71

Xylanolytic Enzymes

Peter Biely
Slovak Academy of Sciences, Bratislava, Slovakia

I. INTRODUCTION insects (8). Most, if not all, fungal plant pathogens

produce and secrete enzymes that degrade cell wall
A. Xylanthe Second Most Abundant Plant
hemicellulose (9). Xylanolytic enzymes also occur in
Cell Wall Component and Natural Polymer
plants. The enzymes participate in the process of cell
wall extension, cell division, seed germination, and
Plant cell walls can be considered to be the main
other morphological and physiological events in
renewable resource formed in the process of photosyn-
plants. During germination endogeneous enzymes cat-
thetic xation of carbon dioxide. They are composed
alyze hydrolysis of these polysaccharides to remove the
of three major polymeric constituents: cellulose, an
physical barrier imposed by the walls on the free diffu-
insoluble skeletal polysaccharide composed of -D-
sion of starch and storage protein-degrading enzymes
glucopyranosyl residues linked -1,4-glycosidically;
through the germinated grains (1012). Enzymes
hemicellulose, a series of matrix and crosslinking het-
degrading hemicellulose polysaccharides were also
eropolysaccharides that include a variety of glucans,
reported to be present in wheat grain (13) and wheat
mannans, arabinans, galactans, and xylans; and lignin,
our (14).
a complex polyphenol. As a part of the carbon cycle,
all three constituents are degraded by specialized
enzyme systems produced by microorganisms. This
C. Application of Xylanolytic Enzymes
chapter is devoted to the enzymes that degrade the
major plant hemicellulose, xylan. After cellulose,
The past two decades have seen a growing interest in
xylan is the most abundant polysaccharide in nature.
microbial enzyme systems that degrade plant xylan, a
Xylanolytic enzymes have been extensively reviewed in
polymer of the ve-carbon sugar D-xylose. Xylose can
the past (15). Books covering xylan and xylanases
be converted to a variety of useful products, including
exclusively have also been published (6, 7).
ethanol, the engine fuel of the future (15, 16). Enzymic
saccharication of agricultural, industrial, and munici-
B. Occurrence of Xylanolytic Enzymes pal wastes may provide sugar syrups for human and
animal consumption, and carbon sources for industrial
Many bacteria, fungi, yeasts, and protozoa are able to fermentations. Xylo-oligosaccharides have a variety of
degrade xylan (1, 8). Some of these are saprophytic soil uses as food additives (17). Plant structural polysac-
or aqueous microorganisms, some grow aerobically, charides provide the major source of nutrients for rum-
some grow at room temperature, and others show ther- inal livestock and also play an important role in animal
mophilicity. Some occur in the digestive track of rumi- fodder. Pretreatment of forage crops with polysacchar-
nant animals and in the intestines of wood-eating ide-degrading enzymes improves the nutritional qual-

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

 ity and digestibility of ruminant fodder and facilitates
composting (18, 19). Enzymic hydrolysis of highly vis-
cous arabinoxylans originating in cereal endosperms
improves nutrient uptake and digestion in broiler
chickens (2023). Other applications of xylanolytic
enzymes include improvement of the baking process
and modication of baked products (2430).
Xylanases are also important components of enzyme
systems used for liquefaction of vegetables and fruit,
and for clarication of juices (3, 18). In all of the
above-listed processes, microbial xylanolytic systems
can be applied together with other enzymes hydrolyz-
ing plant polysaccharides such as amylases, cellulases,
and pectinases. There are, however, applications in
which xylanases should not be contaminated by cellu-
lases to preserve the polymerization degree of cellulose.
Xylanases free of cellulases have applications with
important ecological implications in pulp and paper
industry. They facilitate lignin extraction, reducing
the consumption of toxic chemicals required for pulp
bleaching (3134). The mechanism of this process,
called enzymic prebleaching or bleachbusting, is not
completely understood. However, it is believed that
xylanase attacks the xylan moiety of lignin-carbohy-
drate complexes (31). Xylanases also have potential
in the purication of dissolving pulp from hemicellu-
lose (35, 36), in the recovery of cellulose textile bers
(37), and in paper recycling (38).
D. Chemical Structure of Xylan
Xylan is structurally similar to cellulose, but instead of
D-glucose, its main chain is built from -1,4-linked
xylopyranosyl residues. In contrast to cellulose, how-
ever, the xylan structure is extremely variable and
depends on its source. The structure ranges from an
almost linear unsubstituted chain, e.g., in some grasses,
to highly branched heteropolysaccharides in cereal
seeds. The prex hetero indicates the presence of sugars
other than D-xylose. The main chain is usually substi-
tuted to various degrees by residues of 4-O-methyl-D-
glucuronic acid, D-glucuronic acid, or L-arabinofura-
nose, and in some cases is also esteried by acetyl
groups. The substituents may be also represented by
oligosaccharides and esteried by cinnamic (phenolic)
acids. The principal types of xylan found in plants are
well known and have been extensively reviewed (39
41). They differ in the character of their side chains as
indicated in Table 1.
A hypothetical fragment of a plant xylan which
shows the major structural features found in this
group of hemicelluloses is depicted in Figure 1.
Xylans backbone is built of -1,4-linked D-xylopyra-
nosyl residues. In hardwood xylans the side chains are

-1,2-linked 4-O-methyl-D-glucuronic acid residues
and acetyl groups partially esterifying the two avail-
able hydroxyl groups of xylosyl residues. Owing to

Table 1 Examples of Major Types of Plant Xylans

Approximate ratio
Structural type Source Nature of side chains Xyl:side chain subst.

Linear -1,4-D-xylan Esparto grass Substituents are rare

O-Acetyl-4-O-methyl-D-
glucuronoxylan
O-Acetyl-4-O-methyl-D-
glucuronoxylan
L-Arabino-4-O-methyl-D-
glucuronoxylan
O-Acetyl-L-arabinoxylan
Tobacco stalk
Hardwoods Acetyl, 2-O-
-MeGlcA Xyl:Ac:MeGlcA 10:7:1
Gramineae Acetyl, 2-O-
-MeGlcA Not specied
Legumes Acetyl, 2-O-
-GlcA
Softwoods 3-O-
-L-Araf , 2-O-
-MeGlcA Xyl:Ara:MeGlcA 10:1:3:2
Gramineae 2-O-
-L-Arfa, 3-O-
-L-Araf Xyl:Ara varies from 0.9 to 2.2
(monocots Substitution of Xyl in the main depending on source.
primary walls, chain: single and double Double substitution of Xyl with
ours) Ara is more frequent than single
substitution.
Pericarp L-Arabinan oligomers Not specied

Source: Refs. 4244.

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 Figure 1 Hypothetical plant xylan and the enzymes required for its complete hydrolysis.
the ease of migration of acetyl groups among the
hydroxyl groups, it is difcult to determine their actual
distribution between positions 2 and 3. Softwood
xylans are similar to hardwood xylans, but contain

-1,3-linked L-arabinofuranosyl residues instead of
acetyl groups. Xylans from grasses contain a smaller
proportion of uronic acids, but are more highly
branched with L-arabinofuranosyl residues. As indi-
cated in Table 1, the arabinofuranosyl residues also
can be linked to the main chain by
-1,2-linkages,
often to D-xylopyranosyl residues already substituted
by
-1,3-linked L-arabinofuranosyl residues. In cer-
eals, the ratio of Xyl : Ara is low, ranging from 1 to
2.2 (44). Xylans from graminaceous plants also contain
25% by weight of acetyl groups and 12% of pheno-
lic (cinnamic) acids esterifying the C-5 position of ara-
binose side chains (45). In sugar beet pectins, ferulic
acid esteries the OH-2 of L-arabinofuranose or the
OH-6 of D-galactose (46). Ferulic acid [3(3-methoxy-
4-hydroxy phenyl)-2-propanoic] is the major cinnamic
acid found in cereals and in the Gramineae (47). In
wheat bran, one of 150 of xylose residues is substituted
with L-arabinose esteried with ferulic acid (45).
Ferulic acid is synthesized in plants via the shikimic
pathway and is an intermediate in lignin biosynthesis
(48). In some plants it has been shown to be involved in
the chemical linkages between xylan and lignin, con-
tributing to integrity of the plant cell walls.
Intermolecular crosslinking may result from the dimer-
ization of feruoyl and p-coumaroyl residues or from
the formation of ester linkages between polysacchar-
ides and lignin (49, 50).
Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.
E. Xylanolytic System and Its Components
Depending on its complexity, the complete breakdown
of xylan requires the action of several enzyme compo-
nents which can be considered as a xylanolytic system
(3). As depicted in Figure 1, the more complex the
xylan structure, the more components of the xylanoly-
tic system are required. The crucial enzyme for xylan
depolymerization is endo--1,4-xylanase (-1,4-xylan
xylanohydrolase; EC 3.2.1.8, abbreviated EX). EX
attacks the main chain most easily and therefore
most rapidly at non-substituted regions, generating
nonsubstituted and branched or esteried oligosac-
charides. The main chain substituents are liberated
by corresponding glycosidases or esterases:
-L-arabi-
nosyl residues by
-L-arabinofuranosidases (
-L-ara-
binofuranoside arabinofuranosidase; EC 3.2.1.55), D-
glucuronosyl and 4-O-methylglucuronosyl residues by

