Biotechnology Advances 32 (2014) 316–332

Contents lists available at ScienceDirect

Biotechnology Advances
journal homepage: www.elsevier.com/locate/biotechadv

Research review paper

β-Xylosidases and α-L-arabinofuranosidases: Accessory enzymes for

arabinoxylan degradation
Stijn Lagaert a, Annick Pollet b, Christophe M. Courtin b,⁎, Guido Volckaert a
a
Division of Gene Technology, KU Leuven, Kasteelpark Arenberg 21—box 2462, 3001 Leuven, Belgium
b
Laboratory of Food Chemistry and Biochemistry & Leuven Food Science and Nutrition Research Centre (LFoRCe), KU Leuven, Kasteelpark Arenberg 20—box 2463, 3001 Leuven, Belgium

a r t i c l e i n f o a b s t r a c t

Article history: Arabinoxylan (AX) is among the most abundant hemicelluloses on earth and one of the major components
Received 24 July 2013 of feedstocks that are currently investigated as a source for advanced biofuels. As global research into these
Received in revised form 28 October 2013 sustainable biofuels is increasing, scientific knowledge about the enzymatic breakdown of AX advanced significantly
Accepted 9 November 2013
over the last decade. This review focuses on the exo-acting AX hydrolases, such as α-arabinofuranosidases and β-
Available online 15 November 2013
xylosidases. It aims to provide a comprehensive overview of the diverse substrate specificities and corresponding
Keywords:
structural features found in the different glycoside hydrolase families. A careful review of the available literature
Hemicellulose reveals a marked difference in activity between synthetically labeled and naturally occurring substrates, often
Biomass leading to erroneous enzymatic annotations. Therefore, special attention is given to enzymes with experimental
Xylanolytic enzymes evidence on the hydrolysis of natural polymers.
Hemicellulases © 2013 Elsevier Inc. All rights reserved.
Glycoside hydrolases
Polysaccharide degradation

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 317
2. The xylosidases and arabinofuranosidases of GH 3 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 318
2.1. Substrate specificities . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 318
2.2. Structural data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 318
3. The reducing end xylose-releasing exo-oligoxylanases of GH 8 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 319
3.1. Substrate specificities . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 319
3.2. Structural data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 320
4. The xylosidases of GH 39 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 320
4.1. Substrate specificities . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 320
4.2. Structural data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 320
5. The xylosidases and arabinofuranosidases of GH 43 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 322
5.1. Substrate specificities . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 322
5.2. Structural data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 322
6. The arabinofuranosidases of GH 51 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 324
6.1. Substrate specificities . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 324
6.2. Structural data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 325
7. The xylosidases of GH 52 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 325
7.1. Substrate specificities . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 325
7.2. Structural data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 325
8. The arabinofuranosidases of GH 54 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 326
8.1. Substrate specificities . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 326
8.2. Structural data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 326
9. The arabinofuranosidases of GH 62 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 326
9.1. Substrate specificities . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 326
9.2. Structural data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 327

⁎ Corresponding author. Tel.: +32 16 321917; fax: +32 16 321997.

E-mail address: Christophe.Courtin@biw.kuleuven.be (C.M. Courtin).

0734-9750/$ – see front matter © 2013 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.biotechadv.2013.11.005
 S. Lagaert et al. / Biotechnology Advances 32 (2014) 316–332 317

10. The xylosidases of GH 120 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 327

10.1. Substrate specificities . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 327
10.2. Structural data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 327
11. The enzymes of GH 30 and GH 116 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 327
11.1. Substrate specificities . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 327
12. Synthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 327
Abbreviations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 329
Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 329
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 329

1. Introduction 10 Å apart (Rye and Withers, 2000; Wang et al., 1994) and the reaction
proceeds through a single displacement mechanism. The general acid
Increasing energy costs and environmental concerns have pushed protonates the glycosidic oxygen and the departure of the leaving
the global demand for sustainable renewable fuels. In the European group is accompanied by the nucleophilic attack of a water molecule
Union, Directive 2009/28/EC was implemented in 2010 and sets manda- that has been deprotonated by the general base. In retaining glycosi-
tory goals to achieve a 10% share of renewable energy in the transport dases, the reaction occurs via a two-step double displacement mecha-
sector by 2020. So-called first generation biofuels are made from nism and involves catalytic residues which are approximately 5.5 Å
sugar, starch or vegetable oil extracted from food crops, such as corn, apart (Rye and Withers, 2000; Wang et al., 1994). In the first step, the
sugarcane, soybeans and palms. However, these are under increased general acid protonates the glycosidic oxygen while the second catalytic
scrutiny as they are considered to be responsible for a rise in food prices residue performs a nucleophilic attack at the anomeric carbon. This
and as the land conversion associated with their production may actual- leads to the departure of the leaving group and the formation of a cova-
ly increase carbon dioxide emissions (Fargione et al., 2008; Mitchell, lent intermediate. In the second step, the first catalytic residue now acts
2008). Therefore, the search for beneficial biofuels should focus on as a base and deprotonates an incoming water molecule, which hydro-
sustainable biomass feedstocks, such as waste biomass (e.g. wheat lyzes the glycosyl-enzyme intermediate.
straw) and biomass grown on degraded and abandoned agricultural Different enzymes are needed for the degradation of AX.
lands planted with perennials (e.g. switchgrass) (Fargione et al., 2008; Endoxylanases (EC 3.2.1.8) hydrolyze the backbone in an endo-
Tilman et al., 2009). This is also the aim of the European Commission acting manner, but their activity is frequently hampered by the arabi-
which, in their last proposal for amending the Directive 2009/28/EC,1 nose substitutions (Wong et al., 1988). α-L-Arabinofuranosidases
suggests to cap the share of conventional biofuels in the transport sector (EC 3.2.1.55) cleave arabinose from the backbone and act in synergy
to 5% and to increase the amount of advanced biofuels. Arabinoxylan with endoxylanases. To complete AX degradation, β-xylosidases
(AX) is one of the major components of feedstocks that are currently (EC 3.2.1.37) are needed, which cleave xylose residues from the non-
investigated as a source for advanced biofuels (Fig. 1) (Pauly and reducing end of the xylose chain in an exo-acting manner. β-xylosidases
Keegstra, 2008; Saha, 2003). Unsurprisingly, its enzymatic degradation
is the subject of increasing research efforts. There is a need for more
accurate information on existing and novel AX-degrading enzymes
that can be used in the production advanced biofuels (Dodd and Cann,
2009; Saha, 2003). The properties of these enzymes, such as their
substrate specificity, activity and other biochemical properties need to
be studied and confronted with specific process requirements to evalu-
ate their possible usefulness. In this review, the focus is on diverse
substrate specificities and corresponding structural features found in
exo-acting AX hydrolases.
The AX backbone is composed of β-1,4-linked xylose residues
(Darvill et al., 1980). Arabinose can be substituted at the C(O)2 and/or
C(O)3 positions of the xylose residues (Perlin, 1951a, 1951b) and the
degree of substitution (DS) or arabinose to xylose ratio (A/X) is an
important parameter for AX properties. The arabinosyl residues can
be esterified with hydroxycinnamic acid derivatives, such as ferulic
and p-coumaric acid (Kulkarni et al., 1999; Subramaniyan and Prema,
2002). These hydroxycinnamic acids can from dimers to cross-link
arabinoxylan chains. In addition, ferulic acids can participate in
heterocoupling with monolignols or lignin oligomers, thereby
cross-linking arabinoxylans to lignin. Ferulate cross-linking greatly
impacts lignocellulosic polymer separation and hemicellulose fermen-
tation in general (Grabber et al., 2009; Ishii, 1997).
AX degrading enzymes and other glycosidases hydrolyze the glyco-
sidic bond in a stereoselective way, either with retention or inversion of
the anomeric center (Koshland, 1953; Sinnott, 1990). Both mechanisms
depend on two catalytic residues: a proton donor and a nucleophile/
base. In inverting glycosidases, the catalytic residues are approximately

Fig. 1. Cell-wall polymer (cellulose, hemicellulose and lignin) and hemicellulose composi-
1
Proposal for a directive of the European parliament and of the council, COM (2012) tion for a variety of feedstocks that are currently investigated as a source for advanced
595 (17 October sss). biofuels. Reproduced with permission from Pauly and Keegstra (2008).
318 S. Lagaert et al. / Biotechnology Advances 32 (2014) 316–332

and endoxylanases act in synergy, as endoxylanases generate more re-

ducing ends for β-xylosidases to act on and β-xylosidases remove the
end products that inhibit endoxylanases (Sunna and Antranikian, 1997).
Recently, reducing end xylose-releasing exo-oligoxylanases (EC
3.2.1.156) were discovered which act in an exo-acting manner on the re-
ducing end of the xylose chain. Lately, several comprehensive reviews ap-
peared on xylanases (Collins et al., 2005b; Pollet et al., 2010a). This review
will focus on the exo-acting enzymes.
Glycosidases are classified into glycoside hydrolase families (GH) in
the Carbohydrate Active Enzyme (CAZy) database (www.cazy.org) on
the basis of their amino acid sequence similarities (Cantarel et al.,
2009). Hence, this classification reflects the structural features,
evolutionary relationship and catalytic mechanism of the enzymes
(Cantarel et al., 2009; Gebler et al., 1992). Arabinofuranosidases are pres-
ent in GH 3, 43, 51, 54 and 62, while xylosidases are found in GH 3, 30, 39,
43, 52, 54, 116 and 120. All these families perform hydrolysis with reten-
tion of the anomeric configuration, except for GH 43, which is an
inverting GH. The reaction mechanism for GH 62 is not yet known. The
two characterized reducing end xylose-releasing exo-oligoxylanases
belong to GH 8, which is known to invert the anomeric configuration.

