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Lignocellulose-Degrading Enzymes from

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 Biotechnology Advances 31 (2013) 838–850

Contents lists available at ScienceDirect

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

Research review paper

Lignocellulose-degrading enzymes from termites and their symbiotic microbiota

Jinfeng Ni a,⁎, Gaku Tokuda b,⁎⁎
a
State Key Laboratory of Microbial Technology, Shandong University, 27 Shandanan Road, Jinan, Shandong 250100, China
b
Tropical Biosphere Research Center, COMB, University of the Ryukyus, Nishihara, Okinawa, Japan

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

Available online 23 April 2013 Lignocellulose—the dry matter of plants, or “plant biomass”—digestion is of increasing interest in organis-
mal metabolism research, specifically the conversion of biomass into biofuels. Termites efficiently decom-
Keywords: pose lignocelluloses, and studies on lignocellulolytic systems may elucidate mechanisms of efficient
Termites lignocellulose degradation in termites as well as offer novel enzyme sources, findings which have signifi-
Lignocellulose degradation cant potential industrial applications. Recent progress in metagenomic and metatranscriptomic research
Cellulase
has illuminated the diversity of lignocellulolytic enzymes within the termite gut. Here, we review
Xylanase
Laccase
state-of-the-art research on lignocellulose-degrading systems in termites, specifically cellulases, xylanases,
and lignin modification enzymes produced by termites and their symbiotic microbiota. We also discuss re-
cent investigations into heterologous overexpression of lignocellulolytic enzymes from termites and their
symbionts.
© 2013 Elsevier Inc. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 838
2. The termite intestinal tract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 839
3. Cellulose and cellulase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 839
3.1. Cellulolytic systems in lower termites . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 839
3.2. Cellulolytic systems in higher termites. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 841
4. Hemicellulose and hemicellulase. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 842
4.1. Xylanases from lower termites . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 842
4.2. Xylanases from higher termites . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 843
5. Lignin and its modification enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 844
5.1. Structural changes of lignin during passage of the gut . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 844
5.2. Lignin-modification enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 845
6. Heterologous production of lignocellulases from termites . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 845
6.1. Heterologous production of EGs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 845
6.2. Heterologous production of BGs. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 846
6.3. Heterologous production of xylanases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 847
7. Future perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 848
Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 848
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 848

1. Introduction
Lignocellulose, mainly comprised of cellulose, hemicelluloses, and
lignin, is the most abundant biomass on earth and is recognized as a
potential sustainable source for biofuels and biomaterial production
⁎ Corresponding author. Tel.: +86 531 88363323; fax: +86 531 88362903.
⁎⁎ Corresponding author. Tel.: +81 98 895 8543; fax: +81 98 895 8944.
E-mail addresses: jinfgni@sdu.edu.cn (J. Ni), tokuda@comb.u-ryukyu.ac.jp
(G. Tokuda).
(Himmel et al., 2007; Ragauskas et al., 2006). Termites, which are ef-
ficient lignocellulose decomposers, thrive on dead plant materials and
contribute to carbon mineralization, especially in tropical and sub-
tropical regions (Kudo, 2009; Ohkuma, 2003; Yamada et al., 2005).
Studies to elucidate termite lignocellulose-degrading systems should
identify many cellulose hydrolysis enzymes and enhance our under-
standing of mechanisms of lignocellulose degradation in termites
(Scharf and Tartar, 2008). Such investigations may also contribute
to optimization of plant biomass bioconversion processes. Therefore,
termite biomass degradation has received significant interest in the

0734-9750/$ – see front matter © 2013 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.biotechadv.2013.04.005
 J. Ni, G. Tokuda / Biotechnology Advances 31 (2013) 838–850
last decade. Using “termite” and “enzyme” as key words to search the
National Center for Biotechnology Information's database of biomed-
ical literature (PubMed) we found 238 papers: 230 manuscripts dated
from 2000 to the present (November 27, 2012) and 38 papers pub-
lished prior to 2000 (1978–1999). Such findings indicate the in-
creased research emphasis in the biology of termite enzymes for the
last 10 years.
Termites (Isoptera or Termitoidae) are well-studied social in-
sects, considered to be an epifamily of the cockroach (Blattaria or
Blattodea). The diversity and classification of termites are well docu-
mented elsewhere (Eggleton, 2011; Lo and Eggleton, 2011), so we
have briefly summarized important facts here. Termite group is com-
prised of more than 2600 described species, leaving perhaps 500–
1000 species currently undescribed (Eggleton, 2011). Termites are gen-
erally classified into seven families: Mastotermitidae, Kalotermitidae,
Termopsidae, Hodotermitidae, Rhinotermitidae Serritermitidae, and
Termitidae (Lo and Eggleton, 2011). The Mastotermitidae are the most
primitive family, containing only one wood-feeding species in Australia.
Termites of the Kalotermitidae family consume material from dry wood,
and Termopsidae nest in and feed on wet dead logs. Hodotermitidae are
grass-harvesters, and Rhinotermitidae consume wood, primarily in
temperate zones. Serritermitidae contains few species and only in South
America, suggesting that they may be better classified within the
Rhinotermitidae family (Eggleton, 2011). Termitidae is the largest fam-
ily, consisting of approximately 2000 species and accounting for
almost 75% of all known termites. Termitidae consists of at least
seven sub-families, namely Macrotermitinae, Sphaerotermitinae,
Foraminitermitinae, Apicotermitinae, Termitinae, Syntermitinae,
and Nasutitermitinae (Eggleton, 2011; Lo and Eggleton, 2011).
Termitidae have diverse feeding preferences, the majority feeding
on soil. A few subfamilies such as Termitinae, Nasutitermitinae, and
Syntermitinae contain wood- or litter-feeding termites. Termites of
the sub-family Macrotermitinae cultivate basidiomycete fungi
(Termitomyces sp.) in their nests, and are thus commonly known as
fungus-growing termites.
Based on the presence or absence of flagellated protistan symbionts
in the hindgut of termites, they are conventionally grouped into lower
and higher termites. The first six families, all of which harbor protistan
symbionts in the hindgut, are referred to as “lower termites”. The
remaining family, Termitidae, which lack protistan symbionts in the
hindgut, are traditionally referred to as “higher termites” (Lo and
Eggleton, 2011).
Studies of cellulose digestion in termites and related insects and the
historical importance of these findings have been well-documented
elsewhere (Hongoh, 2011; Lo et al., 2011; Matsui et al., 2009;
Watanabe and Tokuda, 2001, 2010). In this review, we have focused on
the lignocellulolytic system in termites, with special reference to
lignocellulose-degrading enzymes such as cellulases, xylanases, and
laccases produced by termites and their symbiotic microbiota. We also
describe enzyme gene resources, heterologous overexpression systems,
and relevant enzyme properties that hold promise for industrial
applications.
2. The termite intestinal tract
The intestinal tracts of termites are axially structured microenviron-
ments with differences in metabolic activities and microbial community
structures (Köhler et al., 2012). The termite gut generally consists of the
foregut, midgut, and hindgut. The foregut is an esophageal tract of ecto-
dermal origin with an enlarged anterior segment (“the crop”) and a pos-
terior segment (“the gizzard”) that plays a role in mechanical grinding of
ingested wood fragments. The midgut, which in insects is chiefly a secre-
tion site for digestive enzymes and nutrient absorption, is columnar and
uniform with an endodermal origin. The midgut is located posterior to
the foregut. The Malpighian tubules are usually attached to the end of
the midgut to excrete nitrogen wastes to the gut lumen. The hindgut,
839
of ectodermal origin, is the largest organ. The hindgut can be further
subdivided into P1, P2, P3, P4, and P5 segments (Watanabe and
Tokuda, 2010). Among these segments, P3 is typically enlarged to harbor
numerous microorganisms. The gut microbial community contains all
three domains of organisms: Archaea, Bacteria, and Eukaryotes (pro-
tists). Relatively little microbiota is found in the foregut and midgut,
whereas abundant microbiota is found in the hindgut (Hongoh, 2011;
Köhler et al., 2012). The hindgut compartments of higher termites are
more developed and complex than those of lower termites. Except for
Macrotermitinae, Sphaerotermitinae, and Foraminitermitinae, higher
termites have developed a “mixed segment” (half of the gut wall consists
of midgut tissue; the remaining is hindgut tissue) between the mid-
gut and the hindgut, but the precise function of the mixed segment
has not been elucidated. In addition to the intestinal tract, the sali-
vary glands also significantly contribute to the digestive physiology
of termites. Detailed descriptions of termite gut structures are docu-
mented elsewhere (see Bignell, 1994, 2011; Lo and Eggleton, 2011
and references therein).
3. Cellulose and cellulase
Lignocellulose consists of cellulose (20–50%), hemicellulose (15–35%),
and lignin (18–35%). Cellulose, a linear polysaccharide consisting of
β-1,4-linked D-glucopyranosyl units, is the major component of plant
material (20–40%) and the most abundant biomass on earth (Tomme et
al., 1995). Cellulases, found in the gut of lower and higher termites, are
produced by organisms that catalyze the cellulolysis (or hydrolysis)
of cellulose, and three classes have been identified in cellulolysis.
Endo-β-1,4-glucanases (EC 3.2.1.4) hydrolyze cellulose chains in a
non-processive (random) manner, whereas exoglucanases such as
cellodextrinases (EC 3.2.1.74) or cellobiohydrolases (EC 3.2.1.91)
depolymerize cellulose chains from their reducing or non-reducing
ends in a processive or ordered manner. β-Glucosidases (EC 3.2.1.21)
cleave cello-oligosaccharides (especially cellobiose) to liberate glucose.
3.1. Cellulolytic systems in lower termites
Distribution patterns of cellulolytic enzymes in the gut of termites
have been studied extensively, but relevant studies on soil-feeding
termites are limited. The distribution patterns and expression of cel-
lulolytic enzymes in the termite gut varied by termite caste and de-
velopmental stages (Fujita et al., 2008; Shimada and Maekawa,
2010). Thus, this review focuses on mature worker-caste termites
that feed on lignocellulosic materials and have significant digestive
roles among the castes. Generally, lower termites possess strong hy-
drolytic activity (45–85% of total gut activity) against carboxymethyl-
cellulose (CMC) (representing endo-β-1,4-glucanase [EG] activity) in
the salivary glands (Tokuda et al., 2004), whereas these termites pos-
sess stronger cellulolytic activity (40–88%) in the hindgut than in the
salivary glands when microcrystalline cellulose is the substrate (pri-
marily representing cellobiohydrolase [CBH] activity) (Tokuda et al.,
2005). Regarding β-glucosidase (BG), lower termites have stronger
enzymatic activities both in the salivary glands and the hindgut
than higher termites (Slaytor, 2000; Tokuda et al., 2002). The relevant
genes encoding these enzymes have been identified (Lo et al., 2011;
Watanabe and Tokuda, 2010). Based on peptide sequence similarities,
glycoside hydrolases are classified into more than 100 families (see
http://www.cazy.org). Members of the same family frequently have
different substrate specificities but share structural similarities that,
in turn, reflect their evolutionary origins (Henrissat and Bairoch,
1993). According to this classification, all endogenous EGs are affiliat-
ed with the glycoside hydrolase family (GHF) 9 (Leonardo et al.,
2011; Tartar et al., 2009; Watanabe and Tokuda, 2010; Zhang et al.,
2012b), while all endogenous BGs belong to GHF1, except for one
putative endogenous GHF3 BG which has been identified from the
salivary gland EST library of Hodotermopsis sjostedti (Termopsidae)
840 J. Ni, G. Tokuda / Biotechnology Advances 31 (2013) 838–850