-glucuronidase (EC 3.2.1.139), acetic acid, and ferulic
acid and p-coumaric acid residues by corresponding
esterases (EC numbers not assigned). -Xylosidase
(xylobiase or exo--1,4-xylanase) (-1,4-xylan xylohy-
drolase; EC 3.2.1.37) attacks xylo-oligosaccharides
from the nonreducing end, liberating D-xylose.
The hydrolases removing xylan side chains have
been referred to as accessory or auxiliary xylano-
lytic enzymes. Two categories of accessory enzymes
can be recognized: those that operate on both poly-
meric and oligomeric substrates, and those that are
active only on branched or substituted oligosacchar-
ides generated by EXs. There is usually no sharp
boundary between these groups. The enzymes operat-
 ing on polymeric substrates can be distinguished from
those of the second group by their ability to catalyze
precipitation of the originally highly soluble branched
polymeric xylan. This precipitation is due to removal
of substituents that hinder the association of the xylan
main chains by hydrogen bonding (analogous to that
found in cellulose). This precipitation can be used for
specic procedures to detect debranching enzymes in
the absence of depolymerizing endoxylanases.
Accessory enzymes further differ in their requirements
for the ne structure of the xylan main chain and the
position of the group to be removed.
F. Synergism of Xylanolytic Enzymes
The synergistic action of two or more enzymes during
hydrolysis of xylan is evident when the rate of hydro-
lysis of the polysaccharide with two or more enzymes is
faster than the sum of the rates of hydrolysis by indi-
vidual enzymes. Several types of synergism have been
recognized among the xylanolytic enzymes (5153).
The most important type concerns the cooperativity
between the enzymes attacking the main chain (EXs)
and enzymes that liberate side chain substituents, such
as acetyl (54) and arabinofuranosyl residues (51). The
action of EX releases substituted xylo-oligosacchar-
ides, which are more readily diffusible and more favor-
able substrates for accessory enzymes. On the other
hand, the removal of side chain substituents by acces-
sory enzymes creates new sites on the main chain for
productive binding with EX. From some accessory
enzymes the cleavage of the main chain is indispensa-
ble. Individual components of xylanolytic systems are
discussed below.
G. Analysis of Xylan Hydrolysates
It is always important to conrm that the apparent
xylanolytic activity of any preparation is really con-
nected with xylan depolymerization and not with
degradation of some other polysaccharides present as
impurities. This aspect is especially important for plant
enzyme preparations which contain high levels of amy-
lolytic enzymes. Almost all commercial xylan prepara-
tions contain a small amount of starch, which must be
removed by
-amylase treatment before testing for the
presence of xylanases.
The traditional method of analyzing enzymic hydro-
lysates of xylan is by paper and thin-layer chromato-
graphy. A suitable solvent system for separation of D-
xylose and -1,4-xylooligosaccharides (linear or substi-
tuted) on Whatman No. 1 or 3 MM paper is ethyl
Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.
acetateacetic acidwater (18:7:8, always v/v). A simi-
lar efciency of separation can be achieved on thin
layers of microcrystalline cellulose (Merck,
Darmstadt, Germany) in ethyl acetateacetic acid
water (3:2:2) or ethyl acetateacetic acidformic acid
water (18:3:1:5). These systems separate well neutral
oligosaccharides, aldouronic acids, and partially acety-
lated xylo-oligosaccharides. Acetylated xylo-oligosac-
charides show higher mobility than the
corresponding deacetylated products. These systems
may also be used to follow the liberation of 4-O-
methyl-D-glucuronic acid, which migrates in all the
above solvent systems ahead of D-xylose. The use of
aniline-hydrogenphthalate reagent provides a color
differentiation of the acid from xylose and xylo-oligo-
saccharides (orange-brown versus dark-brown colors).
The reagent, which can be poured onto dry chromato-
grams, is prepared by dissolving 1 g of phthalic acid in
50 mL acetone followed by addition of 0.9 mL aniline.
Visualization of reducing sugars is obtained on heating
at 110 C for a few min.
Good separation of neutral products of xylan
hydrolysis is obtained by thin-layer chromatography
on silica gel (Merck) in such solvent systems as 1-buta-
nolethanolwater (10:8:7), 1-propanolethyl acetate
ethanolpyridinewater (7:3:3:2:1) or nitromethane
acetonitrileethanolwater (1:4:3:2), the last being the
fastest system. Reducing sugars can be visualized with
an aniline/diphenylamine reagent, consisting of a solu-
tion of aniline (2 mL) and diphenylamine (2 g) in acet-
one (100 mL) containing 15 mL of 85% H3 PO4 .
Visualization of all sugars, both reducing and nonre-
ducing, is done with 1% orcinol solution in ethanol/
conc. H2 SO4 (9:1). Both reagents can be poured onto
well-dried chromatograms, which is more convenient
than spraying. The solvent system 1-butanolethanol
water (10:5:1) is suitable to follow the reaction of xyla-
nases with 4-nitrophenyl -xylopyranoside. 4-
Nitrophenol and 4-nitrophenyl glycosides also can be
detected directly under UV light (360 nm).
Other, more up-to-date methods of monitoring the
enzymic hydrolysis of xylan include a variety of liquid
chromatographies, such as gel permeation chromato-
graphy, ion exchange chromatography, adsorption
chromatography, reversed-phase chromatography,
and partition chromatography, usually in HPLC set-
ups. All of these methods require the proper choice of
sorbents and eluent, and sophisticated equipment.
Paper and thin-layer chromatography are the most
simple and least expensive separation techniques for
the simultaneous analysis of a large number of sam-
ples.
 II. ENDO-b-1,4-XYLANASE (EC 3.2.1.8)
A. Chemical Reaction Catalyzed
H2 O
R1 -Xyl1-4Xyl1-4Xy11-4Xyl-R2 ! R1 -Xyl1-
4Xyl Xyl-4Xyl-R2
R1 and R2 , substituted or unsubstituted portions of the
main chain of xylan;  reducing end.
B. Classication
-1,4-Xylan xylanohydrolase, EC 3.2.1.8.
C. Properties as Proteins
1. EX Families
A careful comparison of microbial EXs on the basis of
their different physicochemical properties, such as
molecular mass and isoelectric point, was carried out
by Wong et al. (55). This analysis indicated that these
enzymes can be divided into two groups, one consisting
of high-molecular-mass enzymes with low pI values,
and the other consisting of low-molecular-mass
enzymes with high pI values. As usual, there are excep-
tions, particularly concerning pI values. Interestingly,
this grouping of EXs (55) was found to be in agree-
ment with the general classication of glycanases on
the basis of hydrophobic cluster analysis and sequence
similarities (5659). Hydrophobic cluster analysis is
Figure 2 Tertiary structures of endoxylanases of families 10 and 11.
Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.
designed to predict protein folding based on hydropho-
bic/hydrophilic patterns and is used to compare mem-
bers of a protein group of similar functions (60). Acidic
high-molecular-mass EXs (> 30 kDa) were found to be
assigned to glycanase family 10 (59) (formerly family
F), and basic, low-molecular-mass EXs (< 30 kDa), to
glycanase family 11 (formerly family G). While family
10 also contains other glycosyl hydrolases, family 11 is
exclusively made up of EXs.
EXs belonging to the two glycosyl hydrolase families
show remarkable differences in their tertiary structures
as established by crystallography (Fig. 2). Members of
individual families have a similar protein folding pat-
terni.e., a similar three-dimensional structure. These
tertiary structures are more conserved than amino acid
sequences. The most conserved region within a family
appears to be the catalytic domain. EXs of family 11
appear to be smaller, well-packed molecules, formed
mainly of -sheets (6164). These enzymes appear
very small in their native states (65, 66). The catalytic
groups are present in a cleft that accommodates a chain
of ve to seven xylopyranosyl residues.
The tertiary structure of EXs of family 10 (Fig. 2) is
typically an eightfold
= barrel
=8 , resulting in a
salad bowl shape (6770). The substrate binds to a
shallow groove on the bottom of the bowl, which is
shallower than the substrate binding cleft of family 11
EXs (9). This feature, together with the generally
greater conformational exibility of larger enzymes,
may account for the lower substrate specicity and
greater catalytic versatility of family 10 EXs.
 A comparison of the tertiary structure of EXs with
other classied glycosyl hydrolases reveals that the two
EX families evolved from different ancestors. EXs of
family 10 are closely related to the endo--1,4-gluca-
nases of family 5, with which they share some common
catalytic properties (see below). Both enzyme families 5
and 10 belong to a larger clan of enzymes possessing
the eightfold
= barrel architecture (69, 71), known as
clan GH-A (59). EXs of family 11 show a certain
degree of similarity to the low-molecular-mass endo-
-1,4-glucanases of family 12 (formerly H) (72).
2. Multiplicity of Forms
Genetic differences are not the only source of hetero-
geneity among EXs. A multiplicity of forms is fre-
quently observed among the products of one gene.
Several molecular forms of enzymes puried to homeo-
geneity often can be revealed by isoelectric focusing.
The observed multiplicity of forms may be a conse-
quence of several factors. The most common reasons
include differential mRNA processing, partial proteo-
lysis, and variable degrees of amidation and glycosyla-
tion. Additional causes may include autoaggregation
and aggregation with other proteins (51).
3. Multiple Domains
The molecular architectures of EXs range from a single
catalytic domain to a complex arrangement of catalytic
and noncatalytic domains (7376), recently named
modules (73, 74). The catalytic domain (module)
includes the part of enzyme (Fig. 3) that contains the
active site, i.e., the substrate-binding site in the classi-
cal sense, and the catalytic groups. Noncatalytic
Figure 3 Molecular architecture of some endo--1,4-xylanases. (From Ref. 76.)
Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.
domains (modules) of EXs act independently of the
catalytic domain as polysaccharide-binding domains
(carbohydrate-binding modules; CBMs). Catalytic
domains are connected to CBMs to which they are
linked by linker sequences rich in proline and hydroxy
amino acids. The linkers function as exible, extended
hinge regions between functional domains (57).
Originally, it was believed that CBMs increased the
catalytic rate against insoluble substrates. however,
later observations suggested that binding domains
may also facilitate the hydrolysis of soluble polysac-
charides (75, 77).
Surprisingly, there are several EXs that contain
binding modules that bind exclusively to cellulose (cel-
lulose-binding domain; CBD) and not to xylan (57,
74). This is likely related to the biological role of
these enzymes in degrading plant cell walls which are
predominantly built from cellulose closely associated
with hemicellulose. Targeting of EX to cellulose may
bring the enzyme in proximity of its substrate. There is
a family of CBMs (CBD9) that are found only in EXs
(73). CBMs of EX B (XlnB) from Streptomyces lividans
and EX D from Cellulomonas mi appear to be specic
for soluble and insoluble xylan (75, 77). The 3D struc-
ture of this xylan-binding domain (XBD) is very simi-
lar to that of CBD. A single amino acid change
converts the protein from a XBD to a CBD (75). An
example of an EX containing two CBMs has been
found in Pseudomonas uorescens subsp. cellulose (78).
Nature also has designed multifunctional enzymes
with two catalytic domains connected by linker
sequences. Some bacteria produce bifunctional EXs
containing two identical (79) or two different EX
domains (80, 81). Other enzymes possess catalytic
 domains of two different xylanolytic enzymesone
catalyzing the xylan depolymerization, and the other
deacetylation of the xylan main chain (8284). Such
bifunctional enzymes may have an advantage over
enzymes containing a single catalytic domain in the
hydrolysis of native acetylated xylan. Multidomain
xylanases also occur as subunits of cellulosomes and
xylanosomes. These supramolecular extracellular cell-
associated protein complexes of thermophilic clostridia
can efciently degrade cellulose, xylan, and related
plant cell wall polysaccharides (85, 86).
D. Properties as Enzymes
1. Catalytic Properties of EXs Belonging to
Two Families
EXs belonging to glycanase family 10 hydrolyze het-
eroxylans and rhodymenan (a linear algal -1,3--1,4-
xylan) to a greater extent than EXs of family 11 (87,
88). The EXs of family 10 are better able to cleave
some exceptions, also from acetylxylan, than do EXs
of family 11. EXs of family 10 can further hydrolyze
the shortest branched or isomeric xylooligosaccharides
which are released by EXs of family 11 (87).
Characterization of the shortest acidic fragments
released from 4-O-methyl-D-glucurono-D-xylan after
a long-term hydrolysis (88, 89) suggests which lin-
kages are accessible to hydrolysis by the two types
of EXs. EXs of family 10, unlike EXs of family 11,
are capable of attacking the glycosidic linkage next
to the branch and toward the nonreducing end.
While EXs of family 10 require two unsubstituted
xylopyranosyl residues between the branches, EXs
of family 11 require three unsubstituted consecutive
xylopyranosyl residues (Scheme 1). Kinetic data for
cleavage of individual glycosidic linkages of the
xylan main chain are not available, but time course
of product analyses suggest that linkages closer to
substituents are hydrolyzed more slowly than more
distant linkages.

EXs 10 # # # # #
-4Xyl1-4Xyl1-4Xyl1-4Xyl1-4Xyl1-4Xyl1-4Xyl1-4Xyl1-4Xyl1-4Xyl1-
EXs 11 " 2 " 2 2
j j j

1
1
1
MeGlcA MeGlcA MeGlcA

Scheme 1

glycosidic linkages in the xylan main chain that are
near substituents, such as MeGlcA and acetic acid.
The alteration of the xylan main chain by replacing
-1,4-linkages by -1,3-linkages, as it is in rhodyme-
nan, represents a more serious steric barrier for EXs of
family 11 than for EXs of family 10. Consequently,
EXs of family 10 liberate smaller products from 4-O-
methyl-D-glucurono-D-xylan, rhodymenan, and, with
The degradation of a cereal L-arabino-D-xylan with
two EXs of Aspergillus awamori (EXI, 39 kDa, pI 5.7
6.7, most probably a member of family 10, and EXIII,
26 kDa, pI 3.33.5, most probably a member of family
11) (90) is shown in Scheme 2. The cleavage site speci-
cities L-arabinosyl-D-xylan resemble those on 4-O-
methyl-D-glucurono-D-xylan. The L-arabinosyl sub-
stituents represent a more serious steric hindrance for

Araf

1
j
EXI # # # # 2 # #
-4Xyl1-4Xyl1-4Xyl1-4Xyl1-4Xyl1-4Xyl1-4Xyl1-4Xyl1-4Xyl1-4Xyl1-
EXIII " 3 " 3 3
j j j

