Xylanase XYN IV from Trichoderma reesei showing exo- and endo-xylanase activity

Article abstract of DOI:10.1111/febs.12069

A minor xylanase, named XYN IV, was purified from the cellulolytic system of the fungus Trichoderma reesei Rut C30. The enzyme was discovered on the basis of its ability to attack aldotetraohexenuronic acid (HexA-2Xyl-4Xyl-4Xyl, HexA³Xyl₃), releasing the reducing-end xylose residue. XYN IV exhibited catalytic properties incompatible with previously described endo-β-1,4-xylanases of this fungus, XYN I, XYN II and XYN III, and the xylan-hydrolyzing endo-β-1,4-glucanase EG I. XYN IV was able to degrade several different β-1,4-xylans, but was inactive on β-1,4-mannans and β-1,4-glucans. It showed both exo-and endo-xylanase activity. Rhodymenan, a linear soluble β-1,3-β-1,4-xylan, was as the best substrate. Linear xylooligosaccharides were attacked exclusively at the first glycosidic linkage from the reducing end. The gene xyn4, encoding XYN IV, was also isolated. It showed clear homology with xylanases classified in glycoside hydrolase family 30, which also includes glucanases and mannanases. The xyn4 gene was expressed slightly when grown on xylose and xylitol, clearly on arabinose, arabitol, sophorose, xylobiose, xylan and cellulose, but not on glucose or sorbitol, resembling induction of other xylanolytic enzymes from T. reesei. A recombinant enzyme prepared in a Pichia pastoris expression system exhibited identical catalytic properties to the enzyme isolated from the T. reesei culture medium. The physiological role of this unique enzyme remains unknown, but it may involve liberation of xylose from the reducing end of branched oligosaccharides that are resistant toward β-xylosidase and other types of endoxylanases. In terms of its catalytic properties, XYN IV differs from bacterial GH family 30 glucuronoxylanases that recognize 4-O-methyl-d-glucuronic acid (MeGlcA) substituents as substrate specificity determinants. 2012 The Authors Journal compilation

Full text of DOI:10.1111/febs.12069

Xylanase XYN IV from Trichoderma reesei showing
exo- and endo-xylanase activity
1,
1
3
2
ꢀ
ꢁ
ꢀ2
ꢀ
Maija Tenkanen *, Maria Vrsanska , Matti Siika-aho , Dominic W. Wong , Vladimır Puchart ,
1
1
2
€
Merja Penttila , Markku Saloheimo and Peter Biely
1 VTT Technical Research Centre of Finland, Espoo, Finland
2 Institute of Chemistry, Slovak Academy of Sciences, Bratislava, Slovakia
3 United States Department for Agriculture, Agricultural Research Service, Western Regional Research Center, Albany, CA, USA
Keywords
A minor xylanase, named XYN IV, was purified from the cellulolytic sys-
tem of the fungus Trichoderma reesei Rut C30. The enzyme was discovered
on the basis of its ability to attack aldotetraohexenuronic acid (HexA-
2Xyl-4Xyl-4Xyl, HexA3Xyl3), releasing the reducing-end xylose residue.
XYN IV exhibited catalytic properties incompatible with previously
exo- and endo-acting b-1,4-xylanase;
glycoside hydrolase family 30;
Trichoderma reesei; xylan;
xylooligosaccharides
described endo-b-1,4-xylanases of this fungus, XYN I, XYN II and
Correspondence
XYN III, and the xylan-hydrolyzing endo-b-1,4-glucanase EG I. XYN IV
P. Biely, Institute of Chemistry, Slovak
Academy of Sciences, 84538 Bratislava,
was able to degrade several different b-1,4-xylans, but was inactive on
b-1,4-mannans and b-1,4-glucans. It showed both exo-and endo-xylanase
activity. Rhodymenan, a linear soluble b-1,3-b-1,4-xylan, was as the best
substrate. Linear xylooligosaccharides were attacked exclusively at the first
glycosidic linkage from the reducing end. The gene xyn4, encoding
Slovakia
Fax: +4212 5941 0222
Tel: +4212 5941 0275
E-mail: chempbsa@savba.sk
XYN IV, was also isolated. It showed clear homology with xylanases clas-
sified in glycoside hydrolase family 30, which also includes glucanases and
mannanases. The xyn4 gene was expressed slightly when grown on xylose
and xylitol, clearly on arabinose, arabitol, sophorose, xylobiose, xylan and
cellulose, but not on glucose or sorbitol, resembling induction of other
xylanolytic enzymes from T. reesei. A recombinant enzyme prepared in a
Pichia pastoris expression system exhibited identical catalytic properties to
the enzyme isolated from the T. reesei culture medium. The physiological
role of this unique enzyme remains unknown, but it may involve liberation
of xylose from the reducing end of branched oligosaccharides that are
resistant toward b-xylosidase and other types of endoxylanases. In terms of
its catalytic properties, XYN IV differs from bacterial GH family 30 glucu-
ronoxylanases that recognize 4-O-methyl-D-glucuronic acid (MeGlcA)
substituents as substrate specificity determinants.
*Present address
Department of Food and Environmental
Sciences, University of Helsinki, FI 00014,
Helsinki, Finland
(Received 14 September 2012, revised 7
November 2012, accepted 15 November
2012)
doi:10.1111/febs.12069
Introduction
Xylans are the main hemicelluloses in plants, and exist
in plant cell walls both in trees and in annual plants.
Some algae also produce xylans, but there are no reports
regarding microbial xylans. The plant xylans have a back-
bone consisting of b-1,4-linked D-xylopyranosyl units,
which are further substituted with L-arabinofuranose
Abbreviations
Araf, arabinofuranose; EG I, endo-b-1,4-glucanase I from Trichoderma reesei; GH, glycoside hydrolase; HexA, hexenuronic acid;
HexA-2Xyl-4Xyl-4Xyl (HexA3Xyl3), aldotetraohexenuronic acid; MeGlcA, 4-O-methyl-D-glucuronic acid; MeGlcA-2Xyl-4Xyl-4Xyl (MeGlcA3Xyl3),
aldotetraouronic acid; Xyl, D-xylose; Xyl2–Xyl5, b-1,4-xylobiose–b-1,4-xylopentaose; Xyl-4Xyl-4Xyl-Me, methyl b-xylotrioside; Xyl-Me, methyl
b-D-xylopyranoside; XYN, endo-b-1,4-xylanase.
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Novel xylanase from Trichoderma reesei
M. Tenkanen et al.
(L-Araf), 4-O-methyl-D-glucuronic acid (MeGlcA) or
esterified with acetic acid [1]. MeGlcA is normally
linked to the O2 position, but L-Araf and acetic acid
may be linked to O2, O3 or both on the same xylosyl
residue. The structure of xylan varies between plants,
and may even vary in different parts of the same plant.
Algae, such as red seaweed, produce mainly an unsub-
stituted linear xylan with b-1,3- and b-1,4-linkages in
the ratio 1 : 2 [2].
enzymes, and produces all necessary enzymes for xylan
degradation [25]. Several commercial enzyme prepara-
tions are presently manufactured using strains of
T. reesei. The fungus is reported to produce at least
three specific endo-b-1,4-xylanases [26–28]. The two
main xylanases, XYN I and XYN II, with molecular
masses of 19 and 20 kDa and isoelectric points of 5.5
and 9.0, respectively, belong to GH family 11. The
third xylanase, XYN III, is larger, with a molecular
mass of 32 kDa. Its isoelectric point is 9.1, and it has
been classified in GH family 10. T. reesei also pos-
sesses a fourth enzyme that is able to act on the
internal b-1,4-linkages in xylans. This endo-b-1,4-glu-
canase I (EG I) of GH family 7, which cleaves internal
b-1,4-glucosidic linkages in cellulose, is also highly
active on xylans [29,30].
The main enzymes degrading xylans are endo-b-
1,4-D-xylanases (EC 3.2.1.8), which make random cuts
in the xylan backbone. The best known microbial xy-
lanases hydrolyzing all types of xylan belong to the
glycoside hydrolase (GH) families 10 or 11 (http://
www.cazy.org) [3]. Endoxylanases also occur in GH
family 30 [4–10] (originally classified in GH family 5
[11]), GH family 8 [12–14] and GH family 5 [15].
Bacterial GH family 30 xylanases are specialized for
hydrolysis of glucuronoxylan [6–9], while the fungal
enzyme from Bispora sp. was shown to be more ver-
satile [10]. The GH family 5 enzymes appear to be
specialized for hydrolysis of arabinoxylan [15]. The
side groups in xylans are cleaved by a-L-arabinofur-
anosidases (EC 3.2.1.55), a-glucuronidases (EC
3.2.1.139) and acetyl xylan esterases (EC 3.1.1.72).
The formed oligosaccharides are degraded to xylose
by b-xylosidases (EC 3.2.1.37), which remove xylose
units from the non-reducing end of the xylooligo-
saccharides [16]. The side groups restrict the action
of b-xylosidases, and thus only non-substituted oli-
gosaccharides are degraded totally. Some b-xylosid-
ases are reported to act also on polymeric xylan
[17–19].
