Ann. Microbiol., 52, 299-306 (2002)
Some properties of thermostable xylanase
from an Aspergillus niger strain
G. CORAL1*, B. ARIKAN2, M.N. ÜNALDI3, H. KORKMAZ GÜVENMEZ2
1Mersin University, Faculty of Arts and Sciences, Department of Biology,
33342 Çiftliköy-Mersin; 2Çukurova University, Faculty of Arts and Sciences,
Department of Biology, 01330 Balcali-Adana; 3Mustafa Kemal University,
Faculty of Arts and Sciences, Department of Biology, 31040 Hatay, Turkey
Abstract - A thermostable xylanase was isolated from an Aspergillus niger wild type strain
in a liquid Czapek Dox medium, containing oat spelts xylan as the sole carbon source. The
molecular mass of the enzyme was estimated to be about 36 kDa by sodium dodecyl sul-
fate-polyacrylamide gel electrophoresis. The optimum pH for activity was found to be 7.5.
The temperature optimum of the enzyme was found to be 60 °C at pH 7.5. The enzyme
remained stable up to 100 °C, yet lost about 50% of its activity after 15 min at this tem-
Key words: xylanase, Aspergillus niger.
Xylan is the major constituent of hemicellulose and is the second most abundant
renewable resource with a high potential for degradation into useful end products
(Goheen, 1982). Microbial xylanases (1,4-β−D-xylan xylanohydrolase, EC
184.108.40.206) are the preferred catalysts for xylan hydrolysis due to their high speci-
ficity, mild reaction conditions, negligible substrate loss, and side product gener-
ation. However, the cost of the enzymatic hydrolysis of a biomass is one of the
main factors limiting the economic feasibility of this process. Therefore, the pro-
duction of xylanases must be improved by finding more potent fungal or bacteri-
al strains or by inducing mutant strains that can excrete greater amounts of
enzymes, or both (Dekker and Richards, 1976). Filamentous fungi are particular-
ly interesting producers of xylanases since they excrete the enzymes into the
medium and their enzyme levels are much higher than those of yeast and bacteria
(Steiner et al., 1987).
* Corresponding Author. E-mail: firstname.lastname@example.org
In recent years, important applications for xylanases in different industrial
processes have been found. One major area of application is the bleaching of craft
pulp in the paper industry (Zamost et al., 1991; Viikari et al., 1994). Most xylanas-
es known to date are optimally active at or below 50 °C at an acidic or neutral pH.
However, in the process of enzyme-assisted pulp bleaching, the incoming pulp has
a higher temperature and alkaline pH (Zamost et al., 1991) making the use of ther-
mostable alkaline xylanases very attractive (Gessesse, 1998).
Accordingly, the current paper, reports on certain properties of a thermostable
xylanase from the Aspergillus niger Z1 wild-type strain.
MATERIALS AND METHODS
Fungal strain and culture conditions. The Aspergillus niger Z1 wild-type strain
was used as the enzyme source. This fungus was isolated from soil samples accord-
ing to the method of Raper and Fennel (1977). The cultivation was carried out in a
Czapek Dox liquid medium containing oat spelts xylan (Sigma) (1%) as the sole
carbon source. A Erlenmayer flask (1 liter) containing 250 ml of the growth medi-
um was cultured for 5 days at 28 °C on an orbital shaker set at 250 rev min-1. The
mycelia were removed by filtration and the filtrate used for partial purification.
Partial purification of xylanase. Ethanol previously chilled to –20 °C was added
dropwise to the culture filtrate (250 ml) at 4 °C with continuous stirring to a final
concentration of 75%, then the solution was left at –20 °C for 24 h. The resulting
precipitate was collected by decantation and centrifugation. The precipitate was
then dissolved in 30 ml of a phosphate buffer (50 mM, pH 5.0) and the concen-
trated solution dialyzed against the same buffer (pH 5.0) overnight at 4 °C (Bhel-
la and Altosaar, 1984).
Enzyme assay. The xylanase was assayed based on the detection of reducing sug-
ars from oat spelts xylan. A solution of 1 % (wt/vol) oat spelts xylan in a phos-
phate buffer (50 mM, pH 5) was used. An aliquot (0.5 ml) of the partially purified
enzyme preparation was incubated with 0.5 ml of the xylan suspension for 30 min
at 37 °C. The reducing sugars were detected using the dinitrosalicylic acid (DNS)
method of Miller (1959).