2. The xylosidases and arabinofuranosidases of GH 3

2.1. Substrate specificities

With over 4000 sequences, GH 3 is one of the largest families

according to the CAZy database and besides xylosidases and
arabinofuranosidases, it includes β-glucosidases (EC 3.2.1.21), β-N-
acetylhexosaminidases (EC 3.2.1.52), glucan 1,3-β-glucosidases (EC
3.2.1.58), glucan 1,4-β-glucosidases (EC 3.2.1.74) and exo-1,3-1,4-
glucanases (EC 3.2.1.-). A phylogenetic tree of the characterized GH 3
enzymes produced with ClustalX (Larkin et al., 2007) (http://www.
clustal.org/) reveals that, besides two bifunctional xylosidases/β-
glucosidases, all xylosidases and arabinofuranosidases are present in
two clades, A and B (Fig. 2). Clade A is composed of three clusters: one
collecting all the fungal AX degrading enzymes, one all those from plants
and a smaller cluster with bacterial sequences. Clade B gathers the other
bacterial enzymes and one archaeal xylosidase/arabinofuranosidase.
SignalP and TargetP analyses (Emanuelsson et al., 2007) (http://www.
cbs.dtu.dk/services/) indicate all eukaryotic AX hydrolases are secreted,
while all bacterial and archaeal enzymes lack a signal peptide, except for
the enzymes from Prevotella (GenBank ID: ADD92014,2 ADD92015,
ADD92016 and ACN78955) and from an unidentified rumen bacterium
(CAP07659).
Of special interest in GH 3 is the presence of several enzymes report-
ed to be bifunctional. However, this classification is most of the time
based on the ability of these enzymes to cleave several synthetic sub-
strates, such as p-nitrophenylxyloside (pNP-Xyl), pNP-arabinoside
(pNP-Ara) and pNP-glucoside (pNP-Glu). When tested on natural
substrates, most enzymes show only the release of one type of sugar
and are thus from a biological and applied perspective monofunctional
(Table 1). The only true bifunctional AX degrading enzymes, which
can release both xylose and arabinose from natural substrates, are
plant hydrolases from Arabidopsis thaliana (AAM53325), Hordeum
vulgare (AAK38481) and Medicago sativa (ABQ45227).
When looking at the activity of the enzymes on natural substrates, it
is clear that arabinofuranosidase activity is exclusively found in the
cluster of plant glycosidases. These arabinofuranosidases are able to
cleave arabinose from arabinan or arabino-oligosaccharides as well as
from AX. Only the GH 3 arabinofuranosidase from Pyrus pyrifolia
(BAD98523) is not active on AX and liberates only arabinose from
2
All protein ID's refer to GenBank Accession numbers for proteins.
Fig. 2. Phylogenetic tree of the characterized glycoside hydrolase family 3 (GH 3) glycosi-
dases. GenBank Accession numbers are shown succeeded by their activity (the EC number,
excluding 3.2.1.), e.g. xylosidases: 37 and arabinofuranosidases: 55. For bifunctional
enzymes, the activities are separated by a slash. Arabinofuranosidase and xylosidase activ-
ities are indicated with red branches.
arabinan, arabino-oligosaccharides and plant cell wall polysaccharides
(Tateishi et al., 2005). All GH 3 AX hydrolases from microorganisms
show exclusively xylosidase activity on substrates such as xylo-
oligosaccharides (XOS), xylan and AX. Furthermore, AAA80156, one of
the two AX degrading enzymes that are found separated from the two
large clades, does not show xylosidase activity on natural substrates.
Specificity constants of xylosidases towards XOS with a varying
degree of polymerization (DP) were determined for the GH 3 xylosidases
from Hypocrea jecorina (presumably CAA93248) and Talaromyces
emersonii (presumably AAL32053) (Rasmussen et al., 2006). kcat/Km
values increase from DP2 up to DP5 and show a slow decrease at DP6,
suggesting these xylosidases may interact with the substrate at 5
subsites (−1 and +1 to +4). Similar observations were made for the
barley exo-1,3-1,4-glucanases which show markedly higher specificity
constants towards polymeric substrates than towards disaccharides
(Hrmova and Fincher, 1997).
2.2. Structural data
Despite the size of GH 3, little structural data is available. To date,
the structures of only six enzymes have been deposited in the pro-
tein databank (http://www.pdb.org/). No database information is
available for a xylosidase or arabinofuranosidase, although the struc-
ture of a H. jecorina xylosidase (CAA93248) was recently briefly pre-
sented (Sandgren et al., 2009) and a xylosidase from Streptomyces
thermoviolaceus (BAB61064) has been crystallized (Morioka et al.,
2010).
The general fold of GH 3 enzymes is an N-terminal (α/β)8 TIM barrel
fold that is connected through a short linker with an (α/β) sandwich
domain (Harvey et al., 2000). The size of this last domain is different
 S. Lagaert et al. / Biotechnology Advances 32 (2014) 316–332 319

Table 1
Activities of bifunctional arabinoxylan (AX) glycoside hydrolase family 3 (GH 3) enzymes with AX degrading activities on natural substrates. AGP: arabinogalactan protein, AOS: arabino-
oligosaccharides, AXOS: arabinoxylan-oligosaccharides, CWP: cell wall polysaccharides, XOS: xylo-oligosaccharides.

Accession nr. Organism Activity (CAZy) Activity (natural substrates) Remarks Reference

Archaea
AAK43134 Sulfolobus solfataricus P2 3.2.1.37 3.2.1.37 (XOS, xylan) Morana et al. (2007)
3.2.1.55

Bacteria
AAA80156 Erwinia chrysanthemi D1 3.2.1.21 No activity (xylan, cellobiose) Vroemen et al. (1995)
3.2.1.37
ACN78955 Prevotella ruminicola 23 3.2.1.37 3.2.1.37 (XOS, AX) Dodd et al. (2009)
3.2.1.55
CAA91219 Thermoanaerobacter brockii 3.2.1.21 None tested Breves et al. (1997)
3.2.1.37
AAB23220 Thermoanaerobacter ethanolicus JW 200 3.2.1.37 None tested Mai et al. (2000)
3.2.1.55

Eukaryotes (Fungi)
EAA67023 Aspergillus nidulans FGSC A4 3.2.1.37 3.2.1.37 (XOS) 3.2.1.55 (AX) stated, but not apparent Bauer et al. (2006)
3.2.1.55 from data

Eukaryotes (Plants)
AAM53325 Arabidopsis thaliana 3.2.1.37 3.2.1.37 and 3.2.1.55 (AX, AXOS, arabinan) More release of arabinose Minic et al. (2004)
3.2.1.55
AAK96639 Arabidopsis thaliana 3.2.1.37 3.2.1.55 (AX, arabinan) Charles et al. (2006)
3.2.1.55
AAK38481 Hordeum vulgare 3.2.1.37 3.2.1.37 and 3.2.1.55 (AX, AXOS, XOS, AOS) More release of xylose from AXOS Lee et al. (2003)
3.2.1.55
ABQ45227 Medicago sativa subsp. × varia 3.2.1.37 3.2.1.37 and 3.2.1.55 (AX, XOS, AOS, CWP) Xiong et al. (2007)
3.2.1.55
BAE44362 Raphanus sativus 3.2.1.37 3.2.1.55 (AX, arabinan, AGP) Kotake et al. (2006)
3.2.1.55

Unidentified
ACY24766 Uncultured organism 3.2.1.21 3.2.1.37 (xylan, XOS) Beloqui et al. (2010)
3.2.1.37

