L Arabinofuranosidases: the potential applications in biotechnology

REVIEW PAPER
-L-Arabinofuranosidases: the potential applications in biotechnology
Mondher Th. Numan*; Narayan B. Bhosle
National Institute Of Oceanography, Dona Paula, Goa - 403004, India
Abstract: Recently, -L-arabinofuranosidases (EC3.2.1.55) have received increased
attention primarily due to their role in the degradation of lignocelluloses as well as
their positive effect on the activity of other enzymes acting on lignocelluloses. As a
result, these enzymes are used in many biotechnological applications including wine
industry, clarification of fruit juices, digestion enhancement of animal feedstuffs and
as a natural improver for bread. Moreover, these enzymes could be used to improve
existing technologies and to develop new technologies. Production, mechanisms of
action, classification, synergistic role, biochemical properties, substrate specificities,
molecular biology and biotechnological applications of these enzymes have been
reviewed in this article.
Keywords: -L-arabinofuranosidases; lignocelluloses; synergistic role; classification;
applications.
* Corresponding author:
E-mail: mnoman@nio.org
Tel: +91-832-2450234
Fax: +91-832-2450606
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Introduction
Lignocelluloses of plant cell walls are composed of cellulose, hemicellulose,
pectin and lignin. Hemicelluloses are one of the most abundant renewable polymers
on the earth. Moreover, cellulose, hemicelluloses, lignin and pectins are the key
components in the degradation of lignocelluloses. Many enzymes are involved in
the degradation of these polymeric substrates [129]. L-arabinosyl residues are widely
distributed in these polymers as side chains. The presence of these side chains
restricts the enzymatic hydrolysis of hemicelluloses and pectins [93,99,101]. Further,
it also represents a formidable technological barrier that retards the development of
various industrial processes [99]. The use of a single accessory enzyme for partial or
specific modification of lignocelluloses might offer new interesting options for the
utilization of these low cost raw materials [72, 110].

The -L-arabinofuranosidases ( -L-AFases) are accessory enzymes that
cleave
-L-arabinofuranosidic linkages and act synergistically with other
hemicellulases and pectic enzymes for the complete hydrolysis of hemicelluloses and
pectins [77, 113]. These enzymes warrant substantial research efforts because they
represent potential rate limiting enzymes in the degradation of lignocelluloses from
agricultural residues [99]. The action of -L-AFase alone or in combination with
other lignocellulose degrading enzymes represents a promising biotechnological tool
as alternatives to some of the existing chemical technologies such as chlorination in
pulp and paper industry [44,46,74,74], synthesis of oligosaccharides [94,95] and
pretreatment of lignocelluloses for bioethanol production [100,101]. Considering
the potential and future prospects of -L-AFases, this paper reviews the various
aspects of these enzymes with emphasis on their potential for biotechnology.
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Hemicelluloses and pectins
Hemicelluloses and pectins are the matrix polysaccharides of the plant cell
wall. They account for 25-35% of lignocellulose biomass [99]. The hemicellulose
xylans contain a -1,4-linked D-xylose backbone [30]. In many plants, xylan
backbone is substituted by different side chains with L-arabinose, D-galactose, acetyl,
feruloyl, p-coumaroyl and glucuronic residues [1,30]. Xylans from grasses, cereals,
softwood and hardwood differ in their composition. This is basically due to the
differences in the frequency and composition of the side chain substituents of xylans
[30,99,100]. Similarly, arabinoxylans are found in the cell walls of the cereal plants
and grasses belonging to the family Gramineae [1,70]. They contain xylan backbone
that is partially substituted at intervals with -L-arabinofuranose residues [1].
Moreover, wheat arabinoxylan also contain other substituents as shown in Fig.1
[1,30].
Pectins are a family of complex heteropolysaccharides that contain two well-
defined regions called as smooth and hairy (Fig.2) [17,30]. The three pectic
polysaccharides such as homogalacturonan, rhamnogalacturonan I and substituted
galacturonan have been isolated from plant cell walls [28,30]. The dominant feature
of the pectins is the presence of a linear backbone of galacturonic acid containing
varying proportion of methyl ester groups. Pectin polymer backbone is interspersed at
intervals with rhamnose residues carrying the neutral sugars side chains containing
arabinose and galactose that form arabinans, arabinogalactans or galactans (Fig.2)
[17,49]. Pectins are abundant in the soft tissues of citrus fruits [49, 131], sugar beet
pulp and apple [20,28].
