Appl Biochem Biotechnol

DOI 10.1007/s12010-014-1153-2

Partial Characterization of Xylanase Produced

by Caldicoprobacter algeriensis, a New Thermophilic
Anaerobic Bacterium Isolated from an Algerian
Hot Spring
Khelifa Bouacem & Amel Bouanane-Darenfed &
Nawel Boucherba & Manon Joseph &
Mohammed Gagaoua & Wajdi Ben Hania &
Mouloud Kecha & Said Benallaoua & Hocine Hacne &
Bernard Ollivier & Marie-Laure Fardeau

Received: 25 March 2014 / Accepted: 15 August 2014

# Springer Science+Business Media New York 2014

Abstract To date, xylanases have expanded their use in many processing industries, such as
pulp, paper, food, and textile. This study aimed the production and partial characterization of a
thermostable xylanase from a novel thermophilic anaerobic bacterium Caldicoprobacter
algeriensis strain TH7C1T isolated from a northeast hot spring in Algeria. The obtained results
showed that C. algeriensis xylanase seems not to be correlated with the biomass growth profile
whereas the maximum enzyme production (140.0 U/ml) was recorded in stationary phase
(18 h). The temperature and pH for optimal activities were 70 C and 11.0, respectively. The
enzyme was found to be stable at 50, 60, 70, and 80 C, with a half-life of 10, 9, 8, and 4 h,
respectively. Influence of metal ions on enzyme activity revealed that Ca+2 enhances greatly
the relative activity to 151.3 %; whereas Hg2+ inhibited significantly the enzyme. At the best of
our knowledge, this is the first report on the production of xylanase by the thermophilic
bacterium C. algeriensis. This thermo- and alkaline-tolerant xylanase could be used in pulp
bleaching process.
K. Bouacem : A. Bouanane-Darenfed : H. Hacne
Laboratory of Cellular and Molecular Biology (Microbiology group), Faculty of Biology, University of
Science and Technology Houari Boumediene, Bab Ezzouar, Algiers, Algeria
N. Boucherba : M. Kecha : S. Benallaoua
Laboratory of Applied Microbiology, Faculty of Nature Science and Life, University A/Mira of Bejaia,
Targa Ouzemmour 06000, Algeria
A. Bouanane-Darenfed : M. Joseph : W. Ben Hania : B. Ollivier : M.<L. Fardeau (*)
Aix Marseille Universit, CNRS, Universit de Toulon, IRD, Mediterranean Institute of Oceanography
(MIO), UM 110, 13288 Marseille, France
e-mail: marie-laure.fardeau@univ-amu.fr
M. Gagaoua
Maquav, Bioqual Laboratory, INATAA, Universit de Constantine 1, Route de Ain El-Bey,
25000 Constantine, Algerie

Appl Biochem Biotechnol

Keywords Alkalitolerant . Caldicoprobacter algeriensis . Birchwood xylan . Thermostable

xylanase . Partial characterization . Hot spring

Introduction
Hemicelluloses are heterogeneous polysaccharides reported to be the second most abundant
organic structure in the plant cell wall after cellulose. The major hemicellulose polymer in
cereals and hardwood is xylan. Xylan consists of a -1,4-linked D-xylose backbone which can
be substituted with different side-groups such as L-arabinose, D-galactose, acetyl, feruloyl and
p-coumaroyl, and glucuronic acid residues [64].
As xylan is too complex, wide range of enzymes (hydrolases and esterases) must
be implemented in order to achieve its complete degradation. These enzymes contain
those acting at the main chain of xylan as the endoxylanases and -xylosidases and
other so-called debranching enzymes or accessories that address the branches grafted
onto the main chain. Among these debranching enzymes, -L-arabinofuranosidase, glucuronidase, acetyl xylan esterase, and feruloyl/coumaryl esterase were described
[53, 63].
Xylanases are one of the microbial enzymes that have aroused great interest in the
last decade due to their biotechnological potential in many industrial processes. They
represent the largest proportion of the world enzymes market [10, 70, 58]. Xylanases
are produced by bacteria [26, 36], fungi [45, 2], actinomycetes [14, 15, 51], and yeast
[28, 39, 43]. Most of these xylanases are used in food industry [10], in bioethanol
production process [41], and to improve the digestibility of some feeds [74, 62]. Most
of the process steps are performed at high temperatures. In this view of interest,
thermophilic microorganisms gained the attention of the scientific and industrial
communities as they are very useful as a new source of thermostable enzymes [8,
67]. The use of thermophilic microorganisms in the production of thermostable
xylanases has major technical and economic advantages [27]. Several thermophilic
microorganisms are reported capable of producing thermostable xylanases such as
Thermotoga sp. FjSS3-B1 strain [61], Clostridium abosum [55], Pyrodictium abyssi
[4], and Bacillus pumilus [44].
As part of the characterization of biodiversity of the Algerian thermal waters, we isolated a
new thermophilic anaerobic xylanolytic strain, Caldicoprobacter algeriensis TH7C1T from a
hot spring of Hammam Dbagh (Guelma) situated in the northeast of Algeria [13]. In the
present study, an attempt was made to describe the optimization studies related to xylanase
production from C. algeriensis culture. Optimization of enzyme production was carried out
with respect to carbon source, nitrogen source, and the composition of the medium. Partial
characterization of the crude extracellular xylanase and its potential biochemical properties
were also reported.