among several enzymes, with an (α/β)5 sandwich domain in the
Thermotoga neapolitana β-glucosidase (ABI29899) (Pozzo et al., 2010)
and an (α/β)6 domain in the Klyuveromyces marxianus β-glucosidase
(ACY95404) (Yoshida et al., 2010) and barley β-glucan glucohydrolase
(AAD23382) (Varghese et al., 1999). In the N-acetylglucosaminidases
it is often much shorter, e.g. AAA64351 from Bacillus subtilis which has
only an αβα sandwich domain (Litzinger et al., 2010), or is even
completely absent like in AAF93857 from Vibrio cholerae (Stubbs et al.,
2007). In about one tenth of the GH 3 enzymes, a PA14 domain is
inserted in the (α/β) sandwich domain (Yoshida et al., 2010). This
domain was shown to bind glucose and to play a critical role in the
substrate specificity of the K. marxianus β-glucosidase (Yoshida et al.,
2010). However, Pfam analysis (http://pfam.sanger.ac.uk/) shows this
PA14 domain is not widespread within the characterized AX hydrolases
and is limited to β-xylosidases from Prevotella ruminocola (ACN78955),
Prevotella bryantii (ACN78955) and an unidentified organism
(ACY24766). The (α/β)8 TIM barrel and (α/β) sandwich domains can
be further complemented with a third domain at the C-terminus
(Harvey et al., 2000) and the β-glucosidases of T. neapolitana and
K. marxianus both have a C-terminal fibronectin type III domain of
unknown function (Pozzo et al., 2010; Yoshida et al., 2010).
The GH 3 catalytic nucleophile is an aspartate, which is conserved
among all family members and is located in the N-terminal (α/β)8
TIM barrel domain. The catalytic acid is less conserved and difficult if
not impossible to predict from simple alignments (Harvey et al.,
2000). In studied β-glucosidases, a β-glucan glucohydrolase and a
glucosylceramidase, the catalytic acid is a glutamate that is positioned
in the (α/β) sandwich domain and the active site is located at the
interface of the two domains (Chir et al., 2002; Paal et al., 2004; Pozzo
et al., 2010; Varghese et al., 1999; Yoshida et al., 2010). In N-
acetylglucosaminidases, where the (α/β) sandwich domain is often
much reduced, a conserved histidine in the (α/β)8 TIM barrel domain
is suggested to perform the general acid/base function (Litzinger et al.,
2010). The active site of barley β-glucan glucohydrolase and the β-
glucosidases from K. marxianus and T. neopolitana forms a pocket with a
−1 and +1 subsite, with most of the interactions taking place at the −
1 subsite (Pozzo et al., 2010; Yoshida et al., 2010).
Currently, the little structural information that is available of a GH 3
AX hydrolase stems from a β -xylosidase from H. jecorina. This enzyme
was shown to have three domains, the typical GH 3 N-terminal (α/β)8
TIM barrel and (α/β)6 sandwich domain, followed by a third domain
with unspecified fold (Rojas et al., 2005; Sandgren et al., 2009). A
more detailed publication of this structure and the structure of the
crystallized β-xylosidase from S. thermoviolaceus should provide more
information in the near future (Morioka et al., 2010).
3. The reducing end xylose-releasing exo-oligoxylanases of GH 8
3.1. Substrate specificities
GH 8 contains almost 600 sequences, mostly coding for cellulases
(EC 3.2.1.4) and chitosanases (EC 3.2.1.132), but there are also
licheninases (EC 3.2.1.73), xylanases (EC 3.2.1.8) and reducing end
xylose-releasing exo-oligoxylanases (EC 3.2.1.156, further called rex
hydrolases) present. A phylogenetic analysis shows that xylanases
form a distinct clade and rex hydrolases constitute a separate branch
within this clade, suggesting rex hydrolases evolved from xylanases
(Lagaert et al., 2007). For the moment, only two rex hydrolases have
been characterized, Rex from Bacillus halodurans (BAB05824) and
RexA from Bifidobacterium adolescentis (BAF39081) (Honda and
Kitaoka, 2004; Lagaert et al., 2007). As their names imply, they constitu-
tively release a xylose residue from the reducing end of XOS. They don't
hydrolyze xylobiose, have a preference for xylotriose and their specific-
ity constants decrease as the DP of the XOS increases. While RexA shows
low activity on polymeric xylan, Rex is not active on this substrate. They
are not able to hydrolyze xylose or XOS labeled with pNP at the reducing
end (Honda and Kitaoka, 2004; Lagaert et al., 2007, 2011). GH 8 en-
zymes are known to act with inversion of the anomeric configuration
320 S. Lagaert et al. / Biotechnology Advances 32 (2014) 316–332
and for RexA, it was shown that XOS hydrolysis leads to the release
of the β-anomer of xylose and the α-anomer of XOS (Honda and
Kitaoka, 2004). Furthermore, RexA exclusively hydrolyzes XOS in the
β-anomer configuration and the produced α-anomeric XOS must thus
undergo a spontaneous mutarotation before the next xylose residue
can be cleaved off (Honda and Kitaoka, 2004).
3.2. Structural data
The overall topology of GH 8 is an (α/α)6 barrel fold, also present in
the structures of the Pseudoaltermonas haloplanktis xylanase and
B. halodurans rex hydrolase (Fushinobu et al., 2005; Van Petegem
et al., 2003). In the P. haloplanktis xylanase, the active site is situated
in a cleft that accommodates the substrate with six subsites: − 3
to − 1 binds three glycon xyloses, while + 1 to + 3 accommodates
three aglycon xyloses. Modeling and substrate specificity analysis indi-
cates that subsite + 3 is partially blocked by a small loop in a GH 8
xylanase from an uncultured bacterium (ABB71891) (Pollet et al.,
2010b). In the B. halodurans rex hydrolase, this loop has a far more pro-
found effect as its Leu318 and His319 completely block subsite + 2
(Fig. 3) (Fushinobu et al., 2005). His319 is also involved in a direct
hydrogen bond with the β-hydroxyl of the xylose at subsite +1, there-
by contributing to the discrimination of the anomeric configuration of
the XOS substrate. These structural features support the descent of rex
hydrolases from GH 8 xylanases.
The proton donor is a glutamate (Glu70 in B. halodurans rex hydro-
lase numbering) that is completely conserved in GH 8. The general
base, however, is not conserved and, depending on its position, GH 8
is divided in three subfamilies (GH 8a, GH 8b and GH 8c) (Adachi
et al., 2004). The GH 8 endoxylanases and rex hydrolases belong to
subfamily 8a and have an aspartate as a catalytic base (Asp263)
(Collins et al., 2005a; Honda and Kitaoka, 2004). A third conserved cat-
alytically important residue is another aspartate (Asp128) that is in-
volved in sugar-ring distortion and transition-state stabilization
(Collins et al., 2005a; De Vos et al., 2006; Honda and Kitaoka, 2004).
Fig. 3. The substrate binding cleft of glycoside hydrolase family 8 (GH 8) B. halodurans rex
hydrolase. Cartoon representation of B. halodurans rex hydrolase in complex with
xylobiose (yellow) in subsites −1 and −2 (PDB ID: 1WU6), the xylose residue in subsite
+1 is from PDB ID: 1WU5. The Leu and His from the loop that blocks subsite +2 are
colored magenta.
4. The xylosidases of GH 39
4.1. Substrate specificities
GH 39 is a relatively small family (less than 400 sequences) and
the 12 characterized enzymes are mammalian α-L-iduronidases (EC
3.2.1.76) and bacterial xylosidases. Few β-xylosidases were tested on
natural substrates. A B. halodurans xylosidase (BAB04787) releases
xylose from XOS, arabinoxylan-oligosaccharides (AXOS) and
AX, but hydrolysis of xylobiose is barely detectable (Smaali
et al., 2006; Wagschal et al., 2008). The GH 39 xylosidases
from Thermoanaerobacterium saccharolyticum and Clostridium
stercorarium (AAA27369 and AAA23063) also exhibit only limited
activity on xylobiose (Adelsberger et al., 2004; Lee and Zeikus,
1993; Wagschal et al., 2005) and the T. saccharolyticum xylosidase
has a much higher activity towards xylotriose. Neither enzyme
shows any activity towards polymeric AX, but the C. stercorarium
xylosidase binds xylan with enough strength to be used for affinity
purification (Adelsberger et al., 2004; Lee and Zeikus, 1993).
When incubated with xylobiose, the wild type xylosidases from
B. halodurans and T. saccharolyticum both produce detectable amounts
of xylotriose (Lee and Zeikus, 1993; Muzard et al., 2009; Smaali et al.,
2006). When pNP-Xyl is used as a substrate, the enzymes produce a va-
riety pNP-xylobiosides and pNP-xylotriosides (Armand et al., 1996;
Muzard et al., 2009). However, these condensation reactions proceed
with poor regioselectivity, as the products contain β-1,2, β-1,3 and β-
1,4 linkages. In the presence of methanol, the B. halodurans xylosidase
generates methyl D-xylosides from pNP-Xyl, xylobiose and xylotriose
(Muzard et al., 2009). With xylotriose as a donor, the xylosidase
shows a clear preference towards primary alcohols with a low chain
length (C1–C5) as an acceptor.
4.2. Structural data
Several GH 39 xylosidases exist as tetramers in solution (Czjzek
et al., 2004b; Wagschal et al., 2008; Yang et al., 2004). This tetrameric
form is also observed in the crystal structures of the xylosidases from
G. stearothermophilus and T. saccharolyticum (Czjzek et al., 2005; Yang
et al., 2004). Both enzymes have a three domain structure: a catalytic
(β/α)8 barrel domain is linked to a β-sandwich domain which is follow-
ed by a small α-helical domain. The role of the two non-catalytic
domains is unknown, but they may be involved in multimer formation
or carbohydrate binding. All characterized GH 39 xylosidases from ther-
mophilic bacteria contain these three domains (Czjzek et al., 2005).
Catalysis takes place in a pocket at the bottom of a deep cleft (Fig. 4).
Fig. 4. Molecular surface of the glycoside hydrolase family 39 (GH 39) xylosidase from
G. stearothermophilus in complex with 2,5-dinitro-phenyl-β-D-xyloside (PDB ID: 2BFG,
chain A).
 S. Lagaert et al. / Biotechnology Advances 32 (2014) 316–332 321

Although the largest complexed substrate is a 2,5-dinitro-phenyl-β-D-
xyloside that shows interactions at the −1 and +1 subsite, the depth
of the cleft suggests more aglycon subsites (Fig. 4). At least a +2 subsite
is to be expected based on the lower activity on xylobiose compared to
xylotriose.
Glu277 was identified as the catalytic nucleophile in the
T. saccharolyticum xylosidase, while Glu160 was found to be the
acid/base catalyst in the xylosidases of T. saccharolyticum and
B. stearothermophilus (Bravman et al., 2001a; Vocadlo et al., 1998,
2002). These catalytic residues are strictly conserved in GH 39 and the
distance of 5 Å between them is consistent with the retaining mecha-
nism of the enzyme family (Armand et al., 1996; Yang et al., 2004).
The Glu160Ala xylosidase from T. saccharolyticum, but not from
G. stearothermophilus, displays an unusual bell-shaped pH profile,

Table 2
Activities of all glycoside hydrolase family 43 (GH 43) enzymes that were tested on natural substrates. AOS: arabino-oligosaccharides, AX: arabinoxylan, AXOS: arabinoxylan-
oligosaccharides, debr. arabinan: debranched arabinan, GOS: galacto-oligosaccharides, XOS: xylo-oligosaccharides.