1
1
1
Araf Araf Araf

Scheme 2

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

 the formation of productive complexes of the polysac-
charide with EXIII. Only the larger type of the enzyme
is capable of attacking the linkages between substituted
and unsubstituted xylopyranosyl residues.
Studies of the action of EXs on rhodymenan are
complicated by the fact that EXs of family 10 are
also capable of cleaving -1,3-linkages (91, 92).
Generally, EXs of family 10 do not attack the poly-
saccharide at -1,4-linkages which follow -1,3-lin-
kages toward the reducing end (91), liberating
xylotriose, Xyl1-3Xyl-4Xyl, as the shortest isomeric
product EXs of family 11 liberate from rhodymenan
isomeric products larger than xylotriose.
Consistent with the fact that EXs of family 10 are
related to endo--1,4-glucanases and some glycosi-
dases belonging to the clan of glycosyl hydrolases
GH-A (59), EXs of family 10 catalyze hydrolysis of
aryl -D-xylopyranosides and low molecular mass
cellulase substrates. These include aryl -D-cellobio-
sides, aryl -D-lactosides (93, 88), and cellooligosac-
charides (94). The degradation of aryl xylosides, e.g.
of 4-nitrophenyl -D-xylopyranoside, also involves a
complex reaction pathway including glycosyl transfer
reactions leading to xylooligosaccharides and their gly-
cosides (94, 95). The EXs of family 11 do not possess
these catalytic abilities.
2. Mechanism of Substrate Degradation
The hydrolytic reaction takes place in the active site of
the enzyme consisting of the substrate-binding site and
catalytic groups. Consistent with their tertiary struc-
tures, EXs of family 10 appear to have a smaller sub-
strate-binding site than do EXs of family 11. EXs of
family 11 contain a larger number of subsites (ve to
seven) than EXs of family 10 (four to ve subsites). A
subsite is a part of the binding site that interacts with
one xylopyranosyl residue of the substrate. The interac-
tion may not be only attractive, it can also be repulsive,
particularly on the site of the leaving group. The number
Figure 4 Mechanism of hydrolysis of glycosidic linkage with endo--1,4-xylanases.
Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.
of subsites and their binding afnities can be determined
by a procedure that is called subsite mapping (9698)
which is complementary to the image of the substrate
binding site provided by x-ray crystallography (99).
EXs of both families catalyze the hydrolysis of gly-
cosidic linkages with the retention of anomeric cong-
uration (100, 101). The reaction involves a double-
displacement mechanism leading to a new reducing
end with -anomeric conguration (102) (Fig. 4). The
catalytic amino acids in the substrate binding site
divide the subsites to the left (numbered with minus
Roman numbers) and to the right of the catalytic
groups (numbered with positive Roman numbers)
(Fig. 5). The catalytic residues have been identied as
two conserved glutamate residues (in a few cases one
asparate replaces a glutamate), which are located
opposite each other in the active site. One residue
functions as a general acid catalyst protonating the
glycosidic oxygen, while the other functions as a
nucleophile, attacking the anomeric center to form a
covalent enzyme-glycosyl intermediate (Fig. 4). In the
second step a water molecule attacks the intermediate
in a general base-catalyzed process to yield the product
of retained anomeric conguration (99, 103). The
catalytic residues have been identied by chemical
modication, site-directed mutagenesis, and use of
stabilized enzyme-glycosyl intermediate (76, 103).
Glycosyl transfer reactions catalyzed by both types of
EXs at high substrate concentrations are also evidence
for the enzyme-glycosyl intermediate. Using linear -
1,4-xylo-oligosaccharides as substrates, it has been
established that EXs utilize multiple pathways of sub-
strate degradation (104, 105). Unimolecular hydrolysis
takes place at low substrate concentrations. Different
cleavage of the substrate occurs at so-called shifted
binding, which is observed at higher substrate concen-
trations and which is, generally, accompanied by for-
mation of products larger than the substrate. An
example of such reactions of xylotriose catalyzed by
EX from Cryptococcus albidus is shown in Figure 5.
 Figure 5 Possible binding and degradation of xylotriose by EX from Cryptococcus albidus. (A) Productive complexes at low
substrate concentration and probability of their formation; (B) shifted two-one binding of xylotriose at high substrate concen-
tration and subsequent xylosyl transfer to second xylotriose molecule to give xylotetraose, which is nally cleaved to two
molecules of xylobiose. (From Ref. 104.)

At sufciently high concentrations of xylotriose, xylo-
biose initially is generated as the only product of xylo-
triose degradation. Analogous reaction pathways are
also observed with longer oligosaccharides. The main
molecular and catalytic properties of EXs of families
10 and 11 are summarized in Table 2.
3. Xylan-Degrading Endo--1,4-Glucanases
It has been unambiguously established that at least one
type of endo--1,4-glucanase exhibits activity on both
cellulose and xylan (106, 107). The best-known enzyme
in this regard is endoglucanase I from Trichoderma
reesei, a member of family 5 glycosyl hydrolases. The
enzyme catalyzes the hydrolysis of both polysacchar-
ides in the same active site (108). Although the enzyme
belongs to the same clan as EXs of family 10, the
products the enzyme generates from glucuronoxylan
and rhodymenan are more similar to those liberated
by EXs of family 11 (87). In T. reesei the enzyme is
coinduced as a component of the cellulolytic and not
of the xylanolytic system (3).

Table 2 Properties of EXs of Families 10 and 11

Properties EXs of family 10 EXs of family 11

Molecular mass usually > 30 kDa usually < 30 kDa

Isoelectric points usually < 7 usually > 7
Protein fold (catalytic module)
8 two -sheets and one
-helix
Substrate binding site shallow groove deep cleft
Catalytic amino acids two glutamic acid residues (occasionally two glutamic acid residues (occasionally
one glutamate and one asparate) one glutamate and one aspartate)
Number of subsites 45 57
Substrate specicity less specic more specic
heteroxylans active active
aryl--xylosides active inactive
aryl--cellobiosides active inactive
aryl--lactosides active inactive
Stereochemistry of hydrolysis retaining retaining
Multiple reaction pathway at yes yes
high substrate concentrations

