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polymeric substrate and MALDI ToF MS was used for analysis of the
action of AcXEs on neutral and acidic xylooligosaccharides generated
from aspen acetylglucuronoxylan by Clostridium thermocellum GH10
endo-β-1,4-xylanase (CtGH10) [4,6]. Serine-type AcXEs, which occur in
CE families 1, 5 and 6, were capable of liberating acetic acid from singly
and doubly acetylated Xylp residues. Aspartate metalloenzymes, which
are members of the CE4 family, deacetylated effectively only singly 2- or
3-O-acetylated Xylp residues. The CE4 AcXEs require the neighboring
hydroxyl group at positions 3 or 2 non-acetylated or otherwise
unsubstituted [7–9]. Neither the serine-type nor the aspartate-type
AcXEs were capable of liberation of the 3-O-acetyl groups on MeGlcA-
substituted Xylp residues. Studies with oligosaccharides further showed
that a limited number of additional acetyl groups are resistant to AcXEs
in both neutral and acidic xylooligosaccharides, in particular acetyl
groups located on non-reducing-end Xylp residues that may become
non-hydrolyzable by spontaneous migration from position 2 and 3
(National Institute of Health, Bethesda, MD, USA), Dr. J. Hirsch (Institute
of Chemistry, Slovak Academy of Sciences) and Dr. A. Fernandes-
Mayorales (Instituto de Quimica Organica General, CSIC, Madrid,
Spain). 2,3,4-Tri-O-acetate of MeXylp was prepared by a standard
acetylation procedure [20].
Endo-β-1,4-xylanase of GH10 family from C. thermocellum (CtGH10)
(xylanase catalytic module of Xyn Z) was a recombinant enzyme
prepared as described previously [6]. A CE1 family AcXE from
Schizophyllum commune (ScCE1) was purified as described [20]. A
CE4 family AcXE from Streptomyces lividans (StCE4, Ref. [21]) was
supplied by Drs. Claude Dupont and Dieter Kluepfel (Institute of
Armand Frappier, Laval, Canada). A similar enzyme from C. thermocellum
(CtCE4) [8] was provided by Profs. Carlos M.G.A. Fontes (Universidade
Técnica de Lisboa, Portugal) and Gideon J. Davies (University of York,
UK). A CE5 family AcXE from T. reesei (TrCE5) [14] was kindly provided
by Prof. Maija Tenkanen (University of Helsinki, Finland). A family CE6
AcXE from Orpinomyces sp. (OCE6) was from Megazyme Int. (Ireland).
The recombinant CE16 AcE from T. reesei (TrCE16) was produced and
purified as reported by Li et al. [16].
towards position
4 [6]. Studies using monoacetyl and diacetyl
derivatives of 4-nitrophenyl β-D-xylopyranoside have convincingly
demonstrated the occurrence of this type of migration and showed
that the 4-monoacetylated and the 3,4-di-O-acetylated derivatives are
the most abundant in equilibrium [10]. A similar situation could occur
on the non-reducing end Xylp residues of xylooligosaccharides, both
for non-substituted Xylp or Xylp substituted with MeGlcA [11,12].
There is currently only one known microbial esterase that
deacetylates oligosaccharides at the non-reducing end [11]. This
enzyme is the acetyl esterase (AcE) from Trichoderma reesei belonging
to the CE16 family (TrCE16) which is a component of the cellulolytic
system of the fungus [13–16]. Earlier studies of catalytic properties of
the enzyme showed that it does not deacetylate polymeric substrates
like acetylglucuronoxylan but acted on acetylated xylobiose [14].
The enzyme also catalyzes the deacetylation of 4-nitrophenyl β-
xylopyranoside monoacetates and oligosaccharides acetylated at
the non-reducing sugar residue, acting at positions 3 and 4 and, less
effectively, also at position 2 [11,17]. The enzyme also efficiently
catalyzes transacetylation in aqueous medium saturated with vinyl
acetate to position 3 of the non-reducing residues of cello-, manno-
and xylooligosaccharides [11,18]. The acetyl group could be removed
from acetylated saccharide acceptors by the same enzyme in the
absence of an acetyl group donor [11]. In contrast to the formation of
single 3-O-acetylated products in the case of cellooligosaccharides,
transacetylation to Xyl or xylooligosaccharide acceptors afforded a
mixture of acetyl derivatives because the acetyl group migrated to
other positions [10], including position 4. This is in accordance with
the proposed occurrence of the acetyl group at position 4 of the non-
reducing end of neutral and acidic xylooligosaccharides resistant to
AcXEs [6,19]. On the basis of these properties the enzyme was assigned
as an exo-acting deacetylase [19]. Thus, the TrCE16 enzyme appears to
be a good candidate for deacetylation of non-reducing end Xylp
residues in both neutral and acidic xylooligosaccharides resistant to
further deacetylation by AcXEs in families CE1, CE4, CE5 and CE6 [6].