An interesting xylanolytic activity was previously
detected as a contaminant in a T. reesei a-glucuroni-
dase preparation [31]. This enzyme was able to liber-
ate xylose from a xylooligosaccharide, that was not
degraded by XYN I, XYN II or b-xylosidase from
T. reesei. Here we report purification of this enzyme,
named XYN IV, which is an additional specific
xylan-degrading enzyme from T. reesei. The corre-
sponding gene, xyn4, was also isolated, and, based on
amino acid homology, XYN IV was found not to
belong to the two major xylanase families, but to
GH family 30, which is a very diverse family, includ-
ing glucanases, mannanases and xylanases. As the
catalytic properties of the XYN IV purified from a
culture fluid were
a
combination of clear
exo-action and weak endo-action, they were further
confirmed for a recombinant enzyme produced in
Pichia pastoris.
There are only a few reports on specific exo-b-1,4-
xylanases, and information on their catalytic properties
is limited. These enzymes differ from b-xylosidases as
they are inactive on xylobiose. Xylanase II from
Chaetomium thermophile var. coprophile is reported to
produce mainly xylobiose as the final product [20].
Several xylanases from Aeromonas caviae ME-1 are also
exo-acting, but it remains unknown which end of the
oligosaccharides is their target [21–24]. The xylanases
Xyn IV, Xyn V and Xyn X formed exclusively xylo-
biose, xylotetraose or both, respectively [21–24].
Another type of intracellular GH family 8 enzyme was
recently discovered in Bacillus halodurans C-125 [13]. It
was able to hydrolyze xylooligosaccharides with DP
ꢀ 3 by exo-action, releasing xylose units from the
reducing end. However, it did not show activity on
polymeric xylan and was thus named exo-oligoxylanase
[14].
Results
Discovery of the new xylanase
In our previous study of an a-glucuronidase prepara-
tion from T. reesei [31], it was found that the enzyme
did not cleave the glycosidic linkage between hexenu-
ronic acid (HexA) and xylose in the tetrasaccharide
HexA3Xyl3 [32]. Instead, HexA3Xyl3 was converted
into HexA2Xyl2 by liberation of one xylose unit from
the reducing end. This action on HexA3Xyl3 was a
new catalytic activity not exhibited by any of the
known xylan-depolymerizing enzymes from T. reesei,
i.e. the specific endo-b-1,4-xylanases XYN I, XYN II
and XYN III, the xylan-degrading EG I and one b-xy-
losidase. Consequently, this new enzyme was named
XYN IV, and HexA3Xyl3 was used as a substrate
throughout for its purification.
The filamentous fungus Trichoderma reesei is one of
the best producers of cellulolytic and hemicellulolytic
286
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M. Tenkanen et al.
Novel xylanase from Trichoderma reesei
The specific b-1,4-xylanase activity of purified
XYN IV on birch glucuronoxylan was only
16 nkatÁmgÀ1, which is < 1% of the specific activity of
other T. reesei xylanases. The optimum pH for
XYN IV was between pH 3.5 and 4.0 (5 min xylanase
activity assay at 40 °C). The optimal temperature in a
5 min assay at pH 4.0 was between 40 and 50 °C. At
room temperature and at pH values between 3.0 and
5.0 the activity of XYN IV remained unchanged in
24 h incubation. At temperatures higher than 50 °C,
XYN IV lost activity after a few hours (results not
shown). At 40 °C, it was most stable at pH 4.5, and at
50 °C it was most stable at pH 4.0.
Production and purification of XYN IV
T. reesei Rut C30 was cultivated in a similar way as
described previously for production of a-glucuroni-
dase. The two-first chromatographic purification steps
were performed according to the procedure for a-glu-
curonidase, with minor modifications [33]. The major
xylanases of T. reesei were separated from XYN IV in
the two-first steps, resulting in more than 10 000-fold
reduction in the amount of xylanase activity in the
sample. XYN IV was separated from a-glucuronidase
and residual b-xylosidase in the last two steps using
DEAE-Sepharose and gel filtration on Sephacryl S-100.
As XYN IV is a very minor xylanase of T. reesei in
terms of common xylanase assays, its purification degree
and yield were not calculated.
Hydrolysis of xylans by XYN IV
The purified XYN IV was a specific xylanase. It did not
show any action on glucomannan, galactomannan,
b-1,3-b-1,4-glucan, laminarin or carboxymethyl cellu-
lose. Nor did it show any a-arabinofuranosidase,
a-galactosidase, b-mannosidase or b-xylosidase activity
on the corresponding 4-nitrophenyl glycosides as sub-
strates.
Protein properties
The molecular weight of XYN IV is 43.0 kDa accord-
ing to SDS/PAGE (Fig. 1). XYN IV had several iso-
forms with pI values of ꢁ 7 (results not shown). The
N-terminal amino acid sequence was ?-S-Y-A-T-?-S-Q-
Y-?-A-N-I-?-I (? = unclear or very weak signal). The
amino acid sequences of two internal peptides were
G-A-T-S-D-D-E-N-N and T-N-P-N-E-Y-V-Y-A-D-V-
?-S-A-P. These partial sequences were sufficient to
identify the corresponding xyn4 gene in the T. reesei
genome, and the correct amino acid sequence was
obtained based on the gene sequence (see below).
XYN IV was able to act on all three tested xylans
(hardwood glucuronoxylan, wheat arabinoxylan and
rhodymenan, the latter being the most rapidly hydro-
lyzed substrate) (Fig. 2). The main products of glucu-
ronoxylan hydrolysis as shown by TLC were xylose,
xylobiose and an aldotetraouronic acid (Fig. 3).
Longer acidic xylooligosaccharides were also detected
in small amounts. The enzyme also hydrolyzed
1.5
1.25
1
97
67 kDa
45 kDa
43 kDa
0.75
0.5
0.25
0
30 kDa
20 kDa
14.4 kDa
0
2
4
6
8
10
12
Time (h)
Fig. 2. Rate of hydrolysis of various xylans by XYN IV from
T. reesei followed as liberation of reducing sugars expressed as
xylose equivalents. Substrate concentration 10 mgÁmLÀ1; enzyme
concentration 11.4 nkatÁmLÀ1. Black circle, glucuronoxylan; white
circle, wheat arabinoxylan; black triangle, rhodymenan.
S
1
2
S
Fig. 1. SDS/PAGE of purified XYN IV. Samples: S, standards; lane 1,
5 lg XYN IV; lane 2, 2.4 lg XYN IV.
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Glucuronoxylan Rhodymenan Arabinoxylan
acid was also found to be attacked by Pichia stipitis
a-glucuronidase of GH family 115 [37], to give free
uronic acid and Xyl3. The major acidic oligosaccharides
liberated from glucuronoxylan therefore appear to
have the structure Xyl-4(MeGlcA-2)Xyl-4Xyl (MeG-
lcA2Xyl3), which differs from the aldouronic acid liber-
ated by GH family 10 endoxylanases (MeGlcA3Xyl3)
[38]. Treatment of the whole glucuronoxylan hydroly-
sate with b-xylosidase resulted in the disappearance of
xylobiose, an increase in the Xyl concentration, and
conversion of the aldotetraouronic acid MeGlcA2Xyl3
into aldotriouronic acid MeGlcA2Xyl2. However, the
minor products migrating as acidic oligosaccharides
did not change significantly (data not shown). A paral-
lel experiment with glucuronoxylan hydrolysate using
Erwinia chrysanthemi GH family 30 xylanase showed
clearly that all acidic oligosaccharides were converted
to aldotriouronic acid MeGlcA2Xyl2, as described pre-
viously [8]. This suggests that XYN IV does not liber-
ate aldouronic acids of the structure MeGlcA2Xyln, as
bacterial GH family 30 xylanases do. The formation
of MeGlcA2Xyl3 during hydrolysis of glucuronoxylan
is a unique property of the XYN IV enzyme.
Xyl
Xyl2
Xyl3
unknown
MeGlcAXyl3
Xyl4
Xyl5
S
0
5 24 48 72 96 0 5 24 48 72 96 0 5 24 48 72 96
Time (h)
Fig. 3. Hydrolysis by XYN IV of glucuronoxylan, rhodymenan
and wheat arabinoxylan, followed by TLC on silica gels developed
twice in ethyl acetate/acetic acid/2-propanol/formic acid/water
(25 : 10 : 5 : 1 : 15 v/v). Substrate concentration 10 mgÁmLÀ1
;
enzyme concentration 1.4 nkatÁmLÀ1. S, standards. Total sugars
were detected using N-(1-naphthyl)ethylenediamine reagent.
arabinoxylan but to a limited extent, liberating xylose,
Xyl2 and an oligosaccharide. The latter product, pre-
sumably an AraXyl3, showed similar migration as Xyl3
on TLC. The much higher degree of endo-type hydro-
lysis observed in the case of rhodymenan requires a
separate investigation. The endo-action of XYN IV
was also demonstrated using xylan dyed with Remazol
Brilliant Blue (RBB-xylan), a specific substrate for
endoxylanases [34].