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

 E. Qualitative and Quantitative Determination of
Activity
1. Reductometric Assays
The most common way to follow EX activity is to
determine the reducing sugars formed from selected
type of xylan. Two basic procedures for determination
of reducing sugars have been widely used: the
Somogyi-Nelson procedure (109111), and the 2,4-
dinitrosalicylic acid (DNS) test (112114). The meth-
ods are sufcient to prove the presence of the enzyme,
to assay its activity, and to evaluate the extent of poly-
saccharide hydrolysis, which may be the rst criteria
for xylanase differentiation. The two methods for the
determination of reducing sugars have been compared
in an interlaboratory evaluation organized by Finnish
scientists (115). Although the Somogyi-Nelson pro-
cedure is almost 10 times more sensitive than the
DNS method, the latter always gives higher values of
xylanase activity. When standardized with respect to
substrate and procedure, the assay employing the DNS
method afforded reproducible results in various
laboratories with a standard deviation of 17% (115).
Recently Jeffries et al. (116) reported that the differ-
ences between the Somogyi-Nelson and DNS methods
are due to the fact that the rst method is less reactive
and the second method more reactive with xylooligo-
saccharides than with xylose. The prevalence of oligo-
saccharides among the products of xylan hydrolysis is
thus why the Somogyi-Nelson method underestimates,
and the DNS method overestimates enzyme activity.
The most sensitive method for photometric determi-
nation of reducing sugars is that using 2,2 0 -bicinchoni-
nate (117, 118). However, it has not been widely
adopted for determination of xylanase activity.
Nevertheless, the method should be listed here because
of its potential for miniaturization to the scale of a
microplate reader (119).
a. DNS Xylanase Assay (115)
Reagents
1. Birchwood 4-O-methyl-D-glucuronoxylan (Roth
7500, Germany; or Sigma, USA), 1% solution in
0.05 M citrate buffer, pH 5.3 (in the case of fungal
enzymes or other buffers with bacterial enzymes).
The solution is prepared under heating up to boiling
point with stirring until the polysaccharide dissolves.
Clarication by centrifugation is not necessary but
may be useful. Cooled solution can be stored in the
presence of 0.010.02% sodium azide.
Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.
2. D-Xylose, 10 mM calibration solution in 0.05
M citrate buffer, pH 5.3 (or other buffer).
3. DNS reagent prepared according to Sumner
(112) or Miller (113).
Procedure. Preheated xylan solution (1.8 mL; 50 C)
is mixed with 0.2 mL preheated enzyme solution
(50 C) and incubated for 5 min at 50 C. The reaction
is stopped by addition of 3 mL of the DNS reagent.
The mixture is heated for 5 min at 100 C (boiling water
bath) and then cooled in cold water. Absorbance of
samples is measured at 540 nm against the substrate
blank and the values are corrected for absorbance of
the enzyme blank. Calibration samples with known
amounts of xylose are prepared in the presence of the
substrate.
One unit of xylanase activity is dened as the
amount of enzyme liberating 1 mol of xylose equiva-
lent under the experimental conditions in 1 min. (Note:
The volume of the assay can be reduced.)
b. Xylanase Assay Using the Somogyi-Nelson
Method (120)
Reagents
1. Birchwood or beechwood 4-O-methyl-D-glu-
curonoxylan (Roth 7500, Germany; or Sigma, USA),
0.4% solution in 0.1 M acetate buffer, pH 5.4, or 0.2%
solution in 0.05 M acetate buffer (or other buffer). The
solution is prepared under heating up to boiling point
with stirring until the polysaccharide dissolves. The
solution can be stored in the presence of 0.010.02%
sodium azide.
2. D-Xylose, 1 mM calibration solution.
Procedure. Xylan solution (0.25 mL, 0.4%) is mixed
with 0.25 mL enzyme solution, or 0.5 mL of 0.2%
xylan solution is mixed with 1050 L enzyme solution
and incubated at 30 C for an appropriate time. The
reaction is terminated by addition of 0.5 mL of the
Somogyi reagent, mixed well, and heated for 10 min
on a boiling water bath. After cooling in cold water the
samples are vigorously mixed with 0.5 mL of the
Nelson reagent. After 15 min standing with occasional
stirring, cloudy samples are centrifuged to remove the
ne precipitate. Absorbance at 560 nm of the super-
natants is measured. Blanks to correct the values for
reducing power of the substrate and the enzyme are
run in parallel. Calibration is done in the range 0.05
0.5 mol of xylose. One unit of xylanase is dened
similarly as above. (Note: Exact duration of heating
at 100 C with the Somogyi reagent is critical for repro-
ducibility of results.)
 2. Other Xylanase Assays and Specic EX Assays
It should be noted that reductometric assays are not
specic for EXs and, when used with crude xylanolytic
systems or EX preparations containing other xylano-
lytic enzymes, they cover the overall saccharication
ability of the tested enzyme preparations. Frequently,
reductometric xylanase assays may not be sufciently
sensitive or may be hampered by a high background of
reducing sugars in the enzyme preparations. In such
cases, a viscosimetric method of xylanase assay can
be recommended. This method is based on measure-
ment of the decrease of viscosity of a soluble xylan,
e.g., arabinoxylan (121) or soluble xylan derivative,
like carboxymethylxylan (122, 123). Viscosity can be
measured using various viscosimeters or by modern
electronic devices employing vibrating metal rods.
Changes in the resistance of the medium to the vibrat-
ing or rotating rods are recorded and transformed into
activity units. Viscosimetric measurements are impor-
tant to establish the endocharacter of glycanases. Plots
of specic uidity (1=) versus the amount of liberated
reducing sugars give linear relationships, the slopes of
which correspond to the randomness of the xylan
attack by the enzymes (89).
Another specic EX assay employs a soluble cova-
lently dyed xylan, Remazol Brilliant Bluexylan (RBB-
xylan), a substrate that is precipitable from aqueous
solution by ethanol (124). The assay is based on the
photometric determination of enzyme-released dyed
fragments that remain soluble in the presence of etha-
nol. This method allows the determination of xylanase
activity in the presence of larger amounts of reducing
sugars, in membrane fractions of cells, and even in the
presence of viable cells utilizing liberated xylan frag-
ments. Limitations of the method are its sensitivity to
temperature and ionic strength. Testing in 15 labora-
tories showed the method reproducible to
30% rela-
tive standard deviation, suggesting that RBB-xylan
assay could be a useful alternative assay for xylanases
(115). A number of covalently dyed soluble and inso-
luble xylans are available from Megazyme (Wicklow,
Ireland). The company offers also a specic tablet test
for EX activity based on wheat arabinoxylan. The
nephelometric assay of xylanase (125) could be suitable
for differentiation of xylanases with respect to their
performance on insoluble xylan.
a. RBB-Xylan Assay (115, 124)
Reagents
1. RBB-xylan, solution in 0.05 M acetate buffer,
pH 5.4, 56 mg/mL (the concentration should cor-
Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.
respond to 5 mg/mL of undyed xylan) or solution in
0.1 M acetate buffer (1011 mg/mL).
2. Ethanol, 96% or absolute.
Procedure. The enzyme solution or suspension (10
50 L) is mixed in 1.5 or 2.0 mL plastic microtest tubes
with 0.5 mL of preheated (30 C) solution of RBB-
xylan solution (56 mg/mL), or 0.25 mL of enzyme
solution or suspension is mixed with 0.25 mL RBB-
xylan solution in 0.1 M acetate buffer and incubated
at 30 C for an appropriate time. The reaction is termi-
nated by the addition of 1 mL ethanol to precipitate
unhydrolyzed RBB-xylan and its high-molecular-
weight fragments. After standing at room temperature
for 2030 min in closed test tubes, the mixtures are
mixed again and centrifuged at 2500g for 1.53 min.
The absorbance of supernatants is then measured at
595 nm against the respective substrate blank. (Note:
The assay does not give direct data suitable for calcu-
lation of the enzyme activity. However, the assay can
be calibrated by a reductometric assay carried out
under the same experimental conditions with undyed
xylan.
3. Synthetic Substrates
Synthetic substrates of glycosyl hydrolases usually
include chromogenic and uorogenic glycosides
which are widely used to quantify particularly glycosi-
dases. The most common substrates are 4-nitrophenyl
and 4-methylumbelliferyl glycosides. Liberation of 4-
nitrophenol is followed photometrically at 405410 nm
after termination of the enzymic reaction with alkaline
reagents. Fluorimetry is used to follow the liberation of
4-methylumbelliferone (excitation at 365 nm and emis-
sion at 448 nm at pH 1011). To achieve maximal
sensitivity, permitting work in the nM concentration
range of substrates and products, basidication of the
reaction mixtures is required (126, 127).
Similar aryl glycosides of oligosaccharides derived
from polysaccharides can be used as chromogenic and
uorogenic substrates of endoacting glycosyl hydro-
lases. Signicant progress in differentiation of cellulo-
lytic glycanases was achieved by the introduction of 4-
nitrophenyl- and 4-methylumbelliferyl--glycosides or
cellobiose, cellotriose, and lactose (93, 128). Of these
substrates, only 4-nitrophenyl--cellobioside has been
occasionally used for determination of activity of cel-
lobiohydrolase. This assay is not specic, because aryl
-cellobiosides are attacked at the agluconic linkage
also by endo--1,4-glucanases (128) and EXs of family
10 (88). Liberation of the aryl aglycone from cellobio-
 sides occurs also after a two-step hydrolysis of the
substrate by -glucosidase.
Aryl -glycosides of xylo-oligosaccharides have also
been described as potential substrates of EXs
(129135). However, because they have not been com-
mercialized, their use remains very limited in spite of
their great analytical potential and unusually high
sensitivity. 4-Nitrophenyl -xylobioside was used for
characterization of substrate requirements of a non-
specic -xylanase from Cellulomonas mi (129).
4-Methylumbelliferyl --xylobioside and -xylotrioside
were successfully used for detection of EXs in electro-
phoretic gels (130) and for differentiation of EXs (133).
4. Assays for Detection and Screening Purposes
A number of different procedures have been developed
over the years for detection of EXs in gels. The same
principles can be used both for the detection of the
enzyme in electrophoretic gels and for screening of
microorganisms and genetically transformed cells on
solid agar media. The detection and screening techni-
ques employ soluble xylan, insoluble xylan, soluble
covalently dyed xylan, and uorogenic -glycosides
of xylo-oligosaccharides.
In agar gels incorporating insoluble xylan, EX activ-
ity is visualized as the appearance of clearing zones
(136). On solid growth media containing a soluble
xylan-dye conjugate, such as RRB-xylan (124), xyla-
nase-positive colonies are identied by pale-blue or
colorless zones resulting from diffusion of dyed poly-
saccharide fragments generated by EXs (137, 138). The
advantage of these two screening substrates is that the
degradation of the substrate can be followed progres-
sively without any further treatment which might pre-
vent the isolation of the positive colonies directly from
the screening plates. A replica plating is required in the
case of screens employing the soluble xylan complexed
with Congo Red (139, 138) or precipitation of soluble
xylan with ethanol (140). Replica plating is avoided in
plate assays with the uorogenic glycosides 4-methy-
lumbelliferyl -xylobioside (Umb-Xyl2 ) or -xylotrio-
side (Umb-Xyl3 ) (130).
a. Procedures
Detection of microbial producers of xylanase (137,
138). Solid media containing 0.150.2% RBB-xylan
(can be autoclaved) are suitable for the detection of
microbial producers of extracellular EX. Surrounding
colonies of EX producers, pale-blue zones are formed
as a result of diffusion of secreted enzymes and
enzyme-released dyed fragments of RBB-xylan.
Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.
A soluble undyed glucuronoxylan (0.10.2%) can
be used instead of RBB-xylan. The production of EX
is revealed after an appropriate time by ooding the
plate rst with 1 M NaCl and then with 0.1% aqueous
solution of Congo Red (C.I. 22120; Sigma, Aldrich,
Calbiochem). After 15 min the dye solution is poured
off and areas with hydrolyzed polysaccharides are
destained for 10 min with two changes of 1 M NaCl
with a 20-min treatment with 5 M NaCl.
Umb-Xyl2 or Umb-Xyl3 gives the best results when
used in the form of solutions (0.10.5 mM) applied to
the surface of solid growth medium after colonies are
clearly visible. Incorporation of the uorogenic sub-
strates into the solid growth media may not give
straightforward results due to the rapid diffusion of
the uorogenic aglycone in comparison with the rate
of colony growth.
Semiquantitative gel diffusion assay (141, 142). A
solution of 0.150.2% RBB-xylan in an appropriate
buffer is solidied with agar or agarose in petri dishes.
Wells 4 mm in diameter are cut using a cork borer or
suitably shortened disposable plastic micropipette tip
and the gel is removed by suction or by a needle.
Enzyme solutions (10 L) are pipetted into the wells,
and closed dishes are left to incubate at appropriate
temperature for several hours. Diffusion-zone dia-
meters are proportional to the logarithm of enzyme
concentrations.
A soluble glucuronoxylan in combination with the
above described Congo Red complexing can be used
for the same purpose. More sensitive and faster are
similar plate assays with gels containing Umb-Xyl2
or Umb-Xyl3 (0.10.5 mM). Liberation of the uoro-
genic 4-methylumbelliferone around the wells is
observed under UV light (365 nm). Since the liberation
of the aglycone can be a result of -xylosidase action,
enzyme samples should be tested in a parallel plate
containing a specic -xylosidase substrate, 4-methy-
lumbelliferyl -xyloside.
Detection of EX in electrophoretic gels (142). A
warm 1.5% aqueous solution (10 mL) of RBB-xylan
is mixed well with 3% molten agar in appropriate buf-
fer (20 mL) and poured between two glass plates sepa-
rated by 0.75 to 1.0-mm spacers and left to solidify.
The glass plates can be covered by transparent plastic
sheets, so the gel can be easily stored after glass
removal. If not used immediately, the agar gel should
be supplied with 0.010.02% sodium azide. Native
PAGE or isoelectrofocusing gels (agarose or polyacry-
lamide) are washed twice for 510 min (depending on
 their thickness) with a buffer corresponding to pH
optimum of the tested EXs. They are then brought
into contact with the RBB-xylan containing detection
gel. The presence of EX leads to a change in the shade
of the blue background, due to a diffusion of enzyme-
released dyed fragments. The agar detection gel is then
separated from the electrophoretic gel and, if needed,
further incubated in a wet chamber. It is then dipped
into the solvent ethanol0.05 M acetate buffer (pH 5.4)
2:1 (v/v). Zones with enzyme-degraded substrate are
destained as a result of the solubilization of dyed frag-
ments. The background with unhydrolyzed substrate
remains unchanged. The dyed fragments liberated
from the blue detection gel diffuse into the separation
gel, where they mark the position of the enzyme.
Individual xylanase forms can be subsequently isolated
from the separation gel, e.g., by electroelution.
The use of RBB-xylan enables visual monitoring of
substrate hydrolysis, which is important for appropri-
ate timing of contact between the substrate and
enzymes. The detection of EXs by means of Congo
Red (143) requires preliminary experimentation or
knowledge of enzyme activity to terminate the reaction
at the appropriate time.
The fastest procedure for the detection of EXs in
electrophoretic gels can be achieved using Umb-Xyl2
or Umb-Xyl3 (130). Either uorogenic substrate at a
concentration of 0.10.5 mM can be incorporated into
a 0.75 to 1-mm thick agar or agarose detection gel
which is laid upon the rebuffered separation gel. A
simpler approach is to bring the washed separation
gel into contact with a solution of substrates on a
at glass plate. Fluorescence appears rapidly under
UV light (
360 nm) in the regions of EXs. The uor-
escence intensity can be enhanced by ammonium
hydroxide vapors.
III. b-XYLOSIDASE (EC 3.2.1.37)
A. Chemical Reactions Catalyzed
Xyl1-4Xyl ! 2 Xyl Xylnn ! Xyln1 Xyl
NPh--Xyl ! Xyl NPh-OH
-Xylosidase hydrolyzes xylobiose and higher linear -
1,4-xylo-oligoaccharides to monomer and also releases
xylose from branched or substituted xylo-oligosacchar-
ides produced by the action of EXs. -Xylosidases vary
in their afnity toward short and long oligosacchar-
ides. Those which prefer xylobiose as a substrate are
referred to as -xylosidase, and those which prefer
Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.
longer oligosaccharides are referred to as exo--xyla-
nases. A careful literature search suggests, however,
that in spite of the existence of several glycosyl hydro-
lase families into which -xylosidases have been
assigned, and in spite of the fact that the enzymes
behave as both retaining and inverting hydrolases,
there is no sharp boundary between -xylosidase and
exo--xylanase with respect to their substrate prefer-
ence. Therefore, this section considers all enzymes cat-
alyzing successive removal of -xylosyl residues from
non-reducing termini.
B. Classication
-1,4-Xylan xylohydrolase EC 3.2.1.37.
C. Properties as Proteins
-Xylosidases are, with few exceptions, larger enzymes
than EXs. Their molecular mass ranges between 26
and 360 kDa (144), and they are monomeric or dimeric
proteins as shown by SDS-PAGE. Molecular masses
exceeding 120 kDa usually correspond to dimeric/tet-
rameric aggregates. All thus far isolated enzyme spe-
cies showed acidic pI values (3.256.1). The properties
of these enzymes vary considerably, which complicates
their classication. Moreover, -xylosidase belongs to
those xylanolytic enzymes which exhibit variable cellu-
lar localization. In several species of bacteria and
yeasts, the enzyme is localized strictly intracellularly
and is not glycosylated. In these organisms, xylo-oli-
gosaccharides generated outside the cell by EXs must
enter the cell by transport systems localized in plasma-
membrane (3). In other organisms, like Aureobasidium
pullulans, -xylosidase is found outside and inside the
cells. It remains to be established whether some organ-
isms produce two different -xylosidases. In laboratory
cultures of mycelial fungi, -xylosidase remains asso-
ciated with mycelia during early stages of growth and
is released into the media later either by true secretion
or as a result of cell lysis.
Only a small fraction of known -xylosidases have
been classied on the basis of hydrophobic cluster
analysis and comparison of amino acid sequences.
They have been assigned to four glycosyl hydrolase
families: 3, 39, 43, and 52 (58, 59). Family 3, which
contains mainly -glucosidases (EC 3.2.1.21), includes
a nonspecic -glucosidase/-xylosidase from Erwinia
chrysanthemi (145) and the best-known fungal -xylo-
sidases from Aspergillus and Trichoderma species
(146, 147). A common feature of the members of
this family is their retaining character. -Xylosidases
 of thermophilic bacteria have been assigned to family
39 (148150). All -xylosidases of this family are
localized intracellularly and belong to retaining glyco-
syl hydrolases. Their molecular mass is between 55
and 60 kDa.
Family 43 contains 50 to 60-kDa -xylosidases of
several Bacillus spp. and from Butyrivibrio brosolvens.
The latter enzyme has been shown to operate by a
mechanism which results in inversion of the anomeric
conguration (151). If the unequivocal relationship
between family classication and molecular mechan-
ism is valid, other members of family 43 should also
operate with retention of the anomeric conguration.
The amino acid sequences of two Bacillus stear-
othermophilus (7080 kDa of unprocessed precursor)
-xylosidases (152, 153) are so unique that they were
grouped into a special family, assigned as family 52.
The stereochemistry of hydrolysis by these enzymes
has not been established yet. Three-dimensional struc-
tures are not available for any members of these four
families. Some characteristic properties of individual
-xylosidase families are listed in Table 3.
D. Properties as Enzymes
There is limited knowledge about structure-function
relationships in the glycosyl hydrolase families con-
taining -xylosidases. All -xylosidases thus far
described hydrolyze not only xylobiose and xylooligo-
saccharides but also aryl -D-xylopyranosides.
Consequently, 4-nitrophenyl -D-xylopyranoside has
become the most common substrate for -xylosidase
assay. There are, however, examples of aryl -xylosi-
dases which do not attack xylo-oligosaccharides.
Therefore, it is always important to test the enzymes
directly on xylobiose and xylo-oligosaccharides.
Several -xylosidases are reported also to attack poly-
meric xylan in an exofashion. Because of the limited
Table 3 Families of Glycosyl Hydrolases Containing -Xylosidases
Common microbial
Enzyme family producers
3 Erwinia, Aspergillus,
Trichoderma
39 Thermophilic bacteria
43 Butylvibrio, Baccillus
52 Bacillus stearothermophilus
Source: http://expasy.hcuge.ch/cgi-bin/list/glycosid.txt.
Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.
information, it is not possible to correlate the catalytic
properties of enzymes with their glycosyl hydrolase
families. Therefore, only the properties of best
known -xylosidases will be described.
One of the best-characterized -xylosidases is an
intracellular enzyme from Bacillus pumilus (154, 155).
It shows absolute specicity for -xylopyranosyl resi-
dues but does not catalyze glycosyl transfer reactions.
This suggests that the enzyme belongs to family 43 of
glycosyl hydrolases (Table 3). The best-studied fungal
-xylosidases are extracellular enzymes produced by
Aspergillus and Trichoderma spp., i.e., the enzymes
from family 3. Their retaining character is revealed in
glycosyl transfer reactions in high concentrations of
xylo-oligosaccharides and 4NPh--Xyl (156159).
These enzymes do not show high specicity for the
glycan moiety. -Xylosidases from several Tricho-
derma strains exhibit
-L-arabinofuranosidase activity
(160162). This has been conrmed by cloning the T.
reesei -xylosidase gene in yeast (147). The
-L-
arabinofuranosidase from T. reesei hydrolyzes only