Accession nr. Organism Activity (natural substrates) Reference

Exo-α-1,5-arabinofuranosidase
ADB43999 Uncultured bacterium 3.2.1.- (debr. arabinan) Wong et al. (2008)
BAA90772 Streptomyces chartreusis GS901 3.2.1.- (debr. arabinan) Matsuo et al. (2000)
BAC68753 Streptomyces avermitilis MA-4680 3.2.1.- (debr. arabinan) Ichinose et al. (2008)

β-1,3-xylosidase
BAF98235 Vibrio sp. XY-214 3.2.1.- (β-1,3-XOS) Umemoto et al. (2008)

Xylanase
AAB95326 Caldicellulosiruptor sp. Rt69B.1 3.2.1.8 (xylan) Morris et al. (1999)
AAD30363 Caldicellulosiruptor sp. Tok7B.1 3.2.1.8 (xylan) Gibbs et al. (2000)
ACZ98594 Cellulosilyticum ruminicola CGMCC 1.5065 3.2.1.8 (xylan) Cai et al. (2010)

Galactan 1,3-β-galactosidase
ABN51896 Clostridium thermocellum ATCC 27405 3.2.145 (GOS, galactan) Ichinose et al. (2006b)
BAC69820 Streptomyces avermitilis MA-4680 3.2.145 (galactan) Ichinose et al. (2006a)
BAH29957 Irpex lacteus NBRC 5367 3.2.145 (GOS, galactan) Kotake et al. (2009)
BAD98241 Phanerochaete chrysosporium 3.2.145 (GOS, galactan) Ichinose et al. (2005)

β-xylosidase
ABR49445 Alkaliphilus metalliredigens QYMF 3.2.1.37 (XOS) Jordan et al. (2013)
BAB07402 Bacillus halodurans C-125 3.2.1.37 (AXOS) Smaali et al. (2006); Wagschal et al. (2012)
CAA29235 Bacillus pumilis IPO 3.2.1.37 (XOS) Panbangred et al. (1984); Xu et al. (1991)
AAC97375 Bacillus pumilis PLS 3.2.1.37 (xylan) La Grange et al. (2000)
AAB08024 Bacteroides ovatus V975 3.2.1.37 (XOS) Whitehead and Hespell (1990)
BAF39209 Bifidobacterium adolescentis ATCC 15703 3.2.1.37 (XOS, AXOS) Lagaert et al. (2011)
ADC85541 Bifidobacterium animalis subsp. lactis BB-12 3.2.1.37 (XOS) Gilad et al. (2010)
AAA63610 Butyrivibrio fibrisolvens GS 113 3.2.1.37 (XOS) Sewell et al. (1989)
AAT98625 Geobacillus stearothermophilus T-6; NCIMB 40222 3.2.1.37 (XOS) Shallom et al. (2005)
ABC75004 Geobacillus thermoleovorans IT-08 3.2.1.37 (XOS, xylan) Wagschal et al. (2009b)
CAA89208 Prevotella bryantii B14 3.2.1.37 (XOS, xylan) Gasparic et al. (1995)
AAB97967 Selenomonas ruminantium GA192 3.2.1.37 (XOS, xylan) Whitehead and Cotta (2001)
XP_391644 Gibberella zeae PH-1 3.2.1.37 (AXOS) Carapito et al. (2009)
BAC75546 Penicillium herquei IFO 4674 3.2.1.37 (AXOS) Ito et al. (2003)

α-L-arabinofuranosidase
CAB13699 Bacillus subtilis subsp. subtilis str. 168 3.2.1.55 (AX, AXOS) Bourgois et al. (2007)
BAF39204 Bifidobacterium adolescentis ATCC 15703 3.2.1.55 (AX, AXOS) Lagaert et al. (2010)
AAO67499 Bifidobacterium adolescentis DSM 20083 3.2.1.55 (AX, AXOS, arabinan) Lagaert et al. (2010); van den Broek et al. (2005)
XP_391670 Gibberella zeae PH-1 3.2.1.55 (AXOS) Carapito et al. (2009)
CAA40378 Paenibacillus polymyxa ATCC 842 3.2.1.55 (AX) Morales et al. (1995)
ABB92159 Uncultured bacterium 3.2.1.55 (AX, arabinan) Wagschal et al. (2007)
CAL81199 Humicola insolens DSM 18000 3.2.1.55 (AX, AXOS) McKee et al. (2012); Sørensen et al. (2006);
Sørensen et al. (2007)

Bifunctional β-xylosidase/α-L-arabinofuranosidase
ACF39706 Uncultured bacterium 3.2.1.37 (XOS, xylan) and Wagschal et al. (2009a)
3.2.1.55 (AX, AOS, arabinan)

Arabinanase
BAA20372 Bacillus subtilis IFO3134 3.2.1.99 (arabinan) Sakamoto et al. (1997)
CAB15969 Bacillus subtilis subsp. subtilis str. 168 3.2.1.99 (arabinan) Inácio and de Sá-Nogueira (2008)
CAA99586 Bacillus subtilis subsp. subtilis str. 168 3.2.1.99 (arabinan) Proctor et al. (2005)
BAB64339 Bacillus thermodenitrificans TS-3 3.2.1.99 (arabinan) Takao et al. (2002)
ACE84667 Cellvibrio japonicus Ueda 107 3.2.1.99 (arabinan) McKie et al. (1997)
ACE73676 Geobacillus stearothermophilus T-6 3.2.1.99 (arabinan) Alhassid et al. (2009)
ABQ46657 Thermotoga petrophila RKU-1 3.2.1.99 (arabinan) Squina et al. (2010)
ADB43998 Uncultured bacterium 3.2.1.99 (arabinan) Wong et al. (2009)
AAG27441 Aspergillus aculeatus CBS 101.43 3.2.1.99 (debr. arabinan) Skjøt et al. (2001)
EAA58736 Aspergillus nidulans FGSC A4 3.2.1.99 (debr. arabinan) Bauer et al. (2006)
EAA58810 Aspergillus nidulans FGSC A4 3.2.1.99 (debr. arabinan) Bauer et al. (2006)
AAA32682 Aspergillus niger CBS120.49/N400 3.2.1.99 (arabinan) Flipphi et al. (1993a); Veen et al. (1991)
BAD15018 Penicillium chrysogenum 31B 3.2.1.99 (AOS) Sakamoto et al. (2005)
322 S. Lagaert et al. / Biotechnology Advances 32 (2014) 316–332
suggesting an auxiliary acid/base (Vocadlo et al., 2002). Likely, Glu165 is
responsible for this behavior, as its rotameric forms can be within
hydrogen bonding distance from Glu160 (Czjzek et al., 2005).
5. The xylosidases and arabinofuranosidases of GH 43
5.1. Substrate specificities
Besides xylosidases and arabinofuranosidases, GH 43 contains
arabinanases (EC 3.2.1.99), galactan 1,3-β-galactosidases (EC 3.2.1.145),
xylanases (EC 3.2.1.8), exo-α-1,5-L-arabinofuranosidases (EC 3.2.1.-)
and a β-1,3-xylosidase (EC 3.2.1.72). All characterized enzymes are
from fungal or bacterial origin, although the CAZy database also presents
GH 43 sequences from archeae and higher plants (A. thaliana and
Z. mays). Like GH 3, this family is known for its many bifunctional
enzymes and a phylogenetic tree shows a quasi random distribution of
arabinofuranosidase, xylosidase and bifunctional arabino-furanosidase/
xylosidase activities (not shown). However, as is also the case in GH 3,
this is mostly the result of the broad substrate specificity towards
synthetic pNP- or methylumbelliferyl (MUF)-linked monosaccha-
rides. When looking at the experimental evidence, all except one
cleave only a single type of sugar from natural substrates (Table 2). The
one enzyme that displays bifunctional xylosidase/arabinofuranosidase
activity (ACF39706) releases xylose from XOS and xylan and arabinose
from arabino-oligosaccharides, arabinan and AX (Wagschal et al.,
2009a). A phylogenetic tree of GH43 sequences with known enzyme
activities on natural substrates show 5 clades with a much less random
distribution (Fig. 5). Xylosidases and arabinofuranosidases are both
present in clades A and C, while exo-α-1,5-L-arabinofuranosidases,
galactan 1,3-β-galactosidases and arabinanases are found in clades B, D
and E, respectively.
Clade A contains two clusters, one with β-xylosidases (cluster A1)
and one with arabinofuranosidases and xylanases (cluster A2), although
the occurrence of xylanase activities in GH 43 should be considered with
some reservations. Two of the four xylanases are from Caldicellulosiruptor
sp. Rt69B.1 and Tok7B.1 (AAB95326 and AAD30363, respectively). Both
enzymes contain a GH 10 and GH 43 domain (Gibbs et al., 2000;
Morris et al., 1999). As GH 10 is a family that exclusively holds xylanases,
the reported xylanase activity most likely originates from this domain. A
Fig. 5. Phylogenetic tree of the glycoside hydrolase family 43 (GH 43) enzymes with char-
acterized activity on natural substrates. Nomenclature as in Fig. 2.
third enzyme (CAA40378 from Paenibacillus polymyxa) showed xylanase
activity using a zymogram analysis (Gosalbes et al., 1991). However, a
later study could not detect xylanase activity and observed only arabi-
nose release from AX (Morales et al., 1995). The last GH 43 xylanase is
only described recently and limited experimental information is avail-
able (Cai et al., 2010). In this case, xylanase activity was also only demon-
strated with zymogram analysis. To add to the confusion, CAZy lists this
enzyme as a xylosidase instead of a xylanase and suggests the sequence
is only a fragment of the mature protein, although we could not find a
literature reference for this hypothesis. In conclusion, we believe more
experimental evidence is needed to firmly state xylanase activity linked
to a GH 43 domain.
In clade C three clusters can be detected. Two arabinofuranosidases
form cluster 1 (C1), cluster 2 (C2) contains both arabinofuranosidases
and xylosidases and cluster 3 (C3) groups xylosidases. Interestingly,
the bifunctional xylosidase/arabinofuranosidase (ACF39706) in cluster
2 is positioned between xylosidases and arabinofuranosidases, suggest-
ing it may be an intermediate in the evolution of the former to the latter.
In the same regard, the β-1,3-xylosidase (BAF98235 from Vibrio sp. XY-
214) is located at the base of cluster 3, where it branches of from
arabinofuranosidases. Combined with rotation of the substrate between
GH 43 arabinofuranosidases and xylosidases (see Section 5.2), this may
suggest that the β-1,3-xylosidase is an intermediate form in the evolu-
tion of these two activities.
Specificity constants towards XOS with different lengths were
determined for the xylosidases from B. adolescentis (BAF39209),
S. ruminantium (AAB97967) and G. thermoleovorans (ABC75004). kcat/
Km values are similar for DP 2–6 for the B. adolescentis enzyme, while
the S. ruminantium xylosidase shows a preference towards xylobiose
and specificity constants decrease with larger chain lengths (DP 2–6)
(Jordan et al., 2007; Lagaert et al., 2011). The activity of the
G. thermoleovorans xylosidase was only evaluated on xylobiose
and xylotriose, but this enzyme also showed a higher substrate
preference towards xylobiose than xylotriose (Wagschal et al.,
2009b). These observations indicate that XOS interact with GH 43
xylosidases at two subsites (− 1 and + 1).
The substrate specificities towards specific arabinose linkages in AX
are characterized for a number of GH 43 arabinofuranosidases. The two
enzymes that form cluster C1 (CAL81199 and AAO67499) both cleave
the arabinose at the C(O)3 position from xyloses with substitutions at
the C(O)2 as well as the C(O)3 position (Lagaert et al., 2010; McKee
et al., 2012; Sørensen et al., 2006, 2007; van den Broek et al., 2005).
On the other hand, BAF39204 (part of cluster C2) and CAB13699 (part
of A2) exclusively hydrolyze arabinose linkages from xyloses that have
only one arabinose residue linked to their C(O)2 or C(O)3 positions
(Bourgois et al., 2007; Lagaert et al., 2010). These two types of
arabinofuranosidases work in synergy, as removal of C(O)3-linked arab-
inoses from disubstituted xyloses by the first type of enzymes, such as
belonging to cluster C1, makes more C(O)2-linked arabinoses available
for hydrolysis by the second type of enzymes, such as those belonging
to cluster C2 or A2 (Lagaert et al., 2010; Sørensen et al., 2006; Van
Laere et al., 1999).
5.2. Structural data
Three enzymes from cluster C3 (BAF98235, AAB97967 and
AAT98625) exist as a tetramer in solution (Brunzelle et al., 2008; Brüx
et al., 2006; Umemoto et al., 2008). AAB97967 and AAT98625 were
shown to be dimers of dimers with stronger interactions between the
monomers composing the dimers than between the dimers that form
the tetramers. Another enzyme from this cluster (BAB07402) appears
as a dimer (Smaali et al., 2006), while enzymes from other clusters
and clades are monomeric (Alhassid et al., 2009; Squina et al., 2010;
Vandermarliere et al., 2009; Wagschal et al., 2007, 2009a). A multimeric
quaternary structure may therefore be limited to the enzymes of
cluster C3.
 S. Lagaert et al. / Biotechnology Advances 32 (2014) 316–332 323

