Biochemical and catalytic properties of an endoxylanase purified from the culture filtrate of Sporotrichum thermophile

Article abstract of DOI:10.1016/S0008-6215(03)00291-X

An endo-β-1,4-xylanase (1,4-β-D-xylan xylanoxydrolase, EC 3.2.1.8) present in culture filtrates of Sporotrichum thermophile ATCC 34628 was purified to homogeneity by Q-Sepharose and Sephacryl S-200 column chromatographies. The enzyme has a molecular mass of 25,000 Da, an isoelectric point of 6.7, and is optimally active at pH 5 and at 70°C. Thin-layer chromatography (TLC) analysis showed that endo-xylanase liberates mainly xylose (Xyl) and xylobiose (Xyl₂) from beechwood 4-O-methyl-D-glucuronoxylan, O-acetyl-4-O-methylglucuronoxylan and rhodymenan (a β-(1→4)-β(1→3)-xylan). Also, the enzyme releases an acidic xylo-oligosaccharide from 4-O-methyl-D-glucuronoxylan, and an isomeric xylotetraose and an isomeric xylopentaose from rhodymenan. Analysis of reaction mixtures by high performance liquid chromatography (HPLC) revealed that the enzyme cleaves preferentially the internal glycosidic bonds of xylooligosaccharides, [1-³H]-xylooligosaccharides and xylan. The enzyme also hydrolyses the 4-methylumbelliferyl glycosides of β-xylobiose and β-xylotriose at the second glycosidic bond adjacent to the aglycon. The endoxylanase is not active on pNPX and pNPC. The enzyme mediates a decrease in the viscosity of xylan associated with a release of only small amounts of reducing sugar. The enzyme is irreversibly inhibited by series of ω-epoxyalkyl glycosides of D-xylopyranose. The results suggest that the endoxylanase from S. thermophile has catalytic properties similar to the enzymes belonging to family 11.

Full text of DOI:10.1016/S0008-6215(03)00291-X

Carbohydrate Research 338 (2003) 1881ꢀ1890
/
www.elsevier.com/locate/carres
Note
Biochemical and catalytic properties of an endoxylanase purified from
the culture filtrate of Sporotrichum thermophile
Petros Katapodis,a Maria Vrsˇanska´,b Dimitris Kekos,a Wim Nerinckx,c Peter Biely,b
Marc Claeyssens,c Basil J. Macris,a Paul Christakopoulosa,*
a Department of Chemical Engineering, National Technical University of Athens, 5 Iroon Polytechniou Ave, Zografou Campus, 15700 Athens, Greece
b Institute of Chemistry, Slovak Academy of Sciences, 842 38 Bratislava, Slovak Republic
c Department of Biochemistry, Fysiology, Microbiology, Faculty of Sciences, Ghent University, K.L., Ledeganckstraat 35, B-9000 Ghent, Belgium
Received 1 November 2002; received in revised form 9 June 2003; accepted 21 June 2003
Abstract
An endo-b-1,4-xylanase (1,4-b-D-xylan xylanoxydrolase, EC 3.2.1.8) present in culture filtrates of Sporotrichum thermophile
ATCC 34628 was purified to homogeneity by Q-Sepharose and Sephacryl S-200 column chromatographies. The enzyme has a
molecular mass of 25,000 Da, an isoelectric point of 6.7, and is optimally active at pH 5 and at 70 8C. Thin-layer chromatography
(TLC) analysis showed that endo-xylanase liberates mainly xylose (Xyl) and xylobiose (Xyl2) from beechwood 4-O-methyl-
glucuronoxylan, O-acetyl-4-O-methylglucuronoxylan and rhodymenan (a b-(104)-b(103)-xylan). Also, the enzyme releases an
acidic xylo-oligosaccharide from 4-O-methyl- -glucuronoxylan, and an isomeric xylotetraose and an isomeric xylopentaose from
D-
/
/
D
rhodymenan. Analysis of reaction mixtures by high performance liquid chromatography (HPLC) revealed that the enzyme cleaves
preferentially the internal glycosidic bonds of xylooligosaccharides, [1-3H]-xylooligosaccharides and xylan. The enzyme also
hydrolyses the 4-methylumbelliferyl glycosides of b-xylobiose and b-xylotriose at the second glycosidic bond adjacent to the
aglycon. The endoxylanase is not active on pNPX and pNPC. The enzyme mediates a decrease in the viscosity of xylan associated
with a release of only small amounts of reducing sugar. The enzyme is irreversibly inhibited by series of v-epoxyalkyl glycosides of
D-xylopyranose. The results suggest that the endoxylanase from S. thermophile has catalytic properties similar to the enzymes
belonging to family 11.
# 2003 Elsevier Ltd. All rights reserved.
Keywords: Endoxylanase; Family 11; Sporotrichum thermophile
Xylan found in almost all parts of green plant cell walls
is the second most abundant polysaccharide in nature.
In recent years, microbial enzymes depolymerizing this
polysaccharide found applications in food industry,
animal feed, and paper and pulp industries.1ꢀ3
sequence homologies EXs have been classified into two
groups mainly, families 10 and 11.4,5 Previous studies
with endo-xylanases of Streptomyces lividans (families
10 and 11),6 Trichoderrma reesei (two family 11
xylanases),7 Cryptococcus albidus (family 10),8 Thermo-
myces lanuginosus (family 11)9 and two family 10 endo-
xylanases from Thermoascus aurantiacus10,11 suggested
that the enzymes belonging to family 10 exhibit greater
catalytic versatility or lower substrate specificity than
enzymes of family 11. In addition, the activity of EXs of
family 10 is less hampered by the presence 4-O-methyl-
The enzymes acting on the xylan backbone are
classified in two groups: endo-b-1,4-xylanases (E.C.