Temperature, pH optima and heat stability. In all the determinations, the
xylanase activity was measured using oat spelts xylan as the substrate. To esti-
mate the temperature, pH optima and heat stability, the activity was determined
by carrying out the above standard assay at several temperatures or pH values.
After the incubating at different temperatures or pH’s, the xylanase activity was
measured under standard conditions according to a previous paper (Coral and
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). To
determinate the homogenity and molecular weight, the enzyme preparations and
known molecular weight markers were subjected to electrophoresis according to
300 G. CORAL et al.
the method of Bollag and Edelstein (1991) using 10% acrylamide gel. Oat spelts
xylan (0.2%) was incorporated into the separating gel prior to the addition of
ammonium persulfate and polimerization. After the electrophoresis, the gel was
stained for 1 h with Coomassie Blue R dye in a methanol-acetic acid-water solu-
tion (4:1:5, by volume) and then destained in the same solution without the dye.
Zymogram analysis. For the activity staining of the xylanase activity, The SDS
was removed by washing the gel at room temperature in solutions containing 50
mM Na2HPO4, 50 mM NaH2PO4 (pH 7.2), isopropanol 40% for 1 h and 50 mM
Na2HPO4, 50 mM NaH2PO4 (pH 7.2) for 1 h, respectively. The renaturation of
the enzyme proteins was carried out by placing the gel overnight in a solution
containing 50 mM Na2HPO4, 50 mM NaH2PO4 (pH 7.2), 5 mM β-mercap-
toethanol, and 1 mM EDTA at 4 °C. The gel was then transferred onto a glass
plate, sealed in film, and incubated at 37 °C for 4-5 h. The gel was stained in a
solution of 1% Congo Red for 30 min, and destained in 1 M NaCl for 15 min.
Any clear bands indicated the presence of xylanase activity (Saul et al., 1990;
Ikeda et al., 1992; Lee et al., 1994).
RESULTS AND DISCUSSION
The molecular weight was determined by SDS-PAGE, as described in Materials
and Methods above. The analyses of the enzyme by SDS-PAGE only revealed a
single band indicating xylanolytic activity in the gel. The molecular weight of this
band was estimated to be around 36 kDa (Fig. 1).
To estimate the optimum temperature of the enzyme, the activity was determined
at several temperatures between 40 °C to 100 °C. The optimum temperature was
observed to be around at 60 °C (Fig. 2).
The pH optimum was determined in two buffer systems. It was observed that the
enzyme did show activity in an alkali pH. The optimum pH of the enzyme was
found to be 7.5 (Fig. 3).
The thermostability of the enzyme was studied by heating the enzyme at different
temperatures (60-100 °C) for 15 min. The enzyme remained stable up to 100 °C,
with 49.2% of the original activity retained after heat treatment at 100 °C for 15
min (Fig. 4).
Commercial applications of xylanases demand the identification of highly
stable enzymes that remain active under routine handling conditions. Many
advantages, such as reduced contamination risk and faster reaction rates, have
been proposed for the use of thermophiles in biotechnology processes. In gener-
al, parameters such as temperature, pH, and enzymatic stability are important for
the industrial applicability of any enzyme (Kulkarni et al., 1999).
Ann. Microbiol., 52, 299-306 (2002) 301
1 2 3 1 2 3
FIG. 1 – Separation (A) and activity pattern (B) of Aspergillus niger Z1 xylanase in
SDS-Polyacrylamide gel. The electrophoresis was carried out in an SDS-Poly-
acrylamide gel containing 0.2 % oat spelts xylan (SIGMA). The gel was stained
as described under Materials and Methods. Lane 1 and 2 contains the partially
purified xylanase from Aspergillus niger Z1, lane 3 contains the molecular
weight markers: 116000, 97400, 66000, 45000, and 29000 Dalton respectively.
Relative activity (%)
FIG. 2 – Optimal temperature range of xylanase from Aspergillus niger Z1.
302 G. CORAL et al.
Relative activity (%)
FIG. 3 – Optimal pH range of xylanase from Aspergillus niger Z1. Two buffer systems
were used to determine the optimal pH range of the enzyme: Citrate-Phosphate
Buffer (0.1 M Citric acid, 0.2 M Na2HPO4, pH 3.0 to 7.0) and Tris Buffer (0.08
M Tris, 0.1 M HCl, pH 7.5 to 9.0).
Relative activity (%)
FIG. 4 – Heat stability of xylanase from Aspergillus niger Z1.