Materials and Methods

Substrates and Chemicals
Unless specified, all substrates, chemicals, and reagents were of the analytical grade
or highest available purity and were purchased from Sigma Chemical Co. (St. Louis,
MO, USA).

Appl Biochem Biotechnol

Isolation of Microorganism
The preliminary study of bacterial strains isolated from Algerian hot spring [13] revealed that
they produce thermostable enzymes and especially xylanases. Among them, C. algeriensis
TH7C1T, which was isolated from a terrestrial hot spring in Guelma (70 25 E, 36 27 N), a
region situated in the northeast of Algeria.
The medium (TH) used for isolation contained (in g/l): NH4Cl (1.0), K2HPO4 (0.3),
KH2PO4 (0.3), KCl (0.1), MgCl26H2O (0.5), CaCl22H2O (0.1), NaCl (0.5), yeast extract
(2.0), biotrypcase (2.0), CysteineHCl (0.5), together with sodium acetate (2 mM), and Balch
trace element solution (10 ml) [6]. The pH was adjusted to 7.2 using 10 M KOH solution, and
the medium was boiled and cooled at room temperature under a stream of O2-free N2 gas.
Aliquots of 5 ml were dispensed into Hungate tubes, degassed under N2CO2 (80:20 v/v), and
subsequently sterilized by autoclaving at 120 C for 20 min. Before inoculation, 0.1 ml of
10 % (w/v) NaHCO3, 0.1 ml of 2 % (w/v) Na2S9H2O, and 20 mM glucose were injected from
sterile stock solutions into the tubes. Enrichments were performed as described by [13] in
Hungate tubes or serum bottles inoculated with 10 % of sample and incubated at 70 C.
Optimum Growth Conditions
The pH, temperature, and NaCl concentration ranges for growth of C. algeriensis TH7C1T
were determined using basal medium supplemented with 20 mM glucose. The different pH
(5.09.0) of the medium was adjusted by injecting in Hungate tubes aliquots of anaerobic
stock solution of 0.1 M HCl, 10 % NaHCO3, or 8 % Na2 CO3. Water baths were used for
incubating bacterial cultures from 45 to 90 C. NaCl requirement was determined by directly
weighing NaCl in Hungate tubes before dispensing medium. Cultures were subcultured at least
twice under the same experimental conditions before determination of growth rates and use of
substrates [13].
Enzyme Production
Birchwood xylan (10 g/l) was tested at a final concentration of 20 mM in 5 ml growth medium
without glucose then incubated at 70 C, pH 7.2 for 18 h. Before assay, the cells were
separated by centrifugation at 1,0000xg for 20 min. The clear supernatant was used as crude
enzyme preparation.
Analytical Methods
The xylanase activity was assayed using 10 mg/ml birchwood xylan in 50 mM sodium
phosphate buffer at pH 7.0. The reaction mixture consisted of 0.9 ml of substrate and 0.1 ml
of the crude enzyme. This mixture is incubated in a water bath at 60 C for 10 min as described
by [72]. The reducing sugars are determined by the method of Miller [42], using xylose as a
standard. The enzyme reaction is stopped by adding 1.5 ml of a solution based on
dinitrosalicylic acid (DNS) and immersing in boiling water for 5 min. After cooling, the
intensity of the color was measured by optical density at 540 nm. One unit (U) of xylanase was
defined as the amount of enzyme required to release 1 mol reducing sugar as xylose
equivalent in 1 min under the above assay conditions [5].
The cell growth was estimated by soluble protein estimation following Bradfords method
with bovine serum albumin (BSA) as the standard [16] and by measuring the optical density at
600 nm.