The structure of the major xylooligosaccharide
generated from wheat arabinoxylan was solved as Xyl-
4(Ara-3)Xyl-4Xyl (Ara2Xyl3) by applying pure a-arabi-
nofuranosidase and b-xylosidase on arabinoxylan
hydrolysate in which XYN IV was denaturated by
heating. The oligosaccharide was resistant to b-xylosi-
dase and was converted to arabinose and xylotriose by
a-arabinofuranosidase. Xylotriose was hydrolyzed to
xylose in the second step by b-xylosidase. The oligo-
saccharide also showed identical chromatographic
mobility as the authentic compound. As shown below,
XYN IV generates the same tetrasaccharide from
Ara3Xyl4, which excludes the structure Ara3Xyl3.
The structure of aldotetraouronic acid liberated
from glucuronoxylan was established by enzyme-
assisted analysis (Fig. 4). The compound did not serve
as a substrate of the GH family 67 a-glucuronidase of
Aspergillus tubingensis [35], which requires MeGlcA to
be linked to the non-reducing xylopyranosyl residue of
the xylooligosacccharide [35,36]. The aldotetraouronic
acid was hydrolyzed by b-xylosidase to yield a product
that is one xylose residue shorter, MeGlcA2Xyl2, that
was readily hydrolyzed by GH family 67 a-glucuroni-
dase to MeGlcA and xylobiose. The aldotetraouronic
Action on linear xylooligosaccharides
XYN IV showed a typical exo-action on linear b-1,4-xy-
looligosaccharides. The liberation of xylose was accom-
Fig. 4. Enzyme-assisted elucidation of
the structure of the main aldouronic
acid liberated from glucuronoxylan by
T. reesei XYN IV.
288
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M. Tenkanen et al.
Novel xylanase from Trichoderma reesei
panied by formation of shorter oligosaccharides, which
were further digested to xylose and Xyl2 (Table 1). An
example of hydrolysis of 20 mM Xyl5 followed by TLC
is shown in Fig. 5A. Longer incubations provided clear
evidence that Xyl2 is subject to slow hydrolysis. No
products larger than the substrate were observed in
reaction mixtures of 20 mM oligosaccharides, indicating
no significant transglycosylation activity of XYN IV.
The question of whether XYN IV attack of linear
xylooligosaccharides occurs at the reducing or non-
reducing end was answered using [1-3H]-reducing end-
labeled b-1,4-xylooligosaccharides. The radioactive
label enabled determination of the initial bond cleav-
age frequencies of the substrates. [1-3H]-xylotriose,
-xylotetraose and -xylopentaose were attacked by the
enzyme exclusively at the first glycosidic linkage from
the reducing end, as [1-3H]-xylose was found to be the
only radioactive product generated from all labeled
substrates (Fig. 6A). The bond cleavage frequencies of
the first linkage from the reducing end were 1.0 in all
three cases, which means that the oligosaccharides
were strictly degraded from the reducing end by releas-
ing the monomer. The pattern of attack of oligosac-
charides was found to be the same at 0.025, 0.25, 5
and 20 mM substrate concentration, indicating that the
enzyme forms only one type of productive complex
with linear substrates, suggesting a small number of
subsites of the substrate-binding site. This is in con-
trast to xylanases of GH families 10 and 11, which
form productive complexes with two substrate
molecules, resulting in shifted substrate binding and
catalysis of glycosyl transfer reactions at a 20 mM
concentration of linear xylooligosaccharides [39,40].
Determination of the kinetic parameter ko/Km at a
0.025 mM concentration of [1-3H]-labeled substrates
(Table 2) provided unambiguous evidence that xylotri-
ose and xylotetraose are the best substrates for the
enzyme, and that xylobiose serves as a poor substrate.
Based on the kinetic parameters and bond cleavage
frequencies, it may be deduced that the enzyme sub-
strate-binding site consists of one subsite on the right
of the catalytic groups (+I) and at least two subsites to
the left of the catalytic groups (ÀI and ÀII). Calcula-
tion of subsite affinities [41] based on the ko/Km
parameters (with a bond cleavage frequency of 1.0 in
all cases) gave the following values: subsite ÀII,
4.1 kcalÁmolÀ1; subsite ÀIII, À0.05 kcalÁmolÀ1; subsite
ÀIV, À1.5 kcalÁmolÀ1. Thus subsite ÀII appears to be
decisive for binding of linear xylooligosaccharides. The
affinities of subsites +I and ÀI, or the sum of affinities
of these two subsites, could not be calculated on the
basis of obtained data. However, the fact that xylobi-
ose is a very poor substrate suggests that the value will
be very low.
Table 1. Products of hydrolysis of various substrates at 20 and
2 mM concentrations as indicated, with XYN IV at a concentration
1.4 nkatÁmLÀ1. Aliquots were taken at intervals for TLC for product
analysis and semi-quantitative evaluation of the hydrolysis rate.
Disappearance
Hydrolysis of modified and non-linear
xylooligosaccharides
Compound
Cleavage products
of the substrate
Xyl-Me
Xyl-4NPh
Xyl2
No hydrolysis
No hydrolysis
Xyl
–
–
Table 1 also shows the action of XYN IV on a series
of glycosides and acidic oligosaccharides. The product
analysis and semi-quantitative evaluation of the rate of
hydrolysis was performed by TLC. XYN IV did not
attack methyl and 4-nitrophenyl b-D-xylopyranoside.
These substrates are perhaps too small to be bound
productively in the substrate-binding site, or may
contain an unfavorable hydrophobic aglycon. Xyl-
4Xyl-Me hydrolysis was as slow as that of Xyl2, but
produced mainly methanol and Xyl2. As may be
expected, the substrate that is one xylopyranosyl resi-
due longer, Xyl-4Xyl-4Xyl-Me, was hydrolyzed much
faster to give Xyl2 and methyl b-D-xylopyranoside. A
cleavage mode that allowed liberation of a fragment
larger than Me-Xyl from the reducing end was not
observed. This result indicates that the subsite of the
enzyme substrate-binding site to the right of the cata-
lytic groups (subsite +I) recognizes the methyl glyco-
side part of the substrate similarly to the reducing-end
xylopyranose. Additional information regarding the
(+)
+++
+++
Xyl3
Xyl + Xyl2
Xyl4
(Xyl3 + Xyl) ?
Xyl2 + Xyl
Xyl5
(Xyl4 + Xyl) ?
(Xyl3 + Xyl) ? etc.
Xyl2 + MeOH
Xyl2 + Me-Xyl
+
Xyl-4Xyl-Me
(+)
Xyl-4Xyl-4Xyl-Me
Xylotetraitol (Xyl4-ol)
+++
(+)
(Xylitol + Xyl3)a
?
Xyl2 + Xyl + Xylitol
Xyl2 + 4NPh-OH
Xyl2 + MeUmb-OH
Xyl3 + MeUmb-OH
Xyl + HexA2Xyl2
Xyl + MeGlcA2Xyl2
Xyl + MeGlcA2Xyl3
Ara2Xyl3 + Xyl
Xyl-4Xyl-4NPh
Xyl-4Xyl-MeUmbb
Xyl-4Xyl-4Xyl-MeUmbb
++
+
+
HexA3Xyl3
++
++
++
+
b
MeGlcA3Xyl3
MeGlcA3Xyl4
b
b
Ara2Xyl4
b
Ara2Xyl3
No hydrolysis
–
b
a
Xyl3 was not observed as an intermediate due to fast hydrolysis.
b
Substrate concentration 2 mM. (+), poor substrate; +++, excellent
substrate.
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289

Novel xylanase from Trichoderma reesei
M. Tenkanen et al.
A
B
Xyl
Xyl
Xyl
2
3
Fig. 5. TLC analysis (on microcrystalline
cellulose) of the products of hydrolysis of
20 mM Xyl5 by XYN IV isolated from the
growth medium (A) and of 20 mM Xyl4 by
recombinant XYN IV (B) in 0.05 M sodium
acetate buffer (pH 4.0). Enzyme
Xyl
Xyl
4
5
0
0.25 0.5
1
2
4
S
0
0.5
2
8
24 48
concentration 1.4 nkatÁmLÀ1. S, standards.
Time (h)
A
B
Fig. 6. (A) Liberation of [1-3H]-xylose as the
only radioactive product of hydrolysis of
0.025 mM [1-3H]-labeled Xyl3 (black circle),
Xyl4 (white square) and Xyl5 (black triangle)
by XYN IV isolated from the growth
medium. (B) Liberation of [1-3H]-xylose as
the only radioactive product of hydrolysis of
0.025 mM [1-3H]-labeled Xyl4 (white square)
by recombinant XYN IV. The concentration
of the enzyme was in the range
0.35–7.0 nkatÁmLÀ1. The slope of the
function corresponds to the initial bond
cleavage frequency of oligosaccharides,
which, in this case, is equal to 1.0.
were cleaved by XYN IV exclusively at the aglyconic
linkage.
Table 2. Kinetic parameters ko/Km of XYN IV for b-1,4-xylooligo-
saccharides obtained at 0.025 mM substrate concentration. The
enzyme concentration varied from 0.35 to 7 nkatÁmLÀ1
.