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The -L-AFases
The
-L-AFases ( -L-arabinofuranoside arabinofuranohydrolases, EC
3.2.1.55) are the enzymes involved in the hydrolysis of L-arabinose linkages. These
enzymes have been purified from several bacteria, fungi and plants [51,73,93]. They
form a part of the array of glycoside hydrolases required for the complete degradation
of arabinose containing polysaccharides [99,115]. The action of these enzymes
accelerates the hydrolysis of the glycosidic bonds by more than 1017 fold, making
them one of the most efficient catalysts known [98,107]. Such enzymatic hydrolysis
release soluble substrates which are utilized by both prokaryotic and eukaryotic
microorganisms [77]. The -L-AFases specifically catalyze the hydrolysis of terminal
nonreducing- -L-1,2-,
-L-1,3-, and -L-1,5-arabinofuranosyl residues from
different oligosaccharides and polysaccharides [99,101,112]. Whereas, the nature of a
glycone sugar can influence the catalytic activity of other arabinose releasing
enzymes, the -L-AFases do not distinguish between the saccharides link to the
arabinofuranosyl moiety and thus exhibit wide substrate specificity [93,97]. Effective
hydrolysis of -L- arabinofuranosyl residues from various pectic, homo-
hemicellulosic polysaccharides (branched arabinans, debranched arabinans),
heteropolysaccharides (arabinogalactans, arabinoxylans, arabinoxyloglucans,
glucuronoarabinoxylans, etc.) and different glycoconjugates is carried out by the -L-
AFases [8,112]. Moreover, most microbial -L-AFases are secreted into the culture
media thus; they are likely to attack polysaccharides [84].
The synergistic role of -L-AFases
The importance of -L-AFases has come from the fact that, arabinose side
chains on hemicelluloses and pectins participate in cross-linking within the plant cell
wall structure. The presence of these side chains also affects the form and functional
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properties of hemicelluloses and pectins [29]. They reduce the interaction between
polymers chains due to their inherently more flexible water-hungry furanose
conformations. Moreover, the L-arabinofuranoside substitutions on xylan strongly
inhibit the action of xylan degrading enzymes (Fig.1), thus, preventing the complete
degradation of the polymer to its basic xylose units [99,107]. Similarly, L-
arabinofuranoside substitutions in pectin (Fig.2) prevent the complete degradation of
this polymer to its basic units. The
-L-AFases act synergistically with other
hemicellulases and pectinases for the complete degradation of hemicelluloses and
pectins, respectively [4,29,67,102]. Moreover, in some cases, -L-AFases possessing
-xylosidase activity or xylanases with -L-arabinofuranosidase activity also have
been described [73,74,83,121]. Furthermore, some -L-AFases with both exo- and
endo-activity on arabinan, one of the major constituents of pectins, has been
reported [11, 87].
The role of -L- AFases in the degradation of arabinose containing polymers
is well known. They have a cooperative role facilitating the action of other
lignocelluloses degrading enzymes [118,120]. This has been confirmed for -L-
AFase from Thermomonospora fusca that worked in truly synergistic relationship
with endoxylanase from the same bacterium releasing 0.6mg and 0.3mg of reducing
sugars from oat spelt xylan and ball-milled wheat straw, respectively [4]. -L-AFase
played an important role to increase the release of reducing sugars from these
lignocelluloses. However, other authors report the synergistic action of these
enzymes with other pectinases and hemicellulases on lignocelluloses. For instance,
the two enzymes -L- AFases (kabfA and kabjB) from Aspergillus kawachii acted
synergistically with xylanase in the degradation of arabinoxylan releasing higher
amounts of ferulic acid in the presence of feruloyl esterase [68]. Furthermore,
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Hashimoto and Nakata [51] showed that hemicellulose from soy sauce materials
was decomposed synergistically by xylanase, -xylosidase and -L- AFase produced
by Aspergillus oryzae HL15 during moromi(a) fermentation. They also suggested that
-L-AFase of A. oryzae HL15 was very closely involved not only in releasing
arabinose, but also xylose, into moromi mash. The same effect has been shown when
these enzymes act synergistically on arabinoxylan. Moreover, an exo-arabinanase,
Abnx from Penicillium chrysogenum released very little arabinobiose from arabinan,
as the action of Abnx was inhibited by the arabinofuranose unit linked as a side
chain [102]. When Abnx acted, in combination with either -L-AFases (AFQ1 or
AFS1), from the same fungus, the arabinose contents in the reaction mixtures were
higher than the sum of those by the two enzymes acting separately [102].