Appl Biochem Biotechnol

Activity of Crude Enzyme on Various Carbohydrate Substrates

The effect of various carbon sources on the xylanase production was assessed by culturing the
C. algeriensis TH7C1T in the TH medium (pH 7.2) at 70 C. Either birchwood xylan, orange
peel, starch, xylose, cellulose, lactose, galactose, maltose, arabinose, wheat straw, barley or
carboxymethylcellulose was used as carbon source (10 g/ml) individually in liquid medium
TH then the xylanase activity was estimated.
Effects of Nitrogen Concentration on Xylanase Production
To study the influence of nitrogen concentration for the production of xylanase, several
combinations have been used: (1) 1 g of NH4Cl with 2 g of yeast extract and 2 g biotrypcase,
(2) 2 g of NH4Cl with 1.5 g of yeast extract and 1.5 g of biotrypcase, and (3) 3 g NH4Cl with
1 g of yeast extract and 1 g of biotrypcase.
Growth and Xylanase Production Profiles
Xylanase production was performed in laboratory fermenter with a working volume of
500 ml. The culture conditions were as follows: TH medium with 10 g/l of xylan, pH
7.2, and temperature 70 C under the anaerobic conditions during 23 h. Culture
samples were collected at 2 h intervals for the stationary phase and then 1 h during
the cultivation period. Immediately after collection, the samples were centrifuged at
10,000xg for 20 min at 4 C. Supernatants were analyzed for xylanase activity as
described above.
Partial Characterization
Effect of pH on Xylanase Activity
The optimal pH for xylanase activity was obtained by assaying the enzyme activity in
the pH range 4.013.0. Six different buffers (50 mM) were used for this study: citrate
buffer (pH 4.05.5), phosphate buffer (pH 6.07.5), TrisHCl buffer (pH 8.08.5),
glycine-NaOH buffer (pH 9.010.5), bicarbonate-NaOH buffer (pH 11.011.5), and
Na2HPO4-NaOH buffer (pH 12.013.0). The relative activity was determined by the
above described assay method.
Effect of Temperature on Xylanase Activity
Determination of enzyme activity for optimum temperature, the crude enzyme sample was
incubated at various temperatures at pH 11.0 in the range of 50 to 90 C at intervals of 10 C.
The sample was then taken for enzymatic assay as described earlier.
Thermostability
The thermostability of xylanase was determined after incubation of the crude enzyme
in the absence and presence of the substrate at temperatures of 50, 60, 70, and
80 C. The samples were removed after incubation for varying times (from 0 to
15 h) at intervals of 1 h. The residual xylanase activity was determined using the
standard assay.

Appl Biochem Biotechnol

Effects of Various Metal Ions and Other Additives on Xylanase Activity

The effects of various metal ions on xylanase activity were evaluated by incubating the
reaction mixtures containing 10 mg/ml of the birchwood xylan with Mg2+, Ca2+,Mn2+, Fe2+,
Zn2+, Cu2+, NH4+, Hg2+, K+, Ni2+, and Cd2+ at the concentration of 5 mM. To evaluate the
effects of chelating agents and surfactants on the activity of the xylanase, ethylene diamine
tetra acetate (EDTA), dithiothreitol (DTT), sodium dodecyl sulfate (SDS), and
phenylmethanesulfonyl fluoride (PMSF) were tested. The reaction mixtures were incubated
at 60 C for 30 min before the residual activity was measured. The enzyme activity assayed in
the absence of metal ions and reagents was defined as 100 %.