HexA3Xyl3, MeGlcA3Xyl3 and MeGlcA3Xyl4 were
shortened to HexA2Xyl2, MeGlcA2Xyl2 and MeG-
lcA2Xyl3, respectively, by liberation of one xylose resi-
due from the reducing end (Table 1). Similar behavior
of XYN IV on the side-chain sugar was observed with
3-O-L-arabinofuranose-substituted xylooligosaccharides.
Of Ara2Xyl4 and Ara3Xyl4, only the former oligosac-
charide was attacked to give Ara2Xyl3 and xylose
(Table 1).
Xylooligosaccharide
ko/Km (MÀ1 sÀ1
)
Xylobiose
5.1 9 101
4.7 9 104
4.3 9 104
3.7 9 103
Xylotriose
Xylotetraose
Xylopentaose
‘plus’ subsites of the substrate-binding site was
obtained using xylotetraitol as substrate. The
a
reduced tetrasaccharide was hydrolyzed much more
slowly than Xyl4. The first step was formation of xyli-
tol and Xyl3, which, because it is a much better sub-
strate than xylotetraitol, was cleaved immediately at
the reducing end to xylose and Xyl2.
Stereochemical course of hydrolysis of glycosidic
linkages
Xyl-4Xyl-4Xyl-Me was found to be a suitable sub-
strate for the 1H-NMR study of the stereochemical
course of hydrolysis of glycosidic linkages by XYN IV.
The glycoside does not have reducing end, and
Aryl glycosides of xylobiose and xylotriose, Xyl-4Xyl-
NPh, Xyl-4Xyl-MeUmb and Xyl-4Xyl-4Xyl-MeUmb,
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M. Tenkanen et al.
Novel xylanase from Trichoderma reesei
consequently does not produce NMR signals in the
anomeric region of the spectrum that interfere with
monitoring of the newly formed reducing end. The
compound was rapidly hydrolyzed to the b-anomer
Xyl2 and Xyl-Me. The b-anomer was slowly mutaro-
tated to the a-anomer. This result confirms that
XYN IV, similar to other GH family 30 enzymes, is a
retaining glycoside hydrolase that uses a double dis-
placement reaction mechanism for hydrolysis of glyco-
sidic linkages.
acids around the proton donor (Asn-Glu-Pro-Asp,
NEPD) are conserved, but this appears not to be the
case for the nucleophile amino acid. Of six aromatic
amino acids contributing to substrate binding in bacte-
rial enzymes (several tryptophans and tyrosines) that
are conserved in bacterial enzymes, only one is
conserved in fungal enzymes, and two are replaced
with other aromatic amino acids in XYN IV (data not
shown). The most interesting finding relevant to the
mode of action of fungal GH family 30 xylanases was
that Arg293 in Erwinia chrysanthemi and Arg303 in
Bacillus subtilis, which are responsible for recognition
of the MeGlcA residue in the substrate via ionic inter-
action with the MeGlcA carboxyl group [42,43] and
are conserved in all bacterial GH family 30 xylanases,
do not occur in fungal enzymes (Fig. 7).
Isolation and sequence analysis of the xyn4
cDNA
In parallel with purification and characterization of
XYN IV, we studied the regulation of protein expres-
sion in T. reesei. The xyn4 cDNA was incidentally iso-
lated in a screen of Saccharomyces cerevisiae in which
cloning of transcription factors activating the T. reesei
pdi1 promoter was attempted. Among the cDNA clones
obtained in the screening, four were found to be derived
from the same gene, and one of them was sequenced. A
sequence database search revealed that the cDNA has
homology with GH family 30 xylanases from Aeromo-
nas caviae and Erwinia chrysanthemi. Comparison of
the putative amino acid sequence encoded by the cDNA
with peptide sequencing data from the isolated
XYN IV enzyme revealed that the cDNA encodes this
xylanase. Thus the gene was named xyn4.
A BLAST search of the T. reesei genome database
(http://genome.jgi-psf.org/Trire2/Trire2.home.html)
retrieved two gene models with significant homology
to XYN IV. One of them (e_gw1.30.36.1) shows high
homology with fungal b-1,6-glucanases, and the func-
tion of the second one (e_gw1.28.211.1) cannot be pre-
dicted based on homology.
The xyn4 expression pattern
The expression level of xyn4 as a function of culture
conditions was investigated in T. reesei strain QM9414
by Northern hybridization. No xyn4 expression was
detected on glucose, suggesting that the gene is under
catabolite repression. xyn4 mRNA was not detectable
in mycelia grown on sorbitol, or in a glucose depletion
sample, which is unusual for T. reesei cellulases and
hemicellulases [44]. A low xyn4 mRNA level was
detected in mycelia grown on xylose or xylitol, and
a clearly higher level was detected in mycelia grown on
L-arabinose and L-arabitol. The highest expression
levels were detected on cellulose and the three xylans
that were tested. When mycelia were grown with added
sorbitol and either sophorose or xylobiose in the cul-
ture, the xyn4 gene was clearly induced, more strongly
with xylobiose than with sophorose.
The xyn4 cDNA encodes a protein of 465 amino
acids. The N-terminal sequence obtained from
XYN IV matches the xyn4 gene from amino acid posi-
tion 18 onwards, and thus the signal peptide is 17
amino acids in length. The predicted molecular weight
of the mature XYN IV is 50.3 kDa. T. reesei XYN IV
has clear homology with the GH family 30 xylanases
XynA from Erwinia chrysanthemi (25% identity, 41%
similarity [4]), XynD and Xyn3 from Aeromonas caviae
(24% identity, 40% similarity [5]), GH family 30 xy-
lanase from Bacillus subtilis (25% identity, 41% simi-
larity [7]) and Paenibacillus barcinonensis Xyn30D
(23% identity, 37% similarity [9]) (Fig. 7). The closest
homology was observed with XYLD from Bispora sp.
MEY-1 (37% identity, 54% similarity) [10] and with
the N-terminal part of a putative xylanase from the
fungus Leptosphaeria maculans (37% identity, 54%
similarity; Genbank accession number AAO49459.1)
that has not been studied at the enzyme level. The
Leptosphaeria maculans GH family 30 xylanase is
much longer than XYN IV. Alignment of the T. reesei
XYN IV with bacterial GH family 30 xylanases sug-
gests that the proton donor and the nucleophile are
Glu201 and Glu293, respectively (Fig. 7). The amino
In many respects, the expression pattern of xyn4
resembles that of the other xylanase genes xyn1 and
xyn2, the b-xylosidase gene bxl1 and the a-glucuroni-
dase gene glr1 (Table 3) [44], but is closest to that of
bxl1. Both xyn4 and bxl1 are expressed on L-arabinose
and are strongly induced by xylobiose, whereas xyn1
and xyn2 are not. The low expression level detected
on xylose and xylitol is a particular feature of the
xyn4 gene (Table 3). None of the 12 cellulase and
hemicellulase genes studied previously [44] was
expressed in a xylose culture. Surprisingly, no xyn3
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291

Novel xylanase from Trichoderma reesei
M. Tenkanen et al.
Fig. 7. Amino acid sequence alignment of the N-terminal parts (~320 amino acids) of the following GH family 30 xylanases:
Leptosphaeria maculans Xyl38 (accession number Q873Z0), Bispora sp. MEY-1 XylD (accession number D6MYS9), Trichoderma reesei
XYN IV (accession number AAP64786.1), Aeromonas caviae W-61 Xyn3 (accession number P70733), Paenibacillus barcinonensis Xyn30D
(accession number AEY82463.1), Bacillus subtilis XynC (accession number Q45070) and Erwinia chrysanthemi XynA (accession number
Q46961). The amino acid numbering shown on the right side is according to the native protein sequences. Catalytic glutamic acids acting as
catalytic acid/base and nucleophile, are shown in bold and underlined. The amino acids corresponding to arginine conserved in bacterial
sequences (e.g. Arg293 of Erwinia chrysanthemi XylA) and responsible for recognition of the MeGlcA residue in the substrate [42,43] are
shown in bold, underlined and boxed. Aromatic amino acids lining the catalytic cleft and conserved in bacterial proteins are shaded in gray.
Identical amino acids are indicated by asterisks, similar amino acids are indicated by colons, and conserved polar or non-polar amino acids
are indicated by dots.
expression on any of the studied carbon sources was
detected in the strain studied here. The expression pat-
tern of the xyn3 gene is known only for strain PC-3-7
[45], being expressed on cellulose, sorbose and sopho-
rose but not on xylose, xylan or xylooligosaccharides.
enzyme showed identical catalytic properties to the
enzyme isolated from the T. reesei growth medium. It
hydrolyzed xylans and linear and acidic xylooligosac-
charides to the same products as XYN IV purified
from T. reesei. The exo-action of the recombinant
enzyme on Xyl4 is shown in Fig. 5B. The experiment
with [1-3H]-reducing end-labeled xylooligosaccharides
confirmed strict exo-action from the reducing end on
linear xylooligosaccharides (Fig. 6B). Similar to the
enzyme isolated from the culture medium, the recom-
binant enzyme showed a higher specific activity on
rhodymenan than on glucuronoxylan or arabinoxylan.