3.2.1.8, xylan xylanohydrolase, EX) and exo-b-1,4-
xylanases (E.C. 3.2.1.37,
D-xylan xylanohydrolase).
Based on hydrophobic cluster analysis and amino acid
D
-glucuronic acid (MeGlcA), acetic acid and a-
arabinofuranose on the xylan chains.6,10,11 Further-
more, the presence of b-(103) linkages, such as in
L-
* Corresponding author. Tel.: ꢁ
/
30-3210-7723231; fax: ꢁ
/
/
30-3210-7723163.
E-mail address: hristako@orfeas.chemeng.ntua.gr (P.
Christakopoulos).
rhodymenan, leads to a more serious steric barrier for
EXs of family 11 than for family 10 EXs.6,9 In
0008-6215/03/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/S0008-6215(03)00291-X

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P. Katapodis et al. / Carbohydrate Research 338 (2003) 1881ꢀ1890
/
consonance with these considerations, EXs of family 10
liberate from 4-O-methyl- -glucuronoxylan, rhodyme-
contained 25% of the initial crude enzyme activity. The
results of the purification are summarized in Table 1.
D
nan and, with some exceptions, also from acetylxylan
and arabinoxylan, smaller products than those formed
with EXs of family 11.6,10,11
General properties *
/
Isoelectric focusing in the pH
range of 3.0ꢀ9.5 of the purified endo-xylanase revealed
/
a pI of about 6.7 (Fig. 1(A)). The molecular mass of
endo-xylanase was found to be 25 kDa as determined by
SDS-PAGE electrophoresis (Fig. 1(B)).
Almost in most cases, the glycosidase catalytic
mechanism involves two essential carboxyl residues: a
proton donor and a nucleophile. The importance of two
completely conserved and catalytically important gluta-
mate residues in family 11 glycoside hydrolases5,12 has
been demonstrated previously by site-directed mutagen-
esis. Similarly, two conserved glutamate residues are
involved in family 10 glycosidic hydrolases.13
The endo-xylanase was optimally active at pH 5 and
70 8C. Approximately, 24% of the maximum activity
was retained at 80 8C. It was retained 80% of its activity
after 24 h incubation at pH 5 and 50 8C. The enzyme
kept 63 and 55% of its activity, at pH 4 and 6,
respectively. Thermostability of xylanase was deter-
mined by measuring the residual activity, after incuba-
tion of the enzyme at 60 and 70 8C (pH 5) and the
xylanase exhibited half lives of 150 and 15 min,
respectively.
Epoxyalkyl glycosides used as affinity labeling com-
pounds of glycoside hydrolases target mainly for the
nucleophylic residue,14 yielding a covalent ester bond
between protein and the glycoside. v-Epoxyalkyl
D-
xylopyranosides were shown to only inactivate enzymes
from family 11.15
Mode of action on xylan, Xyln and MeUmb (Xyl)n*
/
The relationship between the change in specific fluidity
and release of reducing sugar during hydrolysis of xylan
is shown in Fig. 2. The enzyme mediated a rapid
The thermophilic fungus, Sporotrichum thermophile
produces high levels of xylanases, when grown on a
cheap carbon source such as corn cob. The main
purpose of the present study was to investigate the
biochemical and catalytic properties of a purified
endoxylanase from this organism and to establish its
relationship with the recognized endo-xylanase families.
Production of xylanase *S. thermophile ATCC
/
34628, when grown on corn cob in a 7 L stirred tank
fermentor at 50 8C and pH 5 for 4 days, produced 56 U
of xylanase per mL of the culture filtrate. Also, among
the five strains of S. thermophile tested in our labora-
tory, ATCC 34628 produced the highest xylanase
activity.
Purification of endoxylanase *
eluted from an anion exchanger (Q-Sepharose), at pH
8.5 using a linear gradient of 0ꢀ0.5 M NaCl. During
chromatography, a major fraction X-I was separated.
This fraction contained 35% of the total amount of
endoxylanase applied to the column.
In the second purification step, the concentrated
fraction X-I was applied on a Sephacryl S-200 column
as described in Materials and Methods. At this stage,
the enzyme had a specific activity of 875 U/mg protein
towards birchwood xylan, was purified 13-fold, and
/
The crude enzyme was
Fig. 1. Isoelectric focusing (A) and SDS-PAGE (B) of purified
endo-b-1,4-xylanase from S. thermophile ATCC 34628. (A)
Lane 1: standard protein markers in the order of increasing pI:
b-lactoglobulin A (5.20), bovine carbonic anhydrase B (5.85),
human carbonic anhydrase B (6.55), horse myoglobin (6.85),
horse myoglobin (7.35); lane 2: purified xylanase; (B) Lane 3:
purified xylanase, lane 4: standard protein markers in the order
of increasing molecular mass: trypsin inhibitor (20 kDa),
carbonic anhydrase (30 kDa), ovalbumin (43 kDa), albumin
(67 kDa) and phosphorylase b (94 kDa).
/
Table 1
Purification of a xylanase from S. thermophile
Step
Total protein a (mg)
Total activity b (U)
Specific activity a (U/mg)
Purification (fold)
Yield (%)
Culture filtrate
Ultrafiltration
Q-Sepharose
3400
2750
405
64
224,000
201,000
71,000
56,000
66
73
1.0
1.1
100.0
89.7
31.7
25.0
175
875
2.7
13.3
Sephacryl S-200
a
Protein was measured by the bicinchoninic acid method according to Pierce instruction manual No 23235X.