Results and Discussion

Activity of the Crude Enzyme on Various Carbohydrate Substrates
C. algeriensis strain TH7C1T was found to be able to use a wide range of carbon sources.
However, the more favorable carbon sources in the present study for xylanase production were
birchwood xylan, arabinose, xylose, glucose, galactose, and maltose (Fig. 1). Therefore,
birchwood xylan was the most suitable carbon source for xylanase induction. The recorded
observations for xylanase activity in the production media, supplemented by sugars as
additives, indicate that fructose, starch, and lactose inhibited xylanase production strongly to
minimum productivity equal to 11.9 U/ml which could be caused by catabolism repression
matter. The xylanase of C. algeriensis TH7C1T showed an interesting activity with carboxymethylcellulose (CMC) as a substrate. Xylanase examination in CMC substrate needs to be
conducted because there are several xylanases which are able to hydrolyze not only xylan but
also cellulose [68]. Most xylanases were reported as inducible enzymes. Some studies showed
the use of different kinds of commercial extracted xylan as well as xylan containing agricultural wastes for inducing xylanolytic enzyme production [21, 22, 54, 64, 65]. Many other
studies showed that various agricultural wastes could be used as substrates for producing

Fig. 1 Effect of carbon source on xylanase production

Appl Biochem Biotechnol

xylanolytic enzymes by various microorganisms. These studies also reported that an inducer
for certain microorganism could be an inhibitor for others [11, 46, 49]. The use of purified
xylan as an inducer increases the cost of enzyme production.
Effects of Nitrogen Concentration on Xylanase Production
The C. algeriensis strain TH7C1T exhibits better activity with 2.0 g yeast extract and 2.0 g
biotrypcase (140.0 U/ml). The xylanases activities tested with (1) 2.0 g NH4Cl; 1.5 g yeast
extract; and 1.5 g biotrypcase and (2) 3.0 g NH4Cl; 1.0 g yeast extract; and 1.0 g biotrypcase
exhibited very low activity values of 32.4 and 14.1 U/ml, respectively.
Growth and Xylanase Production Profiles
The time course of growth and production of extracellular xylanase by C. algeriensis
was studied using birchwood xylan as a carbon source during 23 h (Fig. 2). The
growth of culture started rapidly and the highest biomass was reached at 22 h, after
this it decreased slowly. The soluble protein increased and followed a similar trend to
that observed for optical density.
The results showed that the production of xylanase by C. algeriensis increased
gradually and reached its highest value (140.0 U/ml) at 18 h. This production is
independent of growth course as already reported for many microorganisms [19, 52,
76, 54]. Nevertheless, the obtained activity (140.0 U/ml) can be considered relatively
high in comparison to those described for the majority of thermophilic strains. For
example, the Rhodothermus marinus strain has an activity of 1.84.0 U/ml [20, 32]
while xylanase of Bacillus stearothermophilus strain T-6 has an activity of 2.3 U/ml
[35]. A search for microorganisms producing high levels of xylanase activity resulted
in the isolation of several strains (Table 1).

Fig. 2 Time course of growth and enzyme production by C. algeriensis in birchwood xylan defined medium
(pH 7, 60 C), black triangle; absorbance, black circle; xylanase activity, black square; soluble proteins. Data are
the average of three replicates

Appl Biochem Biotechnol

Table 1 Production of xylanases from various microorganisms
Organism

Substrate

Thermotoga maritima
MSB8

0.25 % oat spelt xylan 90 C, pH 6.2, 500 M 27

NaCl

Streptomyces sp. Ab 106

Xylan 10 g/l

55 C, pH 7.5, 5 days

[66]

Bacillus arseniciselenatis
DSM 15340

Oat spelt xylan

65 C, pH 8.0, 48 h

113

[33]

Providencia sp. Strain X1

Weat bran

Shake flask 60 C,
pH 9.0, 48 h

36

[54]

Streptomyces sp. CA24

0.5 % xylan

Shake flask 60 C,
pH 9.0, 120 h

255

[51]

Bacillus pumilus MTCC

8964
Talaromyces thermophilus

0.5 % oat spelt xylan

241

[37]

oat spelt xylan

Shake flask 60 C,
pH 6.0, 48 h
Shake flask 40 C,
pH 7.0, 5 days

6.25

[29]

Bacillus sp. A Q-1

0.5 % oat spelt xylan

Shake flask 60 C,
pH 7.0, 12 h

55.2

[71]

Jonesia denitrificans
BN13

Birchwood xylan,
7 g/l

4 l fermentation, 37 C, 10.8
pH 7.0, 2 days

[14, 15]

Bacillus sp. strain XE

Birchwood xylan
(0.5 %)

Fermentation 55 C,
pH 7.5.