The aldouronic acids MeGlcA3Xyl3 and MeGlcA3Xyl4
were shortened by the recombinant enzyme, with the
removal of one xylose residue from the reducing end.
Recombinant XYN IV
The unique catalytic properties of XYN IV, namely
the combination of weak endo-action with clear exohy-
drolytic activity, prompted us to verify the catalytic
properties of XYN IV isolated from the T. reesei
growth medium using a recombinant enzyme. The
XYN IV gene was expressed in Pichia pastoris, and
the enzyme product was purified. The recombinant
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M. Tenkanen et al.
Novel xylanase from Trichoderma reesei
hydrolyze MeGlcA3Xyl3 to MeGlcA2Xyl2 and xylose;
however, in contrast to XYN IV, the enzyme behaves
as a typical endo-acting xylanase [46]. The exoxylanase
from Bacillus halodurans (GH family 8) was shown to
attack linear xylooligosaccharides in a similar manner
but is an inverting hydrolase [13,14].
Table 3. Expression patterns of five T. reesei genes involved in
xylan degradation.The data on xyn1, xyn2, glr1 and bxl1 are from
Margolles-Clark et al. [44]. The signal intensities based on visual
judgment varied from non-detectable (–) to very strong (+++).
Carbon source
xyn1
xyn2
xyn4
bxl1
glr1
Glucose
–
–
–
–
–
One of the major products formed by XYN IV from
various xylans and xylooligosaccharides was xylose.
All linear xylooligosaccharides studied were exclusively
cleaved at the first glycosidic linkage from the reducing
end. These data suggest that the XYN IV may be a
unique exoxylanase. However, this mode of action
may also be an activity of the enzyme that is limited
to the tested substrates, which differ significantly from
those that the enzyme encounters in nature, as xylans
and derived xylooligosaccharides are often consider-
ably acetylated.
Glucose depletion
Cellulose
–
++
++
–
–
++
++
+
+
+
–
++
–
+++
++
++
+
L-arabinose
L-arabitol
++
–
++
–
+++
–
–
Xylose
–
Xylitol
–
–
+
–
–
Lenzing xylan
Glucuronoxylan
Oat spelt xylan
Sorbitol
++
+
–
+++
++
++
–
++
+(+)
++
–
+
(+)
+
–
++
+
+++
–
(+)
+(+)
(+)
Sorbitol+sophorose
Sorbitol+xylobiose
+
+
++
++
–
–
++
–
The possibility that the low endo-action of XYN IV
may be the result of minor contamination of the
exoxylanase by an endoxylanase was the main reason
why XYN IV isolated from the culture fluid was com-
pared with a recombinant T. reesei XYN IV expressed
in Pichia pastoris. The catalytic properties of both
enzyme preparations were found to be identical, con-
firming that both the endo- and exo-action are inher-
ent properties of this T. reesei GH family 30 xylanase.
These properties are different from the properties of
bacterial GH family 30 appendage-dependent endoxy-
lanases that recognize MeGlcA substituents as sub-
strate specificity determinants [6–9].
The endo-action of the recombinant XYN IV was also
confirmed on RBB-xylan. These results firmly excluded
the possibility that any of the catalytic properties of
XYN IV isolated from the growth medium of T. ree-
sei, such as the weak endo-action, are due to contami-
nation by another type of xylanase.
Discussion
HexA3Xyl3 is a substrate for xylanases that
catalyze cleavage from the reducing end
The specific b-1,4-xylanase activity of XYN IV on
glucuronoxylan was extremely low, just a fraction of
the values exhibited by the T. reesei XYN I and
XYN II on the same polysaccharide substrate. In
addition, the action pattern of XYN IV appears to be
different from GH family 10 xylanases, which always
produce MeGlcA3Xyl3 from glucuronoxylan, whereas
GH family 11 xylanases produce MeGlcA3Xyl4 [38].
The main acidic oligosaccharide produced by XYN IV
or by XynC from S. commune is MeGlcA2Xyl3 [46].
XYN IV also acted on arabinoxylan, releasing mainly
xylose, xylobiose and one major Ara-substituted
xylooligosaccharide, the structure of which was estab-
lished as Ara2Xyl3. As in the case of the main ald-
otetraouronic acid generated from glucuronoxylan, the
Ara-containing tetrasaccharide also contains Araf
linked to the middle xylopyranosyl residue of Xyl3.
Information on the substrate-binding site of
XYN IV was obtained by determining the bond cleav-
age frequencies and kinetic parameters ko/Km of the
labeled xylooligosaccharides. The enzyme appears to
have a substrate-binding site composed of three sub-
sites: ÀII, ÀI and +I (Fig. 8). Subsite ÀII showed the
Here we show that the non-natural hexenuronic acid-
containing xylooligosaccharide, tetrasaccharide Hex-
A3Xyl3, may serve as a specific substrate for screening
of exoxylanases operating on the reducing end because
this oligosaccharide is not attacked by a-glucuronidases.
In contrast, the natural oligosaccharide MeGlcA3Xyl3
is attacked by a-glucuronidases [33, 35–37] and thus is
not suitable for screening of exoxylanases in the pres-
ence of a-glucuronidases. In the present work, use of
HexA3Xyl3 enabled discovery of
a new type of
xylanase from T. reesei RUT-C30. The enzyme
catalyzed release of Xyl from the reducing end of
HexA3Xyl3.
T. reesei XYN IV: exo- versus endo- mode of
action
XYN IV exhibited completely different catalytic prop-
erties to previously described endo-b-1,4-xylanases of
this fungus, or any other currently classified xylanase.
An exception is a non-classified XynC from Schizo-
phyllum commune. This enzyme has the ability to
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293

Novel xylanase from Trichoderma reesei
M. Tenkanen et al.
highest affinity for xylopyranosyl residues. This interac-
tion appears to be decisive for hydrolysis of low-mole-
cular-mass substrates. For unsubstituted b-1,4-linked
xylooligosaccharide, there appears to be either a seri-
ous steric barrier or a xylose repulsive force at the posi-
tion of the hypothetical subsite +II. In the case of
polymeric substrates, this repulsive force must be over-
come by additional enzyme–substrate interactions. This
idea is supported by the facts that the endo-action of
the enzyme is undisputable, and that the structure of
the main aldouronic acid produced from glucuronoxy-
lan is MeGlcA2Xyl3, suggesting accommodation of the
MeGlcA-substituted xylopyranosyl residues at hypo-
thetical subsite ÀII. Based on the ability of XYN IV to
shorten the tetrasaccharide Ara3Xyl4 to Ara2Xyl3, it
may be assumed that subsite ÀII can also accommo-
date 3-O-L-arabinosylated xylopyranosyl residues.
the lack of substrate binding at hypothetical subsite ÀIII.
Cleavage of 4-nitrophenyl-b-D-xylobioside and 4-me-
thylumbelliferyl-b-D-xylobioside at the aglyconic bond,
in contrast to the resistance of 4-nitrophenyl-b-D-xylo-
side and 4-methylumbelliferyl-b-D-xyloside (Table 1),
may be explained by a strong affinity of subsite ÀII
for the xylopyranosyl residue.
XYN IV as a non-transglycosylating xylanase
The retaining character of XYN IV as an enzyme
belonging to GH family 30 was confirmed by determi-
nation of the anomeric configuration of the newly
formed xylobiose from methyl b-xylotrioside by
1H-NMR spectroscopy. In this regard, it is interesting
to note that no glycosyl transfer reactions were observed
even at high substrate concentration. Given the exis-
tence of three subsites of the enzyme substrate-binding
site, it is difficult to imagine how the transfer reaction
may take place when the acceptor site is available to
just the monomer, which is released after cleavage of
the glycosidic linkage to the reducing-end xylose of the
substrate. If the transfer reaction is catalyzed at high
substrate concentrations, such transfer will always lead
to resynthesis of the substrate molecule. Such a reac-
tion may be demonstrated by incubation of the
enzyme with radioactively labeled xylose and unlabeled
xyloligosaccharide. The radioactive xylose will be
incorporated into the resynthesized substrate mole-
cules. However, such experiments have not yet been
performed. It is well known that glycosyl transfer reac-
tions are catalyzed by both GH family 10 and 11
endoxylanases at higher substrate concentrations. Under
Additional considerations concern the subsites of
the substrate-binding site around the catalytic groups
of XYN IV. Xylobiose, which is an excellent substrate
for b-xylosidases, was hydrolyzed three orders of mag-
nitude more slowly than longer oligosaccharides. This
result suggests that the sum of the affinities of subsites
ÀI and +I must be negative, resulting in repulsion of
xylobiose. The fact that conversion of xylotetraose to
xylotetraitol led to a considerable reduction in the rate
of hydrolysis of the same glycosidic linkage suggests
that the reducing-end xylopyranose in linear oligosac-
charides plays an important role in binding of the sub-
strate at subsite +I, and that the binding affinity of
this subsite is positive. The fact that Xyl4 was almost
as good a substrate as Xyl3 (Table 1) is consistent with
Araƒ
such conditions, the enzymes form
a productive
complex with two substrate molecules, a so-called
termolecular (ternary) shifted complex [39–41,47,48].
Such enzymes have a larger number of subsites around
the catalytic groups than XYN IV.