Xylanase activity was assayed using birchwood xylan with reducing sugar detection by the DNS method.
b

P. Katapodis et al. / Carbohydrate Research 338 (2003) 1881ꢀ
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Fig. 2. Plot of increase in specific fluidity (1/nsp) vs. the release
of reducing sugars during the hydrolysis of birchwood xylan
(4% w/v) by purified endo-b-1,4-xylanase of S. thermophile
ATCC 34628 (2.5 U).
decrease in the viscosity of xylan solution, with the
release of only small amounts of reducing sugar.
The hydrolysis pattern of xylotriose, xylotetraose,
xylopentaose and xylohexaose at the concentration
given and by the same amount of enzyme was studied
Fig. 3. Pattern of xylo-oligosaccharides released from soluble
xylan by endo-b-1,4-xylanase from S. thermophile ATCC
34628.
RXyl values 0.36, a chromatographic mobility compa-
tible with aldopentauronic acid (Fig. 4). Aldopentauro-
nic acid, isolated from such polysaccharide hydrolysate,
showed a 13C NMR spectrum identical with that of the
shortest acidic oligosaccharides liberated from glucur-
onoxylan by endo-xylanases of family 11, which is
identical with Xylb1-4(MeGlcAa-1,2)-Xylb-1,4-Xylb-
1,4-Xyl.6
by HPAECꢀPAD analysis of the products released after
/
1 h, as described in Section 1. The enzyme effected
almost complete degradation of xylohexaose but only 35
and 24% of xylopentaose and xylotetraose, respectively
was hydrolyzed during the same reaction time. The
enzyme was not active on xylotriose. Examination of the
hydrolysis products of xylo-oligosaccharides indicates
that the enzyme has an endo character (Table 2).
Clearly, the xylanase attacks mainly the internal glyco-
sidic bonds releasing xylotriose from xylohexaose,
xylobiose and xylotriose from xylopentaose and xylo-
biose from xylotetraose.
The main products of rhodymenan hydrolysis were
xylose, xylobiose and an isomeric oligosaccharide with
b-(10/3) linkage, which had a chromatographic mobi-
lity of an isomeric xylotetraose (RXyl 0.43) (Fig. 5). This
isomeric xylotetraose does not occur among the pro-
ducts of rhodymenan hydrolysis by EXs of family
10.8,10,11
Hydrolysis products generated by the action of
xylanase on soluble beechwood xylan were analyzed
by HPAECꢀPAD (Fig. 3). Xylotriose, xylotetraose,
/
xylopentaose and xylohexaose were the predominant
end products, followed by higher xylooligosaccharides
(ꢀ
Action on polysaccharides *
released from polysaccharides by endo-xylanase were
analyzed by TLC. From beechwood 4-O-methyl-
/X6).
/The hydrolysis products
D-
glucuronoxylan the enzyme liberated mainly xylose,
xylobiose, and acidic oligosaccharides which had the
Table 2
Hydrolysis products released from xylo-oligosaccharides by
the purified xylanase
Substrate (0.5 mM)
X
X2 X3 X4 X5
Xylotriose
ꢀ
/
ꢀ
/
Xylotetraose
Xylopentaose
Xyloexaose
20 62
18
51
56
Fig. 4. TLC analysis of the hydrolysis products released from
ꢀ
/
49
23
ꢀ
21
/
4-O-methyl-D-glucuronoxylan by endo-b-xylanase from S.
ꢀ
/
ꢀ
/
thermophile ATCC 34628. Position of stantdards (S) and time
of incubation time (h) are indicated.

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P. Katapodis et al. / Carbohydrate Research 338 (2003) 1881ꢀ1890
/
Fig. 5. TLC analysis of the hydrolysis products released from
rhodymenan (b-(104)-b-(103)-xylan) by endo-b-1,4-xyla-
/
/
nase from S. thermophile ATCC 34628. Position of stantdards
(S) and time of incubation time (h) are indicated. IsoXyl4
correspond to isomeric tetrasaccharide containing at least one
Fig. 7. Two-dimensional chromatography of products of
hydrolysis of beechwood acetylxylan by endo-b-1,4-xylanase
from S. thermophile ATCC 34628. The hydrolysate was
chromatographed first in direction 1, then the dried chroma-
togram was exposed to ammonium hydroxide vapours to
deacetylated all acetylated products of hydrolysis, and then the
compounds were chromatographed in direction 2.
b-(10/3) linkage.
When endo-xylanase was tested on beechwood O-
acetyl-4-O-methylglucuronoxylan, the acetylated xylo-
oligosaccharides which showed chromatographic mobi-
lity higher than Xyl and found in the hydrolysates by
xylanase of family 10,8,10,11 were not observed among
the hydrolysis products (Fig. 6). The liberated acetylated
and non-acetylated products could be resolved by two-
dimensional TLC, with deacetylation of compounds
between the two developments (Fig. 7). This pattern of
products is similar to that obtain with Xln B and Xln C
from S. lividans, EXs of family 11.6 Non-acetylated
products, mainly Xyl and Xyl2, lay on diagonal (slope
approx 1.0) and monoacetylated products (acetylated
xylotriose; ACX3) lying on the line with slope approx
0.65. The acetylated products like acetylated Xyl2
generated in significant quantities by EXs of family 10
were not observed.8,11
Action on oligosaccharides and the architecture of the
substrate binding site *Bond-cleavage frequencies of
/
oligosaccharides determined as initial product ratios of
degradation of [1-3H]-xylooligosaccharides at 0.25 mM
concentration, are shown in Fig. 8. The enzyme attacks
xylotriose exclusively at the first glycosidic bond from
the reducing end. In higher oligosaccharides, like
xylotetraose and xylopentaose, the enzyme shows a
strong preference to attack the second glycosidic bond
from the reducing end.