-L-arabinofuranosides and is not able to liberate
L-arabinose from arabinoxylan or arabinose-
substituted xylo-oligosaccharides. Aryl -xylosidase
activity of Trichoderma strains is inhibited by
D-xylose. Fungal -xylosidases were shown to have a
specic requirement for structural arrangement of
branched oligosaccharides. The enzyme from A. awa-
mori was tested on different arabinose-substituted
xylooligosaccharides. The enzyme was able to remove
terminal xylopyranosyl residues from the nonreducing
end of branched oligosaccharides only when two con-
tiguous unsubstituted xylopyranosyl residues were
linked to single- or double-substituted xylopyranosyl
residues (163). This is similar to the behavior of -
xylosidase from T. reesei (159, 164). The enzyme
does not liberate -1,4-xylopyranosyl residues linked
to an
-1,3- or -1,3-substituted xylopyranosyl residue
Molecular mass range Stereochemistry Other enzymes
of listed enzymes of hydrolysis in the family
60122 kDa retaining -glucosidase
5560 retaining
-L-iduronidase
5060 inverting
-L-arabinanase
EX
7080 (precursor) unknown none
 (compounds of the type Xyl-4[Ara-3]Xyl- or Xyl-
4[Xyl-3]Xyl-). In such compounds the substituent is
apparently so close to the site of the enzymic attack
(i.e., glycosidic oxygen) that the formation of the
enzyme-substrate complex is sterically impaired. The
-1,4-glycosidic linkage becomes susceptible to the
enzyme attack only if the substituent is linked to a
more distant C-2 position (Scheme 3).
Araf Xyl

1 non-hydrolyzed 1
j substrates j 1. 4-Nitrophenyl--D-xylopyranoside, 4 mM solu-
3 3
Xyl1-4Xyl1-4Xyl- Xyl1-4Xyl1-4Xyl-
Xyl1-4Xyl1-4Xyl- Xyl1-4Xyl1-4Xyl-
" 2 " 2
j hydrolyzed j

1 substrates 1
MeGlcA Xyl
Scheme 3
E. Determination of b-Xylosidase Activity
The activity of -xylosidases, which hydrolyze xylo-
biose, xylo-oligosaccharides, eventually linear portions
of xylan, can be determined reductometrically using
xylobiose and xylotriose, which are poor substrates
for EXs. The sensitivity of assays can be increased by
eliminating the background of oligosaccharides by
their reduction to sugar alcohols with NaBH4 . The
reductometric procedures described in the section
devoted to EXs can be adapted for the use of xylobiitol
or xylotriitol as substrates. Xylobiase activity can also
be determined using xylobiose as a substrate in an
enzyme-coupled assay which employs NAD -depen-
dent glucose dehydrogenase. The dehydrogenase is
not specic for glucose and catalyzes conversion of
generated xylose to xylonolactone (165, 166). The for-
mation of NADH is followed at 340 nm to estimate -
xylosidase activity. Two side reactions, the oxidation
of xylobiose to lactone by the dehydrogenase and
hydrolysis of the lactone by -xylosidase, have to be
taken into consideration.
However, the most common assay of -xylosi-
dase employs 4-nitrophenyl--D-xylopyranoside as
a substrate. There are a great variety of condi-
tions under which the assay can be done involving
the substrate concentration, pH and type of the
assay buffer, and various alkaline reagents to
terminate the reaction and to shift the absorption
maximum of liberated 4-nitrophenol to a visible
Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.
region of the spectrum. In a similar assay, phe-
nyl--D-xylopyranoside is used as a substrate
(167). Liberated phenol is determined using the
phenol reagent from the well-known Lowry proce-
dure for protein determination.
1. -Xylosidase Assay Using 4-Nitrophenyl--
D-Xylopyranoside/Na2 CO3
a. Reagents
tion in 0.05 M acetate buffer, pH 4.5 (or other buffer).
2. 2 M solution of Na2 CO3 .
3. 4-Nitrophenol, 2 mM stock solution in the acet-
ate buffer; 0.2 mM calibration solution in the acetate
buffer.
b. Procedure. Preheated substrate solution (0.9
mL, e.g., at 50 C) is mixed with 0.1 mL appro-
priately diluted enzyme solution and incubated for
suitable time at 50 C. The reaction is terminated by
addition of 1 mL of 2 M Na2 CO3 . The absorbance
is measured at 410 nm against the substrate blank.
The values have to be corrected for enzyme blanks
if they show absorbance at 410 nm. Calibration
with 4-nitrophenol is done in the range of 0.02
0:2 mol per assay mixture. One unit of -xylosi-
dase activity is dened as the amount of enzyme
liberating 1 mol of 4-nitrophenol in 1 min. (Note:
The assay can be miniaturized to the scale of a
microplate reader.)
2. -Xylosidase Assay Using 4-Nitrophenyl--
D-Xylopyranoside/Na2 B4 O7
a. Reagents
1. 4-Nitrophenyl--D-xylopyranoside, 10 mM
solution in 0.05 M acetate buffer, pH 5.0 (or other
buffer).
2. Saturated solution of Na2 B4 O7 .
3. 4-Nitrophenol, 1 mM stock solution in the acet-
ate buffer; 0.1 mM calibration solution in the acetate
buffer.
b. Procedure. Preincubated substrate solution
(0.1 mL 50 C) is mixed with 0.1 mL tested enzyme
sample (50 C) in an Eppendorf test tube and incubated
for appropriate time at 50 C. After addition of 1.0 mL
saturated solution of Na2 B4 O7 , the absorbance is mea-
sured at 410 nm against a substrate blank. Calibration
is done with 4-nitrophenol in the range of 0.01
0:1 mol. One unit of -xylosidase activity is dened
as the amount of enzyme liberating 1 mol of 4-nitro-
 phenol in 1 min. (Note: This assay is carried out in
microcentrifuge test tubes and is suitable for determi-
nation of cell-bound -xylosidase activity. The cells
used as the enzyme source must be separated by cen-
trifugation before measurement.)
F. Assays for Screening Purposes
Detection of -xylosidase activity in solid growth
media or in electrophoretic gels usually involves
incorporation of chromogenic and uorogenic sub-
strates, 4-nitrophenyl--D-xylopyranoside (15 mM)
or 4-methylumbelliferyl--D-xylopyranoside (0.10.5
mM). Activity is revealed by ooding the growth
media or gels with alkaline solutions (1 M Na2 CO3 ,
alkaline buffers) or exposing them to ammonium
hydroxide vapors. Replica plating is required before
alkalication when working in acidic pH regions.
The liberation of 4-methylumbelliferone is revealed
under UV light (360 nm). Alkalication of the solid
medium or gel, e.g., exposure to ammonium hydroxide
vapors, increases the uorescence. As mentioned with
uorogenic substrates of EXs, uorogenic glycosides
for screening purposes give better results when applied
as overlay at the later stages of colony development
than when incorporated into growth media before
inoculation.
IV. a-GLUCURONIDASE, 4-O-METHYL-a-
GLUCURONIDASE (EC 3.2.1.139)
A. Chemical Reactions Catalyzed
Xyl-R Xyl-R
2 sequencing (146, 176). The sequences show a high
j !

1
MeGlcA MeGlcA
-R 1-4Xyl; 1-4Xyl1-4Xyl; 1-4Xyln
One of the best substrates
Xyl1-4Xyl1-4Xyl Xyl1-4Xyl1-4Xyl
2
j !

1
MeGlcA MeGlcA

Reducing end
Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.
B. Classication

-D-Glucuronidase (EC 3.2.1.139).
C. Occurrence of a-Glucuronidase

-Glucuronidase activity has been detected in cellulo-
lytic and xylanolytic systems of a variety of fungi (168
176), actinomyces, and bacteria (177184). Two
reports mention
-glucuronidase in snail digestive
juice (184, 185). There is no report on the occurrence
of the enzyme in plants. In most microorganisms
-
glucuronidase was found to be secreted into culture
medium. However, there are examples in which the
enzymes has been found conned to the cells (172,
180, 181).
D. Properties as Proteins

-Glucuronidase has been puried from a relatively
small number of microorganisms. All eukaryotic
-glu-
curonidases, with the exception of the enzyme from
Agaricus bisporus (196), are monomeric enzymes with
molecular masses between 91 and 150 kDa. Their iso-
electric points are acidic, between values 4.6 and 5.3
(146). The
-glucuronidase puried from the snail
Helix pomatia (185) also shares these properties.
Bacterial
-glucuronidases are usually found as dimers
with subunits of a molecular mass
70 kDa. pI values
of bacterial enzymes are similar to those of fungal
enzymes.
-Glucuronidase from the thermophilic bac-
terium Thermotoga maritima shows a variable oligo-
meric structure depending on the salt concentration
(183).
So far only ve amino acid sequences of

-glucuronidases are known, all based on gene
degree of similarity (4060% identity) and have been
assigned to a separate glycosyl hydrolase family 67,
which does not contain any other hydrolases. A
three-dimensional structure of
-glucuronidase has
not been reported; however, the enzyme from
Bacillus stearophilus has been successfully crystallized
and subjected to x-ray analysis (186).
E. Properties as Enzymes

-Glucuronidases seem to belong to those accessory
xylanolytic enzymes that do not operate on polymeric
substrates. With exception of
-glucuronidase from
Thermoascus aurantiacus (170),
-glucuronidases failed
to liberate 4-O-methyl-D-glucuronic (MeGlcA) acid or
 D-glucuronic acid at an appreciable rate from poly-
meric substrates. In a few cases, an extremely low
rate was found. However, it should be stressed that
even a slight contamination of
-glucuronidase by
EX and -xylosidase would generate polysaccharide
fragments that would serve as
-glucuronidase sub-
strates.
-Glucuronidases, regardless of their eukaryo-
tic or prokaryotic origin, are very selective toward the
structure of aldouronic acids. They are capable of
removing MeGlcA or GlcA from aldouronic acids in
which MeGlcA or GlcA residues are linked to a single
xylopyranosyl residue or to a nonreducing terminal
xylopyranosyl residue of xylo-oligosaccharides. Such
fragments can be generated from glucuronoxylan by
the action of EXs of family 10 or by the action of a
combination of EXs of family 11 and -xylosidase
(Scheme 4) (88, 187).
Xyl1-4Xyl1-4Xyl Xyl Xyl1-4Xyl1-4Xyl1-4Xyl Xyl1-4Xyl1-4Xyl1-4Xyl
2 2
j j

1
1
MeGlcA MeGlcA
Hydrolyzed compound Nonhydrolyzed compounds which become substrates after removal
of xylopyranosyl residues left from the branch by -xylosidase
An interesting question related to the substrate spe-
cicity of
-glucuronidase is whether the enzyme is
specic for GlcA or MeGlcA. The enzyme from
Aspergillus niger hydrolyzed GlcA-Xyl3 about two
times faster than MeGlcXyl3 (172). This example sug-
gests that the 4-O-methyl group in the acid is not deci-
sive for the action of the enzymes.
A surprising property of microbial
-glucuronidases
is their inability to hydrolyze 4-nitrophenyl-
-D-glu-
curonide, a compound that could otherwise serve as
a convenient chromogenic substrate. The only
-glu-
curonidase capable of hydrolyzing this compound was
the snail enzyme (185). Microbial
-glucuronidases
require uronic acid to be linked to at least one xylo-
pyranosyl residue, which can be in the form of an aryl
glycoside as in 4-nitrophenyl-2-O-(methyl-4-O-methyl-

-D-glucuronosyl)--D-xylopyranoside (188).
Optimal pH values for bacterial
-glucuronidases
are in the range of 5.46.5 and in the range of 3.5
Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.
5.0 for fungal enzymes. Km values for aldouronic
acids as substrates were reported in the range of
0.140.95 mM.
The stereochemistry of hydrolysis of glycosidic link-
age by
-glucuronidase was recently investigated with
puried Aspergillus tubingensis enzyme, which belongs
to family 67 of glycosyl hydrolases. By NMR spectro-
scopy it has been established that the enzyme uses a
single displacement reaction mechanism, leading to
inversion of the anomeric conguration (189). Based
on this nding, inverting character can be expected
with other
-glucuronidases.
F. Determination of a-Glucuronidase Activity
As mentioned above, the activity of
-glucuronidase
cannot be determined directly on glucuronoxylan or
2 2
j j

1
1
MeGlcA MeGlcA
Scheme 4
a related polymeric substrate. Glucuronoxylan can
be used for the
-glucuronidase assay only in the
presence of xylan-depolymerizing enzymes which
generate aldouronic acids of the required structures
(190). The most common substrate for
-glucuroni-
dase assays are linear aldouronic acids obtained by
enzymic or acid hydrolysis of glucuronoxylan. From
such aldouronic acids, liberation of MeGlcA is
usually followed by determination of the reducing
power of the free uronic acid by a modication of
the Somogyi copper reagent and the Nelson phos-
phomolybdenic acid reagent according to Milner
and Avigad (191). The method is quite specic for
uronic acids and suffers only slightly from the pre-
sence of neutral reducing sugars. The assay could be
affected by some salts (citrate, phosphate), however.
Alternative methods include HPLC determination of
free MeGlcA (173, 174) or the equivalent amount of
xylo-oligosaccharides (169), or GLC of trimethylsilyl
 oxim derivatives of uronic acids (178, 179, 182, 184).
Unfortunately, there is no direct
-glucuronidase
assay available that would utilize a chromogenic sub-
strate. As we have already mentioned, 4-nitrophenyl-