Furthermore, Morales et al [88] reported that the two -L-AFases i.e. AF64 and AF53
from Bacillus polymyxa facilitate the action of the endoxylanase on oat spelt xylan
and wheat bran arabinoxylan. An increase in the production of smaller
xylooligosaccharides has occurred because of the cooperative action of -L-AFases
used in these experiments. -L-AFases also act synergistically with endo-arabinanase
and cinnamoyl esterase (CinnAE) from Aspergillus niger. When sugar-beet pulp
(SBP) incubated with mixture of the former enzymes, the esterase was able to release
14 times more of the alkali-extractable ferulic acid present in the whole pulp as free
acid than CinnAE alone [70].
(a) : Moromi is a fermenting mixture or mash of rice, water, koji (malted
soybeans) and A. oryzae which produced during the traditional fermentation of
soy sauce and in production of sake the traditional alcohol beverage in Japan.
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Classification of arabinose- releasing enzymes
Kaji [60] classified -L-AFases on the basis of their sources and substrate
specificity. Beldman et al [8] classified arabinose-releasing enzymes depending on the
mode of action and their substrate specificity. However, both classifications were not
effective as they were too broad to define the substrate specificities of these enzymes.
Moreover, newly isolated enzymes have shown different modes of action than those
enzymes classified before. Because of this, further subclasses and a new class need to
be added to the existing system of classification proposed by Beldman et al [7]. In
view of this, three subclasses of the existing arabinoxylan- -L-
arabinofuranohydrolases class could be introduced [38,122,123] and designated
Subclass (1) AXHB-md 2, 3, Subclass (2) AXHB-m 2,3 and Subclass (3) AXHd3.
Subclass (1) AXHB-md 2,3 includes enzymes that release arabinose from both
singly and doubly substituted xylose, and able to hydrolyze p-nitrophenyl -L-
arabinofuranoside at a rate similar to that for oligosaccharide substrates. This subclass
was exemplified by the enzyme arabinoxylan arabinofuranohydrolase isolated from
germinated barley [38].
Subclass (2) AXHB-m 2,3 includes enzymes that hydrolyze arabinose residues
from C2 or C3 linked to a single-substituted xylose residue and do not hydrolyze p-
nitrophenyl
-L-arabinofuranoside. The enzyme isolated from Bifidobacterium
adolescentis [122] represents this subclass.
Subclass (3) AXHd3: which include enzymes that are able to release only C3-
linked arabinose residues from double-substituted xylose residues but do not
hydrolyze p-nitrophenyl -L-arabinofuranoside. This subclass was represented by the
enzyme isolated from Bifidobacterium adolescentis [123].
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Recently, new types of -L-AFases have been isolated with properties that
have not been reported earlier. Such enzymes could not be assigned to any one of
arabinose releasing enzyme classes. These enzymes have the ability to act on both
interior -1,5 backbone as well as -1,3–side chains of arabinan and debranched
arabinans in addition they are able to act on p-nitrophenyl -L-arabinofuranoside.
In view of this, these enzymes should be assigned into a new class represented by -
L-AFase isolated from the thermophilic bacterium PRI-1686 [11] and Tm-AFase
from the hyperthermophilic bacterium Thermotoga maritima MSB8 [87].
The most recent classification scheme based on amino acid sequences, primary
structure similarities and hydrophobic cluster analysis has classified -L-AFases into
five glycosyl hydrolases families (GHs) i.e. GH3, GH43, GH51, GH54, and GH62
[23,52]. This classification is useful to study evolutionary relationship, mechanistic
information and structural features of these enzymes [25].
Mechanisms of action of -L-AFases
Like other glycoside hydrolases, -L-AFases mediate glycosidic bond
cleavage via acid/base-assisted catalysis employing two major mechanisms, giving
rise to either an overall retention or an inversion of the anomeric configuration
[26,136]. In both mechanisms, as shown in Fig. 3, the hydrolysis usually requires two
carboxylic acids, which are conserved within each glycoside hydrolases family [98]
and proceed through an exocarbonium ion-like transition state [92,98,107].
