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Partial Characterization of Xylanase Produced

by Caldicoprobacter algeriensis, a New ThermophilicAnaerobic 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 Hacène &

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 resultsshowed that C. algeriensis xylanase seems not to be correlated with the biomass growth profilewhereas 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. Theenzyme 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 greatlythe 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.

Appl Biochem BiotechnolDOI 10.1007/s12010-014-1153-2

K. Bouacem : A. Bouanane-Darenfed : H. HacèneLaboratory 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. BenallaouaLaboratory 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 Université, CNRS, Université de Toulon, IRD, Mediterranean Institute of Oceanography(MIO), UM 110, 13288 Marseille, Francee-mail: [email protected]

M. GagaouaMaquav, Bioqual Laboratory, INATAA, Université de Constantine 1, Route de Ain El-Bey,25000 Constantine, Algerie


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Keywords Alkalitolerant . Caldicoprobacter algeriensis . Birchwood xylan . Thermostablexylanase . 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 incereals and hardwood is xylan. Xylan consists of a β-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 containthose acting at the main chain of xylan as the endoxylanases and β-xylosidases and

other so-called debranching enzymes or accessories that address the branches graftedonto the main chain. Among these debranching enzymes, α -L-arabinofuranosidase, α -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 thelast decade due to their biotechnological potential in many industrial processes. Theyrepresent the largest proportion of the world enzymes market [10, 70, 58]. Xylanasesare 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 industrialcommunities as they are very useful as a new source of thermostable enzymes [8,67]. The use of thermophilic microorganisms in the production of thermostablexylanases has major technical and economic advantages [27]. Several thermophilicmicroorganisms are reported capable of producing thermostable xylanases such asThermotoga 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 ahot spring of Hammam D’ bagh (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. Partialcharacterization of the crude extracellular xylanase and its potential biochemical propertieswere also reported.

Materials and Methods

Substrates and Chemicals

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

Appl Biochem Biotechnol


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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), aregion situated in the northeast of Algeria.

The medium (TH) used for isolation contained (in g/l): NH4Cl (1.0), K 2HPO4 (0.3),KH2PO4 (0.3), KCl (0.1), MgCl2·6H2O (0.5), CaCl2·2H2O (0.1), NaCl (0.5), yeast extract (2.0), biotrypcase (2.0), Cysteine – HCl (0.5), together with sodium acetate (2 mM), and Balchtrace element solution (10 ml) [6]. The pH was adjusted to 7.2 using 10 M KOH solution, andthe 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 N2 – CO2 (80:20 v/v ), andsubsequently 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 ) Na2S·9H2O, and 20 mM glucose were injected from

sterile stock solutions into the tubes. Enrichments were performed as described by [13] inHungate 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(5.0 – 9.0) of the medium was adjusted by injecting in Hungate tubes aliquots of anaerobicstock 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 directlyweighing 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 mediumwithout glucose then incubated at 70 °C, pH 7.2 for 18 h. Before assay, the cells wereseparated by centrifugation at 1,0000 xg 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 mlof 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 astandard. 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, theintensity of the color was measured by optical density at 540 nm. One unit (U) of xylanase wasdefined as the amount of enzyme required to release 1 μ mol reducing sugar as xyloseequivalent in 1 min under the above assay conditions [5].

The cell growth was estimated by soluble protein estimation following Bradford’s methodwith bovine serum albumin (BSA) as the standard [16] and by measuring the optical density at 600 nm.



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Activity of Crude Enzyme on Various Carbohydrate Substrates

The effect of various carbon sources on the xylanase production was assessed by culturing theC. 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 mediumTH 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, severalcombinations 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 with1 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, pH7.2, and temperature 70 °C under the anaerobic conditions during 23 h. Culturesamples were collected at 2 h intervals for the stationary phase and then 1 h duringthe cultivation period. Immediately after collection, the samples were centrifuged at 10,000 xg 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 inthe pH range 4.0 – 13.0. Six different buffers (50 mM) were used for this study: citrate

buffer (pH 4.0 – 5.5), phosphate buffer (pH 6.0 – 7.5), Tris – HCl buffer (pH 8.0 – 8.5),glycine-NaOH buffer (pH 9.0 – 10.5), bicarbonate-NaOH buffer (pH 11.0 – 11.5), and

Na2HPO4-NaOH buffer (pH 12.0 – 13.0). The relative activity was determined by theabove described assay method.

Effect of Temperature on Xylanase Activity

Determination of enzyme activity for optimum temperature, the crude enzyme sample wasincubated 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 enzymein the absence and presence of the substrate at temperatures of 50, 60, 70, and80 °C. The samples were removed after incubation for varying times (from 0 to15 h) at intervals of 1 h. The residual xylanase activity was determined using thestandard assay.