-glucuronide does not serve as substrate of micro-
bial
-glucuronidases. Recently, a new -xylosidase-
coupled
-glucuronidase assay has been introduced
that uses 4-nitrophenyl-2-O-(4-O-methyl-
-D-glu-
curonosyl)--D-xylopyranoside (NPh-Xyl-MeGlcA)
(188). Liberation of MeGlcA from the compound
yields an equimolar amount of 4-nitrophenyl--xylo-
pyranoside. In the presence of excess of -xylosidase
to hydrolyze the xyloside, the amount of liberated
4-nitrophenol becomes a measure of
-glucuronidase
activity (Scheme 5).
NPh-Xyl-MeGlcA ! MeGlcA NPh-Xyl

-glucuronidase
Scheme 5
1. Determination of
-Glucuronidase Activity
Using the Milner-Avigad Copper Reagent
a. Reagents
1. A mixture of aldotriuronic and aldotetrauronic
acid (ratio
8 : 2) (Megazyme, Ireland), a solution 2
mg/mL in 0.05 M acetate buffer pH 5.0.
2. Copper reagent prepared according to Milner
and Avigad (191).
3. Arsenomolybdate reagent of Nelson reagent (110).
4. D-Glucuronic acid, 1 mM calibration solution
in 0.05 M acetate buffer (calibration in the range 0.02
0:2 mol).
b. Procedure. Enzyme (0.04 mL) is added to a
preheated solution of aldouronic acids (0.16 mL,
40 C) and incubated at 40 C for an appropriate time.
The reaction is stopped by adding 0.6 mL of the copper
reagent. The mixture is heated in a boiling water bath
for 10 min and then cooled in an ice-water bath.
Subsequently 0.4 mL of the Nelson reagent is then
added and after mixing the absorbance at 600 nm is
measured against the substrate blank. One unit of
-
glucuronidase is dened as the amount of enzyme lib-
erating 1 mol of glucuronic or 4-O-methylglucuronic
acid per 1 min under the selected assay conditions.
(Note: The original procedure (191) involves dilution
Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.
of the developed reaction mixture with water. Some
authors stop the reaction by boiling and clarify the
mixture by centrifugation before addition of the cop-
per reagent (146).)
2. -Xylosidase-Coupled Assay of
-
Glucuronidase (188)
a. Reagents
1. 4-Nitrophenyl-2-O-(4-O-methyl-
-D-glucuro-
nosyl)--D-xylopyranoside (NPh-Xyl-MeGlcA), pre-
pared by alkaline deesterication of its methyl ester
(191, 192), 4 mM solution in 0.05 M acetate buffer,
pH 5.0.
! NPh-OH Xyl
-xylosidase
2. Recombinant -xylosidase of Aspergillus niger
produced in Saccharomyces cerevisiae (191), a solution
2 U/mL of 0.05 M acetate buffer, pH 5.0 (one unit of
-xylosidase is dened as the amount of enzyme liber-
ating 1 mol of 4-nitrophenol from 4-nitrophenyl--D-
xylopyranoside).
3. Saturated solution of Na2 B4 O7 .
4. 4-Nitrophenol, 1 mM solution in the acetate
buffer, calibration in the range 0.010:1 mol.
b. Procedure. 0.1 mL of 4 mM solution of NPh-
Xyl-MeGlcA is mixed with 0.1 mL of the -xylosidase
solution (200 mU per reaction mixture), preheated to
37 C, and the reaction is started with addition of 52
0 L of the tested enzyme solution. After 10 min or
longer incubation at 37 C 1 mL saturated solution of
Na2 B4 O7 is added and the absorbance is measured at
410 nm. An alternative procedure is to add 0.1 mL
enzyme solution to 0.05 mL double-concentrated sub-
strate and -xylosidase solution in 0.1 M acetate buf-
fer. One unit of
-glucuronidase is the amount of
enzyme required to release 1 mol of 4-nitrophenol.
(Note: The assay was applied to three different
-glu-
curonidases. Their Km values for NPh-Xyl-MeGlcA
were in the range 0.210.58 mM, which is in the
range of Km values of
-glucuronidases for aldouronic
acids. The new substrate is still not commercially avail-
able.)
 G. Assays for Screening Purposes of hardwood holocellulose, e.g., birchwood or beech-
wood holocellulose (198, 199). An analogous polysac-
No direct in situ detection of
-glucuronidases has charide of a lower polymerization degree (average DP
been reported.
25) and acetyl content of 13% can be obtained as a
nondialyzable fraction of a water-soluble, noncellulo-
sic polymer by steaming birch at 200 C for 10 min
(200). An aqueous thermochemical treatment of hard-
V. ACETYLXYLAN ESTERASE (3.1.1.72)
wood leads to a similar material with a higher acetyl
A. Chemical Reactions Catalyzed content (201). Since none of the above-mentioned
AcXE substrates are commercially available and their
isolation is difcult or dependent on availability of
H2 O
special equipments, some authors have replaced nat-
R1 -Xyl1-4Xyl1-4Xyl1-4Xyl1-4Xyl-R2 !
ural acetylxylan by a chemically acetylated polysac-
2 3
charide. A widely used method of chemical
j j
acetylation of xylan was published by Johnson et al.
Ac Ac
(202). There are also examples of the use of fully che-
mically acetylated hardwood xylan as a substrate for
R1 -Xyl1-4Xyl1-4Xyl1-4Xyl1-4Xyl-R2 2AcH
AcXE (203, 204). As will be shown below, a majority
of AcXEs can be assayed on a variety of aryl acetates
H2 O
which serve as substrates of nonhemicellulolytic acet-
Xyl1-4Xyl ! Xyl1-4Xyl AcH
ylesterases and lipases.
2
j
C. Classication
Ac
H2 O
Acetylxylan esterase (EC 3.1.1.72).
Xyl1-4Xyl ! Xyl1-4Xyl AcH
3
D. Properties as Proteins
j
Ac Reducing end
AcXEs show the greatest diversity in molecular orga-
nization among all components of microbial xylanoly-
Acetylxylan esterases (AcXEs) are components of tic systems. On the basis of amino acid sequence
microbial xylanolytic and cellulolytic systems which similarity AcXEs have been assigned to seven families
are able to remove acetyl groups esterifying D-xylopyr- of 13 so far recognized families of carbohydrate
anosyl residues of xylan main chain at positions 2 or 3 esterases (73, 205) (Table 4).
(193195). AcXEs also deacetylate partially acetylated The best-known fungal AcXEs from Aspergillus,
xylo-oligosaccharides. The enzyme action on polysac- Penicillium, Schizophyllum and Trichoderma spp. can
charide substrates creates new sites on the xylan main be found in carbohydrate esterase families 1 and 5
chain, suitable for productive binding with depolymer- together with bacterial acetylxylan esterases. The
izing EXs. This effect is reected in a clear coopera- majority of these represents one catalytic domain of
tivity of the two enzymes during acetylxylan bifunctional acetylxylan-degrading enzymes. Diverse
degradation, a typical example of enzyme synergy AcXEs of the anaerobic fungus Neocallimastix patri-
(54, 196, 197). Acetylxylan degradation with EXs pro- ciarum (214) can be found in families 2, 3, and 6.
ceeds faster and to a higher degree in the presence of AcXEs produced by Streptomyces are grouped in
AcXEs (54). Deacetylation of xylo-oligosaccharides family 4 together with similar AcXE modules of bacter-
makes the oligosaccharides fully susceptible to the ial bifunctional enzymes. Their sequences are similar to
action of -xylosidase. those of nodulating proteins (NodB) from Rhizobium
spp. and with some yeast enzymes, identied as chito-
B. AcXE Substrates oligosaccharide and chitin deacetylases (205).
Fungal AcXEs, and some bacterial enzymes, are
The substrate similar to the native O-acetyl-4-O- usually monomeric enzymes, while some AcXEs from
methyl-D-glucurono-D-xylan present in plant cell thermophilic anaerobic bacteria occur as oligomers of
walls can be prepared by dimethylsulfoxide extraction smaller subunits (224). As already mentioned, numer-

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

 Table 4 Families of Carbohydrate Esterases Containing Acetylxylan Esterases

Microbial producer of Mw Other enzymes

Family acetylxylan esterase (kDa) pI in the family References

1 Aspergillus niger 30.5 3.1 None 206

Schizophyllum communea 30 3.4 203, 207
Penicillium purpurogenum AcXE I 48 7.8 208
Pseudomonas uorescens 209
Catalytic domains of bifunctional 210212
enzymes of anaerobic bacteria
2 Ruminal anaerobic bacteria and None 213, 214
fungi
3 Ruminal anaerobic bacteria and None 213215
fungi
4b Streptomyces lividans 34 9.0 Chitin and chito- 216, 217
Streptomyces thermoviolaceus 34.3 oligosaccharide 218
deacetylase
Catalytic domains of bifunctional 82, 212, 219
enzymes of anaerobic bacteria
5 Trichoderma reesei 34 6, 8, 7.0 Cutinase 220, 221
Penicillium purpurogenum AcXE II 23 7.8 208, 222
6 Anaerobic ruminal fungi 214, 223
7 Thermoanaerobacterium 32, 26 4.2, 4.3 224, 225
Thermotoga neapolitana
a
Assignment based on a sequence of fty NH2 -terminal amino acids (226).
b
This may be the only family, members of which do not attack synthetic low-molecular-mass substrates, such as aryl acetates (226).
Source: http://afmb.cnrs-mrs.fr/
pedro/CAZY/db.html.

ous enzymes have modular architecture. Some of
AcXEs, such as those from Pseudomonas uorescens
(209) or Trichoderma reesei (221), contain cellulose-
binding domains (CBDs) separated from the catalytic
domain by linker regions. Others, like AcXE from
Streptomyces lividans (217), contain a xylan-binding
domain (77). AcXE of Cellulomonas mi is a part of
a bifunctional enzyme which contains both CBD and
XBD (82). The two-catalytic module architecture,
represented by various combinations of EXs linked
to AcXEs, is very common particularly among acetyl-
xylan-degrading bifunctional enzymes of ruminal
anaerobic bacteria (212). AcXE domains of such
bifunctional enzymes can be found in carbohydrate
esterase families 1, 2, 3, and 4 (Table 4). With the
bifunctional EX-AcXE enzyme from Clostridium
thermocellum (83) it has been demonstrated that the
two catalytic domains act synergistically in the degra-
dation of acetylxylan. This observation again suggests
that the production of an enzyme having both deb-
ranching and depolymerizing activity can be a great
advantage for a microorganism competing for a
carbon source with microorganisms using separate
enzyme components.
AcXEs existing as single-domain enzymes have a
molecular mass in the range of 2348 kDa and acidic
or neutral pI values. There does not seem to be a spe-
cial relationship between these two parameters and
their assignment into carbohydrate esterase families
(Table 4).
Three-dimensional structures are known only for
AcXEs belonging to family 5. Crystallization has
been reported for AcXEII from P. purpurogenum
(227) and AcXE from T. reesei (228). The protein
from P. purpurogenum shows an
= hydrolase fold,
similar to that of cutinase (227). Cysteine residues form
ve S-S bridges which makes the structure very rigid
and tightly packed. The substrate binding site appears
to be small and able to accommodate only an acetyl
group, thus giving a relatively high substrate specicity
to the enzyme.
E. Properties as Enzymes
1. Substrate Specicity
Substrate specicity of AcXEs is one of the least-clar-
ied aspects of this xylanolytic component. There is
very limited knowledge on the structure-function rela-