(Yuki et al., 2008). Detailed cellulase gene structures of termites are
reviewed by Lo et al. (2011).
Compared to host cellulases, symbiotic protistan communities in
lower termites produce more complex cellulolytic enzymes. Meta-
transcriptomic sequencing techniques have provided comprehensive
information about cellulolytic enzymes from the protistan symbionts of
five genera of termites, Mastotermes darwiniensis (Mastotermitidae),
Neotermes koshunensis (Kalotermitidae), H. sjostedti, Reticulitermes
speratus (Rhinotermitidae), Reticulitermes flavipes, and Coptotermes
formosanus (Rhinotermitidae) in addition to the closely-related wood
roaches Cryptocercus punctulatus (Scharf and Tartar, 2008; Sethi et al,
2013; Tartar et al., 2009; Todaka et al., 2007, 2010a; Xie et al., 2012). In
the protistan cellulolytic system, GHF5 EGs, GHF7 EGs, and GHF7 CBHs
appear to function as a core enzyme set that might be conserved across
protistan generations during the history of symbiosis between termites
and intestinal protists. On the other hand, the GHF45 EGs might supple-
ment these core cellulases; this family is absent from N. koshunensis and
forms multiple clades in the phylogenetic tree (Todaka et al., 2010a).
The symbiotic protists primarily express GHF3 BGs, which are pre-
dicted to perform similar glucose release functions as endogenous
GHF1 enzymes (Scharf and Tartar, 2008; Tartar et al., 2009; Todaka
et al., 2007; Xie et al., 2012).
As shown in Fig. 1, lower termites produce both EGs and BGs primar-
ily in the salivary glands. These endogenous cellulases probably hydro-
lyze amorphous regions of cellulose during its passage through the
midgut. A combination of endogenous EG and BG can release glucose
from filter paper (Zhang et al., 2010) and pine lignocelluloses (Scharf
et al., 2011). An essential contribution of an endogenous EG to termite
survival and fitness has been verified with RNAi investigations (Zhou
et al., 2008b). Partially degraded cellulosic fragments being moved to
the hindgut are endocytosed by the symbiotic protists. Because no (or
only a trace of) endogenous cellulases enter the hindgut (Fujita et al.,
2010; Nakashima et al., 2002; Watanabe et al., 2006), cellulolysis in
food vacuoles is conceivably accomplished synergism of multiple cellu-
lolytic enzymes that originated from protists. Because ingested cellulos-
ic materials are masticated and fragmented (less than 20 μm in the
foregut; Fujita et al., 2010), this micro-fragmentation of food could
have increased surface area, facilitating access of cellulolytic enzymes
and stimulating endocytosis by symbiotic protists. In addition, the pres-
ence of CBHs in the protistan cellulolytic system apparently confers