From the electrophoresis of the partially purified enzyme solution, two pro-
tein bands were observed after the gel was stained with Coomassie Brillant Blue,
whereas, only a single band showing xylanolytic activity was detected after activ-
ity staining with Kongo Red (Fig. 1). The molecular weight of the single protein
Ann. Microbiol., 52, 299-306 (2002) 303
band was calculated to be about 36 kDa. Microbial xylanases are single subunit
proteins with molecular masses whitin a the range of 8 - 145 kDa (Kulkarni et al.,
1999). In a previous study, a purified xylanase enzyme preparation also showed a
single protein band on SDS-PAGE, and the molecular weight of this enzyme was
found to be 24 kDa (Sardar et al., 2000). According to the existing data, the
molecular weights of the endo-1,4-β-xylanase (EC 220.127.116.11) and exo-1,4-β-xylosi-
dase (EC 18.104.22.168) of Aspergillus niger were found to be 14 and 122 kD respec-
tively (Frederick et al., 1981; Uhlig, 1998).
The optimum temperature of the current enzyme was found to be 60 °C at pH
7.5. (Fig. 2). This value agrees well with previous the literature. The optimum
temperature for xylanases from bacterial and fungal sources has been found to
vary between 40 and 60 °C (Kulkarni et al., 1999). The heat stability of the cur-
rent enzyme matched that of a mesophilic organism. Fungal xylanases are gener-
ally less thermostable than bacterial xylanases. Yet, fungi that are mesophilic in
origin and produce thermostable xylanases include Ceratocystis paradoxa, a
xylanase that is stable at 80 °C for 1 h (Dekker and Richards, 1975) and Tricho-
derma harzianum, a xylanase that can retain 52% of its optimum activity for 4 h
at 100 °C (Fadel, 2001). The thermostable xylanase previously identified from
Aspergillus is the only one reported from a thermotolerant Aspergillus strain at 37
°C showing a maximum activity at 80 °C (Mendicuti et al., 1997).
It was found that the xylanase of Aspergillus niger Z1 exhibited the activity
within a neutral to alkaline pH range. The optimum pH of the enzyme was found
to be 7.5 at 37 °C. (Fig. 3) Other xylanases from different organisms show an
optimum pH within a range of 4.0-7.0. However, certain xylanases from
Aspergillus kawachii and Penicillium herque exhibit an optimum pH more on the
acidic side (pH 2.0 - 6.0) (Funaguma et al., 1991; Ito et al., 1992). Endoxylanase
I and II from Aspergillus awamori show an optimum pH at 5.5 – 6.0 and 5.0,
respectively (Kormelink et al., 1992). The optimal pH value for the activity of a
commercial xylanase from an Aspergillus niger strain was found to be 4.0 (Fred-
erick et al., 1981). From the enzyme data on Aspergillus niger, the optimum pH’s
for the xylanases are within a range of 4.0 - 5.0 (Uhlig, 1998). Ideally, for indus-
trial application, xylanases should have a neutral to alkaline pH optimum and
good thermal stability. Alkaline xylanases have gained importance due to their
application in the development of eco-friendly technologies used in the paper
and pulp industries as these enzymes are able to hydrolyse xylan, which is solu-
ble in alkaline solutions (Horikoshi, 1996). The xylanases from Cephalosporium
are the only ones reported from an alkaliphilic fungus with activity within a board
pH range of 6.5 - 9.0 (Bansod et al., 1993). The current results showed that, the
Z1 xylanase had a low activity in an acidic pH range yet, a good activity within a
board pH range from 6.5 - 9.0. The xylanase enzyme in the present stduy has
properties quite different from those produced by the other Aspergillus sp. So, the
significance of this work is that, certain properties of the present xylanase are
appropriate in different industrial applications, especially in the process of
enzyme-assisted pulp bleaching.
304 G. CORAL et al.
Bansod S.M., Dutta -Choudhary M., Srinivasan M.C., Rele M.V. (1993). Xylanase active
at pH from an alkatolerant Cephalosporium sp. Biotechnol. Lett., 15: 965-970.
Bhella R.S., Altosaar I. (1984). Purification and some properties of extracellular α-amy-
lase from Aspergillus awamori. Can. J. Microbiol., 31:149-154.
Bollag D.M., Edelstein S.J. (1991). Protein methods. Wiley-Liss Publication.
Coral G., Çolak Ö. (2000). The isolation and characterization of glucoamylase enzyme of
an Aspergillus niger natural isolate. Turk. J. Biol., 24: 601-609.
Dekker R.F.H., Richards G.N. (1975). Purification, properties and mode of action of
hemicellulase-produced by Ceratocystis paradoxa. Carbohydr. Res., 39: 97-114.