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

 tionship of these enzymes, particularly considering that
they have been assigned to seven different families
(Table 4). The situation is complicated because there
are esterases and lipases which cannot be strictly con-
sidered as components of xylanolytic systems, but
which could participate in the later stages of acetylxy-
lan degradation, by deacetylation of xylooligosacchar-
ides. It is known that nonhemicellulolytic esterases and
lipases operate quite efciently on low-molecular-mass
acetylated carbohydrases (226, 229). As a consequence,
we shall limit the discussion here to the catalytic prop-
erties of enzymes that deacetylate polymeric substrates
as such or coinduced with other plant cell walls hydro-
lyzing enzymes.
Several lines of evidence suggest that there are two
major types of acetylesterases that participate in acet-
ylxylan degradation (197). They differ in their afnity
toward polymeric and oligomeric substrates. The
enzymes that perform well on polymeric substrates
are capable of causing precipitation of xylan from
solution due to deacetylation (207, 226). The removal
of acetyl groups results in the association of xylan
molecules into insoluble aggregates. This property is
very typical of AcXEs of family 4 (Table 4), which
appear to be most specic for acetyl xylan and do
not hydrolyze low-molecular-mass substrates such as
4-nitrophenyl- or 4-methylumbelliferyl acetate (226).
The enzymes of other families share properties compa-
tible with general acetylesterases and acylesterases
(226). They are active on a variety of synthetic aryl
acetates and acylates (226). This second group of
enzymes do not perform well on polymeric acetylxylan
but are more active on acetylated oligosaccharides gen-
erated from acetylxylan by EXs (197, 226, 230). Thus
EXs would synergistically facilitate the release of acetic
acid in concert with this type of acetylesterases.
Additional studies are also required to dene the spe-
cicity of AcXEs with respect to the type of polysac-
charide that they deacetylate. There are indications
Scheme 6
Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.
that at least some AcXEs are quite specic for acetyl-
xylan (197, 230).
2. Specicity for the Position of the Acetyl
Group in the Main Chain
Xylose residues in xylan are acetylated at positions 2
and/or 3, and the acetyl groups are more or less evenly
distributed between these positions. This raises ques-
tions of whether the AcXEs are capable of releasing
acetyl groups bound only at one position or at both
positions, and to what extent is deacetylation inu-
enced by the migration of acetyl groups between posi-
tions 2 and 3. Studies of three AcXEs on acetylated
methyl glycosides showed the regioselective removal of
acetyl group from positions 2 and 3 (229, 231, 232).
AcXE from S. commune AcXE exhibited a preference
for position 3 (229), while the T. reesei AcXE deacety-
lated the model compounds sequentially, rst at posi-
tion 2 and then rapidly at position 3 (231). S. lividans
AcXE worked almost simultaneously on positions 2
and 3, essentially catalyzing a double deacetylation of
compounds at both positions (232). Two methyl xylo-
pyranoside diacetates, which had a free hydroxyl
group at position 2 or 3, i.e., the derivatives that
most closely mimicked monoacetylated xylopyranosyl
residues in acetylxylan, were deacetylated by 1 or 2
orders of magnitude faster than 2,3,4-tri-O-acetyl- or
2,3-di-O-acetyl--D-xylopyranoside. These observa-
tions explained the double deacetylation. The second
acetyl group was removed immediately after the rst
one. An implication of these results is that the deace-
tylation mechanism of positions 2 and 3, when the
neighboring positions 3 and 2 are nonacetylated,
might involve an enzyme-catalyzed formation of a
ve-member transition state intermediate (Scheme 6).
Such intermediates are believed to be involved in the
spontaneous migration of acetyl group along the gly-
copyranoid ring in aqueous media. If this hypothetical
 mechanism is experimentally conrmed, the question
of regiospecicity of AcXE at positions 2 and 3 will
become irrelevant.
3. Reaction Mechanism
The inactivation of AcXEs with phenyl methyl sulfonyl
uoride (221, 227) and the occurrence of the serine
sequence GXSXG in active site of AcXEs (195) suggest
that AcXEs utilize a catalytic mechanism similar to
lipases and serine proteases. This mechanism involves
two major elements, a nucleophilic serine and a general
acid-base catalyst, usually a histidine. It remains to be
established whether all types of AcXEs contain the
serine motif (GXSXG) in their catalytic site.
G. Determination of AcXE Activity
The most specic AcXE assay employs as substrates
hardwood acetylxylans extracted either by DMSO or
obtained by steaming wood. The naturally occurring
acetylxylan can be replaced by chemically acetylated
xylan. In the presence of EXs, both types of polysac-
charides are also suitable for enzymes that prefer acety-
lated xylo-oligosaccharides as substrates. The amount
of released acetic acid can be determined by HPLC
chromatography, or enzymically using the Boeringer
Test Combination No. 148,261. Practically all HPLC
procedures employ for analysis of acetic acid a Bio-
Rad (Richmont, CA) Aminex HPX-87H column
eluted with 0.01 N H2 SO4 (193, 202), connected to a
refractometric detector. Glycerol may be recom-
mended as internal standard. The conditions of
AcXE assay on acetylxylan vary considerably among
different laboratories. Typical conditions include a
substrate concentration in the range of 0.210%, pH
in the range of 5.07.0, and temperature in the range of
3075 C.
Since most AcXEs exhibit general acetylesterase
activity [the exception is family 4 (226)], their activity
also can be determined using chromogenic or uoro-
genic acetylesterase substrates such as 4-nitrophenyl
acetate,
- or -naphthyl acetate, and 4-methylumbel-
liferyl acetate. Examples of selected AcXE assays are
given below.
1. AcXE Assay on Acetylxylan (202, 233)
a. Reagents
1. Acetylxylan obtained from hardwood by
DMSO extraction or steaming, or chemically acety-
lated xylan, 10% solution or suspension in 0.4 M
potassium phosphate buffer, pH 6.0 or 6.5.
Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.
2. 1 N H2 SO4 .
3. Acetic acid, 0.1.0% (w/v).
4. Glycerol, 1% (w/v).
b. Procedure. One volume of acetylxylan solu-
tion or suspension is mixed with one volume of enzyme
solution and incubated at 50 C. At time intervals, 0.2-
mL aliquots are taken, mixed with 10 L of 1 N
H2 SO4 to stop the reaction, claried by a short centri-
fugation, and supernatants analyzed for acetic acid by
HPLC. Standards of acetic acid are analyzed under the
same conditions. Glycerol can be used as an internal
standard at 0.1% or 0.2% concentration. One unit of
AcXE is dened as the amount of enzyme liberating
1 mol of acetic acid per minute. (Note: Termination
of the reaction is done by heating of the sample at
100 C in case the acetic acid is determined enzymically
(200).)
2. Acetylesterase Assay on 4-Nitrophenyl
Acetate (233)
a. Reagents
1. 4-Nitrophenyl acetate, saturated solution in 0.2
M potassium phosphate buffer, pH 6.5 (freshly pre-
pared solution).
2. 4-Nitrophenol, 1 mM standard solution in 0.2
M potassium phosphate buffer, pH 6.5.
b. Procedure. A volume of 1050 L appropri-
ately diluted enzyme solution is mixed with 1 mL clar-
ied saturated solution of 4-nitrophenyl acetate in a 1
cm light-path microcuvette and incubated at 22 C.
Absorbance at 410 nm is measured at time intervals
against a substrate blank. Alternatively, the absor-
bance can be followed continuously. The amount of
substrate hydrolyzed is calculated from a calibration
curve prepared using 4-nitrophenol in the 0.05
0:3 mol/mL range in 0.2 M phosphate buffer. The
reaction cannot be terminated chemically because of
the labile nature of the substrate. One unit of acetyles-
terase activity is dened as the amount of enzyme lib-
erating 1 mol of 4-nitrophenol in 1 min.
3. Acetylesterase Assay on 4-Nitrophenyl
Acetate (202)
a. Reagents
1. 4-Nitrophenyl acetate, 50 mM solution in
DMSO (stable at 4 C), 2 mM solution in water pre-
pared freshly by diluting 1 volume of the DMSO solu-
tion with 24 volumes of water.
2. 0.1 M potassium phosphate buffer, pH 6.5.
 3. 4-Nitrophenol calibration solution in 0.05 M
phosphate buffer.
b. Procedure. Enzyme present in 1.5 mL of 0.1 M
phosphate buffer is mixed with 1.5 mL of 2 mM solu-
tion of 4-nitrophenyl acetate and incubated at 25 C.
The increase in absorbance at 410 nm is measured
against a substrate blank. Enzyme activity is calculated
from the slopes of absorbance versus time and calibra-
tion values of 4-nitrophenol obtained under the same
conditions. One unit of acetylesterase activity is
dened as above.
4. Acetylesterase Assay on
-Naphthyl Acetate (234)
a. Reagents
1.
-Naphthyl acetate, 1 mM solution in 0.05 M
sodium citrate buffer, pH 5.3.
2. Fast Corinth V Salt (Sigma F-6389), 0.1% solu-
tion in 1 M acetate buffer, pH 4.3, containing 10%
Tween.
3.
-Naphthol, calibration solution in the citrate
buffer.
b. Procedure. Enzyme solution (0.2 mL) is incu-
bated with 1.8 mL
-naphthyl acetate solution at 50 C
for 10 min, after which 1 mL dye solution is added.
The absorbance at 535 nm is read exactly 10 min after
addition of the dye. The calibration curve is con-
structed under the same conditions.
E. Fluorimetric Acetylesterase Assay on 4-
Methylumbelliferyl Acetate (235)
a. Reagents. 4-Methylumbelliferyl acetate, 10
mM solution in methanol. 0.5 M potassium phosphate
buffer, pH 6.5.
b. Procedure. 4-Methylumbelliferyl acetate solu-
tion (10 L) is mixed with 10 L phosphate buffer
and made up to 100 L with the enzyme sample
and water. After an appropriate time of incubation
at 40 C, the mixture is diluted with 2.9 mL distilled
water. The uorescence is measured at an excitation
wavelength of 330 nm and an emission wavelength at
445 nm using 4-methylumbelliferone as the calibra-
tion standard. One unit of esterase is dened as
1 mol of the product formed per minute. (Note: A
much less sensitive acetylesterase assay using 4-methy-
lumbelliferyl acetate is a photometric assay which
exploits the fact that 4-methylumbelliferone shows
considerable absorbance at 354 nm (not an absorp-
tion maximum) while its acetate does not absorb at
this wavelength (224).)
Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.
H. Assays for Screening and Detection Purposes
All substrates used for AcXE assays can also be
employed for detection of the enzyme in gels.
Detection of AcXE using polymeric acetylxylan is
based on the precipitation of the polysaccharide due
to deacetylation (207). This approach is applicable
only in those cases when the AcXEs located in the
gel are free of EXs, which would prevent the precipita-
tion by hydrolyzing the deacetylated polymer. Such a
situation occurs after separation of EXs from AcXEs
by electrophoretic methods. This means that precipita-
tion of acetylxylan can be used for screening purposes
only in the case of naturally occurring and recombi-
nant strains that do not produce EXs.
For initial screens of microorganisms and transfor-
mants producing AcXEs belonging to other families
than family 4 (Table 4), it is possible to use chromo-
genic and uorogenic aryl acetates, such as
- or -
naphthyl acetate and 4-methylumbelliferyl acetate
(214). An alternative is to screen for the production
of enzymes clearing of milky solid media containing
insoluble per-O-acetylated carbohydrates, e.g., penta-
O-acetyl-D-glucose (236). Subsequent screening of
positive colonies for the ability to deesterify acetylxy-
lan itself is necessary. The principles outlined above
can be used for detection of AcXEs in electrophoretic
gels.
1. Detection of AcXE in Gels Using
Acetylxylan as a Substrate (233)
An approximately 1-mm-thick at 1.52.0% agarose
gel containing 0.52.0% of acetylxylan (DMSO
extracted or obtained by steaming wood) and 0.01%
sodium azide in 0.1 M phosphate buffer, pH 6.5, is
prepared by casting between glass plates. Visualiza-
tion of AcXE is carried out by superimposing the
detection and rebuffered separation gel in a plastic
bag and incubating at 3050 C until cloudy areas
appear. The precipitate forms in both the separation
and detection gels because of cross-diffusion of
enzyme and substrate. The method fails when esterase
is located close to EXs. Gels with precipitates are
photographed in diffuse light similarly as immunopre-
cipitation bands.
2. Detection of Acetylesterase in Gels by
-
Naphthyl Acetate (237)
A solution of 20 mg
-naphthyl acetate in 4 ml of
N,N 0 -dimethylformamide is mixed with 36 mL of a
solution of 20 mg of Diazo Blue R (Sigma) in 0.05
 M phosphate buffer, pH 7.4. An electrophoretic or
isoelectrofocusing gel is washed with 0.1 M phosphate
buffer, pH 7.4, for 15 min. The gel is then placed in a
freshly prepared substrate-dye solution and incubated
for 1 h at 40 C. The reaction is stopped by decanting
the staining solution and covering the gel with a solu-
tion of 2% acetic acid.
3. Detection of Acetylesterase Using 4-
Methylumbelliferyl Acetate (233)
A at detection agarose gel containing 0.1% 4-methy-
lumbelliferyl acetate in 0.1 M phosphate buffer is
freshly prepared. The gel is placed in contact with a
washed electrophoretic gel and incubated at room tem-
perature until a bright uorescent zone appears under
UV light at 360 nm on both separation and detection
gels. An alternative procedure is to bring the separa-
tion gel into contact with a saturated solution of 4-
methylumbelliferyl acetate in 0.1 M phosphate buffer
on a glass plate.
VI. a-L-ARABINOFURANOSIDASE (EC
3.2.1.55)
A. Chemical Reactions Catalyzed
Xyl1-4Xyl1-4Xyl-R
3 2
j ! Xyl1-4Xyl1-4Xyl-R Ara