The ko/Km parameters (Fig. 8) indicate that the
enzyme hydrolyzes xylotriose about 70 times slower
Fig. 8. Bond cleavage frequencies of [1-3H]-xylooligosacchar-
ides and their ko/Km parameters during initial attack by endo-
b-1,4-xylanase from S. thermophile ATCC 34628. Symbols
(k) and (m) correspond to xylopyranosyl and labeled redu-
Fig. 6. TLC analysis of the hydrolysis products released from
O-acetyl-4-O-methyl-
nase from S. thermophile ATCC 34628. Position of standards
D-glucuronoxylan by endo-b-1,4-xyla-
(S) and time of incubation time (h) are indicated.
cing-end D-xylose residues, respectively.

P. Katapodis et al. / Carbohydrate Research 338 (2003) 1881ꢀ
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than xylotetraose and xylotetraose about 2.5 times
slower that xylopentaose. These data suggest that the
enzyme substrate binding site consists of five distinct
subsites. This assumption was confirmed by calculating
the affinities for several putative subsites around the
catalytic groups (Fig. 9).The enzyme has an asymmetric
distribution of binding affinities right and left of the
catalytic groups. Strong affinity to bind xylopyranosyl
Fig. 10. Schematic representation of the productive enzyme
glucuronoxylan complex of S. thermophile ATCC 34628 endo-
b-1,4-xylanase. Only subsites ꢂ
/
3 and ꢁ2 can accommodate a
/
residues is exhibited by subsites ꢂ
affinity was found at the hypothetical subsite ꢁ
affinities of subsites ꢂ1 and ꢁ1 were not determined
/
3, ꢂ
/
2 and ꢁ
/
2. No
substituted xylopyranosyl residue.
/3. The
/
/
structure of xylan (three xylopyranosyl units in one
turn).
due to lack of additional experimental data. The non-
appreciable rate of hydrolysis of xylobiose by the
enzyme suggests that the sum of affinities of the two
subsites adjacent to the catalytic groups will be negative.
Hence, endo-b-1,4-xylanase of S. thermophile appears to
have a substrate binding site interacting with five
xylopyranosyl residues (Fig. 10). The structure of the
shortest acidic fragment liberated from glucuronoxylan
Action on artificial substrates *The useful criterion
/
for differentiating the xylanases of families 10 and 11 is
their action on 4-nitrophenyl-b-xylopyranoside and 4-
nitrophenyl-b-cellobioside. Both these substrates were
hydrolyzed at the aglyconic by xylanases of family 10.
The action of endo-xylanase from S. thermophile was
tested on both substrates at low and high substrate
concentrations. No evidence has been obtained for the
liberation of 4-nitrophenol from any of the two sub-
strates either at 40 or 100 mM. The enzyme hydrolysed
MeUmb-Xyl2 at the aglyconic linkage while MeUmb-
Xyl3 is hydrolysed mainly in the middle. Kinetic
parameters determined for their hydrolysis are sum-
marised in Table 3. The affinity of XYLI towards
MeUmb-Xyl3 was 1000 times higher than that towards
MeUmb-Xyl2 (Table 3) in contrast with the family 10
xylanase from T. aurantiacus which exhibited almost the
same affinity for the two substrates.11 The affinity of the
enzyme towards CNPX2 was in the same range with that
calculated for MeUmb-Xyl2 (Table 3).
suggests that only subsite ꢂ
/
3 and ꢁ2 are able to
/
accommodate xylopyranosyl residues carrying 4-O-
methylglucuronosyl substituents (Fig. 10). Recently,
the structure of a family 15 carbohydrate-binding
module (CBM) in complex with xylopentaose has been
solved by X-ray crystallography.16 The data show that
xylopentaose adopts a threefold helical structure in the
binding cleft of CBM, consistent with the known
Inactivation experiments *
vation kinetics of the S. thermophile xylanase by (S)-4,5-
epoxypentyl-b- -xyloside are shown in Fig. 11(a). Semi-
/As an example, the inacti-
D
logarithmic plotting of the residual activity against time
is shown in Fig. 11(b), giving the values of the apparent
rate constants (ki) for different inhibitor concentrations.
The inactivation constants (ki, Ki) were calculated
graphically either by hyberbolic (Fig. 11(c)) or linear
(Fig. 11(d)) fitting of the data obtained.17 The results are
summarized in Table 4. All the v-epoxyalkyl glycosides
used were found to inactivate the enzyme.
This is the first report on the purification and
characterization of an endoxylanase from the culture
Fig. 9. Subsite affinities of S. thermophile ATCC 34628 endo-
b-1,4-xylanase. The subsite affinities were calculated by the
method of Suganuma and coworkers (1978) using the bond-
cleavage frequencies of all determined endo-b-1,4-xylanase
cleavage modes. The histogram of the subsite interaction
energy with xylopyranosyl residues of linear 1,4-b-xylooligo-
saccharides shows the average values. The calculated values for
individual subsites are given in the corresponding columns. 1
Table 3
Kinetic constants of endo-xylanase from S. thermophile for
model substrates
Substrate
Km (mM)
Kcat (minꢂ1
)
Kcat/Km
CNPX2
MeUX2
MeUX3
0.50
0.45
0.53
8
4
16
9
calꢃ4.184 J. The negative numerals are used to mark the
/
subsites left of catalytic groups and positive numeral those
right of catalytic groups. The arrow indicates the position of
catalytic groups.