Fig. 1. Cellulolytic systems in lower and higher termites. Lower termites secrete endogenous endo-β-1,4-glucanases (EGs) and β-glucosidases (BGs) in the salivary glands. These enzymes
are probably mixed with wood particles ingested by the termites and may act primarily against amorphous cellulose. Chewed wood particles are partially degraded during passage
through the midgut. Remaining wood particles enter the hindgut, where they are digested in the food vacuoles of symbiotic flagellates with protistan EGs, BGs, and cellobiohydrolases
(CBHs) that are highly active against crystalline cellulose. These protists also produce hemicellulolytic enzymes. Such cellulolytic systems of wood-feeding higher termites have been
chiefly studied in Nasutitermitinae, which secrete endogenous BGs both in the salivary glands and the midgut, whereas endogenous EGs are secreted only in the midgut. These enzymes
also act primarily against amorphous cellulose, but may have an undescribed mechanism that enhances digestibility of crystalline cellulose (Tokuda et al., 2012). Details of this endoge-
nous cellulolytic system are depicted in Fig. 3. The hindgut harbors symbiotic bacteria and archaea. Among them, Treponema and Fibrobacteres are primarily involved in cellulolysis with
EGs and BGs. Like these cellulases, cellobiose- and cellodextrin-phosphorylases (GHF94) appear to be involved in cellulolysis, but there is no evidence that CBHs are present in the hind-
guts of higher termites. Nevertheless, the hindgut precipitates contain activity against crystalline cellulose (Tokuda and Watanabe, 2007). Many different hemicellulolytic enzymes are
also produced by these bacterial symbionts.
 J. Ni, G. Tokuda / Biotechnology Advances 31 (2013) 838–850
efficient digestibility of crystalline cellulose in the hindgut. This dual
cellulose digestion system has been proposed as a model of efficient cel-
lulose hydrolysis in lower termites (Nakashima et al., 2002; Tokuda et
al., 2007; Watanabe and Tokuda, 2010).
3.2. Cellulolytic systems in higher termites
Distribution of cellulase activities in higher termites is more variable.
The reason for this is somewhat obscure, but ecological events may
have affected cellulolytic systems during the evolution of higher termites
(Fig. 2). In most wood-feeding termites of the subfamily Nasutitermitinae,
EG activities are confined to the midgut, but Nasutitermes lujae share an
almost equal amount of EG activity both in the midgut and the hindgut
(Slaytor, 2000). In addition, some species of Termitinae have most EG
activity in the hindgut, which is presumably attributable to the symbiotic
amoeba (Slaytor, 2000) whose nature has not been clarified yet. Similar to
the distributions of EG activities, distribution of BG activities is also
variable. Some termites, including Nasutitermes spp., have BG activity
primarily in the salivary glands and the midgut (Slaytor, 2000; Tokuda
et al., 1997). Other termites possess up to 99.5% BG activity in the hind-
gut (Slaytor, 2000). Distribution of cellulase activities in the fungus-
growing termites (subfamily Macrotermitinae) is more complicated
(Rouland-Lefèvre, 2000) partially due to varying dependence on the
symbiotic fungi among species as a nutritional carbon source (Hyodo
et al., 2003). Contributions of the hindgut microorganisms to cellulose
digestion in higher termites have long been neglected, but wood-
fiber associated cellulase activities were reported in the hindgut of
Nasutitermes takasagoensis and Nasutitermes walkeri (Tokuda and
Watanabe, 2007). These cellulase activities were significantly de-
creased if antibiotics were administrated to termites. These bacterial
cellulolytic activities in the hindgut accounted for ~ 50% compared to
cellulase activity in the midgut if microcrystalline cellulose was the
substrate (Tokuda and Watanabe, 2007). Thus, there is strong evi-
dence that the endogenous cellulolytic system in the midgut of
higher termites is more important than that of lower termites. As with
lower termites, endogenous EGs in higher termites are affiliated with
GHF9 and are secreted from the midgut, except for the fungus-growing
Lower termites
Macrotermitinae
Sphaerotermitinae
Loss of protists/
Fungus-growing
habit acquired
Bacterial comb- Foraminitermitidae
growing habit
acquired?
Transition to
soil/humus-feeding
Apicotermitinae
habit
Syntermitinae
Wood/litter-feeding habit
re-acquired in some Termitinae
species?
Acquisition of cellulolytic
amoeba in some soil- Nasutitermitinae
feeding species
Fig. 2. Possible ecological events that could have affected the cellulolytic systems in
higher termites. Higher termites (i.e. family Termitidae) have currently been thought
to exist in seven subfamilies. Phylogenetic relationships shown here were based on In-
ward et al. (2007) and Lo and Eggleton (2011). Although not illustrated here,
Termitinae and Nasutitermitinae might be polyphyletic. Ecological events potentially
affecting the cellulolytic systems during the evolution of higher termites are indicated
to the left of the tree. These events were assumed from Slaytor (2000), Inward et al.
(2007) and Garnier-Sillam et al. (1989).
841
termite Odontotermes formosanus, in which EG genes are still expressed
in the salivary glands (Tokuda et al., 2004). Endogenous GHF1 BGs of
N. takasagoensis were expressed and secreted in the midgut and the sal-
ivary glands (Tokuda et al., 2009, 2012), whereas the fungus-growing
termite Macrotermes barneyi expressed an endogenous GHF1 BG gene
primarily in the midgut (Wu et al., 2012).
Recent metagenomic analyses have clarified the presence of diverse
cellulase genes in the hindgut microflora of higher termites. Among 45
GHFs discovered in the hindgut microbial communities of Nasutitermes
sp., seven GHFs (i.e. GHF5, GHF8, GHF9, GHF44, GHF45, GHF51, and
GHF74) might be acting as EGs, whereas GHF1 and GHF3 might partici-
pate in cellulolysis as BGs (Warnecke et al., 2007). In contrast, many
GHF94 genes that encode cellodextrin or cellobiose phosphorylases
were present in the metagenomic library, suggesting that several bacteria
degrade cello-oligosaccharides via phosphorylation without depending
on BGs. The presence of some of these GHFs in the hindgut was further
confirmed by a proteomic analysis (Burnum et al., 2011). Most of these
GHF genes were assumed to be derived from Spirochetes (genus Trepone-
ma) or Fibrobacteres. Metagenomic analysis did not identify potential
CBHs or components of cellulosomes (complex structures of cellulases
and carbohydrate-binding proteins that adhere to the cell surface of an-
aerobic cellulolytic bacteria via docking proteins, cohesins and dockerins
(Lynd et al., 2002)). However, these results do not exclude the possibility
of efficient degradation of crystalline cellulose by hindgut microflora. A
third mechanism of cellulose digestion is emerging from studies of
Fibrobacter spp., which can disrupt crystalline structure of cellulose with-
out exoglucanases or processive EGs (Ransom-Jones et al., 2012). A pro-
tein complex in the outer membrane of the cell is predicted to bind to
cellulose fibers, cleave individual cellulose chains, and transport them
to the periplasmic space where EGs cleave these cellulose chains
(Wilson, 2009). Indeed, interactions between the intestinal bacteria and
lignocellulosic fragments were observed under an electron microscope
in the anterior paunch (P3) of N. takasagoensis (Tokuda et al., 2005).
One GHF5, EG, has been found in the hindgut metagenomic library of an-
other xylophagous termite, Microcerotermes sp. (subfamily Termitinae)
(Nimchua et al., 2012), from which cellulolytic Bacillus subtilis was isolat-
ed (Taechapoempol et al., 2011). Functional screening of a fosmid library
from microbial DNA extracted from the gut of the fungus-growing ter-
mite Macrotermes annandalei failed to identify CBHs and EGs. Rather,
BG genes were found, but their affiliations were not determined (Liu et
al., 2011). With EST analysis of the symbiotic fungi Termitomyces sp.
from Macrotermes gilvus, identification is confirmed for all necessary cel-
lulolytic enzymes, CBHs (GHF6 and GHF7), EGs (GHF5, 44, 61), and BGs
(GHF1 and GHF3) (Johjima et al., 2006).
As mentioned above, the endogenous cellulolytic system of wood-
feeding higher termites is thought to contribute to cellulose digestion
more significantly than lower termites. Fig. 3 illustrates the endogenous
cellulolytic system of Nasutitermes as a representative of wood-feeding
higher termites based on Tokuda et al. (2012). Masticated cellulosic frag-
ments would be first attacked by salivary BGs that do not enter the
midgut, but the actual role of salivary BGs is ambiguous. A salivary BG
tends to more preferentially hydrolyze β-1,3-glucans (found in fungal
cell walls) than β-1,4 linkages generally recognized in cellulose chains
(Uchima et al., in press). A recent study on labial gland secretion of
termites indicated the presence of p-arbutin that is hydrolyzed by BG to
produce hydroquinone, a precursor of p-benzoquinone, an irritating de-
fensive secretion (Sillam-Dussès et al., 2012). In addition, a possible func-
tion of BG as a proteinaceous pheromone and reproductive suppressor
has also been suggested (Korb et al., 2009; Matsuura et al., 2009). It is
probable that salivary BGs in higher termites play roles other than
cellulolysis. Ingested cellulosic particles are fragmented into less than
150 μm in the foregut. The midgut epithelium secretes both EGs and
BGs that make essential contributions to cellulose digestion. Luminal con-
centrations of these enzymes are extremely high (~1500 U/ml for EG and
~80 U/ml for BG), which may be important for primarily hydrolyzing
amorphous regions of cellulose. Although a purified or recombinant EG
842 J. Ni, G. Tokuda / Biotechnology Advances 31 (2013) 838–850

Fig. 3. Endogenous digestive system in N. takasagoensis. Wood fragments ingested by the termites may first mix with the salivary BGs. The wood particles in the foregut are broken
down to 150 μm in diameter and enter the midgut. The midgut tissue secretes both EGs and BGs into the luminal space. Based on the midgut luminal volume (approx. 75 nl), the
concentrations of EG and BG activities are approximately 1500 units/ml and 80 units/ml, respectively (one unit is the amount of enzyme that produces 1 μmol of reducing sugar or
glucose/min). It is presumed that amorphous regions of cellulose exposed on the surface of the wood particles are digested primarily by these enzymes, and released glucose might
be absorbed across the midgut tissue. Finally, the partially digested wood particles are moved to the hindgut via the mixed segment. The role that the mixed segment plays in di-
gestion has yet to be clarified. PM, peritrophic membrane.