1
Araf
R 0 -Xyl1-4Xyl1-4Xyl-R
32
j ! R 0 -Xyl1-4Xyl1-4Xyl-R Ara

1
Araf

-L-Arabinofuranosidase is a component of hemi-
cellulolytic systems that participates in hydrolysis of
plant polysaccharides built from or containing nonre-
ducing terminal
-L-arabinofuranosyl residues (44,
238). Such polysaccharides have been recognized as
part of pectic substances, gum exudates, and wood
and cereal hemicelluloses (239). The pectic substances
mainly include L-arabinan and various types of L-ara-
Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.
bino-D-galactans. Other natural substrates of
-L-ara-
binofuranosidase are glycosylated terpenols of grape,
important precursors of grape and wine aromas. Many
of these terpenols are linked to disaccharide moieties in
which the nonreducing terminal sugar is frequently L-
arabinofuranose. Thus,
-L-arabinofuranosidase can
be used for enhancement of wine avor by the release
of free terpenols (240242).
Since the enzymes degrading arabinans and arabi-
nogalactaus were specically covered in Chapter 70,
here we shall focus on
-L-arabinofuranosidases
involved in the degradation of polysaccharides con-
taining main chains built from -1,4-linked xylopyra-
nosyl residues, specically, softwood arabinoglu-
curonoxylans and cereal arabinoxylans. Some of
these enzymes, particularly those which attack low-
molecular-mass fragments of these polysaccharides,
participate in the degradation of all types of L-arabi-
nose-containing polysaccharides. However, there are

-L-arabinofuranosidases specic for arabinoxylans.
In the softwood polysaccharide the L-arabinofurano-
syl residues are linked to the main chain
-1,3- or
-
1,2 glycosidically as single substituents. Cereal arabi-
noxylans contain, in addition to
-1,3- and
-1,2-
linked L-arabinofuranosyl residues as single substitu-
ents, doubly arabinosylated xylopyranosyl residues of
the main chain.
B. Properties as Proteins
Fungal
-L-arabinofuranosidases occur mostly as
monomeric enzymes with molecular masses between
32 and 84 kDa and pI values between 3.2 and 7.5
(44). The majority of described fungal enzymes are
products of Aspergillus spp. (146). and have pI values

3:5. Bacterial and Streptomyces
-L-arabinofurano-
sidases frequently occur as oligomers, form dimers up
to octamers, with subunits of molecular mass between
33 and 75 kDa. pI values were reported in the range of
3.89.0.
Some
-L-arabinofuranosidases show modular
architecture similar to other xylanolytic enzymes.
Interesting in this regard is
-L-arabinofuranosidase
from T. reesei (244, 245). Two different forms of the
enzyme have been isolated from a culture uid of the
fungus. The 25-kDa form corresponds to the catalytic
domain only (245). The 53-kDa form, the rst form of
the enzyme described (244), also contains a noncataly-
tic xylan-binding domain (245). The large 53-kDa form
can be converted to the shorter one by proteolytic
digestion (245).
-L-Arabinofuranosidase from P.
uorescens was reported to contain a cellulose-binding
 domain in the N-terminal portion of the polypeptide
chain (246). A xylan-binding domain was detected in
C-terminal region of
-L-arabinofuranosidase B from
S. lividans (247). The enzymes containing the noncata-
lytic polysaccharide-binding domains are specic for
release of L-arabinofuranose from polymeric sub-
strates.
C. Properties as Enzymes
Microbial
-L-arabinofuranosidases can be divided
into three different groups according to substrate spe-
cicities (239). The groups seem to coincide with three
different glycosyl hydrolase families into which
-L-
arabinofuranosidases belong (Table 5).
The enzymes that perform best on short
-1,5-
linked arabinooligosaccharides and are active on 4-
nitrophenyl-
-L-arabinofuranoside (NPh-Araf ) and
inactive on polymeric substrates are grouped in family
51. They include both fungal and bacterial
-L-arabi-
nofuranosidases of molecular mass 5772 kDa, which
catalyze the hydrolysis of the glycosidic linkage with
retention of the anomeric conguration (248). These
are apparently the enzymes designed to degrade more
arabinans rather than arabinoxylans.
Family 54 includes mainly fungal
-L-arabinofura-
nosidases (M.m. 4953 kDa) which exhibit somewhat
broader substrate specicities than the enzymes of
family 51. The 54 family
-L-arabinofuranosidases
attack not only NPh-Araf and arabinose-containing
oligosaccharides, but also polymeric substrates such
as arabinoxylans and arabinoglucuronoxylans (239).
These enzymes catalyze hydrolysis of
-1,5-,
-1,3-
and also
-1,2-linked terminal L-arabinofuranosyl resi-
dues with retention of the anomeric conguration
(248).
The family 62 of
-L-arabinofuranosidases (Table
5) includes enzymes which preferentially attack poly-
meric substrates, i.e., arabinoxylans. The best-known
members of this family are enzymes from Aspergillus
spp., assigned as AXHs (249),
-L-arabinofuranosi-
dase B from Streptomyces lividans (247), and an
enzyme from Ps. uorescens subsp. cellulosa having a
modular architecture (246). The enzymes show a clear
preference for
-1,2-linked and
-1,3-linked
-L-arabi-
nofuranosyl residues of arabinoxylan main chain.
However, the enzymes of this family do not remove

-L-arabinofuranosyl residues from doubly substituted
xylopyranosyl residues.
An arabinofuranose-releasing enzyme of novel sub-
strate specicity was isolated from Bidobacterium
adolescentis DSM 20083 (250). This enzyme specically
releases terminal arabinofuranosyl residues linked
-
1,3 to xylopyranosyl residues of main chains that
also carry
-1,2-linked arabinofuranose. Wood and
McCrea (251) reported the occurrence of a hydrolase
releasing feruloylated L-arabinofuranosyl residues
from wheat straw arabinoxylan. Additional evidence

Table 5 Families of
-L-Arabinofuranosidases and Differences in Their Properties

Producing Molecular Mechanism of

Family microorganism mass (kDa) Hydrolyzed substrates hydrolysis Ref.

51 A. niger (enz. A) 5772 NPh-
-Araf , arabino- Retaining 205

Bacillus oligosaccharides (enzymes are
Streptomyces inactive on polymers)
Thermotoga
Clostridium
54 A. niger (enz. B) 4953 NPh-
-Araf , arabino- Retaining 205, 253
T. reesei oligosaccharides, arabinoxylan
Ewericella nidulans (attack mainly on
-1,3-linked
Synchocystis sp. Araf , no attack on double
arabinosylated xylopyranosyl
residues.
62 Aspergillus (AXH) 4759 arabinoxylan (the enzymes remove Unknown 246, 247,
Streptomyces (enz. B),
-1,3- and
-1,2-linked L-Araf 249
Ps. uorescens residues, no attack on double
arabinosylated xylopyranosyl
residues)

Source: Refs. 44, 205.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

 for hydrolysis of substituted arabinofuranosyl residues
has been reported for
-L-arabinofuranosidase from
A. terreus (252). The mode of action of enzymes
operating on high-molecular-mass arabinoxylans and
their relationship to glycosyl hydrolase families is
illustrated in Scheme 7. Based on the complexity of
structures of plant arabinoxylan, one can anticipate
the discovery of arabinofuranosidases with other
substrate specicities.
Scheme 7
D. Determination of
-L-Arabinofuranosidase
Activity
As indicated in Table 5, two families of
-L-arabino-
furanosidases can be assayed easily using the chromo-
genic substrate 4-nitrophenyl
-L-arabinofuranoside
or the uorogenic substrate 4-methylumbelliferyl
-L-
arabinofuranoside. One should be aware of the fact
that these substrates can also be attacked by -xylosi-
dases of family 43, which exhibit aryl
-L-arabinofur-
anosidase activity (Table 3). Therefore, it is imperative
to conrm the identity of the enzyme by determining
its ability to release arabinose from arabino-sub-
stituted xylans or xylooligosaccharides (5).
-L-
Arabinofuranosidases of family 62 must be assayed
on natural substrates, arabinoxylans, and oligosac-
charides derived from arabinoxylans. Released arabi-
nose is determined using HPLC or as reducing sugar.
Examples of determination of
-L-arabinofuranosi-
dase activity are given below.
1.
-L-Arabinofuranosidase Assay Using 4-
Nitrophenyl
-L-Arabinofuranoside (244)
a. Reagents
1. 4-Nitrophenyl
-L-arabinofuranoside, 2 mM
solution in 0.05 M sodium citrate phosphate buffer
Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.
(pH 4.0) or in other buffer depending on the pH opti-
mum of the assayed enzyme.
2. 1.0 M sodium carbonate.
b. Procedure. 4-Nitrophenyl
-L-arabinofurano-
side solution (0.9 mL) is mixed with 0.1 mL of the
enzyme sample, incubated at 50 C or an appropriate
time, and the reaction is stopped by addition of 0.5 mL
of 1 M solution of Na2 CO3 . Liberated 4-nitrophenol is
measured spectrophotometrically at 400 nm. One unit
of
-L-arabinofuranosidase activity is dened as the
amount of enzyme liberating 1 mol of 4-nitrophenol
in 1 min. (Note: The literature offers a variety of mod-
ication of the above procedure with respect to buffer,
substrate concentration, and the reaction terminating
agent. Termination of the reaction using a saturated
solution of sodium tetraborate is recommended.)
2. Fluorimetric Assay of
-L-
Arabinofuranosidase (254)
a. Reagents
1. 4-Methylumbelliferyl
-L-arabinofuranoside,
10 mM solution in dimethyl formamide.
2. 0.02 M acetate buffer, pH 4.5.
3. 0.2 M sodium carbonate buffer, pH 10.0.
4. 4-Methylumbelliferone.
b. Procedure. Enzyme solution, 520 L, is made
up to 200 L with 0.02 M acetate buffer (pH 5.5) con-
taining 4 L of 10 mM solution of the substrate in
dimethyl formamide. To monitor the enzymic activity
20-L aliquots of the reaction mixture are diluted with
2 mL sodium carbonate buffer, and the liberated 4-
methylumbelliferone is determined uorimetrically.
 One unit is dened as the amount of enzyme that
releases 1 mol of 4-methylumbelliferone per minute.
3. Assay of 1,4--Arabinoxylan
Arabinofuranohydrolase (249)
a. Reagent. Arabinoxylan (wheat, rye, or oats;
Megazyme), 0.1% solution in 0.05 M acetate buffer,
pH 5.0.
b. Procedure. The solution of oats spelt arabi-
noxylan is mixed with a small volume of enzyme solu-
tion, incubated at 30 C for an appropriate time, and
analyzed for L-arabinose by HPLC.
4. Assay of 1,4--Arabinoxylan
Arabinofuranohydrolase (247)
a. Reagents
1. Arabinoxylan (wheat, rye, or oats; Megazyme),
1.0% solution in 0.05 M acetate buffer, pH 5.0.
2. p-Hydroxybenzoic acid anhydride (PANBAH)
reagent for determination of reducing sugars (256).
b. Procedure. 0.9 mL of 1.0% arabinoxylan solu-
tion is mixed with 0.1 mL appropriately diluted
enzyme and incubated at 55 C. The reaction is stopped
by transferring 0.1 mL of the sample to 0.3 mL of the
PANBAH reagent and heating for 5 min at 95 C. The
reducing sugars are determined spectrophotometrically
at 405 nm using L-arabinose as standard. One unit if
enzyme activity is dened as the amount of enzyme
that releases 1 mol of arabinose per minute.
E. Assays for Detection and Screening Purposes
of a-L-Arabinofuranosidases of Families 51
and 54

-L-Arabinofuranosidases which hydrolyze aryl
-L-
arabinofuranosides can be detected in electrophoretic
gels or in screening growth media using two sub-
strates: uorogenic 4-methylumbelliferyl
-L-arabino-
furanoside (255), or chromogenic 5-bromo-3-indolyl-

-L-arabinofuranoside (254). Specic procedures how
to apply these glycosides can be found in the section
devoted to endo--1,4-xylanases. With the rst
substrate, which is commercially available, the
presence of the enzyme results in the liberation of 4-
methylumbelliferone, which shows a strong uores-
cence under UV light (
360 nm). The uorescence
can be enhanced by alkaline agents, e.g., exposing the
gels to ammonium hydroxide vapors. The hydrolysis
of 5-bromo-3-indolyl glycoside results in the forma-
Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.
tion of an intensely colored indigo precipitate. The
glycoside was suggested for use in cloning experi-
ments in which
-L-arabinofuranosidase will be used
as a reporter gene (254).
ACKNOWLEDGMENT
The author thanks Dr. Timothy D. Leathers from the
Biopolymer Unit of the USDA, ARS, in Peoria, IL, for
generous help with English in this chapter.
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