Dekker R.F.H., Richards G.N. (1976). Hemicellulases: their occurrence, purification,
properties and mode of action. Adv. Carbohydr. Chem. Biochem., 32: 277-352.
Fadel M. (2001). High level xylanase production from sorghum flour by a newly isolate of
Trichoderma harzianum cultivated under solid state fermentation. Ann. Microbiol.,
Frederick M.M., Frederick J.R., Fratzke A.R., Reilly P.J. (1981). Purification and char-
acterization of a xylobiose and xylose producing endo-xylanase from Aspergillus
niger. Carbohydr. Res., 97(1): 87-103.
Funaguma T., Naito S., Morita M., Okumara M., Sugiura M., Hara A. (1991). Purifica-
tion and some properties of xylanase from Penicillium herquei Banier and Sartory.
Agric. Biol. Chem., 55: 1163-1165.
Gessesse A. (1998). Purification and properties of two thermostable alkaline xylanases
from an alkaliphilic Bacillus sp. Appl. Environ. Microbiol., 64(9):3533-3535.
Goheen D.W. (1982). Chemicals from wood and other biomass. Part I: Future supply of
organic chemicals. J. Chem. Educ., 58: 465-468.
Horikoshi K. (1996). Alkaliphiles: from an industrial point of view. FEMS Microbiol.
Rev., 18: 259-270.
Ikeda T., Yamazaki H., Yamashita K., Shinke R. (1992). The tetracycline inducible
expression of α-amylase in Bacillus subtilis. J. Ferment. Bioeng., 74: 58-60.
Ito K., Ogassawara J., Sugimoto T., Ishikawa T. (1992). Purification and properties of
acid stable xylanases from Aspergillus kawachii. Biosci. Biotechnol. Biochem.,
Kormelink F.J.M., Gruppen H., Wood T.M., Beldman G. (1992). Mode of action of xylan
degrading enzymes from Aspergillus awamori. In: Visser J., Beldman G., Kusters-
van-Someren M.A., Varagen A.G.J., eds, Xylans and Xylanases. Elsevier, Amster-
dam, London, New York,Tokyo, pp. 141-147.
Kulkarni N., Shendye A., Rao M. (1999). Molecular and biotechnological aspects of
xylanases. FEMS. Microbiol. Rev., 23(4): 411-456.
Lee S., Morikawa M., Takagi M., Imankawa T. (1994). Cloning aapT gene and charac-
terization of its product, α-amylase, pullulanase (aapT) from thermophilic and alka-
liphilic Bacillus sp. strain XAL601. Appl. Environ. Microbiol., 60: 3761-3773.
Mendicuti C., Laura P., Trejo-Aguilar B.A., Aguilar O.G. (1997). Thermostable xylanas-
es produced at 37 °C and 45 °C by a thermotolerant Aspergillus strain. FEMS
Microbiol. Lett., 146: 97-102.
Miller G.L. (1959). Use of dinitrosalicylic acid reagent for determination of reducing
sugar. Anal. Chem., 31: 426-428.
Raper K.B., Fennel D.I. (1977). The Genus Aspergillus. Krieger R.E. Publishing Com.,
Huntington, New York.
Ann. Microbiol., 52, 299-306 (2002) 305
Sardar M., Roy I., Gupta M.N. (2000). Simultaneous purification and immobilization of
Aspergillus niger xylanase on the reversibly soluble polymer EudragitTM L-100.
Enzyme Microbial. Tech., 27: 672-679.
Saul D.J., Williams L.C., Grayling R.A., Chamley L.W., Love D.R., Berquist P.L. (1990).
celB, a gene coding for a bifunctional cellulase from the extreme thermophile “Cal-
docellum saccharolyticum”. Appl. Environ. Microbiol., 56(10): 3117-3124.
Steiner W., Lafferty R.M., Gomes I., Esterbauer H. (1987). Studies on a wild type strain
of Schizophyllum commune: Cellulase and xylanase production and formation of the
extracellular polysaccharide schizophyllan. Biotechnol. Bioeng., 30: 169-170.
Uhlig H. (1998). Industrial Enzymes and Their Applications. John Willey and Sons INC.,
Viikari L., Kantelinen A., Sundquist J., Linko M. (1994). Xylanases in bleaching: from
an idea to industry. FEMS Microbiol. Rev., 13: 335-350.
Zamost B.L., Nielsen H.K., Starnes R.L. (1991). Thermostable enzymes for industrial
applications. J. Ind. Microbiol., 8: 71-82.
306 G. CORAL et al.