of this termite shows trace activity against microcrystalline cellulose
(Hirayama et al., 2010; Tokuda et al., 1997), the midgut crude enzyme
can hydrolyze highly crystalline cellulose to some extent (Tokuda et al.,
2012). However, a detailed mechanism of this phenomenon has yet to
be elucidated. Short cello-oligosaccharides released by these enzymes
would be degraded in the ectoperitrophic space where BGs accumulate
and glucose products would be absorbed across the midgut wall. As
shown in Fig. 1, remaining cellulosic fragments enter the mixed segment
and then the hindgut, but as mentioned before, the role of the mixed seg-
ment in cellulose digestion is unclear. Cellulosic fragments would be fur-
ther hydrolyzed by prokaryotic cellulases in the hindgut. However, the
detailed mechanism underlying the cellulolytic system requires further
study. In addition, it is likely that other wood-feeding higher termites, es-
pecially those belonging to Termitinae, have different cellulolytic systems
than Nasutitermes (Slaytor, 2000). Hence, further explorations of xyloph-
agous species are required to fully understand cellulolytic systems in
higher termites. In fungus-growing termites, clear conclusions are not
available regarding the mechanism of cellulose digestion. For example,
in the case of O. formosanus, intestinal cellulase activity may be insuffi-
cient to produce the necessary amount of glucose required for respiration
(Tokuda et al., 2005) and the symbiotic fungus is likely a sole carbon
source based on the stable carbon isotope ratio (Hyodo et al.,
2003). In spite of these studies, its cellulolytic mechanism has been
investigated recently, suggesting an “acquired enzyme hypothesis”—
that lignocellulolytic enzymes acquired from the symbiotic fungi act co-
operatively with endogenous digestive enzymes in termites (Deng et
al., 2008a,b).
4. Hemicellulose and hemicellulase
Hemicellulose is a general term for major noncellulosic polysaccha-
rides in plant cell walls which are the second most common polysaccha-
rides on earth. Cellulose is composed of only glucose, whereas main
chains of hemicellulose are composed of xylose, or glucose and mannose,
which is often acetylated or shortly branched with arabinose, galactose, or
other acidic sugars. Chemical compositions of hemicelluloses vary across
plant species: large differences are documented to exist between gymno-
sperms and angiosperms. Usually, hemicelluloses of gymnosperms con-
sist primarily of acetylated glucomannan, arabinoglucronoxylan, and
galactoglucomannan. In contrast, hemicelluloses of angiosperms primari-
ly contain non-acetylated glucuronoxylan and glucomannan. Thus, the di-
gestion of hemicelluloses requires a large set of poly- and di-saccharidases
which include xylanases (EC 3.2.1.8), β-xylosidases (EC 3.2.1.37),
α-glucuronidase (EC 3.2.1.131), α-arabinofuranosidase (EC 3.2.1.39)
and acetylxylan esterase (EC 3.1.1.72) (Juturu and Wu, 2012; Liu et al.,
2011; Saha, 2003). Xylan, a linear polymer of β-xylopyrannosyl units
linked by β-1,4-glycosidic bonds, is the major component of hemicellu-
lose. Xylanases and β-xylosidases cleave the backbone of xylan to release
xylose (Ahmed et al., 2009). Addition of xylanase to a cellulase mixture
enhances enzyme accessibility to cellulose in a lignocellulosic substrate
via increases in fiber swelling and fiber porosity, significantly improving
hydrolytic efficiency (Hu et al., 2011). Glucose residues present in hemi-
cellulose are also susceptible to cellulases (Scharf et al., 2011). Although
multiple hemicellulolytic enzymes could collaboratively act against ligno-
cellulose with cellulases, studies on hemicellulose degradation in termites
are limited compared to what is known about cellulose digestion. Thus,
we have primarily focused on xylanases.
4.1. Xylanases from lower termites
In lower termites examined to date, the majority of xylanase activities
are confined to the hindgut (e.g. Arakawa et al., 2009; Smith et al., 2009;
Zhou et al., 2007), although a report suggests that (1) host GHF9 + GHF1
enzymes together can effectively release glucose from beechwood xylan,
and that (2) a host laccase could enhance this (Scharf et al., 2011). The
presence of numerous xylanolytic bacteria and yeasts is reported from
 J. Ni, G. Tokuda / Biotechnology Advances 31 (2013) 838–850 843

the gut of M. darwiniensis, Zootermopsis angusticollis (Termopsidae), and
Neotermes castaneus (Slaytor, 2000). Purification and molecular cloning
of major xylanases from C. formosanus revealed that these xylanases
were affiliated with GHF11 and their origin was attributed to one of
three intestinal protistan species, Holomastigotoides mirabile (Table 1)
(Arakawa et al., 2009). Recent transcriptomic analyses indicated the ab-
sence of any hemicellulases in host tissues of H. sjostedti (Yuki et al.,
2008), R. flavipes (Tartar et al., 2009), and C. formosanus (Zhang et al.,
2012b) except for GHF35 β-galactosidases in C. formosanus and a GHF2
β-mannosidase from H. sjostedti. In contrast, meta-transcriptomic analy-
ses of hindgut prokaryotic communities in lower termites provided a
number of hemicellulolytic enzymes including GHF10 and GHF11
xylanases (Table 1) (Tartar et al., 2009; Todaka et al., 2007, 2010a;
Cairo et al., 2011; Xie et al., 2012). Todaka et al. (2010a) suggest that
GHF10 xylanases are members of the core enzyme set and GHF11
xylanases assist these core enzymes. Primitive termites M. darwiniensis,
N. koshunensis as well as the wood-roach C. punctulatus lack GHF11
xylanases. However, GH10 xylanase is much less abundant in
Rhinotermitidae, the most apical lineage of lower termites, such as R.
speratus, R. flavipes, and C. formosanus (Tartar et al., 2009; Todaka et al.,
2010a; Xie et al., 2012) and GHF11 might play a central role in xylan
lysis in these termites. This is consistent with reports suggesting that
GHF11 xylanase purified from C. formosanus accounted for 87% of total
xylanase activity (Arakawa et al., 2009). In addition, GHF11 xylanase
and GHF1 β-xylosidase/β-glucosidase were reported from Reticulitermes
santoensis gut bacterial isolates (Matteotti et al., 2011, 2012). Contribu-
tion of bacterial xylanases to hemicellulose degradation in lower termites
has yet to be verified.
4.2. Xylanases from higher termites
Except for the possible presence of bifunctional cellulase/xylanase in
the midgut of Trinervitermes trinervoides (subfamily Nasutitermitinae)
(Potts and Hewitt, 1974), there are no reports of endogenous xylanases
from litter- or wood-feeding higher termites. However, the midgut of
fungus-growing termites often contains abundant xylanase activity and
significant differences in these distribution patterns occur among species
(Rouland-Lefèvre, 2000). There are two hypotheses to explain the origin
of midgut xylanase activity: 1) the enzyme is acquired from symbiotic
fungi, 2) the enzyme is endogenous (Rouland-Lefèvre, 2000; Slaytor,
2000). Accordingly, some xylanases with different molecular weight
(36, 56, 22.5 kDa) have been purified from the fungus-growing termite
Macrotermes bellicosus and its symbiotic fungus (Table 1) (Matoub and
Rouland, 1995) a xylanase with big molecular weight over 80 kDa was
also purified from symbiotic fungus of M. subhyalinus (Faulet et al.,
2006). A clear picture of origin of the midgut xylanases has yet to be elu-
cidated, but a recent study indicated the presence of a GHF11 xylanase in
the gut microbiota of the fungus-growing termite, M. annandalei
(Table 1) (Liu et al., 2011). GH11 xylanase was also existed in the gut
microbiota of higher termite Nasutitermitidae (Brennan et al., 2004).
According to recent metagenomic analyses of the hindgut microbiota
in wood-feeding higher termites Nasutitermes sp. and Microcerotermes
sp., these metagenomic libraries seem to contain hemicellulolytic enzyme
genes more abundantly than cellulase genes (Nimchua et al., 2012;
Warnecke et al., 2007). In these termites, GHF8, GHF10, GHF11, and
GHF43 xylanases were obtained from the metagenomic libraries of the
hindgut microflora, and the presence of an apparent xylanosome operon
was suggested in Microcerotermes sp. (Nimchua et al., 2012). On the
other hand, GHF10 xylanases were detected with proteomic analysis
of Nasutitermes sp. GHF8 was also detected, but substrate specificity
(xylanase or cellulose) has not been defined at this time (Burnum et
al., 2011). A thermostable microbial GHF10 xylanase was found in
bacteria Saccharopolyspora pathumthaniensis S582 isolated from a grass-
feeding termite of another subfamily Apicotermitinae, Speculitermes sp.
(Sinma et al., 2011). An EST analysis of the fungal symbiont Termitomyces
sp. of the fungus-growing termite M. gilvus indicated that the fungus also
produces hemicellulolytic enzymes including xylanases (Johjima et al.,
2006). Thus, symbiotic microbiota in higher termites has a potential
role in degrading hemicelluloses and hence may be rich reservoirs of
hemicellulolytic genes.

Table 1
Xylanases purified or sequenced from termites or symbiotic microorganisms.

