Professional Documents
Culture Documents
Article Caldicobacter 2014 PDF
Article Caldicobacter 2014 PDF
DOI 10.1007/s12010-014-1153-2
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
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.
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.
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
Substrate
Thermotoga maritima
MSB8
Xylan 10 g/l
55 C, pH 7.5, 5 days
[66]
Bacillus arseniciselenatis
DSM 15340
65 C, pH 8.0, 48 h
113
[33]
Weat bran
Shake flask 60 C,
pH 9.0, 48 h
36
[54]
0.5 % xylan
Shake flask 60 C,
pH 9.0, 120 h
255
[51]
241
[37]
Shake flask 60 C,
pH 6.0, 48 h
Shake flask 40 C,
pH 7.0, 5 days
6.25
[29]
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]
Birchwood xylan
(0.5 %)
Fermentation 55 C,
pH 7.5.
100
[57]
Wheat bran
arabinoxylan
Fermentation 60 C
pH 8,2, 24 h.
2995
[7]
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
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 %
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].
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
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
Fig. 7 Effects of metal ions and other additives on the activity of the crude enzyme of C. algeriensis
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.
References
1. Abou, H. M., Nordberg, K. E., Bartonek-Roxa, E., Raghothama, S., Simpson, P., Gilbert, H., Williamson,
M., & Holst, O. (2000). Biochemical Journal, 345, 5360.
2. Ahmad, Z., Butt, M. S., & Riaz, M. (2013). Pakistan Journal of Agricultural Science, 50, 433437.
3. Akhavan Sepahy, A., Ghazi, S., & Akhavan Sepahy, M. (2011). Journal of Enzyme Research. doi:10.4061/
2011/593624 .
4. Andrade, C. M. C., Aguiar, W. B., & Antranikian, G. (2001). Applied Biochemistry and Biotechnology, 91,
655669.
5. Bailey, M. J., Biely, P., & Poutanen, K. (1992). Journal of Biotechnology, 23, 257270.
6. Balch, W. E., Fox, G., Magrum, L., Woese, C., & Wolfe, R. (1979). Microbiological Reviews, 43, 260.
7. Bataillon, M., Nunes-Cardinali, A. P., Castillon, N., & Duchiron, F. (2000). Enzyme and Microbial
Technology, 26, 187192.
8. Beg, Q., Bhushan, B., Kapoor, M., & Hoondal, G. (2000). Journal of Industrial Microbiology &
Biotechnology, 24, 396402.
9. Brenger, J.-F., Frixon, C., Bigliardi, J., & Creuzet, N. (1985). Canadian Journal of Microbiology, 31, 635
643.
10. Bhat, M. (2000). Biotechnology Advances, 18, 355383.
11. Biely, P. (1985). Trends in Biotechnology, 3, 286290.
12. Bok, J. D., Goers, S. K., Eveleigh, D. E. (1994) In Enzymatic Conversion of Biomass for Fuel Production,
pp. 54-65. Edited by M. E. Himmel, J. O. Baker & R. P. Overend. ACS Symposium series 566. Washington,
DC: American Chemical Society.
13. Bouanane-Darenfed, A., Fardeau, M.-L., Grgoire, P., Joseph, M., Kebbouche-Gana, S., Benayad, T.,
Hacene, H., Cayol, J.-L., & Ollivier, B. (2011). Current Microbiology, 62, 826832.
14. Boucherba, N., Gagaoua, M., Copinet, E., Bettache, A., Duchiron, F., Benallaoua, S., Boucherba, N.,
Gagaoua, M., Copinet, E., Bettache, A., Duchiron, F., & Benallaoua, S. (2014). Applied Biochemistry and
Biotechnology. doi:10.1007/s12010-013-0709-x .
15. Boucherba, N., Said, B., Estelle, C., Hakim, H., & Duchiron, F. (2011). Process Biochemistry, 46, 519525.
16. Bradford, M. M. (1976). Analytical Biochemistry, 72, 248254.
17. Cannio, R., Di Prizito, N., Rossi, M., & Morana, A. (2004). Extremophiles, 8, 117124.
18. Cesar, T., & Mra, V. (1996). Enzyme and Microbial Technology, 19, 289296.