100

[57]

Bacillus sp. strain SPS-0

Wheat bran
arabinoxylan

Fermentation 60 C
pH 8,2, 24 h.

2995

[7]

Paenibacillus campinasensis Birchwood xylan

G1-1

Shake flask 37 C,
pH 7.0, 96 h

143

[76]

Caldicoprobacter
algeriensis

Fermentation 70 C,
pH 7.2, 18 h

140

This study

Birchwood xylan
10 g/l

Cultivation conditions

Xylanase activity References

(U/ml)
[73]

Characterization of the Extracellular Xylanase from Strain TH7C1T

Effect of Temperature and pH
The effect of temperature on the xylanolytic activity of C. algeriensis strain is shown in
(Fig. 3). We found that the optimum temperature is 70 C which is higher than that of
Paenibacillus sp. (55 C) reported by Zhao et al. [75]. Okazaki et al. [50] have reported
similar results in strains belonging to the Bacillus genus. In agreement to our study, xylanases
from C. abosum [55], Clostridium stercorarium [56], and Clostridium thermocellum [30]
show the same optimum temperatures of 70 C. Xylanases, that are optimally active at higher
temperatures, are produced by the extreme thermophilic anaerobic bacteria belonging to the
order Thermotogae. These xylanases show optimal activities at temperatures between 90 and
105 C [38].
Concerning the pH, the xylanolytic activity is important between the pH 6.0 and 12.0, with
a maximum activity at pH 11.0 (Fig. 4). These results are consistent with those reported in the
literature for the xylanases of Bacillus halodurans PPKS-2 [52]. For example, xylanase from
Paenibacillus macquariensis showed maximum activity at pH 8.6. However, it exhibited
enzyme activity over a broad pH range of pH 4.011.0 [40]. Otherwise, the xylanases of
C. abosum [55], Bacillus arseniciselenatis DSM 15340 [33], Bacillus mojavensis AG137 [3],
and Streptomyces sp. CA24 [51] have also an optimum pH between 8.0 and 9.0. On contrary,

Appl Biochem Biotechnol

Fig. 3 Effect of temperature on the activity of the crude xylanase from C. algeriensis sp. nov. strain TH7C1T.
The temperature profiles were determined by assaying xylanase activity at temperatures between 50 and 90 C at
pH 11.0. The activity of the enzyme at 70 C was taken as 100 %

the optimum pH of the Thermotoga sp. FjSS3-B1 strain [60] and Sulfolobus solfataricus [48]
was 5.3. Most xylanases isolated so far are optimally active at acidic or neutral pH [47, 12, 73,
25]. New alkaline xylanases were reported to be therefore needed for example in pulp and
paper industry [47].
Ideally, for industrial application, xylanases should have a neutral to alkaline pH optimum
and good thermal stability. Alkaline xylanases have gained importance due to their application

Fig. 4 Effect of pH on the activity of the crude xylanase from Caldicoprobacter algeriensis sp. nov. strain
TH7C1T. Six different buffers (50 mM) were used evaluated in the pH range of 413 at 60 C using birchwood
xylan: citrate buffer (white square) (pH 4.05.5), phosphate buffer (white diamond) (pH 6.07.5), TrisHCl
buffer (white circle) (pH 8.08.5), Glycine-NaOH buffer () (pH 910.5), bicarbonate-NaOH (x) (pH 1111.5),
and Na2HPO4-NaOH buffer (+) (pH 12.013.0). Xylanase activity was. The activity of the enzyme before
incubation was taken as 100 %