Enzyme Origin MW (kDa) Glycoside hydrolase family Reference

Endoxylanase Purified from symbiotic fungus of M. bellicosus 36 n.d. Matoub and Rouland (1995)
Exoxylanase Purified from M. bellicosus 56 n.d. Matoub and Rouland (1995)
Exoxylanase Purified from symbiotic fungus of M. bellicosus 22.5 n.d. Matoub and Rouland (1995)
Xylanase From termite Nasutitermes sp. gut library (XYL6419) n.d. GH11 Brennan et al. (2004)
Xylanase Purified from symbiotic fungus of M. subhyalinus 80–87 n.d. Faulet et al. (2006)
Endo-1,4-beta-xylanase Purified from symbiotic flagellatesof Coptotermes formosanus 19, 17, 18 GH11 Arakawa et al. (2009)
Endo-1,4-beta-xylanase Cloned from a gut microbiota fosmid library of M. annandalei 64.5 GH11 Liu et al. (2011)
Xylanase Purified from bacteria from higher termite 36 GH10 Sinma et al. (2011)
Xylanase Purified from bacteria from higher termite 205 GH10 Dheeran et al. (2012)
Endo-1,4-beta-xylanase Cloned from genomic library of bacteria from R. santoensis 29 GH11 Matteotti et al. (2012)
Xylanase Cloned from hindgut fosmid library from Microcerotermes sp. n.d. Nimchua et al. (2012)
GH8, 10, 11

Endo-1,4-beta-xylanase From protist of C.formosanus through 454 pyrosequencing 34 GH10 Xie et al. (2012)
Endo-1,4-beta-xylanase From EST analysis of symbiotic fungus of fungus-growing termite n.d. GH10, 11 Johjima et al. (2006)
*Xylanase From hindgut microbiota metagenomic analysis of Nasutitermes n.d. GH8, 10, 11, 43 Warnecke et al. (2007)
*Xylanase From cDNA library of protist of R. speratus n.d. GH10, 11, 43 Todaka et al. (2007)
*Xylanase From EST analysis of several lower termites n.d. GH8, 10, 11, 43 Todaka et al. (2010)
*Xylanase From metatranscriptomic analysis of R. flavipes n.d. GH10, 11 Tartar et al. (2009)
*Xylanase From metaproteomic analysis of C. gestroi n.d. GH10, 11, 43 Cairo et al. (2011)
*Xylanase From transcriptome analysis of C. formosanus n.d. GH8, 10, 11 Zhang et al. (2012)
n.d.: not determined.
a
Obtained from “omics” study; xylanase activity has not been confirmed.
Xylanases found from lower termites are highlighted in gray.
844 J. Ni, G. Tokuda / Biotechnology Advances 31 (2013) 838–850

5. Lignin and its modification enzymes
5.1. Structural changes of lignin during passage of the gut
Lignin has a complex amorphous and recalcitrant structure,
consisting of p-hydroxyphenylpropane monomer units joined by
ether or C\C bonds (Cornwell et al., 2009). Lignins comprise 20–30%
of wood's dry weight, and gymnosperms primarily contain guaiacyl
lignin, whereas angiosperms mainly contain syringyl lignin. Here we
summarize the structural changes of lignin which have been well
documented within the Coptotermes species. Early studies on lignin
degradation in termites have been elegantly described in a review by
Breznak and Brune (1994). In such studies, 14C-[lignin]-lignocellulose
was administrated to Coptotermes acinaciformis but no significant
14
release of respired CO2 was detected. A preliminary study with C.
formosanus by Kyou et al. (1996) suggested that milled-wood-lignin
was specifically endocytosed by one of three symbiotic protists,
Holomastigotoides hartmanni and their microscopic observation
suggested that its surface was likely degraded (Kyou et al., 1996).
Gel permeation chromatography of termite fecal lignin indicated a
modification of the lignin–carbohydrate complex, but side chain and
aromatic ring cleavages of ingested lignin molecules were not detected
with infrared and UV spectrometry. A simple β-O-4′ type lignin
model compound (1-(4-O-benzylguaiacyl)-3-hydroxy-2-(4-methyl-
umberlliferyl)-propanone) was fed to C. formosanus for one week and
4-methyl-umbelliferone measurements indicated that ~45% of the
compound was degraded by the termites (Hirai et al., 2000). In contrast,
degradation of oligomers (3- to 5-mer) synthesized from coniferyl alco-
hol, which was fed to C. formosanus for 20 days, was not detected with
HPLC (Hirai et al., 2000), although possible oxidations of the lignin olig-
omers in another termite H. sjostedti were detected by the same exper-
iment. Moreover, a comparative analysis of lignin between a dietary
wood pieces and feces of C. formosanus using solid-state CP/MAS 13C
NMR revealed that the host and the gut symbionts had little or no ability
to degrade natural lignin: no alterations in aromatic ring carbons and aryl
methoxyl carbons were noted although wood polysaccharides were
significantly degraded (Hyodo et al., 1999). Termites are thought to be
capable of degrading phenylpropanoid lignin monomers or dimers but
incapable of mineralizing larger lignin moieties. Recently, investigations
into the structural details of lignin changes during passage through the
termite gut have been initiated and are described below.
A current scheme of the termite lignolytic system is illustrated in
Fig. 4. In a series of recent publications, Ke and colleagues analyzed
structural changes of lignin in the gut of C. formosanus via a combination
of spectroscopic techniques and NMR, with a special emphasis on pyrol-
ysis gas chromatography–mass spectrometry analysis. Because pyroly-
sis is conducted at 210–610 °C, the structure of large lignin molecules
is difficult to differentiate from resulted pyrolytic fragments. Neverthe-
less, such data are useful for ascertaining structural modifications of lig-
nin based on pyrolytic patterns and semi-quantitative, high-resolution
structures. With these methods, initial structural changes already
occurred at the termite mastication step (Ke et al., 2012a). Further mod-
ifications appeared to continuously occur in the termite foregut and the
midgut (Ke et al., 2010). These modifications were characterized by lig-
nin ring decarboxylation and side chain oxidation. Cleavage of some
aromatic rings is presumed to occur in the hindgut based on the pyro-
lytic analysis of short aromatic model compounds fed to termites (Ke
et al., 2011b). No potential lignin degradation enzymes have been
found from metatranscriptomes of the symbiotic protistan community
in the hindgut of C. formosanus (Xie et al., 2012), whereas in general, a
role has been suggested for bacteria in lignin degradation (Bugg et al.,
2011). Apparently, termites harbor intestinal bacteria with aromatic
degrading capabilities (Bugg et al., 2011; Le Roes-Hill et al., 2011). Clas-
sification of these bacteria was coincident with classes of previously
identified bacteria with in vitro lignin-degradation abilities. The meta-
bolic pathway of bacterial lignin degradation is still unclear, but reports
of bacteria that produce peroxidases suggest that such bacteria may be
involved in these lignin modifications (Bugg et al., 2011; Le Roes-Hill et
al., 2011). Analysis of termite feces revealed modifications on lignin
alkyl side groups, ring hydroxyl groups, and ring methoxyl groups
with conservation of the abundant β-O-4′ interunit lignin linkage and
retention of the original aromatic properties (Ke et al., 2011a,c).
Therefore, it is conceivable that the termite digestive system does not
reduce aromatic moieties in large lignin molecules but only modifies
some functional groups (Ke et al., 2011c). Indeed, other research
indicates that total lignin increased from 28.9% in wood to 59.2% in

Lignocellulose
(Lignin + cellulose + hemicellulose)

Laccase Lignin ring decarboxylation and side

Aldo-keto reductases chain oxidation etc., at the chewing step,
Catalases and in the foregut and the midgut

Lignocellulose with reduced glucan

content: conservation of the abundant
Exposure of cellulose (and β-O-4’ interunit lignin linkage and
hemicellulose) fibers retention of the original aromatic
properties

Some presumed aromatic ring

Cellulases and hemicellulases cleavage by the hindgut bacteria
(no enzymes involved in this
process have been isolated)

Feces
(Total lignin proportion increased without reducing aromatic
moieties)