Xylβ1-4Xylβ1-4Xylβ1-4Xyl*
MeGlcA
Xylβ1-4Xylβ1-4Xyl*
β
β
Xylβ1-4Xyl 1-4Xyl 1-4Xyl-ol
β
Xylβ1-4Xyl 1-4Xyl—Me
Position of XYN IV among xylanases and its
physiological function
Xylβ1-4Xylβ1-4Xylβ1-4Xyl*
β
β
Xyl 1-4Xyl 1-4Xyl*
Currently all known bacterial GH family 30 xylanases
are ‘appendage-dependent’ endoxylanases [49]. Their
activity is strictly dependent on the presence of MeGlcA
or GlcA side residues [6–9,49,50]. The decisive enzyme
–substrate interaction for this substrate specificity was
shown to be ionic interaction of one conserved argi-
nine with the carboxyl group of the MeGlcA side resi-
due [42,43]. The fungal GH family 30 xylanase from
Bispora sp. also hydrolyzes arabinoxylan, with xylose
and xylobiose as the main products of hydrolysis [10].
Similar to the Bispora sp. enzyme, T. reesei XYN IV
-II -I
+I
Fig. 8. Hypothetical subsites in the substrate-binding site of
XYN IV, and productive complexes with the following substrates
from bottom to top: Xyl3, Xyl4, methyl b-xylotrioside, xylotetraitol,
aldotetraouronic acid MeGlcA3Xyl3 and arabinoxylotetraose
Ara2Xyl4. The larger arrow indicates the enzyme catalytic groups,
and the asterisks indicate the reducing end of oligosaccharides.
The smaller arrow marks the points of cleavage.
294
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M. Tenkanen et al.
Novel xylanase from Trichoderma reesei
also does not recognize MeGlcA side chains as sub-
strate specificity determinants. This maybe due to lack
of the positively charged Arg close to the active site
that is conserved in bacterial enzymes (e.g. Arg293 of
Erwinia chrysanthemi) but is replaced by Gln or Glu in
fungal enzymes (Fig. 7). The glutamic acid acting as
the catalytic acid/base and the surrounding residues
are quite conserved between bacterial and fungal
enzymes. In contrast, a region around the catalytic
nucleophile (Glu293 in XYN IV) does not show any
significant homology with bacterial enzymes. The low
degree of conservation of aromatic amino acids lining
the substrate-binding site in bacterial enzymes also
supports the view that the fine architecture of the sub-
strate-binding site of XYN IV differs from that of bac-
terial enzymes. This is mainly reflected in a strict exo-
action action of XYN IV on short linear xylooligosac-
charides, in contrast to negligible activity of bacterial
enzymes on substrates that do not contain uronic acid
side chains. The abundance of xylose in xylan hydroly-
sates produced by the Bispora sp. GH family 30 xylan-
ase [10] suggests that the Bispora sp. enzyme has a
similar exo-acting mode of action on xylooligosaccha-
rides to T. reesei XYN IV. The fungal GH family 30
xylanases clearly form a distinct sub-family as indi-
cated previously [11]. However, the overall structure of
fungal GH family 30 xylanases does not differ very
much from bacterial enzymes. Of the GH family 30
xylanases, only the enzymes from Erwinia chrysanthemi
and Bacillus subtilis have been crystallized and have
been shown to consist of two domains: a catalytic
domain and a putative xylan-binding domain com-
posed of nine b-strands [42,43,51]. The auxiliary
domain was suggested to be present in all GH fam-
ily 30 enzymes, regardless their catalytic properties
[11]. The modeled structure of the Bispora sp. enzyme
confirms this suggestion [10]. In the case of the Paeni-
bacillus barcinonesis enzyme, this domain was shown
to be indispensable for the enzyme activity [9]. Its dele-
tion abolished the enzyme activity [9]. All these facts
suggest that the putative xylan-binding domain is also
present in XYN IV and is crucial for its activity.
tion group? The recognition group does not seem to be
MeGlcA, at least not to such an extent as in the case of
bacterial GH family 30 xylanases. The present study
provides evidence for the fact that, on xylooligosaccha-
rides, the enzyme behaves mainly as a xylose monomer-
producing catalyst. There is no apparent structural
explanation for these catalytic activities of XYN IV.
The major role of the enzyme may therefore be
release of xylose from the reducing end of xylooligosac-
charides carrying a substituent close to the non-reduc-
ing end. This catalytic property is not exhibited by any
known xylanolytic enzyme except the Bacillus halodu-
rans intracellular xylanase, which does not recognize
polymeric xylan as a substrate [13]. In this regard, the
enzyme may be an important enzyme for saccharifica-
tion of the major plant hemicellulose. However, it is
difficult to explain the more pronounced endo-action of
XYN IV on rhodymenan. Rhodymenan does not occur
in the habitats of the fungus and is therefore not consid-
ered a natural substrate. It is obvious that further study
is required to clarify the role of XYN IV in the life cycle
of T. reesei. Of special interest remains elucidation of
the mode of action of XYN IV on native partially acet-
ylated plant xylans. The fact that all endoxylanases
were designed by nature to attack partially but signifi-
cantly acetylated polysaccharides is often neglected.
Acetylation of xylan is not restricted to hardwood
xylan; considerable acetylation has also been reported
for xylans from cereals and annual plants, including
Arabidopsis thaliana [54–56].
Experimental procedures
Substrates
Hardwood xylan with a reduced content of MeGlcA (Xyl:
MeGlcA ratio of ꢁ 27 : 1) was obtained from Lenzing
AG (Lenzing, Austria). 4-O-Methylglucuronoxylan from
birchwood (Xyl:MeGlcA ratio of ꢁ 9 : 1, referred to
throughout as glucuronoxylan) was purchased from Roth
(Karlsruhe, Germany). Rhodymenan, an algal unsubstitut-
ed b-1,3-b-1,4-xylan, was generously supplied by A.I. Usov
(Zelinski Institute of Organic Chemistry, Russian Academy
of Sciences, Moscow, Russia). RBB-xylan was prepared as
described previously [34]. Wheat arabinoxylan with a Ara:
Xyl ratio of 41 : 59, glucomannan from konjac, galacto-
mannan from locust beans and barley b-glucan were pur-
chased from Megazyme (Wicklow, Ireland). Oat spelt
arabinoxylan and laminarin were obtained from Sigma
(St. Louis, MO) and carboxymethyl cellulose was obtained
from Fluka (Lausanne, Switzerland).
The exo-oligoxylanase of Bacillus halodurans, which
also acts from the reducing end of the xylooligosaccha-
rides, is an intracellular GH family 8 enzyme, but is
inverting and inactive towards polymeric xylan [13,14].
Homologous GH family 30 Xyn3 from Aeromo-
nas caviae strain W-61 has been reported to be a spe-
cific endo-1,4-b-xylanase that produces mainly long
oligosaccharides with six or more xylose units [52,53].
Several questions concerning XYN IV remain open.
What is the specific role of the enzyme in plant cell-wall
degradation? Does the enzyme have a substrate recogni-
b-1,4-xylooligosaccharides, from xylobiose (Xyl2) to
xylopentaose (Xyl5), were obtained from Megazyme.
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M. Tenkanen et al.
Xylotetraitol was prepared by reduction of the corre-
sponding tetramer using NaBH4. [1-3H]-reducing end-labeled
b-1,4-xylooligosaccharides (specific radioactivity ꢁ 50 MBqÁ
lmolÀ1) were prepared by custom catalytic tritiation per-
formed at Nycom Ltd (Prague, Czech Republic), followed
by chromatographic purification.
to 133 mSÁcmÀ1 by addition of (NH4)2SO4. The adjusted
sample was loaded on a phenyl Sepharose FF column
(1100 mL, height 11 cm; GE Healthcare, Little Chalfont,
UK) equilibrated by 25 mM sodium acetate buffer pH 5.5,
containing 0.85 M (NH4)2SO4. Elution of XYN IV was per-
formed by reducing the salt concentration linearly from
0.85 to 0.6 M. The flow rate was 90 mLÁminÀ1. Fractions of
450 mL were collected and analyzed for the presence of
XYN IV. Active fractions were pooled (8550 mL) and
concentrated by ultrafiltration to 1200 mL.
A xylotriose with an a-1,2-linked hexenuronic acid at the
non-reducing end xylose unit (HexA3Xyl3) was isolated by
anion exchange chromatography and gel filtration after
enzymatic hydrolysis of alkali-cooked birch pulp [32].
MeGlcA-substituted oligosaccharides (MeGlcA3Xyl3, MeG-
lcA3Xyl4) and arabinose-substituted xylooligosaccharides
(Ara2Xyl3, Ara2Xyl4 and Ara3Xyl4) were obtained as
described previously [17,40].