Appl Biochem Biotechnol

in the development of eco-friendly technologies used in the paper and pulp industries as these
enzymes are able to hydrolyse xylan, which is soluble in alkaline solutions [31], indicating the
potential industrial use of this enzyme.
Thermostability
Thermostability is a very important parameter, in consideration of the fact that most industrial
processes using the xylanolytic enzymes occur at relatively high temperatures property.
Stability of the enzyme was the most important factor in studying characteristics.
Thermostability without Substrate The thermostability was studied by incubating the supernatant up to 15 h at temperatures of 50 to 80 C (Fig. 5). The activity of the crude enzyme was
characterized by different half-life of 10, 9, 8, and 4 h at 50, 60, 70, and 80 C, respectively.
After 9 h of incubation at 70 C, the residual activity was 5.84 %. The xylanase activity of our
strain is more thermostable than xylanase of C. thermocellum with a half-life time of 36 min at
70 C [30]. Xylanase from S. solfataricus has a half-life of 47 min at 90 C [17] while the
P. abyssi xylanase has a half-life of 100 min at 105 C [4].
Thermostability in the Presence of Substrate Thermostability of xylanases was higher in the
presence of birchwood xylan, the half-life time was 10 h at 50 C and 4 h at 80 C. The relative
activity was 2.65 % after 14 h incubation at 80 C (Fig. 6). Indeed, the substrate had a
protective effect on the enzyme which makes it more thermostable. Several studies described
this protective effect of the substrate to the enzyme [23, 18, 59].

Effects of Various Metal Ions and Other Additives on Xylanase Activity

The effects of metal ions and chemicals on the enzyme activity are summarized in Fig. 7. As
shown in Fig. 7, the activity of C. algeriensis xylanase was not significantly inhibited by the
presence of different metal ions. The same results were obtained by earlier studies with the

Fig. 5 Thermostability profiles of xylanase (without substrate) from C. algeriensis at different temperatures.
Black square; 50 C, black triangle; 60 C, black triangle; 70 C, black circle; 80 C. Data are the average of three
replicates

Appl Biochem Biotechnol

Fig. 6 Thermostability profiles of xylanase from Caldicoprobacter algeriensis in the presence of the substrate at
different temperatures. Black square; 50 C, black triangle; 60 C, black triangle; 70 C, black circle; 80 C. Data
are the average of three replicates

xylanases produced by Thermotoga thermarum [60] and B. halodurans PPKS-2 [52].

However, our results showed that the enzyme was strongly inhibited by Hg2+ which decreased
its activity down to 66.0 %. This effect might be due to the presence of catalytically important
cysteine as recently reported by [52]. The same effect of Hg2+ was observed in C. stercorarium
[9]. Otherwise, the xylanase activity was significantly stimulated by Ca2+ ions (51.3 %). This
result suggests that this metal ion protected the enzyme against thermal denaturation and
played a vital role in maintaining the active conformation of the enzyme at higher temperatures, as there are probably two carbohydrate binding modules (CBMs) with Ca2+ [1]. Similar
effects were also observed with the xylanases produced by T. thermarum [60]. Inconsistent
results were reported for other bacterial xylanases from Bacillus subtilis cho40, Geobacillus
thermoleovorans, and Paenibacillus campinasensis, whose activities were not affected by
Ca2+ [34, 69].

Fig. 7 Effects of metal ions and other additives on the activity of the crude enzyme of C. algeriensis

Appl Biochem Biotechnol

Regarding the effect of chemical reagents, xylanases from C. algeriensis resist to SDS
while a total inactivation has been reported in several xylanases produced by microorganisms
[23, 32, 24, 33]. Addition of EDTA, a chelating agent, to the reaction mixture has no effect on
the enzyme. This could lead to the conclusion that the crude enzyme is not a metalloenzyme.

Conclusion
An extracellular xylanase from C. algeriensis sp. nov. strain TH7C1T was produced and
partially characterized in this study. The time course for xylanase accumulation by the
C. algeriensis sp. nov. strain TH7C1T in submerged anaerobic fermentation showed that the
highest xylanase activity reached 140.0 U/ml in an optimized medium with mix of birchwood
and oats spelt xylan used as substrates after 18 h of cultivation. The crude xylanase was
optimally active at pH 11.0 and 70 C. Overall, the findings indicate that the thermo- and
alkaline-tolerant xylanase presents promising properties for the pulp and paper industry.
Further studies, some of which are currently underway, are needed to find the purification to
the homogeneity and the biochemical characterization of the pure enzyme.
Acknowledgments We wish to express our gratitude to Abdelhak Kouchah (Hyproc Shiping Company) for his
valuable help during the preparation of this work.

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