Fig. 4. A scheme of lignin modification in the gut of termites. Modifications of lignin and hemicellulose at the chewing steps and in the gut are thought to result in unlocking of
cellulose fibers from lignocellulose and enabling efficient cellulose digestion. However, lignin is thought to retain the original aromatic properties throughout the gut passage.
 J. Ni, G. Tokuda / Biotechnology Advances 31 (2013) 838–850
feces of C. formosanus with observations of similar chemical changes of
lignin (Hyodo et al., 1999). Glucan content was significantly reduced
during gut passage (Li et al., 2012). Such lignin modifications likely
facilitate the enzymatic hydrolysis of wood ingested by termites (Li et
al., 2012), but genes encoding lignolytic enzymes or pure enzymes
have not been isolated from termite gut bacteria.
5.2. Lignin-modification enzymes
Lignin mineralization is extensively studied in white-rot and
brown-rot fungi and the following enzymes are known to participate in
lignin degradation: lignin peroxidase (LiP) (EC 1.11.1.14), manganese
peroxidase (MnP) (EC 1.11.1.13), versatile peroxidase (VP) (EC
1.11.1.16), and laccase (EC 1.10.3.2) (Bugg et al., 2011). Lignin is
depolymerized and mineralized by radical reactions that are triggered
by oxidation with these enzymes. In recent years, cellobiose dehydroge-
nase (CDH) (EC 1.1.99.18) has been shown to participate in the
lignocellulolytic process, possibly via the production of free radicals
(Harreither et al., 2009). On the other hand, LiP, MnP, VP, and CDH have
not been identified in insects, whereas laccases are commonly involved
in melanization and sclerotization of the cuticular layers in insects.
Recently, laccases have been documented as candidates for lignin-
modification enzymes (“lignases”) in termites. Although “lignases” gen-
erally represent enzymes that efficiently degrade or mineralize lignin,
this does not appear to be true in termites, according to the aforemen-
tioned studies. From R. flavipes, cDNAs encoding putative laccases
were found in a host EST library (Scharf and Tartar, 2008; Tartar et al.,
2009), while strong oxidative activity against pyrogallol, a monomeric
lignin model compound, was detected in the salivary glands and foregut
extract of this termite (Tartar et al., 2009). Further studies on these
genes indicated that these laccases were primarily produced in the
salivary glands and that recombinant laccases, which were expressed
in Trichoplusia ni larva using a Baculovirus-expression vector, showed
trace or no activity against widely-used laccase substrates such as
2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) and
syringaldazine as well as melanin precursors but preferentially oxidized
lignin monomers such as 2,6-dimethylphenol (DMP) and pyrogallol in
the presence of H2O2 (Coy et al., 2010). These enzymes also act against
soluble lignin derivatives (Coy et al., 2010), but a detailed mode of ac-
tion is unclear. Addition of the salivary laccase reduces glucose produc-
tion from an enzymatic reaction by endogenous EG and BG of R. flavipes
when pine wood sawdust is used as a substrate, whereas it enhances
the activity of a protistan GHF11 xylanase when using beechwood
xylan as a substrate (which contains lignin) (Scharf et al., 2011; Sethi
et al., 2013). Of note, the simultaneous actions of endogenous (salivary
glands and/or midgut) and symbiotic cellulases (hindgut) are unlikely
in vivo (Tokuda et al., 2007). Previous studies suggested that lignin
modifications similar to those in the gut of C. formosanus were observed
in the gut of R. flavipes (Ke et al., 2010) but degradation activity against
the β-O-4′ type lignin model compound in the Rhinotermitid R. speratus
was reduced, compared to activity observed C. formosanus (~13%)
(Hirai et al., 2000). Moreover, an early study using R. flavipes also
indicated no significant change of lignin content in wood particles
during passage through the gut, but suggested the possibility of
chemical lignin modifications (Breznak and Brune, 1994). Therefore,
putative laccases in R. flavipes might be unable to degrade large
insoluble lignin molecules and contribute to functional group
modifications of lignin or detoxification of lignin monomers that
have antimicrobial activity (Borneman et al., 1986). These activities
may also be related to a potential ability of termites to degrade
toxic polycyclic aromatic hydrocarbons, a finding that was recently
been described (Ke et al., 2012b). In addition, aldo-ketoreductases and
catalases have been shown to enhance lignocellulose degradation in
vitro (Sethi et al., 2013). These two enzymes alone, or together can signif-
icantly enhance glucose liberation from pine-wood lignocelluloses by
combining to termite endogenous GHF9, GHF1, and protistan GHF7
845
(Sethi et al., 2013). However, such collaborative actions have yet to be
established to occur in vivo.
Details of possible lignin modifications have yet to be elucidated in
higher termites. No genes encoding lignolytic enzymes were identified
in the metagenomic library from the hindgut microflora of wood-
feeding Nasutitermes (Warnecke et al., 2007). Still, some knowledge ex-
ists regarding the putative lignocellulytic enzymes from fungus-growing
termites, in which lignin degradation is thought to be accomplished
through symbiosis with the white-rot fungi, Termitomyces sp. Little effect
of delignification without the aid of Termitomyces was suggested in
O. formosanus (Li et al., 2012). Predominant laccase activities against
DMP and ABTS were detected in the fungus combs and fungi isolated
from the nests of three genera of fungus-growing termites (i.e.
Macrotermes, Odontotermes, and Microtermes) (Taprab et al., 2005). The
relevant genes involved in plant cell wall degradation were identified
from the symbiotic fungi of fungus-growing termites (Johjima et al.,
2006). A small amount of laccase activity was also detected in the gut
of O. formosanus (the laccase activities were in this order: symbiotic
fungus > fungus comb > worker alimentary tracts) (Pan et al., 2009).
The laccase purified from the fungus combs of O. formosanus has been
characterized (Zhou et al., 2010b): the molecular mass of the laccase
was 65 kDa, and the optimum pH and temperature were pH 4.0 and
10 °C with ABTS as the substrate, respectively (Vmax and Km were
3.62 μmol min−1 mg−1 and 119.52 μM, respectively). These character-
istics are not significantly different from other fungal laccases (Zhou
et al., 2010b). With these fungi, lignin is progressively degraded in
the combs and degrees of such degradation differ among various
Termitomyces species or strains (Hyodo et al., 2003). In contrast,
the gut pH of Macrotermitinae ranges from 5.0 to 8.5 (Bignell and
Eggleton, 1995), which may not be a suitable physical condition for
the fungal laccases and may account for the small amount of activity.
6. Heterologous production of lignocellulases from termites
For industrial applications of termite lignocellulolytic enzymes, re-
combinant enzyme productions (overexpressions) in heterologous
hosts are useful and necessary. In the last several years, increasing in-
vestigations have been reported regarding the heterologous expres-
sions of lignocellulolytic enzymes obtained from termites and their
symbiotic microbiota. However, an expression of CBH has yet to be
achieved; although, this is apparently an essential enzyme for crystal-
line cellulose digestion. Next, we summarize what is known about re-
combinant enzymes and compare enzymatic properties of EGs, BGs,
and xylanases from termites or their symbionts.
6.1. Heterologous production of EGs
Comparisons of enzymatic properties of recombinant EGs from ter-
mites are shown in Table 2. Recombinant production (overexpression)
of termite GHF9 enzymes was previously difficult, although the function-
al expression of NtEG has been detected using the Congo-red plate assay
(Tokuda et al, 1999). Successful overexpression of termite GHF9 EG was
first reported in 2005 with Escherichia coli (Ni et al., 2005). The
overexpressed mutant EG was obtained by family shuffling of EG
genes from four different termite species (R. speratus, N. takasagoensis,
C. formosanus, and C. acinaciformis). The mutant enzyme had 20–30-fold
higher activity against CMC and had properties similar to those of the na-
tive enzymes (Ni et al., 2005). Using the adapted mutant cDNAs as paren-
tal genes combined with native-form cDNAs, further family shuffling was
performed and a mutant EG with improved thermostability was
obtained. This mutant EG had high specific activity (889 units/mg) and
the thermostability was 10 °C higher than parental enzymes (Ni et al.,
2007a). Furthermore, activity related amino acid mutations of a GHF9
EG were identified (Ni et al., 2010).
Currently, a number of termite EGs have been reported to be
expressed in different hosts. EG (CfEG3a) of C. formosanus, in both
846 J. Ni, G. Tokuda / Biotechnology Advances 31 (2013) 838–850

Table 2
Enzymatic properties of purified recombinant EGs from termites and their symbionts.

