test, and an equation linking the measured response
to the independent variables, was established (data not shown).
The statistical model was validated with respect to ␤-glucanase
production under the optimized conditions predicted by the model.
An experimental response of 2.2 U ml−1 (1.85 U mg−1) was obtained
using 8% barley flour, 1.5% yeast extract, 0.5% casein peptone, 0.2%
oligoelements solution, pH 5.5, 37 ◦C and 150 rpm.
3.2. Purification of ˇ-glucanase and N-terminal amino acid
sequence
A ␤-glucanase was purified from the supernatant of A. niger
US368 grown in the optimized culture medium. The purification
procedure, the purification degree, and production yield of each
Fig. 1. Zymogram of ␤-1,3;1,4-glucanase activity in culture medium containing
barley flour (lane 1) and culture medium containing soybean meal (lane 2).

F. Elgharbi et al. / Carbohydrate Polymers 98 (2013) 967–975
971
Table 1
Summary of the purification protocol of the ␤-glucanase produced by A. niger US368.
Steps
Total protein (mg)
Total activity (U)
Specific activity (U mg−1
)
Purification (-fold)
Yield (%)
Culture supernatant
Concentrated crude extract
Gel filtration
1080
167.02
6.2
2000
780
207.8
1.85
4.67
33.33
1
100
39
10.39
2.52
14.24
step are summarized in Table 1. After purification, the ␤-glucanase
was enriched about 14.24-fold, with a yield of 10.39% (Table 1).
The purified enzyme showed a specific activity of 33.33 U mg−1
and displayed a single protein band with a molecular mass of
about 32 kDa on SDS–PAGE (Fig. 3a). Zymogram activity staining
revealed one clear zone of glucanase activity (Fig. 3b), and the puri-
fied enzyme was also noted to exhibit a single protein band on
native PAGE (data not shown), thus indicating that the ␤-glucanase
extracted from A. niger US368 was a monomeric enzyme.
niger US368 showed high pH stability within the range of pH 3–10
after 1 h incubation at 37 ◦C (Fig. 4c). It also displayed a marked
thermostability, retaining 100% and 70% of activity after incuba-
tion at 60 ◦C for 45 min and 90 min, respectively. The half-lives
recorded for the purified enzyme at 50 ◦C, 60 ◦C, 65 ◦C, and 70 ◦C
were 150 min, 135 min, 90 min, and 30 min, respectively. At 80 ◦C,
the enzyme was noted to be inactive and unstable (Fig. 4d).
The sequence determined for the first 9 N-terminal amino acids
of the purified ␤-glucanase, namely A-G-T-N-P-P-I-G-V, showed
uniformity, strongly indicating that it was isolated in a pure form.
3.3. Optimum pH, temperature, and stability
The effects of pH and temperature on the activity of the purified
␤-1,3;1,4-glucanase are shown in Fig. 4a and b, respectively. The
enzyme was noted to exhibit significant activity at 65 ◦C and in the
pH range of 3–6.5, with maximal activity being recorded in the pH
range of 4–5. No enzyme activity was detected at pH values higher
than 6.5 (Fig. 4a).
At pH 5, the purified enzyme was substantially active in the tem-
perature range of 40–80 ◦C, with maximal activity recorded at 60 ◦C
(Fig. 4b).
The pH stability and thermostability profiles of the enzyme
are shown in Fig. 4c and d, respectively. The glucanase from A.
Fig. 4. (a) Effect of pH on the activity of the ␤-glucanase purified from A. niger US368.
The relative activities were determined according to the standard assay with buffers
solutions at 100 mM as follows: glycine–HCl buffer (pH 3–4), sodium acetate buffer
(pH 4–6), phosphate buffer (pH 6–7) and Tris–HCl buffer (pH 7–9). (b) Effect of tem-
perature on the activity of the ␤-glucanase purified from A. niger US368. The relative
activities were determined according to the standard assay at pH 5 and tempera-
tures varying from 40 to 80 ◦C. (c) pH stability of the ␤-glucanase purified from A.
niger US368. The relative activities were determined according to the standard assay
Fig. 3. (a) SDS–PAGE of proteins during the purification of ␤-1,3;1,4-glucanase.