The signature structure of GH 43 enzymes is a catalytic domain with a
five-bladed β-propeller fold (Nurizzo et al., 2002). The crystal structure
of two xylosidases (AAB97967 from S. ruminantium and AAT98625
from G. stearothermophilus, both part of cluster C3) and one arab-
inofuranosidase (CAB13699 from B. subtilis, cluster A2, and CAL81199
from H. insolence, cluster C1) have been published (Brunzelle et al.,
2008; Brüx et al., 2006; McKee et al., 2012; Vandermarliere et al.,
2009). All four enzymes contain an additional C-terminal β-sandwich
domain. Multiple alignment of characterized GH 43 enzymes indicates
that this second domain is present in the whole family except in the
clusters A1, E1 and clade B (not shown). In cluster A2 this domain is clas-
sified in the CAZy database as a carbohydrate binding module of family 6
(CBM 6). CBM 6 modules have two binding sites that can interact with a
variety of different substrates, like xylan and XOS, cellulose and cello-
oligosaccharides, lamarin and glucans (Fernandes et al., 1999; Henshaw
et al., 2004). At least in the structure of the GH 43 arabinofuranosidase
of B. subtilis, aromatic residues of CBM 6 that play a crucial role in carbo-
hydrate binding are lacking in the first binding site and a loop blocks the
second site, suggesting a loss of substrate binding during evolution
(Vandermarliere et al., 2009). However, not all GH 43 enzymes have
lost the binding capabilities of this module. The β-sandwich domain
from the exo-1,5-α-L-arabinofuranosidase from Streptomyces avermitilis
(BAC68753 from clade B) binds branched arabinan and AX and its re-
moval leads to a decreased activity against debranched arabinan
(Ichinose et al., 2008). The crystal structure of this enzyme shows arabi-
nose bound at three sites in this C-terminal domain that belongs to CBM
42.
The different nature of the activities found in GH 43, e.g. arab-
inanases (endo-hydrolysis of a linear backbone), xylosidases and exo-
1,5-α-L-arabinofuranosidases (exo-hydrolysis of a linear backbone)
and arabinofuranosidases (exo-hydrolysis of backbone substituents)
requires different substrate orientations in the active site (Fig. 6). In
xylosidases and exo-1,5-α-L-arabinofuranosidases, the active site is a
pocket that harbors the sugar residue at the non-reducing end and the
rest of the substrate backbone is positioned at right angles to the
enzyme surface. The substrate is positioned similarly in arabinanases,
but here the active site doesn't form a pocket but a cleft and allows
the substrate to continue along − 2 and − 3 subsites. In contrast, in
arabinofuranosidases, the xylose backbone is positioned perpendicular
to the previous substrates and is bound in a long groove on the enzymes
surface and the active site is a pocket at the bottom of this groove
(Vandermarliere et al., 2009). The structure of the H. insolens
arabinofuranosidase (CAL81199) was recently published and reveals
the structural determinants for the specificity towards C(O)3-linked
arabinose on disubstituted xyloses (McKee et al., 2012). In addition to
a deep pocket that houses the active site, the arabinofuranosidase hous-
es a shallower adjacent pocket that binds to the C(O)2-linked arabinose
of these xylose residues.
Three residues are completely conserved in family 43, two aspar-
tates and a glutamate. Despite the different activities and substrate
orientations, these residues are similarly positioned in the active site.
In the xylosidase from G. stearothermophilus Asp15 and Glu187 were
demonstrated to be the general base and general acid, respectively
(Shallom et al., 2005). The third catalytic residue, Asp128, is thought
to be involved in pKa modulation and the orientation of the catalytic
acid and the substrate (Brüx et al., 2006; Nurizzo et al., 2002). It has
been noted that glycoside hydrolases with five-bladed β-propeller
folds never conform to the general distances between the catalytic
residues of inverting and retaining enzymes (Brunzelle et al., 2008).
Indeed, although GH 43 enzymes act with inversion of the anomeric
configuration (Jordan et al., 2007; Kersters-Hilderson et al., 1976; Pitson
et al., 1996), the distance between the general acid and general base is

Fig. 6. Different orientations of substrates in glycoside hydrolase family 43 (GH 43) enzymes. A. Superposition of different substrates bound in the active site of GH 43 enzymes. Active site
residues are shown in green. Orange: arabinohexaose bound to C. japonicus arabinanase (PDB ID: 1GYE), magenta: arabinose in the −1 subsite and arabinobiose in the +1 to +2 subsite of
Streptomyces avermitilis exo-1,5-α-arabinofuranosidase (PDB ID: 3AKH), blue: xylobiose from G. stearothermophilus β-xylosidase (PDB ID: 2EXJ), yellow: xylotetraose bound to B. substilis
arabinofuranosidase (PDB ID: 3C7O), this substrate does not contain the glycon arabinose that would be located in the −1 subsite. B. Molecular surface of the C. japonicus arabinanase
(1), the B. substilis arabinofuranosidase (2) and the G. stearothermophilus xylosidase (3).
324 S. Lagaert et al. / Biotechnology Advances 32 (2014) 316–332

approximately 7 Å, significantly less than the 10 Å normally observed in

inverting enzymes (Brüx et al., 2006; Rye and Withers, 2000; Wang
et al., 1994). Of particular interest is the study of McKee et al. (2012),
which showed that the conversion of Tyr166, which neighbors the cat-
alytic Asp167 in the H. insolens arabinofuranosidase, to alanine intro-
duces endo-xylanase activity while the enzyme keeps its, albeit
lowered, arabinofuranosidase activity. The Tyr166Ala mutation disrupts
the lip of the active site pocket, thereby allowing the xylan backbone
into the active site.

6. The arabinofuranosidases of GH 51

6.1. Substrate specificities

With the exception of four characterized endoglucanases (3.2.1.4),

GH 51 exclusively holds arabinofuranosidases. Despite the relatively
moderate size of this family (over 700 sequences), it contains the largest
number of studied arabinofuranosidases (35 enzymes) and most of
these enzymes were characterized on natural substrates (Table 3). A
few arabinofuranosidases cleave a variety of monosaccharides linked
to pNP or MUF, but in contrast to GH 43, no bifunctionality is claimed
on this basis. There is, however, one bifunctional enzyme from
A. thaliana (AAF19575) that releases both xylose and arabinose from
AX and AXOS (Minic et al., 2004). A phylogenetic tree puts all GH 51
sequences in three groups: clade A, clade B and group C (Fig. 7). Clade Fig. 7. Phylogenetic tree of characterized enzymes from glycoside hydrolase family 51 (GH
A is further divided into four clusters: A1, A2, A3 and A4. Two bacterial 51). Nomenclature as in Fig. 2.
enzymes are present in cluster A1, while all plant and fungal GH 51
arabinofuranosidases are grouped in clusters A2 and A3, respectively. remain in the cytoplasm, except in Streptomyces lividans (AAA50393),
The GH 51 endoglucanases form cluster A4. Cluster B and group C gather Streptomyces chartreusis (BAA90771), Cytophaga xylanolytica (AAC38456
the remaining bacterial arabinofuranosidases and the fungal arab- and AAC38457) and Cellvibrio japonicus (ACE86344).
inofuranosidase from Aspergillus nidulans (EAA65870). SignalP and Most GH 51 arabinofuranosidases release arabinose from AX, AXOS,
TargetP analysis indicates that all plant and fungal arabinofuranosidases arabinan and arabino-oligosaccharides (Table 3). Nonetheless, a num-
are secreted with the exception of the arabinofuranosidase from ber of the enzymes from clade B show specificity towards arabinan
A. nidulans. In contrast, based on the same analysis, the bacterial enzymes (oligosaccharides) and are not active on AX(OS) (ABP67153, ABI34800

Table 3
Activities of glycoside hydrolase family 51 (GH 51) arabinofuranosidases on natural substrates. AOS: arabino-oligosaccharides, AX: arabinoxylan, AXOS: arabinoxylan-oligosaccharides.