Source organism Specific activity Optimal temperature Optimal pH Km Vmax Expression host Reference
(U/mg) (°C) (CMC) (CMC)
(mg/ml) (U/mg)

Chimeric termite EGs

A18 544 55 7 10.7 1074 E.coli Ni et al. (2005)
PA68 560 55 6.9 12.7 889 E.coli Ni et al. (2007a)

Reticulitermes speratus
RsEG (GHF9) 1200 45 5.5 2 1429 A.orizae Hirayama et al. (2010)

Nasutitermes takasagoensis
NtEG (GHF9) 1392 65 6 4.67 1667 A.orizae Hirayama et al. (2010)

R.flavipes
Cell-1 (GHF9) n.d. n.d. n.d. 14.66 0.838 E.coli Zhou et al. (2010a)
Cell-1 (GHF9) 1.3 50–60 6.5–7.5 9.93 1.056 T.ni⁎ Zhou et al. (2010a)

Coptotermes formosanus
CfEG3a (His-tagged) (GHF9) 328 37 5 2.2. 590 E.coli Zhang et al. (2009, 2011)
CfEG5 (GHF9) 325 43 5.6 5.6 548 E.coli Zhang et al. (2011)

Protists from C.formosanus

CFP-EG1 (GHF5) 105 70 6 1.9 148.2 E.coli Inoue et al. (2005)

Protists from R.speratus

RsSymEG1 (GHF7) 603 45 6.5 1.97 769.6 A.orizae Todaka et al. (2010b)

One unit (U) is defined as the amount of enzyme that releases 1 μmol of reducing sugar (glucose equivalent) per minute. Activities against carboxymethylcellulose (CMC) are
shown for comparisons. n.d, not determined. In addition to above-mentioned cellulases, 26 GHF5, 7 GHF9, and 2 GH45 bacterial EG genes from the metagenomic library in the hind-
gut of Nasutitermes sp. and one GHF5 EG from the metagenomic library in the hindgut of Microcerotermes sp. were expressed in E.coli and P.pastoris and their activity was confirmed
(Nimchua et al., 2012; Warnecke et al., 2007).
⁎ Baculovirus was used as an expression vector.

native form (nCfEG) and C-terminal His-tagged form (tCfEG) was
expressed in E. coli using pET28 vector (Zhang et al., 2009). The re-
combinant nCfEG and tCfEG showed cellulolytic activity against
CMC with specific activities of 328 and 385 units/mg respectively.
Another EG (CfEG5) from C. formosanus was also expressed in E. coli
with a specific activity of 325 units/mg (Zhang et al., 2011). The re-
combinant CfEG5 was active against filter-paper cellulose, resulting
in mostly cellobiose and cellotriose. A native form of EGs from R.
speratus and N. takasagoensis (RsEG and NtEG) was also produced in
the filamentous fungus Aspergillus oryzae, which is a more suitable
host for mass production than E. coli (Hirayama et al., 2010). The
activities of purified recombinant EGs against CMC were significantly
higher than that of any other EGs reported (1200 and 1392 units/mg
for RsEG and NtEG, respectively). These specific activities of recombi-
nant endogenous termite EGs were approximately 100 times higher
than commercially available Trichoderma EGs (Hirayama et al.,
2010). These EGs were also expressed in the yeast Pichia pastoris
using vector pBGP3, which allowed one-step purification of heterolo-
gous proteins with N-terminal 6 × His and myc-epitope tags (Uchima
and Arioka, 2012). An endogenous salivary EG from R. flavipes was
also expressed using both Baculovirus and E. coli expression systems
to compare their properties (Zhou et al., 2010a). As a result, the
Baculovirus-mediated expression of EG was more readily obtained in
solubilized form and was more active than the enzyme expressed in
E. coli. Recombinant Baculovirus/T. ni-expressed enzyme activity was
comparable to the native enzyme, and the optimal activity was
obtained at pH 6.5–7.5 and 50–60 °C. In addition, enhanced activity
was documented up to 70 °C in the presence of CaCl. Km and Vmax
for the Baculovirus-mediated expression of EG against CMC were
0.993% w/v and 1.056 μmol/min/mg, respectively.
As for protistan EGs, a GHF5 EG gene was overexpressed in E.coli,
and its optimal pH and temperature were 5.8–6.0 and 70 °C, respective-
ly (Inoue et al., 2005). Km and Vmax for CMC were 1.90 mg/ml and
148.2 units/mg protein, respectively. Also, a GHF7 EG from a symbiotic
protist of R. speratus was expressed in A. oryzae (Todaka et al., 2010b).
The recombinant EG had relatively high specificity (603 units/mg)
and its optimal pH and temperature were 6.5 and 45 °C, respectively.
Km and Vmax for CMC were 1.97 mg/ml and 769.6 units/mg protein,
respectively.
Recombinant EGs are useful for studying cellulose metabolism in ter-
mites and have the potential for being engineered as new biocatalysts for
cellulose-based biofuel production. The recombinant cellulase should
also be useful for designing and screening inhibitors to develop target-
specific and environment-friendly bio-termiticides (Zhang et al., 2011;
Zhou et al., 2008a,b).
6.2. Heterologous production of BGs
BG is critical for cellulose degradation and glucose production in the
termite. GHF1 BGs from termites and their symbionts have been success-
fully overexpressed in prokaryotic and eukaryotic hosts (Table 3). NkBG,
obtained from the salivary glands of N. koshunensis (Tokuda et al., 2002),
was first heterologously expressed in E. coli (Ni et al., 2007b). The enzy-
matic properties of the recombinant BG are mostly consistent with the
native enzyme. Km and Vmax against cellobiose were 3.8 mM and
220 units/mg, respectively. The optimum pH and thermostability were
5.0 and 45 °C respectively. However, the specific activity of the recombi-
nant enzyme was 156.7 U/mg, which is almost 3-fold higher than that of
the partially purified BG of N. koshunensis (Tokuda et al., 2002), and it
preferentially hydrolyzed laminaribiose (consisting of β-1,3-linked glu-
cose) over cellobiose (consisting of β-1,4-linked glucose). To construct
a more efficient mass production system, NkBG was expressed in A.
oryzae (Uchima et al., 2011). Specificity against p-nitrophenyl-β-D-
glucoside (pNPG) was 12.4 U/mg. Km and Vmax against pNPG were
0.77 mM and 16 units/mg, respectively. The other enzyme proper-
ties were similar to the enzyme expressed in E. coli. Interestingly,
glucose stimulated NkBG activity; in contrast, other BGs are often
inhibited by glucose, the end product of enzymatic hydrolysis. For
example, activity of the commercially available BG, Novozyme188,
is inhibited by 0.6 M glucose (Uchima et al., 2012), but NkBG activity
was 100% at this glucose concentration, compared to activity measured
without glucose. Moreover, incubation of NkBG with 200 mM glucose
delayed enzyme inactivation using heat (55 °C) (Uchima et al., 2011).
 J. Ni, G. Tokuda / Biotechnology Advances 31 (2013) 838–850 847

Table 3
Enzymatic properties of purified recombinant BGs (GHF1) from termites and their symbionts.

Source organism Specific Optimal Optimal Km Vmax Relative act. (%) at Expression Reference
activity temperature pH (mM) (U/mg) glucose conc. 0.6 Ma host
(U/mg) (°C)

Neotermes koshunensis
NkBG (cellobiose) 156. 7 45 5 3.8 220 n.d. E.coli Ni et al. (2007b)
NkBG (pNPG) 12.4 40 5 0.77 16 110 A.oryzae Uchima et al. (2011)

Reticulitermes favipes
RfBGluc-1 (cellobiose) n.d. 40 6.5–7 1.44 638 n.d. T.nib Scharf et al. (2010)
RfBGluc-1 (pNPG) n.d. 40 6.5–7 1.66 22.92 n.d. T.nib Scharf et al. (2010)

Coptotermes formosanus
β-glucosidase (cellobiose) n.d. 49 5.6–6.2 2.3 462.6 85 E.coli Zhang et al. (2012a)

Macrotermes barneyi
MbmgBG1 206 (cellobiose) 45 (pNPG) 5 (pNPG) n.d. n.d. n.d. E.coli Wu et al. (2012)

Nasutitermes takasagoensis
G1sgNtBG1 (pNPG) n.d. 50 6 n.d. n.d. 70 P.pastris Uchima et al. (2013)
G1mgNtBG1 (pNPG) 5.83 65 5.5 0.67 8 50 P.pastris Uchima et al. (2012)

Bacerial isolate from R.santoensis

BglB (pNPG) 0.441 40 6 n.d. n.d. n.d. E.coli Matteotti et al. (2011)
BglB (pNPX) 2.112 n.d n.d n.d. n.d. n.d. E.coli Matteotti et al. (2011)

Symbiont of Globitermes sulphureus

bgl-gs1 (pNPG) 110 90 6 0.18 61.6 n.d. E.coli Wang et al. (2012)

One unit (U) is defined as the amount of enzyme that releases 1 μmol of glucose (from cellobiose as substrate) or p-nitrophenol (from p-nitrophenyl-β-D-glucopyranoside; pNPG or
p-nitrophenyl-β-D-xylopyranoside; pNPX as substrate) per minute. n.d, not determined.
a
Activity of Novozyme 188, a commercially β-glucosidase, was found to be completely inhibited in the presence of 0.6 M glucose (Uchima et al., 2012).
b
Baculovirus was used as an expression vector.