3995
7538

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P. Katapodis et al. / Carbohydrate Research 338 (2003) 1881ꢀ1890
/
Fig. 11. Inactivation kinetics of the S. thermophile endo-b-1,4-xylanase with (S)-4,5-epoxypentyl-b-
Residual activity as a function of time and inhibitor concentration. The enzyme (80 mM) was incubated with different concentration
of (S)-4,5-epoxypentyl-b- -xyloside [1 (open circles), 2 (black squares), 3 (open triangles), 4 (black circles), 5 (open squares), 6 mM
D-xyloside [XOC5(s)]. (a)
D
(black triangles) in 50 mM phosphate buffer, pH 5.6, at 25 8C]. Samples were withdrawn at the indicated time intervals for
measurement of residual (Res.) activity; (b) semi-logarithmic plot; (c) hyperbolic plot; (d) linearized plot.
medium of S. thermophile ATCC 34628 grown on corn
cob as carbon source. The level of xylanase produced by
S. thermophile on corn cob as carbon source was approx
60 higher than that produced on wheat straw.18
The enzyme was purified to homogeneity by a simple
procedure which included mainly ion-exchange and gel
filtration chromatographies. The molecular mass (25
kD) of purified endo-xylanase purified from S. thermo-
phile was within the range detected for xylanases
belonging to the family 11.19 A low-molecular mass
xylanase can be useful in pulp bleaching, since smaller
enzymes can more readily access the fiber wall structure
and alter more efficiently pulp properties.20
oligosaccharides containing at least four xylose residues
and attacks mainly the internal glycosidic bonds.
The xylanase from S. thermophile hydrolyzes beech-
wood xylan to a mixture of xylo-oligosaccharides with
xylotriose, xylotetraose, xylopentaose and xylohexaose
being the major products. This makes the purified
xylanase potentially suitable for use in the production
of xylo-oligosaccharides.26 Xylo-oligosaccharides pre-
pared from various sources of xylans such as wheat
bran,27 birchwood, or corncob28 can selectively be used
by the beneficial intestinal microflora, Bifidobacteria,
and are thus expected to be used as a valuable food
additives. These sugars comprise mainly xylobiose,
xylotriose, xylotetraose, and higher oligosaccharides.
The nature of the fragments liberated from 4-O-
methylglucuronoxylan, rhodymenan, and acetyl-4-O-
methylglucuronoxylan is fully compatible with the fact
Like fungal xylanases from Neocallimastix frontalis,21
Trichoderma reesei,22 Aspergillus oryzae23 and Fusarium
oxysporum24,25 the presently studied enzyme showed
low activity on xylotriose. The xylanase hydrolyzes xylo-
Table 4
Inactivation of the xylanase from S. thermophile
Xꢀ
/
Oꢀ
/
C3(S)
Xꢀ
/
Oꢀ
/
C3(R)
Xꢀ
/
Oꢀ/C4(S)
Xꢀ
/
Oꢀ/C4(R)
Xꢀ
/
Oꢀ/C5(S)
Xꢀ
/
Oꢀ
/
C5(R)
X2ꢀ
/
Oꢀ
/
C3
1/Ki 110
ki 0.0047
168
0.0031
43
0.0220
54
0.0310
125
0.0030
205
0.0116
240
0.0067
Association constants (1/Ki, Mꢂ1) and inactivation rates (ki, hꢂ1) were determined for the alkyl epoxides derived from
and xylobiose.
D-xylose

P. Katapodis et al. / Carbohydrate Research 338 (2003) 1881ꢀ
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that the purified xylanase from S. thermophile belongs
to family 11. The enzyme does not liberate short
fragments from the polysaccharides, as in the case of
the xylanases from family 10. This is in agreement with
its relatively low activity towards short xylo-oligosac-
charides such as MeUmb-Xyl2 a substrate around 1000
times less preferred than MeUmb-Xyl3. The identifica-
tion of the shortest acidic fragment released from 4-O-
methylglucuronoxylan leads to the assumption that the
enzyme, in contrast to EXs of family 10, cannot attack
the xylosidic linkage towards the non-reducing end next
to the branch. Similarly to other xylanases of family 11,
the S. thermophile xylanase requires three unsubstituted
consecutive xylopyranosyl residues (Fig. 10) for pro-
ductive binding.
epoxyalkyl-derived xylo-oligosaccharides, even at high
concentration and with prolonged incubation times.15
Since v-epoxyalkyl xylosaccharide inactivations must
occur via prior recognition of the carbohydrate moiety
at the minus subsites,15 hydrolases belonging to family
11 also should have a good ligand-recognition capability
at the minus subsites (in comparison with those of
family 10). This is in agreement with the fact that the
enzymes belonging to family 10 exhibit lower glycon
specificity than enzymes of family 11 since cellobiosides,
lactosides and
sides containing a cleavable aglycon are hydrolysed with
considerable turn-over numbers.35
D
-glucopyranosyl-b-(1,4)-
D-xylopyrano-
Finally, based on the catalytic properties and the
partial subsite mapping, the endo-xylanase purified
from S. thermophile can be classified in family 11 of
glycosyl hydrolases.