Accession nr. Organism Active on Inactive on References

ACB54691 Anoxybacillus kestanbolensis AC26SARI AX, AXOS, arabinan, AOS Canakci et al. (2008)
AB758288 Aureobasidium pullulans ATCC 20524 AX Ohta et al. (2013)
ABC55452 Bacillus pumilus ARA AX Pei and Shao (2008)
CAA99576 Bacillus subtilis subsp. subtilis str. 168 AX, AXOS, arabinan, AOS Bourgois (2008); Inacio et al. (2008)
CAA99595 Bacillus subtilis subsp. subtilis str. 168 AXOS, arabinan, AOS AX Bourgois (2008); Hoffmam et al. (2013);
Inacio et al. (2008)
BAF40305 Bifidobacterium adolescentis ATCC 15703 AX, AXOS, arabinan Lagaert et al. (2010)
AAO84266 Bifidobacterium longum B667 AX,AXOS, arabinan, AOS Lagaert (2013); Margolles and de los
Reyes-Gavilán (2003)
ABP67153 Caldicellulosiruptor saccharolyticus DSM 8903 AOS AX, arabinan Lim et al. (2010)
ACE86344 Cellvibrio japonicus Ueda107 AX, AXOS, arabinan, AOS Beylot et al. (2001)
AAN05450 Clostridium cellulovorans AX, AXOS, arabinan Kosugi et al. (2002)
AAC28125 Clostridium stercorarium NCIMB 11754 AX, AXOS Adelsberger et al. (2004)
ZP_00503782 Clostridium thermocellum ATCC 27405 AX, AXOS, arabinan, AOS Taylor et al. (2006)
AAC38456 Cytophaga xylanolytica AX, arabinan Kim et al. (1998); Renner and Breznak (1998)
AAC38457 Cytophaga xylanolytica AX, arabinan Kim et al. (1998)
ABI34800 Geobacillus caldoxylosilyticus TK4 arabinan, AOS AX Canakci et al. (2007)
AAD45520 Geobacillus stearothermophilus T-6 NCIMB 40222 arabinan, AX (lower) Gilead and Shoham (1995)
BAA90771 Streptomyces chartreusis GS901 AX, arabinan, AOS AXOS Matsuo et al. (2000)
AAA61708 Streptomyces lividans 66 AX, AXOS, arabinan Manin et al. (1994)
ACY69989 Streptomyces sp. S9 ACCC 41168 AX Shi et al. (2010)
CAA76421 Thermobacillus xylanilyticus D3 AX, AXOS Debeche et al. (2000); Rémond et al. (2008)
AAD35369 Thermotoga maritima MSB8 arabinan AX Miyazaki (2005)
AAF19575 Arabidopsis thaliana AX, AXOS, arabinan Minic et al. (2004)
BAB21568 Aspergillus awamori IFO4033 AX, AXOS, arabinan, AOS Kaneko et al. (1998b)
EAA65870 Aspergillus nidulans FGSC A4 AOS Bauer et al. (2006)
AAC41644 Aspergillus niger CBS 120.49/N400 AOS AX Flipphi et al. (1993b)
ADZ98861 Chrysosporium lucknowense C1 AOS Kühnel et al. (2011)
AAK21879 Hordeum vulgare AX, AXOS, arabinan, AOS Lee et al. (2001)
CAL81200 Meripilus giganteus CBS 521.91 AX, AXOS Sørensen et al. (2006)
BAG71680 Penicillium chrysogenum 31B AX, arabinan, AOS Sakamoto and Kawasaki (2003); Sakamoto et al. (2013)
ABO93602 Penicillium purpurogenum MYA-38 AX, arabinan, AOS Fritz et al. (2008)
 S. Lagaert et al. / Biotechnology Advances 32 (2014) 316–332
and AAD35369) (Canakci et al., 2007; Lim et al., 2010; Miyazaki, 2005)
or show a much lower activity towards AX(OS) (AAD45520 and
CAA99595) (Bourgois, 2008; Gilead and Shoham, 1995). In general,
when comparing activity on branched arabinan and debranched
arabinan, activity on the branched form is much higher, suggesting
hydrolysis of the C(O)2- and C(O)3-linkages between arabinose substit-
uents and backbone residues (Beylot et al., 2001; Lagaert et al., 2010;
Matsuo et al., 2000; Miyazaki, 2005; Sakamoto and Kawasaki, 2003;
Taylor et al., 2006). Specific cleavage of arabinose substituents was
demonstrated with H1-NMR for CAA99576 from B. subtilis (Bourgois,
2008). The Aspergillus enzymes are the only GH 51 enzymes we found
which show a higher activity on the α-1,5-linked arabinose main
chain (Bauer et al., 2006; de Vries and Visser, 2001; Kaneko et al.,
1998b).
Like in GH 43, the substrate specificity towards specific arabinose
substitutions in AX or AXOS is determined for a number of GH 51
enzymes. Arabinofuranosidases from Meripilus giganteus and Penicillium
chrysogenum (CAL81200 and BAG71680, respectively, both cluster A3)
and B. subtilis (CAA76421, group C) exclusively hydrolyze arabinose
linked to the C(O)2 or C(O)3 of monosubstituted xylose residues
(Bourgois, 2008; Sakamoto et al., 2013; Sørensen et al., 2006), while
H. vulgare AAK21879 (cluster A2) and C. japonicus ACE86344 (group C)
also show a preference towards these linkages, but are able to release a
small amount of arabinose from disubstituted xyloses (Beylot et al.,
2001; Lee et al., 2001). Furthermore, the crystal structures of B. longum
AAO84266, Clostridium thermocellum ZP_00503782, G. stearothermophilus
AAD45520 (all clade B) and Thermobacillus xylanilyticus CAA76421
(group C) suggest the size of the active site can only harbor the arabinose
from monosubstituted xyloses (see Section 6.2). GH 51 enzymes that
specifically cleave arabinose from disubstituted xylose are limited to clus-
ter A1. Similar to the GH 43 enzymes with activity towards disubstituted
xylose, B. adolescentis BAF40305 exclusively cleaves the arabinose on
position C(O)3 of a disubstituted xylose (Lagaert et al., 2010). No direct
evidence towards linkage preference is present for the other enzyme of
cluster A1, but S. chartreusis BAA90771 releases arabinose from AX and
not from two tested AXOS samples (Matsuo et al., 2000). As both samples
only contained monosubstituted xyloses, it is not unlikely that this
enzyme is also specific towards arabinose on disubstituted xylose
residues.
6.2. Structural data
GH 51 arabinofuranosidases are found as hexamers in solution
(Hövel et al., 2003b; Inacio et al., 2008; Miyazaki, 2005; Taylor et al.,
2006). This multimeric configuration is also seen in the crystal structures
of B. longum AAO84266, Clostridium thermocellum ZP_00503782,
G. stearothermophilus AAD45520, Thermobacillus xylanilyticus
CAA76421, Thermotoga maritime AAD35369 and Thermotoga petrophila
ABQ46651 (Hövel et al., 2003a; Im et al., 2012; Lagaert, 2013; Paës et al.,
2008; Souza et al., 2011; Taylor et al., 2006). GH 51 is a member of the
same glycoside hydrolase clan (A) as GH 39 and shares a similar fold.
The catalytic domain is a N-terminal (β/α)8 TIM barrel, followed by a
β-sandwich domain of unknown function. One of the strands of this
C-terminal domain is formed by approximately 10 N-terminal amino
acids. Multiple alignment of the studied arabinofuranosidases indicates
this two domain architecture is present in all arabinofuranosidases,
although the S. lividans AAA61708 may contain an additional third
domain at its C-terminus.
The catalytic acid/base and nucleophile of the arabinofuranosidase
from G. stearothermophilus are a conserved Glu175 and Glu294, respec-
tively (Shallom et al., 2002a, 2002b). They are separated by approxi-
mately 5 Å, typical for the retaining mechanism displayed in this
family (Hövel et al., 2003a; Pitson et al., 1996). These catalytic residues
line a pocket shaped active site at the bottom of a large, non-linear
groove. The structure with the largest complexed substrate is that of
T. xylanilyticus arabinofuranosidase with arabinose linked to the C(O)3
325
of the middle xylose in xylotriose (Paës et al., 2008). It reveals interac-
tions at four subsites: − 1 (the pocket that harbors the arabinose),
+1, +2 and +2′ (the groove that binds the middle, reducing end and
non-reducing end xylose, respectively). Substrate specificity of GH 51
enzymes seems to be influenced by the variation of the length and flex-
ibility of several loops that surround the active site (Lagaert, 2013). The
β7α7 loop varies considerably in length and in the structure of
B. longum AAO84266, an insertion in this loop forms an α-helix and
bends over subsite + 2′, hindering large and substituted chains
(Fig. 8) (Lagaert, 2013). In the T. xylanilyticus arabinofuranosidase, the
β2α2 loop can adopt an open and closed conformation (Fig. 8) (Paës
et al., 2008). In the closed conformation, Trp99 provides an important
interaction with the arabinosyl moiety. Due to the larger sizes of the
β7α7 loop, the open conformation is sterically impossible in the
arabinofuranosidases from G. stearothermophilus, C. thermocellum and
B. longum (Lagaert, 2013; Paës et al., 2008).
7. The xylosidases of GH 52
7.1. Substrate specificities
GH 52 is a small family, with only 32 bacterial sequences, from
which six enzyme products were characterized. Exclusively xylosidase
activity is observed. The GH 52 enzymes from Aeromonas caviae and
G. stearothermophilus 21 hydrolyze XOS to xylose (Nanmori et al.,
1990; Suzuki et al., 2001). The xylosidase from A. caviae shows
transglycosylation activity and incubation with xylotriose leads initially
to the production of xylotetraose and xylopentaose (Suzuki et al., 2001).
Incubation of the xylosidase from G. stearothermophilus T-6 with pNP-
Xyl leads to the release of pNP and the formation of higher order XOS,
while no free xylose is observed (Bravman et al., 2003).
7.2. Structural data
Although the crystallization and 2 Å data collection of the
G. stearothermophilus T-6 xylosidase was already reported in 2004, no
crystal structures of GH 52 enzymes are available yet (Czjzek et al.,
2004a). The xylosidases from A. caviae and G. stearothermophilus (strain
21 as well as T-6) form dimers in solution, although the xylosidase from
Geobacillus pallidus is suggested to be a trimer (Contreras et al., 2008;
Nanmori et al., 1990; Quintero et al., 2007; Suzuki et al., 2001). Interest-
ingly, isothermal titration calorimetry indicates that the dimeric form of
Fig. 8. Different loop conformations in glycoside hydrolase family 51 (GH 51)
arabinofuranosidases. Cartoon representation of the T. xylanilyticus arabinofuranosidase
with β2α2 in the open (green, PDB ID: 2VRQ, chain A) and the closed conformation
(blue, only loop shown, PDB ID: 2VRQ, chain C). The cartoon representation of the
B. longum arabinofuranosidase is shown in gray and shows the insertion in loop β7α7
that forms an α-helix and bends over the substrate binding cleft (PDB ID: 2Y2W). The
substrate (yellow, PDB ID: 2VRQ, chain A) and Trp99 from T. xylanilyticus are shown in
sticks.
326 S. Lagaert et al. / Biotechnology Advances 32 (2014) 316–332
the G. stearothermophilus T-6 xylosidase only contains a single active
site, formed by the residues of both monomers (Contreras et al., 2008).
This enzyme was shown to perform hydrolysis with retention of the
anomeric configuration using the likely catalytic residues Glu337 and
Glu413 (Bravman et al., 2001b). The enzyme from G. pallidus is formed
by α-helices (44%) and β-sheets (40%), while these values are 30% for
both secondary structures in the G. stearothermophilus T-6 xylosidase
(Contreras et al., 2008; Quintero et al., 2007).
8. The arabinofuranosidases of GH 54
8.1. Substrate specificities
GH 54 is a relatively small family with less than 100 sequences, split
equally amongst bacteria and eukaryota. All 22 currently characterized
enzymes are highly conserved fungal proteins that exclusively display
arabinofuranosidase activity on natural substrates. Solely based on the
hydrolysis of pNP-Xyl and pNP-Ara, the Hypocrea koningii GH 54
enzyme (AAA81024) is called a bifunctional arabinofuranosidase/
xylosidase (Wan et al., 2007a). All enzymes that were tested on AX
and/or arabinan release only arabinose from both substrates (de Wet
et al., 2008; Guais et al., 2010; Kaneko et al., 1998a, 1998b; Kormelink
et al., 1993; Sakamoto et al., 2013; Sakamoto and Kawasaki, 2003;
Takata et al., 2010). When comparing hydrolysis of debranched and
branched arabinan, activity is always lower or non-existent on the
debranched form (Kaneko et al., 1998a, 1998b; Sakamoto and
Kawasaki, 2003; Sakamoto et al., 2013; Takata et al., 2010). Several en-
zymes also cleave arabinose from arabinogalactan (Kaneko et al.,
1998b; Kormelink et al., 1993; Sakamoto and Kawasaki, 2003; Takata
et al., 2010), although the Aurobasidium pullulans arabinofuranosidase
(AAR87863) does not (de Wet et al., 2008). Aspergillus niger AbfB
(AAB53944) was shown to release arabinose from AXOS only
when the arabinose was bound to a singly substituted xylose residue
at the non-reducing end (Kormelink et al., 1993). Similarly, the
arabinofuranosidases from Aspergillus awamori (BAB21567) and H.
jecorina (CAA93243) readily cleave arabinose linked to the C(O)3 posi-
tion of the non-reducing end xylose in xylobiose, while the activity is
absent or much reduced towards arabinose substituted at the C(O)3 po-
sition of the middle xylose in xylotriose (Kaneko et al., 1998a, 1998b).
On the other hand, the GH 54 arabinofuranosidase from P. chrysogenum