Lanes are designated as follows: lane M, molecular mass standard proteins: lane
1, crude culture supernatant; lanes 2 and 3, pooled fraction from the PL aquagel OH
40 column. (b) Zymogram of ␤-1,3;1,4-glucanase. Lane 4, crude culture supernatant;
lane 5, purified enzyme.
at pH values ranging from 3 to 10. Aliquots were taken after 1 h of incubation at 37 ◦
C
and immediately assayed for residual ␤-glucanase activity in standard assay condi-
tions. (d) Thermostability of the purified A. niger US368 ␤-glucanase at pH 5.0 and
temperatures of 50 ◦C (ꢀ), 60 ◦C (ꢁ), 65 ◦C (᭹), 70 ◦C (ꢂ), and 80 ◦C (*).

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F. Elgharbi et al. / Carbohydrate Polymers 98 (2013) 967–975
Table 2
3.8. The ˇ-glucanase is a retaining enzyme
Effects of metal ions on ␤-glucanase activity. The relative activities were measured
at optimum of pH and temperature and enzyme activities without metal ions were
taken as 100%.
The configuration of the anomeric carbon of glucose (end-
product of the ␤-glucan hydrolysis) was determined according
Metal ions (5 mM)
Relative activity (%)
Specific activity (U mg−1
)
to the method described in Section
Saibi & Gargouri, 2011). Finding showed that the amount of ␤-
glucan hydrolysis measured spectrophotometrically (7.5 g ml−1
2 (Genta et al., 2007;
None
Mg2+
Cu2+
Zn2+
Fe2+
100
98
0
98
0
48
89
0
1.85 0.32
1.22 0.25
0
1.22 0.25
0
0.6 0.22
1.11 0.31
0
)
and the amount of glucose determined using GOD method
(6.8 g ml−1) without heating (␤ form), are similar. Whereas, after
boiling, the amount of ␤-glucose is roughly nil (0.054 g ml−1).
Consequently, ␤-glucanase could be considered as a retaining-
enzyme.
Co2+
Ca2+
Mn2+
3.4. Effects of metal ions
4. Discussion
No significant change in enzymatic activity was observed after
extensive dialysis against EDTA, which suggested that divalent
metal ions were not involved in the enzymatic catalysis process.
The effects of cations on enzymatic activity were also investi-
The strain of A. niger US368 was already described for producing
an interesting xylanase (Hmida-Sayari et al., 2012). In the line to test
whether this strain could produce a mixture of hydrolytic enzymes
useful for degradation of water-soluble nonstarch polysaccharides
of several feedstuffs, we demonstrated that it produce also an inter-
esting ␤-glucanase.
In the present study, the production of ␤-glucanase by A. niger
US368 was optimized using statistical experimental designs of
Plackett–Burman and L18C Chakravarty by using barley flour as the
sole carbon source. Hence the production level was substantially
increased from 0.6 to 2.2 U ml−1. This production was comparable
to those from Bacillus licheniformis (Lloberas, Querol, & Bernués,
1988) and Penicillium emersonii (James, Fare, Sagar, Lucas, & DeGray,
1975). However, production was still lower than those attained
from Paecilomyces thermophila (Yang et al., 2008) and Rhizomucor
miehei (Tang et al., 2012).
gated through the addition of 5 mM Mg2+, Fe2+, Zn+2, Ca+2, Cu2+
,
Co2+, and Mn2+ to the reaction buffer. The enzyme was noted
to be completely inhibited by Fe2+, Mn2+ and Cu2+, and partially
inhibited by Mg2+ and Zn2+ (2%), by Ca2+ (11%) and by Co2+ (52%)
(Table 2).