It was reported that low concentrations (2ꢀ5 mg/L) of
/
acidic xylooligomers formed from hardwood xylan with
fungal xylanases affect the growth of tissue cultures of
aspen (Populus tremuloides)29 and pine (Pinus thunber-
gii).30 Furthermore, small quantities of acidic xylan
hydrolysate stimulate rooting of conifer cuttings in
hydroponic culture solution.31 The addition of purified
aldoterauronic acid obtained from birchwood xylan by
treatment with a T. aurantiacus family 10 endoxylanase
at a concentration of 1.6 mg/L demonstrated recogniz-
able and high promotive action towards the induction of
callus and somatic embryogenesis in culture explants of
common mallow (Malva silvestris L) and cotton (Goss-
sypium hirsutum).31 The liberation of aldopentauronic
1. Experimental
1.1. Materials
S. thermophile ATCC 34628 was purchased from ATCC
and maintained on potato dextrose-agar (PDA) med-
ium. Q-Sepharose and Sephacryl S-200 were from
Pharmacia, Sweden. Beechwood 4-O-methyl-
onoxylan was isolated from sawdust as described.7 O-
Acetyl-4-O-methyl- -glucuronoxylan was obtained by
D-glucur-
D
the extraction of beechwood holocellulose with di-
methylsulphoxide.36 Rhodymenan, a b-(1,4)-b-(1,3)-xy-
lan from Rhodymenia stenogona was a gift from Dr A.I.
Usov (Russian Academy of Sciences, Moscow, Russia).
Xylo-oligosaccharides and arabinoxylan were purchased
from Megazyme (Ireland). Methylumbelliferyl b-glyco-
sides of xylobiose (MeUmb-Xyl2) and xylotriose
(MeUmb-Xyl3) were synthesized as will be described
elsewhere (Vrsˇanska´, M., Nerinckx, W., Biely, P. and
Claeyssens, M., unpublished data). [1-3H]-Reducing-end
labelled xylo-oligosaccharides with specific radioactivity
of approx 10 MBq/mmol, were obtained by catalytic
titration of unlabelled compounds by the method of
Evans and coworkers.37 The (R,S)-v-epoxyalkyl-b-
xylopyranosaccharides used in this study as well as the
chromogenic substrate 2-chloro-4-nitrophenyl-b-xylo-
bioside (CNPX2) were synthesized as described pre-
viously.15 All other chemicals used were analytical grade
and purchased either from Sigma, or other companies.
acid from beechwood 4-O-methyl-D-glucuronoxylan by
the endoxylanase from S. thermophile makes it a
potential candidate for the production of acidic xylo-
oligosaccharides.32
Since the isomeric xylotetraose, Xylb1-4Xylb1-
3Xylb1-4Xyl, occurs among the products of rhodyme-
nan hydrolysis by the xylanase of T. aurantiacus, it can
be proposed that the enzyme similarly as other EXs of
family 11,33 attacks in the polysaccharide b-(10
linkages which follow a b-(103) linkage towards the
reducing end.
/
4)
/
The action pattern on acetylxylan becomes an addi-
tional criterion for a more defined classification of EXs.
No acetylated Xyl2 was found in the hydrolysates by
xylanase from S. thermophile a characteristic fragment
of hydrolysis of acetylxylan by EXs of family 10.8
Similar results were observed with XlnB and XlnC of
S. lividans,6 EXII of T. reesei8 and EXI of Schizophyl-
lum commune,34 all members of family 11.
All the v-epoxyaxyalkyl glycosides used were found
to inactivate the enzyme. Similar results were obtained
with two glycoside hydrolases of family 11 (T. lanugi-
nosus Xyn and T. reesei Xyn II).15 In striking contrast
with the family 11 enzymes, the family 10 glycoside
hydrolases from T. aurantiacus (Xyn) and C. thermo-
cellum (Xyn Z) were inactivated by none of the v-
1.2. Production of xylanase
The mineral medium used for the production of
xylanolytic enzymes contained per litre: KH2PO4, 3.0;
K2HPO4, 2.0; MgSO4×
FeSO4×7 H2O, 0.005; MnSO4×
H2O, 0.0014; CoCl2×6 H2O, 0.0002 g. A 5 mL mycelia
/
7 H2O, 0.5; CaCl2×
/
2 H2O, 0.1;
/
/
4 H2O, 0.0016; ZnSO4×
/
7
/

1888
P. Katapodis et al. / Carbohydrate Research 338 (2003) 1881ꢀ1890
/
and spore suspension of S. thermophile from a 6-day old
culture, grown on PDA slope at 45 8C, was inoculated to
five 500 mL Erlenmeyer flasks each containing 200 mL
of the above mineral medium supplemented with corn
cob (27 g/L) as carbon source and (NH4)2HPO4 (7.5 g/
L) as nitrogen source. The flasks were incubated at
50 8C in an orbital shaker operating at 200 rpm for 2
days. The preculture (500 mL) was used to inoculate a 7
L stirred tank fermentor containing 5 L mineral medium
having above composition. Maximum xylanase produc-
tion has obtained when using corn cob (unpublished
data). The fungus was cultured for 4 days at 50 8C and
pH 5.0. Oxygen concentration was controlled to a
minimum of 20% saturation by varying agitation speed.
The fermentation broth was first centrifuged at 10,000g
for 30 min at 4 8C and the clear supernatant was assayed
for xylanase as described below and concentrated by
ultra-filtration on a Minitan system (Millipore, Bedford,
MA, USA) with Minitan membrane plates (10 kD cut
off). The retentate, containing endo-xylanase activity,
was used for further purification of the enzyme.