(BAG71681) is able to release arabinose from single as well as double
substituted xylose in wheat AX (Sakamoto et al., 2013).
8.2. Structural data
ArfB from A. awamori is the only GH 54 enzyme with a determined
structure. The protein is monomeric in the asymmetric unit of the
crystals, similar to the natural occurrence of other GH 54 arab-
inofuranosidases in solution (De Ioannes et al., 2000; de Wet et al.,
2008; Miyanaga et al., 2004). It comprises a catalytic C-terminal domain
with a β-sandwich fold, linked to a domain with a β-trefoil fold which
belongs to CBM 42 (Fig. 9) (Miyanaga et al., 2004). Pfam analysis
shows this two domain architecture is present in all characterized GH
54 arabinofuranosidases, with the exception of EAA29123 from Neuros-
pora crassa and CAL85369 from Penicillium funiculosum. In EAA29123
the second domain is absent, while it is replaced with a CBM 1 domain
that specifically binds cellulose in CAL85369 (Guais et al., 2010).
Based on their position in relation to the arabinose in the catalytic
site of the A. awamori arabinofuranosidase structure and the drop in
activity of mutants, Glu221 and Asp297 are the suggested catalytic
nucleophile and acid/base, respectively (Miyanaga et al., 2004). An
extensive mutagenesis study of the H. koningii arabinofuranosidase
supports this hypothesis and points to a third catalytic residue, most
likely involved in substrate binding (Asp219 in A. awamori numbering)
(Wan et al., 2007b). In the A. awamori crystal structure, Glu221 and
Asp297 are separated by 5.6 Å, in accordance with the retaining
Fig. 9. Structure of the glycoside hydrolase family 54 (GH 54) arabinofuranosidase of
A. awamori. Cartoon representation of the catalytic and CBM 42 domain (orange and
purple, respectively) of the A. awamori arabinofuranosidase in complex with arabinose
(yellow stick representation). The catalytic residues are shown in red, the arabinose bind-
ing residues of CBM 42 in blue (PDB ID: 1WD4).
mechanism displayed by GH 54 enzymes (Miyanaga et al., 2004;
Pitson et al., 1996). They line a concave, negatively charged pocket
that forms the − 1 catalytic subsite (Miyanaga et al., 2004). Eight
conserved cysteine residues are responsible for three disulfide bridges
in the catalytic domain and one in the CBM 42 domain (Miyanaga
et al., 2004).
The CBM 42 domain of the A. awamori arabinofuranosidase contains
three subdomains (α, β and γ) of about 50 amino acid residues and
structures in complex with arabinose, arabinofuranosyl-xylobiose and
arabinotriose are published (Miyanaga et al., 2004, 2006). In all three
cases, binding is observed in the pockets of the β and γ, but not the α
subdomain. Interestingly, CBM 42 binds only the non-reducing end
arabinose side chains and not the arabinose or xylose backbone
(Miyanaga et al., 2006). Arabinose is recognized in the same manner
in both binding pockets. The side chain sugar forms hydrogen bonds
with an aspartate and histidine, while stacked between two tyrosine
residues (Miyanaga et al., 2006). Mutation of these aspartates in the A.
awamori arabinofuranosidase or removal of the whole CBM domain in
the H. jecorina enzyme leads to a strong decrease in affinity for AX
(Miyanaga et al., 2006; Nogawa et al., 1999). Furthermore, while the
activity of these mutants towards pNP-Ara does not change, the activity
towards AX decreases to 1–10% (Miyanaga et al., 2006; Nogawa et al.,
1999).
9. The arabinofuranosidases of GH 62
9.1. Substrate specificities
GH 62 is a small family of highly conserved bacterial and eukaryotic
sequences (71 and 36, respectively), which exclusively comprises
arabinofuranosidases. All GH 62 enzymes release arabinose from AX
(Bauer et al., 2006; Beylot et al., 2001; Kimura et al., 2000; Lange et al.,
2006; Madrid et al., 1996; Sakamoto et al., 2011; Tsujibo et al., 2002;
Vincent et al., 1997). When tested on arabinan, they release arabinose
from the branched, but not from the debranched form (Bauer et al.,
2006; Kimura et al., 2000; Madrid et al., 1996; Tsujibo et al., 2002;
Vincent et al., 1997). Notable exceptions are ACE85320 from C. japonicus
and BAG71682 from P. chrysogenum, which are also inactive on branched
arabinan (Beylot et al., 2001; Sakamoto et al., 2011). While pNP-Ara is
often, if not always, the best substrate for arabinofuranosidases of other
GHs, several GH 62 enzymes display a remarkably low activity on this
substrate (Beylot et al., 2001; Sakamoto et al., 2011; Tsujibo et al.,
2002; Vincent et al., 1997).
 S. Lagaert et al. / Biotechnology Advances 32 (2014) 316–332
Substrate specificities of GH 62 enzymes towards specific arabinose
substitutions in AXOS and AX were determined for ACE85320
from C. japonicus, CAM07245 from Penicillium capsulatum and
BAG71682 from P. chrysogenum. All three enzymes are able to cleave
arabinose from monosubstituted, but not from disubstituted
xyloses in AX or AXOS (Beylot et al., 2001; Lange et al., 2006;
Sakamoto et al., 2011). Furthermore, the C. japonicus and P. capsulatum
arabinofuranosidase show a preference towards C(O)3-linked arabi-
nose compared to C(O)2-linked residues (Beylot et al., 2001; Lange
et al., 2006).
9.2. Structural data
Although the arabinofuranosidase from A. niger 3 M43 has been
crystallized a long time ago, no crystal structure from a GH 62 enzyme
is available (Scott et al., 1997). However, as GH 62 belongs to GH clan
F together with GH 43, the general fold is expected to be a 5-bladed β-
propeller. On the same basis, GH 62 enzymes may be the second
arabinofuranosidase family which acts with inversion of the anomeric
center. Multiple alignment of GH 62 with sequences from GHs 32, 43
and 68 points to the same three catalytic residues as found in GH 43
(Pons et al., 2004). Glu and Asp are thought to be the general acid and
base, respectively, and a second Asp is likely involved in pKa modulation
and orientation of the catalytic acid and the substrate.
Multiple sequence alignment and pfam analysis shows that the char-
acterized members typically contain a single catalytic domain, with the
exception of three bacterial enzymes which contain additional N-
terminal GH and/or CBM domains. ACE85320 from C. japonicus has a
total of three domains, from the N-terminus a CBM 2, CBM 35 and GH
62 domain. Deletion and fusion experiments with the CBM 2 domain
of ACE85320 showed that this domain binds crystalline cellulose
(Kellett et al., 1990). On the other hand, the C. japonicus CBM 35 domain
interacts in a calcium-dependent manner with unsubstituted xylan, but
not with the substituted form or XOS (Bolam et al., 2004). AAC26524
from S. lividans contains one N-terminal domain that belongs to CBM
13 and binds AX (Dupont et al., 1998; Vincent et al., 1997). AAD32559
from Streptomyces chattanoogensis has three modules, a GH 10 domain
linked through a CBM 13 module to the GH 62 domain. No substrate
specificities or binding characteristics have been determined, but as
GH 10 and GH 62 are families with exclusively xylanases and
arabinofuranosidases, respectively, the enzyme is thought to have
both activities (Hernandez et al., 2001).
10. The xylosidases of GH 120
10.1. Substrate specificities
Recently, GH 120 was introduced. It contains 40 bacterial sequences,
from which two enzymes were characterized. T. saccharolyticum
xylosidase ABM68042 acts with retention of the anomeric configuration
and cleaves xylose from both xylobiose and xylotriose (Huang et al.,
2012; Shao et al., 2011). In contrast, B. adolescentis xylosidase
BAF39080 is almost inactive on xylobiose, but readily hydrolyzes XOS
with a higher DP (Lagaert et al., 2011). Furthermore, it releases up to
30% of xylose from AXOS, but hydrolysis of AX is below 1%.
10.2. Structural data
Crystal structures of the T. saccharolyticum xylosidase have been
published recently (Huang et al., 2012). They show that the enzyme is
assembled as a tetramer and contains two domains, folded as a right-
handed β-helix and a β-sandwich domain. Although xylobiose is
the largest complexed substrate in the crystal structures, an open
carbohydrate-binding cleft suggests that the enzyme is able to accom-
modate larger substrates as well. Hydrolysis of substrates is performed
by the retaining mechanisms, accomplished by Asp382 as the
327
nucleophile and a Glu405 as the general acid/base. Interestingly, outside
of the catalytic cleft, the enzyme displays nine xylose-binding sites at its
surface (Huang et al., 2012). It would be worthwhile to compare with
structural data on the B. adolescentis xylosidase to get more insight in
the structural determinants that prevent it to hydrolyze of xylobiose.
11. The enzymes of GH 30 and GH 116
11.1. Substrate specificities
According to the CAZy database, GH 30 contains three xylosidases.
AAK19754 from Phytophthora infestans hydrolyzes pNP-Xyl and pNP-
Glu and is therefore named a bifunctional glucosidase/xylosidase, but
it does not act on any tested natural substrate, such as xylan, AX and
xyloglucan (Brunner et al., 2002). The characterization of ABC55722
from B. adolescentis Int57 and ABX45137 from Bifidobacterium breve
has not been published, but their suggested activities stem from the
comment in their sequence files in the NCBI database. It is currently
unknown on which substrates the xylosidase activities were observed,
although the B. breve enzyme is suggested to hydrolyze kakkalide and
ginsenoside according to deposition ABX45137. We ourselves re-
combinantly expressed an enzyme from B. adolescentis ATTC15703
which has 94 and 93% identity with the GH 30 enzymes from B. breve
and B. adolescentis Int57, respectively. Although it hydrolyzes pNP-Xyl
and pNP-Ara, it does not release xylose, arabinose or glucose from
xylan, AX, XOS, debranched and branched arabinan and xyloglucan
and it lacks xylanase and cellulase activity (unpublished).
Similarly, the GH 116 enzyme from Sulfolobus solfataricus is a sug-
gested bifunctional glucosidase/xylosidase because it hydrolyzes pNP-
and MUF-linked glucosides and xylosides (Cobucci-Ponzano et al.,
2010). However, it lacks activity on any tested oligosaccharide sub-
strate, like XOS and glucooligosaccharides. All these results suggest GH
30 and GH 116 may not contain true xylosidases which are able to
hydrolyze XOS or xylan.
12. Synthesis
In spite of the fact that synthetic substrates come in handy for
screening, determining optimal conditions, etc., the above literature
review suggests that hydrolysis of them may be insufficient to claim a
certain enzyme activity. A number of xylosidases that were only tested
on pNP-linked substrates are most likely arabinofuranosidases and vice
versa. This review therefore focuses on results obtained with natural
substrates, such as XOS, AX and xylan.
An important difference regarding the substrate specificities of
xylosidases is their discriminations between substrates with varying
length. While limited, data on GH 3 xylosidases suggest they display a
preference towards polymeric substrates, although XOS are in the end
completely degraded to xylose. There are indications that the prefer-
ence of these xylosidases is due to a relatively large number of subsites.
On the other hand, two GH 43 enzymes show a preference towards
xylobiose, while a third enzyme is indifferent towards XOS with DP2–
DP6. Likely, GH 43 xylosidases have only one or two substrate binding
subsites. In contrast to GH 3 and GH 43, GH 39 xylosidases and at least
one GH 120 enzyme display very limited activity towards xylobiose,
but readily cleave larger substrates, indicating that these enzymes
have at least three subsites that are responsible for these properties. It
would be interesting to see if combinations of these xylosidases with
different properties can lead to higher yields in AX degradation process-
es. Other interesting candidates in this respect are the rex hydrolases,
which hydrolyze the xylan backbone from the reducing end.
Although only a limited amount of arabinofuranosidases have been
tested for their substrate specificities towards specific arabinose substi-
tutions in AX, all of them fall in two separate classes. Those that are able
to hydrolyze arabinoses from monosubstituted xyloses, both C(O)2- or
C(O)3-linked, and those that cleave the arabinose from the C(O)3
328 S. Lagaert et al. / Biotechnology Advances 32 (2014) 316–332