3.5. Enzyme specificity
The A. niger US368 purified ␤-glucanase was assayed for its abil-
ity to hydrolyze several glucan substrates. Only barley ␤-glucan
was efficiently hydrolyzed. No activity was detected for the enzyme
against laminarin (1,3-␤-glucan) and CM-cellulose (soluble 1,4-
␤-glucan), which strongly suggested that the enzyme was a 1,3
1,4-␤-glucanase (or lichenase) that can presumably be considered
as a member of the EC 3.2.1.73 enzyme category.
The extracellular glucanase produced by A. niger US368 was
purified and the molecular mass was estimated (32 kDa). ␤-
1,3;1–4-glucanases with similar molecular mass values have pre-
viously been purified from Rhizopus microsporus var. microsporus
(36.5 kDa) (Celestino et al., 2006) and Orpinomyces strain PC-2
(27 kDa) (Chen et al., 1997).The purified enzyme was demon-
strated to be an ␤-1,3;1,4-glucanase (EC 3.2.1.73) or lichenase
3.6. Hydrolysis properties of the purified ˇ-glucanase
The hydrolysis products formed during the action of the purified
enzyme on barley ␤-glucan were studied by TLC (Fig. 5a) and HPLC
(Fig. 5b). When barley ␤-glucan was used as a substrate, oligosac-
charides (mainly trisaccharide and tetrasaccharide) followed by
disaccharide (cellobiose) and glucose were formed as the major
products of hydrolysis after a prolonged incubation of 4 h with the
enzyme. This was also confirmed by mass spectroscopic analysis.
Indeed, the ESI-MS of the final hydrolysis products of ␤-glucan by
the purified enzyme revealed the presence of small oligosaccha-
rides and glucose (Fig. 5c). They are G4 (m/z = 664), G3 (m/z = 504;
M+H+), G2 (m/z = 343; M+H+), and G1 (m/z = 162; M−H2O + H+,
m/z = 180; M+H+, m/z = 202; M+Na+).
since it was able to hydrolyzed barley ␤-glucan (33.33 U mg−1
)
and lichenan (16.8 U mg−1) and did not attack laminarin and
CM-cellulose. In fact, most endo-␤-1,3;1,4-glucanases (lichenase)
cleave ␤-1,4 linkages adjacent to ␤-1,3 bonds in glucans, yield-
ing chiefly cellobiosyltriose and cellotriosyltetraose (Chen et al.,
1997; Erfle, Teather, Wood, & Irvin, 1988; Hrmova & Fincher, 2001;
Yoo, Lee, Chang, Lee, & Yoo, 2007). The hydrolysis patterns were
similar to those previously described for ␤-1,3;1,4-glucanase from
Orpinomyces strain PC-2 (Chen et al., 1997), Talaromyces emersonii
(Murray et al., 2001), and Aspergillus japonicus (Grishutin, Gusakov,
Dzedzyulya, & Sinitsyn, 2006). This enzyme represents the first
lichenase studied from A. niger.
The analysis of the N-terminal sequence of ␤-glucanase using
BLAST program (Gish & States, 1993) revealed no similarity with
any of the sequence in the non-redundant databases. This was not,
in fact, unexpected since there was a large heterogeneity on the
lichenases already reported from fungi (Chen et al., 1997). Fur-
thermore, this is the first time a lichenase was studied from A.
niger.
3.7. Kinetic parameters
The Michaelis–Menten constants were determined for barley
␤-glucan and lichenan (Table 3). The Km and Kcat values were
0.62 mg ml−1 and 19.11 s−1 for barley ␤-glucan and 0.38 mg ml−1
and 13.32 s−1 for lichenan, respectively.
The optimal pH and temperature values determined for the puri-
fied ␤-1,3;1,4-glucanase from A. niger US368 are in agreement with
the ones previously reported for several other fungi and bacteria
(Planas, 2000). They are also comparable to those described for
the enzymes currently used in the brewing industry (McCleary &
Nurthen, 1986).