The concentrate was further buffer-exchanged into
20 mM sodium phosphate buffer pH 6.0 by gel filtration on
a Sephadex G-25 column (4 L, height 40 cm). Then the
sample was applied to
a CM-Sepharose FF column
(350 mL, height 18 cm high; GE Healthcare, Little Chal-
font, UK). After washing the unbound impurities from the
column, XYN IV was eluted by an increasing NaCl gradi-
The oligosaccharide methyl glycosides b-D-Xylp-(1,4)-b-
D-Xylp-O-Me (Xyl-4Xyl-Me) (methyl b-xylobioside) and
b-D-Xylp-(1,4)-b-D-Xylp-(1,4)-b-D-Xylp-O-Me
(Xyl-4Xyl-
ent from 0 to 0.4 M. The flow rate used was 45 mLÁminÀ1
,
4Xyl-Me) (methyl b-xylotrioside) [57,58] were generous gifts
from P. Kovac and J. Hirsch (Institute of Chemistry, Slovak
Academy of Sciences, Bratislava, Slovakia). 4-nitrophenyl
and methyl b-D-xylopyranosides (Me-b-Xyl) were obtained
from Sigma. 4-methylumbelliferyl b-xylobioside and b-xylo-
trioside were prepared as described previously [59].
and the fraction size was 90 mL. XYN IV activity was
shown in two peaks, which were first pooled separately but
later combined after final purification step (gel filtration) as
they both contained XYN IV (different isoforms). Both
pooled fractions (1350 and 630 mL) were concentrated by
ultrafiltration to 100 mL.
Final purification of XYN IV was performed by gel fil-
tration on a Sephacryl S-100 HR column (width 5.7 cm,
height 100 cm; GE Healthcare, Little Chalfont, UK). The
eluent used was 50 mM sodium acetate buffer pH 4.5 con-
Production and purification of XYN IV
T. reesei strain Rut C30 was used as an enzyme producer.
It was cultivated in a bioreactor with a working volume
of 28 L. The fermentation conditions were: temperature
29 °C, pH controlled between 6.0 and 6.5 by automatic
addition of NH4OH or H3PO4, pO2 ꢀ 20% controlled by
agitation, aeration 5 LÁminÀ1, cultivation time 75 h. The
culture medium used contained 30 gÁLÀ1 xylan from Lenz-
ing AG, 15 gÁLÀ1 distiller’s spent grain, 5 gÁLÀ1 KH2PO4
and 5 gÁLÀ1 (NH4)2SO4. After cultivation, the mycelium
was separated by centrifugation for 20 min, 4000 g in
Cryofuge 8000 centrifuge (Heraeus, Sepatech, Osterode,
Germany), and the culture filtrate was concentrated to
1.8 L by ultrafiltration (20 kDa cut-off, type ES 625, PCI
Membrane Systems Ltd, Basingstoke, Hampshire, UK).
The concentrated culture filtrate (1800 mL) was adjusted
to pH 4.5 and conductivity 17 mSÁcmÀ1, after which it was
loaded onto a CM-Sepharose FF column (2 L, height 20 cm)
equilibrated with 50 mM sodium acetate buffer pH 4.5 con-
taining 0.14 M NaCl. The chromatografic columns and media
for protein were acquired from Pharmacia, presently GE
Healthcare (Little Chalfont, UK). After washing unbound
materials from the column, bound proteins were eluted using
a linear gradient of 0.14–0.40 M NaCl in 50 mM sodium ace-
tate buffer pH 4.5 at a flow rate of 150 mLÁminÀ1. Fractions
in 450 mL volumes were collected and measured for activity
against HexA3Xyl3. The total volume of combined active
fractions was 3150 mL, containing 0.6 mgÁmLÀ1 protein.
Before the next purification step, the pH of the pool was
adjusted to pH 5.5 and the conductivity was increased
taining 0.1 M NaCl, and the flow rate was 33 mLÁminÀ1
.
The combined active fractions were concentrated and used
for XYN IV characterization.
Auxiliary enzymes
Purified GH family 67 a-glucuronidase from Aspergillus
tubingensis [35] was generously supplied by J. Visser and
R.P. de Vries (Wageningen Agricultural University, The
Netherlands). Purification of GH family 115 a-glucuronidase
from Pichia stipitis was performed as described by Ryab-
ova et al. [37]. The b-xylosidase was Aspergillus niger
enzyme produced in recombinant Saccharomyces cerevisiae
[36]. The GH family 51 a-L-arabinofuranosidase from
A. niger was obtained from Megazyme. The GH family 30
glucuronoxylanase from Erwinia chrysanthemi was supplied
by J.F. Preston (University of Florida, Department of
Microbiology and Cell Science, Gainesville, FL, USA).
Activity assays
The activity of XYN IV during its purification was followed
qualitatively using HexA3Xyl3 as substrate. HexA3Xyl3
(0.5 mgÁmLÀ1) in 0.1 M sodium citrate buffer, pH 4, was
incubated with the enzyme overnight at 40 °C, after which
the reaction was terminated by boiling. The samples were
analyzed for liberated xylose and HexA2Xyl2 by TLC on
296
FEBS Journal 280 (2013) 285–301 ª 2012 The Authors Journal compilation ª 2012 FEBS

M. Tenkanen et al.
Novel xylanase from Trichoderma reesei
silica gel-coated sheets (Merck, Darmstadt, Germany) devel-
oped in ethyl acetate/acetic acid/water (6 : 4 : 1 v/v). Sugars
were visualized using orcinol reagent (Sigma, St. Louis, MO,
USA). b-1,4-xylanase activity was determined as described
by Bailey et al. [60] using 1% 4-O-methylglucuronoxylan as
the substrate. After determination of the pH optimum of
purified XYN IV, the xylanase assay was performed at pH
4.0. The activity is expressed in katals (kat).
ethyl acetate/acetic acid/2-propanol/formic acid/water
(25 : 10 : 5 : 1 : 15 v/v), or on microcrystalline cellulose
(Merck) using ethyl acetate/acetic acid/water (18 : 7 : 10 or
3 : 2 : 2 v/v). On silica gel-coated sheets, sugars were visu-
alized using orcinol or N-(1-naphthyl)ethylenediamine
reagent. On cellulose, reducing sugars were detected using
aniline-hydrogenphthalate reagent. The main aldouronic
acid liberated by XYN IV from glucuronoxylan was purified
from the hydrolysate by preparative TLC. On chromato-
grams, the compound was localized by detection of sugars
on guide strips and eluted from the silica gel by methanol.
The activity of XYN IV on konjac glucomannan, locust
bean galactomannan, barley b-glucan, carboxymethyl cellu-
lose and laminarin was tested by incubating 0.5% polysac-
charide solution in 0.1 M sodium citrate buffer, pH 4.0,
with XYN IV (35 mgÁgÀ1) for 24 h at 40 °C.
Bond cleavage frequencies and kinetic
parameters
The a-L-arabinofuranosidase, a-galactosidase, b-xylosi-
dase and b-mannosidase activities of XYN IV were exam-
ined using the corresponding 4-nitrophenyl glycosides as
substrates [61].
Bond cleavage frequencies [39–41,48,49] of [1-3H]-reducing
end-labeled b-1,4-xylooligosaccharides were determined in
0.05 M sodium acetate buffer, pH 4.0, at 30 °C at four sub-
strate concentrations (0.25, 2, 5 and 20 mM) using varying
enzyme concentrations. Aliquots of the mixture were taken
after various time intervals and analyzed by TLC on cellu-
lose-coated sheets. After detection of guide strips with xylose
and xylooligosaccharides as standards, the corresponding
areas of the chromatograms were cut out and the radioactiv-
ity of the substrate and its hydrolysis products was counted
in toluene scintillation fluid using a 1214 RACKBETA liquid
scintillation counter (LKB Wallac, Turku, Finland). The
fraction of total radioactivity for all compounds was calcu-
lated using
Investigation of protein properties
SDS/PAGE was performed on a 10% gel slab as described
previously [62]. Isoelectric focusing was performed on a 5%
polyacrylamide gel using a Multiphor II system (Pharmacia,
Uppsala, Sweden) with pH gradient 3.5–9.5. Proteins were
stained by silver staining (silver stain kit; Bio-Rad, Her-
cules, CA, USA).
The N-terminal amino acid sequence of the intact
XYN IV and the amino acid sequence of two internal pep-
tides (produced by trypsin degradation and purified by
HPLC using C18 reversed-phase column) were analyzed by
automated Edman degradation at the University of Kuopio
(Finland). Protein content was analyzed as described previ-
ously [63] using bovine serum albumin as standard.
n
X
3H À Xyli=
3H À Xyli
1
where 3H-Xyli is the radioactivity of the product or sub-
strate with degree of polymerization i, and n is the degree
of substrate polymerization. This ratio was plotted against
the extent of the reaction obtained by dividing the radioac-
tivity in all products by the total radioactivity in the sample
The pH optimum of XYN IV was determined at 40 °C in
McIlvaine buffers with pH values between 3.0 and 8.0. The
effect of different temperature (30, 40, 50 and 60 °C) on
enzyme activity was followed at pH 4.0. The stability of
XYN IV was studied at pH 3.0 and 5.0 at room tempera-
ture, 40 and 50 °C, by incubating purified XYN IV for 24 h.
nÀ1
n
X
The slopes of the lines
X
3H À Xyli=
3H À Xyli
1
1
Hydrolysis experiments with xylans and
xylooligosaccharides
nÀ1
X
Various xylans (5 or 10 gÁLÀ1) dissolved in 0.1 M sodium
citrate buffer or 0.05 M sodium acetate buffer, pH 4.0, were
treated with 500 nkatÁgÀ1 (31 mgÁgÀ1) of XYN IV at 40 °C.