1.5. Purification of endoxylanase
1.5.1. Anion exchange chromatography. Two thousand
seven hundred and fifty milligram of crude enzyme in 50
mM histidineꢀ
Sepharose column (20ꢄ
mL of the equilibrating buffer, a linear gradient of 0ꢀ
/
HCl buffer, pH 8.5, was loaded on a Q-
15 cm). After washing with 20
0.5
/
/
M NaCl in 100 mL of the same buffer was applied.
Elution with this buffer (flow rate of 60 mL/h) yielded
two endo-xylanase active fractions, the main fraction X-
I (62% of the total eluted xylanase activity), and the
minor fraction X-II. Fraction X-I was desalted on a PD-
10 column (G-25 Sephadex) and concentrated by freeze-
drying.
1.5.2. Gel filtration. Fraction X-I, dissolved in 2.5 mL 5
phosphate buffer pH 5 was applied on a
100 cm)
mM citrateꢀ
/
Sephacryl S-200 (Pharmacia) column (2.5ꢄ
/
pre-equilibrated and eluted with the same buffer (flow
rate of 60 mL/h). This purification step yielded one
endoxylanase active fraction and its homogeneity was
checked by SDS- (sodium dodecyl sulfate-polyacryla-
mide gel electrophoresis) and IEF-PAGE.
1.3. Assay of xylanase
1.6. Biochemical characterization of purified
endoxylanase
Endo-b-1,4-xylanase activity was assayed using 1% (w/
v) birchwood glucuronoxylan in 100 mM citrateꢀ
/
1.6.1. Determination of purity and molecular mass. These
were determined by SDS-PAGE using the PhastSystem
electrophoresis unit (Pharmacia, Sweden) and a 10ꢀ
phosphate buffer, pH 5.0 and 15 min incubation at
50 8C. The release of reducing sugars was determined by
the DNS method38 and 1 unit (U) of activity was defined
as the amount of the enzyme that liberates 1 mmol of
reducing sugar (xylose equivalent), per minute. The
relationship between reducing sugars and fluidity of a
xylan solution in the presence of endoxylanase, was
determined as follows: A 20 mL reaction mixture
containing 4.0% (w/v) birchwood xylan and the enzyme
/15%
polyacrylamide gel.
1.6.2. Determination of isoelectric point (pI). The IEF-
PAGE and the PhastSystem electrophoresis unit (Phar-
macia, Sweden) were employed. The gel contained
ampholytes covering a pH range of 3.0ꢀ9.5.
/
(2.5 U) in 100 mM citrateꢀphosphate buffer pH 5 was
/
1.6.3. Effect of pH and temperature on the activity and
stability of endoxylanase. The enzyme activity was
incubated at 45 8C. The relative viscosity (Oswald
viscometer) and the amount of reducing sugars released
was estimated at different time intervals. The reciprocal
of the specific viscosity (1/nsp) was calculated by the
studied in the pH 3.0ꢀ
Na2HPO4 buffer, pH 3.0ꢀ
buffer, pH 6.0ꢀ8.0; 100 mM Trisꢀ
7.0ꢀ9.0. The temperature effect was followed between
30 and 80 8C. The stability of the endoxylanase was
studied at pH 3.0ꢀ9.0 range, using the above buffers at 0
and 50 8C.
/
9.0 range: 100 mM citrateꢀ
7.0; 100 mM phosphate
HCl buffer, pH
/
/
/
/
formula 1/nspꢃ
/
to/(tꢂto), where t and to represent the
/
/
time of outflow of the reaction mixture and buffer,
respectively. This was plotted against the reducing
sugars released.
/
1.6.4. Hydrolysis of polysaccharides. The 2.0% (w/v)
solutions of polysaccharides in 50 mM citrateꢀ
1.4. Protein determination
/
Na2HPO4 buffer, pH 5.0 were incubated with appro-
priately diluted enzyme at 50 8C. The aliquots were
analyzed at different time intervals, for hydrolysis
products, by thin-layer chromatography (TLC) on
microcrystalline cellulose (DC-Alufolien Cellulose,
The protein concentration was determined by the
bicinchoninic acid method according to Pierce instruc-
tion manual No 23235X using bovine serum albumin
(BSA) as a standard. A280 was used to monitor protein
in column effluents.
Merck) in the solvent system 3:2:1 EtOAcꢀ
/
AcOHꢀ
/

P. Katapodis et al. / Carbohydrate Research 338 (2003) 1881ꢀ
/1890
1889
water (by vol.). Reducing sugars were visualized by the
anilineꢀhydrogen phthalate reagent.
Dionex ED-40 electrochemical detector (Dionex, USA).
A AcONa gradient (0ꢀ300 mM in 60 mM NaOH) was
/
/
applied during 40 min at a flow rate of 1 mL/min. The
products were quantified on the basis of peak height
using standard (Xyl)n solutions.