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Effects of Various Metal Ions and Other Additives on Xylanase Activity

The effects of various metal ions on xylanase activity were evaluated by incubating thereaction mixtures containing 10 mg/ml of the birchwood xylan with Mg2+, Ca2+,Mn

2+, Fe2+,

Zn2+

, Cu2+

, NH4+

, Hg2+

, K +

, Ni2+

, and Cd2+

at the concentration of 5 mM. To evaluate theeffects of chelating agents and surfactants on the activity of the xylanase, ethylene diaminetetra acetate (EDTA), dithiothreitol (DTT), sodium dodecyl sulfate (SDS), and

phenylmethanesulfonyl fluoride (PMSF) were tested. The reaction mixtures were incubatedat 60 °C for 30 min before the residual activity was measured. The enzyme activity assayed inthe 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 recordedobservations for xylanase activity in the production media, supplemented by sugars asadditives, indicate that fructose, starch, and lactose inhibited xylanase production strongly tominimum 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 carboxy-methylcellulose (CMC) as a substrate. Xylanase examination in CMC substrate needs to beconducted 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 showedthe use of different kinds of commercial extracted xylan as well as xylan containing agricul-tural 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



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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 purifiedxylan 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 biotrypcaseexhibited 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). Thegrowth 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 tothat observed for optical density.

The results showed that the production of xylanase by C. algeriensis increasedgradually and reached its highest value (140.0 U/ml) at 18 h. This production isindependent of growth course as already reported for many microorganisms [19, 52,76, 54]. Nevertheless, the obtained activity (140.0 U/ml) can be considered relativelyhigh in comparison to those described for the majority of thermophilic strains. For

example, the Rhodothermus marinus strain has an activity of 1.8 –

4.0 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 resultedin 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 arethe average of three replicates



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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 reportedsimilar results in strains belonging to the Bacillus genus. In agreement to our study, xylanasesfrom 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 theorder Thermotogae. These xylanases show optimal activities at temperatures between 90 and105 °C [38].

Concerning the pH, the xylanolytic activity is important between the pH 6.0 and 12.0, witha maximum activity at pH 11.0 (Fig. 4). These results are consistent with those reported in theliterature for the xylanases of Bacillus halodurans PPKS-2 [52]. For example, xylanase from

Paenibacillus macquariensis showed maximum activity at pH 8.6. However, it exhibitedenzyme activity over a broad pH range of pH 4.0 – 11.0 [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,

Table 1 Production of xylanases from various microorganisms

Organism Substrate Cultivation conditions Xylanase activity(U/ml)

References

Thermotoga maritimaMSB8 0.25 % oat spelt xylan 90 °C, pH 6.2, 500 μ M NaCl 27 [73]

Streptomyces sp. Ab 106 Xylan 10 g/l 55 °C, pH 7.5, 5 days 8 [66]

Bacillus arseniciselenatis

DSM 15340Oat 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 MTCC8964

0.5 % oat spelt xylan Shake flask 60 °C, pH 6.0, 48 h

241 [37]

Talaromyces thermophilus oat spelt xylan 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, pH 7.0, 2 days

10.8 [14, 15]

Bacillus sp. strain XE Birchwood xylan(0.5 %)

Fermentation 55 °C, pH 7.5.

100 [57]

Bacillus sp. strain SPS-0 Wheat branarabinoxylan

Fermentation 60 °C pH 8,2, 24 h.

2995 [7]

Paenibacillus campinasensisG1-1

Birchwood xylan Shake flask 37 °C, pH 7.0, 96 h

143 [76]

Caldicoprobacter

algeriensis

Birchwood xylan10 g/l

Fermentation 70 °C, pH 7.2, 18 h

140 This study



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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. 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 %

Fig. 4 Effect of pH on the activity of the crude xylanase from Caldicoprobacter algeriensis sp. nov. strainTH7C1T. Six different buffers (50 mM) were used evaluated in the pH range of 4 – 13 at 60 °C using birchwoodxylan: citrate buffer (white square) (pH 4.0 – 5.5), phosphate buffer (white diamond ) (pH 6.0 – 7.5), Tris – HCl buffer (white circle) (pH 8.0 – 8.5), Glycine-NaOH buffer (∆) (pH 9 – 10.5), bicarbonate-NaOH (x) (pH 11 – 11.5),and Na2HPO4-NaOH buffer (+) (pH 12.0 – 13.0). Xylanase activity was. The activity of the enzyme beforeincubation was taken as 100 %



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in the development of eco-friendly technologies used in the paper and pulp industries as theseenzymes 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 super-natant up to 15 h at temperatures of 50 to 80 °C (Fig. 5). The activity of the crude enzyme wascharacterized 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 relativeactivity 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 describedthis 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. Asshown 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 threereplicates



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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 decreasedits 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 Ca

2+

ions (51.3 %). Thisresult 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 tempera-tures, 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, Geobacillusthermoleovorans, and Paenibacillus campinasensis, whose activities were not affected byCa2+ [34, 69].

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. Dataare the average of three replicates

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



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Regarding the effect of chemical reagents, xylanases from C. algeriensis resist to SDSwhile 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 onthe 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 theC. algeriensis sp. nov. strain TH7C1T in submerged anaerobic fermentation showed that thehighest xylanase activity reached 140.0 U/ml in an optimized medium with mix of birchwoodand oats spelt xylan used as substrates after 18 h of cultivation. The crude xylanase wasoptimally 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 tothe homogeneity and the biochemical characterization of the pure enzyme.

Acknowledgments We wish to express our gratitude to Abdelhak Kouchah (Hyproc Shiping Company) for hisvaluable help during the preparation of this work.

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