Liberation of reducing sugars was determined according to
the Somogyi–Nelson procedure [64]. The products of
hydrolysis of various xylans, linear xylooligosaccharides,
their glycosides, hexenuronic acids and aldouronic acids (at
2 or 20 mM substrate concentrations as indicated) with
XYN IV (10–500 nkatÁgÀ1 substrate) in 0.1 M sodium cit-
rate buffer or 0.05 M sodium acetate buffer (both pH 4.0)
were analyzed by TLC on silica gel-coated sheets (Merck)
developed in 1-butanol/ethanol/water (10 : 8 : 5 v/v) or in
3H À Xyli=
3H À Xyli
1
give the initial product ratios, which are equal to the initial
bond cleavage frequencies. A relationship between the nat-
ural logarithm of the ratio of total radioactivity to the
radioactivity of the substrate
n
X
ln
3H À Xyli=3H À Xyln
1
and time was used to determine the ko/Km parameters [41].
The ko/Km parameters and bond cleavage frequencies were
FEBS Journal 280 (2013) 285–301 ª 2012 The Authors Journal compilation ª 2012 FEBS
297

Novel xylanase from Trichoderma reesei
M. Tenkanen et al.
then used to calculate the affinity of hypothetical subsites of
the substrate-binding site for xylopyranosyl residues [41].
Total T. reesei QM9414 RNA was isolated as described
by Chirgwin et al. [71]. Northern hybridization was per-
formed using Hybond N nylon membranes (Amersham,
Vienna, Austria) according to the manufacturer’s instruc-
tions using 0.5 lg of RNA for the cellulose-grown samples
and 5 lg RNA for other samples.
Stereochemistry of hydrolysis
The stereochemical course of cleavage of glycosidic bonds
was followed by 1H-NMR spectroscopy using methyl
b-D-xylotrioside as a substrate. A 0.5 mL aliquot of a
5 mM solution of the xylotrioside in 0.05 M sodium acetate
buffer in D2O, pD 4.5, prepared from deuterized acetate
and acetic acid (Aldrich Chemicals, St. Louis, MO, USA),
was incubated with XYN IV lyophilized twice from D2O
(85 nkatÁmLÀ1) at 35 °C in a NMR test tube. 1H-NMR
spectra were recorded after various times on a Bruker
Preparation of recombinant XYN IV
The xyn4 gene (AAP64786) was synthesized by EZBiolab
(Carmel, IN) as a 1400 bp ORF ligated in the SmaI site of
a modified pUC57 plasmid (GenBank: Y14837; https:ncbi.
nlm.nih.gov/). The gene was sub-cloned in fusion to the
a-mating factor signal peptide in the Pichia expression vec-
tor pGAPZa (Invitrogen, Carlsbad, CA, USA). The a-mat-
ing factor signal peptide was then removed and replaced
with the XYN IV native signal peptide (MKSSISVV-
€
AM 300 spectrometer (Bruker, Tubingen, Germany). The
concentration of the enzyme required for rapid hydrolysis
of the substrate was established in a preliminary experiment
followed by TLC.
LALLGHSAA) using
a DNA linker. The linker was
designed to encode the start Met and native signal peptide
sequence with 5′ BstI and 3′ HindIII overhangs, and was
ligated into BstI–HindIII-digested Xyn IV-pGAPZ. The
resulting construct was further PCR-amplified from the
unique SacI site of xyn4 to insert a stop codon at the 3′
end of the gene. Ligation of the PCR fragment into SacI–
BamHI-digested Xyn IV-pGAPZ yielded the final gene vec-
tor construct with the vector myc epitope and the His-tag
sequence removed. Transformation of the gene vector into
Pichia pastoris was achieved by electroporation, and active
clones grown on YPD-Zeocin plates were identified by
TLC analysis of the hydrolytic activity on Xyl4.
Isolation of the xyn4 gene
The screening of a cDNA expression library [65] con-
structed from T. reesei RUT-C30 was performed essentially
as described previously [66]. The screening construct con-
tained a 1.2 kb fragment from the promoter of the T. reesei
pdi1 gene cloned upstream of the Saccharomyces cerevisiae
HIS3 gene. The T. reesei cDNA expression library was
transformed into yeast strain DBY746 (a, his3D1, leu2-3,
ura3-52, trp1-289, Cyhr) containing the screening construct
as described by Gietz et al. [67]. Transformants were plated
on SC medium lacking Leu, Ura and His [68], and plas-
mids were isolated from clones that were able to grow on
this medium and tranformed into Escherichia coli for
restriction analysis and sequencing. The xyn4 cDNA in the
plasmid of the selected transformant was sequenced from
both strands using internal oligonucleotide primers.
The active transformant was cultured at 30 °C in med-
ium containing 1% yeast extract, 2% peptone, 2% dex-
trose, buffered with 100 mM potassium phosphate, pH 6.0
and supplemented with 1.34% yeast nitrogen base and
0.4 lgÁmLÀ1 biotin. A 500 mL culture was harvested by
centrifugation at 6000 g, and the supernatant was filtered
through a 0.45 lm MachV Supor device (Nalgene, Roche-
ster, NY, USA). The filtrate was concentrated tenfold and
buffer-exchanged with 10 mM HEPES, 25 mM NaCl, pH
8.0, using a PelliconXL BioMax cassette (10 kDa molecular
weight cut-off) on a Labscale TFF system (Millipore, Bed-
ford, MA, USA). The sample was applied to a CM Bio-
Gel A column (0.75 cm 9 30 cm; Bio-Rad, Hercules, CA,
USA) equilibrated with buffer A (10 mM HEPES, 25 mM
NaCl, pH 8.0) at a flow rate of 12 mLÁhÀ1. The column
was then washed with 80 mL buffer A, followed by elution
Expression of the xyn4 gene
For gene expression studies, T. reesei strain QM9414 was
grown in shake flasks (28 °C, 200 rpm) for 3 days in mini-
mal medium [69] with 6% glucose, 6% arabinose, 6%
arabitol, 6% xylose, 6% xylitol, 3% Solka floc cellulose
(James River Corp., Richmond, VA, USA), 3% partially
debranched xylan from beech (Lenzing AG), 3%
4-O-methylglucuronoxylan from birch or 3% arabinoxylan
from oat spelts. When induction of the xyn4 gene was
studied, T. reesei QM9414 was first grown in 2% sorbitol
and xyn4 expression was induced at 72 h by addition of
1 mM sophorose or at 82 h by 2 mM xylobiose. The myce-
lium was harvested at 87 h. A culture grown for 87 h with
2% sorbitol served as a control. The glucose-depleted sam-
ple originated from a 125 h glucose batch fermentation
[70].
using
a linear gradient of 25–500 mM NaCl. Active
fractions were pooled, concentrated and buffer exchanged
to 0.1 M Na acetate, pH 4.0, using an Amicon Ultra-15
concentrator (10 kDa molecular weight cut-off; Millipore,
Bedford, MA, USA).
The purified enzyme was gel-blotted onto a poly(vinyli-
dene difluoride) membrane for N-terminal sequencing. The
result showed correct processing of the signal peptide
producing the predicted mature protein.
298
FEBS Journal 280 (2013) 285–301 ª 2012 The Authors Journal compilation ª 2012 FEBS

M. Tenkanen et al.
Novel xylanase from Trichoderma reesei
11 St John FJ, Gonzales JM & Pozharski E (2010)
Consolidation of glycosyl hydrolase family 30: a dual
domain 4/7 hydrolase family consisting of two
structurally distinct groups. FEBS Lett 584, 4435–4441.
12 Collins T, Meuwis M-A, Stals I, Claeyssens M, Feller
G & Gerday C (2002) A novel family 8 xylanase,
functional and physicochemical characterization. J Biol
Chem 277, 35133–35139.
Acknowledgments
€
ꢀ
ꢀ
ꢀ
Maria Burgermeister, Maria Cziszarova, Riitta Isoni-
emi, Riitta Nurmi, Taina Simoinen, Victor J. Chan
and Helena Simolin are thanked for the skillful techni-
cal assistance. Genencor International Inc., the Finnish
Funding Agency for Technology and Innovation
(Tekes), Finland, and the Slovak Science Grant
Agency VEGA (grant 2/0001/10) are thanked for
financial support. This work was also supported by
the Research & Development Operation Programme
funded by the European Regional Development Fund
(ITMS 26220120054).
13 Fushinobu S, Hidaka M, Honsa Y, Wakagi T, Shoun
H & Kitaoka M (2005) Structural basis for the
specificity of the reducing end xylose-releasing exo-
oligoxylanase from Bacillus halodurans C-125 . J Biol
Chem 280, 17180–17186.
14 Honda Y & Kitaoka M (2004) A family 8 glycoside
hydrolase from Bacillus halodurans C-125 (BH2105) is
a reducing end xylose-releasing exo-oligoxylanase.
J Biol Chem 279, 55097–55103.
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