Retaining -L-AFases are members of GH3, GH51and GH54 families that
cleave the glycosidic bond using a two-step double-displacement mechanism, as
illustrated in Fig.3a. This was also confirmed by the crystal structure studies and
snapshots along the reaction pathway of GH51 described by Hövel et al [56]. In the
first step of the reaction (glycosylation), the acid-base residue acts as a general acid,
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protonating the glycosidic oxygen and stabilizing the leaving group. The nucleophilic
residue attacks the anomeric carbon of the scissile bond, forming a covalent glycosyl-
enzyme intermediate with the opposite anomeric configuration of the substrate. In the
second step (deglycosylation), the acid-base residue, acting this time as a general
base, activates a water molecule that attacks the anomeric center of the glycosyl-
enzyme intermediate from the same direction of the original bond, liberating the free
sugar with an overall retention of the anomeric configuration [36,56].
Inverting -L- AFases that represent GH43 family use a single displacement
mechanism, in which one carboxylate acts as a general base catalyst, deprotonating
the nucleophilic water molecule that attacks the bond, while the other carboxylic acid
acts as a general acid catalyst by protonating the leaving aglycone (Fig. 3b) [107,136].
Production of -L-AFases

The -L-AFases production is influenced by carbon source and composition
of the growth medium. Various carbon sources including monomeric sugars and
complex polysaccharides have been used to assess their effect on the production,
induction, and substrate specificity of -L- AFases (Table 1). For examples, pentoses
D-arabinose, L-arabinose, D-xylose and hexoses D-galactose, D glucose, D-mannose,
L-sorbose have been commonly used. Other sugars cellobiose, lactose, lactulose,
maltose, mellibose, sucrose, trisaccharide, raffinose, D-arabitol, L-arabitol, D-
mannitol, D-sorbitol and xylitol also have been used. Sugar beet pulp (starch free),
wheat bran (starch free), wheat straw, oat meal, rice straw and corn cob are some of
the lignocelluloses that have been used for the production of -L-AFases.
Polysaccharides such as oat spelt xylan, birchwood xylan, beechwood xylan, wheat
arabinoxylan, arabinogalactan, larch wood arabinogalactan, sugar beet arabinan,
galactan CMC, guar gum, gum Arabic and Locust bean gum have also been used.
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Pectins, schizophyllan, starch, xanthan, carboxymethyl cellulose, potato -1,4-
galactan, carob galactomannan, Me- -xyloside, and lactobionic acid are some other
carbon sources utilized for -L-AFases production.
Generally, arabinose-containing substrates are essential for the efficient
production of -L-AFases [9,68]. Monomeric compounds L-arabitol and L-
arabinose induce the genes involved in the production of these enzymes in some
microorganisms [27]. Conversely, other monosaccharides such as glucose and
galactose may inhibit the production of -L-AFases [9,68]. Arabinogalactans and oat
meal were found to be the best inducers for -L-AFase isolated from Bacillus
pumilus PS213 [32]. -L-AFase was produced by Rhodothermus marinus when the
culture was grown on birchwood xylan [46]. L-Arabitol was the inducer for the
production of -L-AFases enzymes araA and araB by the Aspergillus niger mutants
[26]; ABF1 by the Penicillium purpurogenum [15,27] and kabfA and kabjB by the
Aspergillus kawachii [68].
-L-AFase production by Pseudomona cellulosa was
repressed when glucose was used in the production medium [9].
The experiments carried out by Gomes et al [46] indicated that carbon and
nitrogen sources influence the production of -L-AFase by Rhodothermus marinus. In
these experiments, different concentrations of xylan (2–6g/l) and yeast extract (4–
12g/l) were used to increase the enzyme production. The highest enzyme activity (108
nkat/ml) was obtained with medium containing 3g/l and 9g/l of Birchwood xylan and
yeast extracts, respectively. The lowest enzyme activity (86 nkat/ml) was obtained
with medium containing 5g/l and 7g/l of Birchwood xylan and yeast extracts,
respectively [46]. Aspergillus niger 10 showed highest -L-AFase activity (243U/ml)
when grown on a solid state medium with C: N ratio of 15.9. The carbon and nitrogen
sources used were dried skins of grape pomace and casein peptone, respectively [57].
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