position of disubstituted xyloses (Fig. 10). The arabinofuranosidases
from the first group seem to be most widespread, with 2 GH 43, 4
GH 51, 3 GH 54 and 2 GH 62 enzymes. Until now, only three
arabinofuranosidases show hydrolysis of arabinoses from disubstituted
xyloses, two from GH 43 and one from GH 51, while the properties of a
second GH 51 enzyme suggest the same specificity. Both GH families are
very distinct, in structure as well as in hydrolysis mechanism. Since the
GH classification is widely assumed to show evolutionary relationships,
it is likely that the presence of substrate specificities towards C(O)3-
linked arabinose on disubstituted xylose in both families is a result of
convergent evolution. It remains to be seen if all arabinofuranosidases
are part of these two classes, or if other enzymes will be found with
other specificities, e.g. towards C(O)3-linked arabinoses, regardless of
mono- or disubstituted xyloses, or towards C(O)2-linked arabinose on
disubstituted xylose. Nonetheless, for now, all current evidence
suggests that for processes where an intensive enzymatic degradation
of AX is wanted, such as in the production of biofuels, the synergistic
action of enzymes from both classes is needed (Fig. 10). Of particular
interest in this regard is the study of Rasmussen et al. (2012), wherein
synergistic AX degradation between several different types of enzymes
was examined. In addition to the observed synergy between the two
types of arabinofuranosidases, arabinofuranosidases also act synergisti-
cally with β-xylosidases. Optimal hydrolysis required the combined
action of an endoxylanase, the two types of arabinofuranosidases and
a β-xylosidase (Rasmussen et al., 2012).
Since arabinofuranosidases and xylosidases both are crucial
enzymes for an intensive AX degradation, enzymes displaying both
activities have a clear advantage. Hence, it is no surprise that significant
interest went to these bifunctional enzymes and that authors are eager
to claim this property. However, when carefully looking at literature
data on the 23 bifunctional xylosidases/arabinofuranosidases from the
CAZy database, it is clear that most of these display the dual activity

AX

Endoxylanases
GH 5,8, 10 and 11

AXOS (mono-and disubstituted)

di(3)-specific arabinofuranosidases
GH 43, 51, 54 and 62

AXOS (monosubstituted)

mono-specific arabinofuranosidases
GH 43 and 51

XOS

β-xylosidases Rex hydrolases

GH 3, 39, 43, 52, 120 GH 8

xylose

Fig. 10. Schematic overview of arabinoxylan (AX) degradation. Arrows indicate cleavage sites. Endoxylanases cleave the xylan backbone randomly, endoxylanase families are taken from
Collins et al. (2005b).
 S. Lagaert et al. / Biotechnology Advances 32 (2014) 316–332
only on synthetic substrates. If incubated with natural substrates, in
general, they release either xylose or arabinose, but not both. We only
found five exceptions: three plant GH 3, one bacterial GH 43 and one
plant GH 51 enzymes release both arabinose and xylose from natural
substrates. Even in these cases, when a comparison between the two
different activities was made, the enzymes showed a preference
towards the hydrolysis of one type of sugar. For example, the GH 3
and GH 51 bifunctional enzymes from A. thaliana release xylose less
efficiently than arabinose from natural substrates, in contrast, the GH
3 enzyme from H. vulgare releases more xylose than arabinose from
AXOS (Lee et al., 2003; Minic et al., 2004). Crystal structures of these
bifunctional enzymes would greatly aid in explaining why these
enzymes, and not the others, are able to release both xylose and arabi-
nose from natural substrates.
Abbreviations
A/X arabinose to xylose ration
AX arabinoxylan
AXOS arabinoxylan-oligosaccharides
CAZy Carbohydrate Active Enzyme
CBM 6 carbohydrate binding module of family 6
DP degree of polymerization
DS degree of substitution
GH glycoside hydrolase families
MUF methylumbelliferyl
pNP-Ara pNP-arabinoside
pNP-Glu pNP-glucoside
pNP-Xyl p-nitrophenylxyloside
XOS xylo-oligosaccharides
Acknowledgments
Financial support from the European Commission in the Communi-
ties 7th Framework Programme (FP7/2007–2013) for the Biocore Pro-
ject (grant agreement n°FP7-241566) is gratefully appreciated. This
publication reflects only author's views and the Community is not liable
for any use that may be made of the information contained in this
publication. We further acknowledge the financial support from the
‘Instituut voor de aanmoediging van Innovatie door Wetenschap en
Technologie in Vlaanderen’ (IWT) for SBO project funding (IMPAXOS,
IWT/040073/SBO).
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