1.6.5. Isolation and identification of products of
hydrolysis of 4-O-methyl- -glucuronoxylan. A solution
of beechwood 4-O-methyl- -glucuronoxylan (0.5 g in
D
D
25 mL of distilled water, pH 5.0) was incubated for 2
days at 50 8C with 5 U of S. thermophile xylanase. After
this time the polysaccharide was found to be almost
1.6.8. Determination of kinetic parameters for the
hydrolysis of aryl glycosides. Hydrolysis of aryl glyco-
sides was carried out in 50 mM citrateꢀphosphate
/
completely hydrolyzed into a mixture of
D-xylose,
buffer, pH 5.6 at 40 8C using a SPECTRAmax 250
Microplate Spectrophotometer (Molecular Devices,
USA). Reaction after temperature equilibration were
initiated by the addition of 10 mL solution of pure
xylanase (20 mg protein) in a total volume of 210 mL.
xylobiose, small amount of xylotriose and an acidic
oligosaccharide migrating on TLC identically as an
aldopentauronic acid. To isolate the acid, the mixture
was passed through a column of Dowex 1 (acetate
form), which was washed first with distilled water until
no more neutral sugars were found in the eluate.
Aldopentauronic acid was liberated from the column
by 3 M solution of AcOH. The AcOH was eliminated by
evaporation in vacuum and the resulting syrup (75 mg)
was used for 13C NMR spectroscopy (Bruker AVANCE
DPX 300, Germany).
The substrate concentrations ranges used were: 0.1ꢀ
mM for 2-chloro-4-nitrophenyl-b-xylobioside (CNPX2),
4-nitrophenyl-b- -xylopyranoside (pNPX), 4-nitrophe-
nyl-b- -cellobioside (pNPC). The release of pNP or
/
100
D
D
CNP was monitored spectrophotometrically at 405 nm.
Kinetic constants (Km and kcat) were determined based
on the initial rates of hydrolysis.
1.6.6. Bond cleavage frequencies and kinetic parameters
of [1-3H]-xylo-oligosaccharides. The initial bond clea-
vage frequencies of reducing-end labeled oligosacchar-
ides, identical with relative rates of the cleavage of
individual glycosidic linkages, were determined using the
method of Allen and Thoma39 at 0.25 mM substrate
concentration and appropriate enzyme dilutions. The
reactions were performed in 0.02 M acetate buffer (pH
5.0) at 50 8C. Aliquots of the mixtures were removed at
1.6.9. Determination of kinetic parameters for the
hydrolysis of MeUmb(Xyl)n. Reaction mixtures (1.0
mL) consisting of 0.9 mL of 0.1ꢀ/20 mM MeUmb(Xyl)n;
nꢃ2ꢀ3, in 50 mM buffer citrateꢀ
/
/
/phospate, pH 5 and
0.1 mL enzyme (appropriate enzyme dilutions) were
incubated at 25 8C for 4 h. Samples (50 mL) were
removed at different time intervals and analyzed by
HPLC using a NH2-mBondapak column (300ꢄ
(Waters) with 70:30 MeCNꢀwater as mobile phase at a
flow rate of 1.0 mL/min. MeUmb and MeUmb(Xyl)n,
nꢃ1ꢀ3, were identified using a Waters UV detector
/3.9 mm)
time intervals and subjected to TLC in 3:2:1 EtOAcꢀ
AcOHꢀwater (by vol.). After detection of guide strips
with standards with anilineꢀhydrogen phthalate re-
/
/
/
/
/
/
agent, the radioactivity in substrate and products was
measured in a liquid scintillation counter. The ratio of
radioactivity in the substrate and products was deter-
mined and used for graphic determination of the initial
bond cleavage frequencies, i.e., frequencies correspond-
ing to the zero reaction time. From the same experi-
ments ko/Km parameters were determined using a
(Model 440) at 313 nm. The products were quantified on
the basis of peak height using standard MeUmb and
MeUmb(Xyl)n solutions. Analysis of the frequency of
bond cleavage of MeUmb(Xyl)n was carried out as
described previously.13
linearized form of the Michaelisꢀ
/
Menten equation
1.6.10. Inactivation experiments. Sets of reaction mix-
tures containing different concentrations of the epoxides
according to the procedure of Suganuma and cow-
orkers.33 The two parameters obtained for oligosacchar-
ides up to [1-3H]-xylopentaose were sufficient to
calculate the number of substrate-binding subsites and
subsite affinities as described by Suganuma and cow-
orkers.33
(0ꢀ
mM) in a total volume of 100 mL were incubated at 25 8C
in 50 mM citrateꢀphosphate buffer at pH 5. At 30 min
/10 mM) and a constant concentration of enzyme (0.5
/
intervals, 10 mL aliquots were taken to measure the
residual activity using 2-chloro-4-nitrophenyl b-xylobio-
side (CNPX2; 200 mL; 500 mM) as substrate. The
product formation (CNP, 405 nm) was followed con-
tinuously at 20 8C in a microtiterplate reader. The
activity of an enzyme blank (without epoxide) was
followed in the same way. Apparent inactivation con-
stants were obtained and the relevant association (1/Ki)
and inactivation (ki) constants were calculated by
conventional analysis of the data.15
1.6.7. Activity towards xylan, Xyln. Reaction mixtures
(1.0 mL) consisting of 0.9 mL of 0.1% (w/v) xylan or 0.5
mM Xyln, nꢃ
/
2ꢀ
/
5, in 20 mM buffer citrateꢀNa2HPO4,
/
pH 5.0 and 0.1 mL enzyme (10 mg protein) were
incubated at 50 8C for 1 h. Quantitative data were
obtained by analysis of samples (20 mL) using an HPLC
system (Waters 600E) with CarboPac PA1 column and

1890
P. Katapodis et al. / Carbohydrate Research 338 (2003) 1881ꢀ1890
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The authors acknowledge the financial support of the
commission of European Communities through the
INCO Copernicus Programme Project number IC15-
CT-96-1000.
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