In this study we have analyzed the action of TrCE16 on natural
substrates and the interplay of this enzyme with various AcXEs. The
exo-type action of TrCE16 on the non-reducing end sugar residues
was demonstrated on xylooligosaccharides generated from aspen acetyl
glucuronoxylan by the CtGH10 xylanase alone or in the presence of four
different AcXEs (CE1, 4, 5 and 6). Nearly complete deacetylation of
oligosaccharides could be achieved by combining several AcXEs with
TrCE16 AcE.
2.2. Hydrolysis of aspen acetyl glucuronoxylan with enzymes
A solution of aspen acetyl glucuronoxylan (0.5%, w/v) in 0.05 M
Tris/HCl buffer (pH 6.5) was incubated with an endoxylanase
(CtGH10; 0.07 mg/ml) at 40 °C. After 24 h the mixture was heated
at 100 °C for 5 min to denature the enzyme. 50 μl aliquots of the
mixture with heat-inactivated xylanase were mixed with small
volumes of solutions of AcXEs alone or in combination with TrCE16
to give the final enzyme concentrations: ScCE1 (0.13 mg/ml), SlCE4
(0.045 mg/ml), CtCE4 (0.4 mg/ml), TrCE5 (0.13 mg/ml), TrCE16
(0.022 mg/ml). All incubation mixtures containing a combination
of an AcXE and TrCE16 acetylesterase were run in parallel with the
mixtures containing the CtGH10 generated xylooligosaccharides (with
denatured or active GH10) treated with only AcXEs. Therefore, the
products identified by MALDI ToF MS in the mixtures with only
AcXEs, reported in our previous paper [6], are included in this work to
aid a direct comparison of their action in the presence of TrCE16. The
action of TrCE16 on xylooligosaccharides in the absence of AcXEs was
investigated under identical conditions. The mixtures with esterases
were then incubated further for 24 h at 40 °C. After denaturation of
the esterases (5 min, 100 °C), the samples were cooled, and prior to
MALDI ToF MS, they were examined by TLC on silica gel (Merck,
Germany) and vacuum dried. TLC was done in ethyl acetate–acetic
acid–2-propanol–formic acid–water (25:10:5:1:15, v/v) and the
sugars were detected using the N-(1-naphthyl)ethylenediamine
dihydrochloride reagent [22]. The conditions used in the enzyme
treatments afforded samples suitable for MALDI ToF MS analysis, as
described in detail in a recent study [6].
In a separate experiment, using the conditions described above,
acetyl glucuronoxylan was incubated for 48 h with CtCE10 (without
the denaturing step) in combination with ScCE1 or SlCE4 AcXE in the
presence or absence of TrCE16 AcE. This implies that the esterases
acted on the polysaccharide in the presence of an active GH10 xylanase.
The reaction mixtures were heated for 5 min at 100 °C and cooled prior
to MALDI ToF MS analysis [6].
2.3. Action of TrCE16 AcE on acetylated derivatives of methyl β-D-
xylopyranoside
2. Materials and methods
The deacetylation of the triacetate and diacetates of Me-β-Xylp at
10 mM concentration in 0.05 M sodium phosphate buffer (pH 6.0) by
TrCE16 AcE (0.4 mg/ml) at 40 °C was monitored by TLC on silica gel
(Merck, Germany) in ethyl acetate-benzene-isopropanol (2:1:0.1, v/v).
Sugars were detected with the N-(1-naphthyl)ethylenediamine dihy-
drochloride reagent [22].
2.1. Polysaccharides, oligosaccharides, glycosides and enzyme preparations
Aspen acetyl glucuronoxylan was isolated from aspen sawdust by
hot water extraction as described elsewhere [6]. Di-O-acetates (2,3-,
2,4- and 3,4-) of Me-β-Xylp were generous gifts from Dr. P. Kovac
Biely, Peter