Table 3
Kinetic parameters for purified ␤-1,3;1,4-glucanase.
Substrate
Vmax (U ml−1
)
Km (mg ml−1
)
Kcat (s−1
)
Kcat/Km
(ml mg−1 s−1
)
Barley ␤-glucan
Lichenan
34.46 7.315
24.02 3.9
0.62 0.218
0.38 0.121
19.11
13.32
30.82
35.1
The purified enzyme showed a manifest thermostability reach-
ing 90 min and 30 min at 65 ◦C and 70 ◦C, respectively; better,

F. Elgharbi et al. / Carbohydrate Polymers 98 (2013) 967–975
973
Fig. 5. (a) TLC analysis of final products of ␤-glucan hydrolysis by the purified ␤-1,3;1,4-glucanase. Incubation times (h) were indicated. Glucose (G1) and cellobiose (G2)
were used as the standard markers. (b) HPLC elution profile of the final products of glucan hydrolysis by the purified ␤-1,3;1,4-glucanase. Glucose (G1), cellobiose (G2),
cellotriose (G3), cellopentaose (G5) and cellohexaose (G6) were used as the standard markers. (c) Analysis of the end product of the enzymatic hydrolysis of ␤-glucan by
ESI-MS/HPLC after incubation for 4 h. ␤-Glucan was sequentially cleaved into G4 (m/z = 664), G3 (m/z = 504; M+H+), G2 (m/z = 343; M+H+), and G1 (m/z = 162; M−H2O+H+,
m/z = 180; M+H+, m/z = 202; M+Na+).

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F. Elgharbi et al. / Carbohydrate Polymers 98 (2013) 967–975
than those previously purified from several other strains, includ-
ing Rhizopus microsporus var. microsporus (Celestino et al., 2006)
and Orpinomyces strain PC-2 (Chen et al., 1997). Indeed, the half-
lives reported for the ␤-glucanase of Rhizopus microsporus var.
microsporus at the temperatures of 60 ◦C and 70 ◦C were 10 min
and 1 min, respectively, while ␤-glucanase of Orpinomyces strain
PC-2 was reported to retain only 30% of its activity at 55 ◦C after 1 h
of incubation.
No significant change in enzymatic activity was observed after
extensive dialysis against EDTA, which suggested that divalent
metal ions were not involved in the enzymatic catalysis process.
Similar insensitivity toward the divalent metal ions were previ-
ously reported for glucanases produced from R. microsporus var.
microsporus (Celestino et al., 2006), Rhizopus oryzae (Murashima
et al., 2002), and Trichoderma harzianum (Rana, Theodore, Naidu, &
Panda, 2005).
The Km and Vmax values exhibited by the enzyme on barley ␤-
glucan were 0.62 mg ml−1 and 34,46 U ml−1, respectively. A broad
range of Km values have been previously reported for barley ␤-
glucan from different sources (Celestino et al., 2006; Ekinci et al.,
1997; Erfle et al., 1988; Planas, 2000). ␤-1,3;1,4-Glucanase from
Bacillus species was, for instance, described to display Km values
in the range of 1.2–1.5 mg ml−1 on barley ␤ glucan (Planas, 2000).
In fact, the affinity constants described for the barley ␤-glucan
presented in the current study were lower than those previously
reported for Talaromyces emersonii (Murray et al., 2001) and Rhizo-
pus microsporus var. microsporus (Celestino et al., 2006).
The purified ␤-glucanase is a retaining-enzyme. In fact, it is
described that the glycoside hydrolases family 16 act with retention
of configuration (Planas, 2000). According to these findings and the
3D structure of all known retaining ␤-glucanase, one can say that
the distance between the two ␤-glucanase catalytic acids should
be around 5.5◦ A (Thu & David, 2010).
Appendix A. Supplementary data
Supplementary data associated with this article can be
found, in the online version, at http://dx.doi.org/10.1016/j.carbpol.
2013.07.009.
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