GHF1 BG genes from other lower termites, R. flavipes and
C. formosanus, have also been heterologously expressed. RfBGluc-1
was produced in T. ni with a baculovirus-insect expression system
(Scharf et al., 2010). Km and Vmax against pNPG were 1.66 mM and
22.92 μmol/min/mg, respectively, while Km and Vmax against cellobiose
were 1.44 mM and 638 μmol/min/mg, respectively. The temperature
stability of the recombinant RfBGluc-1 increased about 20% in the pres-
ence of 30 mM CaCl2 (Scharf et al., 2010). Recombinant RfBGluc-1,
when combined with a host GHF9 EG, had 100–300-fold higher glucose
production from pine sawdust, compared to EG or BG alone (Scharf et
al., 2011). The rate of glucose production was further enhanced by an
addition of a protistan GHF7 (Sethi et al., 2013).
BG from C. formosanus was functionally expressed in E. coli (Zhang
et al., 2012a). Km and Vmax against cellobiose were 2.33 mM and
462.6 μmol/min/mg, respectively. Glucose had no significant effect
on BG activity when the glucose concentration was increased to up
to 0.3 M. An approximately 6% reduction occurred when glucose
reached 0.4 M. Nevertheless, 71% activity remained when 1.0 M glucose
was present in the reaction mixture. This BG was irreversibly inactivated
by an inhibitor of conduritol B epoxide, a finding which might be useful
in the development of enzyme-specific bio-termiticides.
G1NtsgBG1 and G1NtmgBG1, obtained from the salivary glands and
the midgut of the higher termite N. takasagoensis (Tokuda et al., 2009),
were expressed in the yeast Pichia pastoris (Uchima et al., in press). In con-
trast to monomeric forms of BGs in lower termites, the purified recombi-
nant G1NtsgBG1 and G1NtmgBG1 were assumed to be homotrimeric
with molecular masses of 163 kDa and 169.5 kDa, respectively. These
BGs were relatively thermostable and highly glucose-tolerant. Further-
more, G1NtmgBG1 acted synergistically with Celluclast 1.5 L, releasing
more reducing sugars from Avicel than Novozym 188 combined with
Celluclast 1.5 L, suggesting that this termite BG holds promise for supple-
mental use in cellulose hydrolysis (Uchima et al., 2012). Recently,
MbmgBG1 obtained from the midgut of the fungus-growing termite M.
barneyi was functionally expressed in E. coli with relatively high specific-
ity (206 units/mg against cellobiose) (Wu et al., 2012). Apart from endog-
enous BGs from termites, a highly thermostable GHF1 BG has been
identified from a metagenomic library of the gut of the higher termite
Globitermes sulphureus (Amitermes-group Termitinae) (Wang et al.,
2012). This BG (bgl-gs1) was overexpressed in E. coli and recombinant
BG had an optimal temperature with pNPG at 90 °C which retained
more than 60% of its activity for 120 min at 75 °C. The specific activities
of bgl-gs1 on pNPG and salicin were 110 units/mg and 14 units/mg;
Km and Vmax values were 0.18 mM and 61.6 units/mg for pNPG and
2.59 mM and 71.9 units/mg, respectively.
6.3. Heterologous production of xylanases
Recombinant xylanases and those purified from termite gut bacteria
are listed in Table 4. Recently, a xylanase (XylB8) from Gram-positive
bacteria of Reticulitermes santonensis was cloned by functional activity
screening (Matteotti et al., 2012). XylB8 was affiliated with GHF11
and expressed in E. coli. Recombinant xylanase had maximal activity
against beechwood xylan at a pH of 5.0 and 55 °C. Surprisingly, this
xylanase retained 28% activity after preincubation at 100 °C. Km and
Vmax were 9 mg/ml and 3333 units/mg, respectively. Compared to the
activity against beechwood xylan, it also hydrolyzed birchwood xylan
(96%) and weakly degraded CMC (6%).
A GHF11 xylanase gene was also obtained from a fosmid library of the
gut symbionts of the fungus-growing termite M. annandalei (Liu et al.,
2011). This xylanase (Xyl6E7) was expressed in E. coli. Activity against
beechwood xylan was 771 units/mg; activity against birchwood xylan
was 733 units/mg. The optimal pH was 7.5 and the activity was temper-
ature sensitive. Maximal activity was observed at 55 °C, a preincubation
at 30 °C to 55 °C for 5 min reduced activity to less than 50%. However,
L-cysteine restored activity to more than 60% after preincubation at
55 °C for 30 min.
In addition, xylanases (GHF10 and GHF11) from the metagenomic
library of Microcerotermes sp. were partially characterized (Nimchua
et al., 2012). These xylanases performed optimally at pH 8.0 and
55 °C. They were stable at alkaline pHs and had greater than 70% ac-
tivity at pH 9.0.
Recently, reports of xylanases purified from termite gut bacteria sug-
gest that because the bacteria are culturable, these xylanases could be
attractive for industrial applications. A thermostable xylanase was iden-
tified from a gut bacterium (S. pathumthaniensis S582) of Speculitermes
sp. (Sinma et al., 2011). The optimal temperature and pH of the xylanase
848 J. Ni, G. Tokuda / Biotechnology Advances 31 (2013) 838–850

Table 4
Enzymatic properties of recombinant xylanases and purified xylanase from termite gut bacteria.

Source organism Substrate Specific activity Optimal temperature Optimal pH Km Vmax Expression host Reference
(U/mg) (°C) (mg/ml) (U/mg)

Gram-positive bacteria from Reticulitermes santoensis

mXylB8 (GHF11) Beechwood xylan 1837 55 5 9 3333 E.coli Matteotti et al. (2012)
Gut symbionts from Macrotermes annandalei
Xyl6E7 (GHF11) Beechwood xylan 771 n.d. n.d. n.d. n.d. E.coli Liu et al. (2011)
Birchwood xylan 733 50–55 7.5 6.96 1057.8 E.coli Liu et al. (2011)
Gut symbionts from Microcerotermes sp.
X0012 (GHF11) Birchwood xylan n.d. 55 8 n.d. n.d. E.coli Nimchua et al. (2012)
X1098 (GHF10) Birchwood xylan n.d. 55 8 n.d. n.d. E.coli Nimchua et al. (2012)

Purified bacterial xylanase

Saccharopolyspora pathumthaniensis S582 isolated from Speculitermes sp.
Extracellular xylanase (GHF10) Beechwood xylan 965 70 6.5 3.92 256 Non-recombinant Sinma et al. (2011)
Paenibacillus macerans IIPSP3 isolated from wood-feeding higher termites
Xylanase (possibly GHF10) Beechwood xylan 4170 60 4.5 6 7407 Non-recombinant Dheeran et al. (2012)
Birchwood xylan n.d. n.d. n.d. 6.1 7575 Non-recombinant Dheeran et al. (2012)

One unit (U) is defined as the amount of enzyme that releases 1 μmol of reducing sugar (xylose equivalent) per minute; n.d., not determined.

were 70 °C and 6.5. Km and Vmax were 3.92 mg/ml and 256 units/mg.
The ORF of this xylanase encoded 368 amino acid residues belonging
to GHF10.
Another thermostable xylanase was purified from a gut bacterium
(Paenibacillus macerans IIPSP3) of wood-feeding higher termites (species
not identified) (Dheeran et al., 2012). The optimal temperature was
60 °C, but it was active over a broad range of temperatures (40–90 °C)
and pHs (3.5–9.5). The half-life of xylanase was 6 h at 60 °C and 2 h at
90 °C. This xylanase has a molecular mass of 205 kDa, and the internal
amino acid sequence was not significantly homologous with known
xylanases. A typical feature of this xylanase was not only thermostability
but its high specific activity (Vmax = 7407 units/mg).
Although uncharacterized at present, the protistan GHF11 xylanase
gene was expressed in T. ni larva using an insect Baculovirus-expression
vector system (Sethi et al., 2013), and significantly more xylose was lib-
erated from pine lignocellulose when the enzyme was combined with
host laccase than when the xylanase was tested alone, indicating that
the combination of hemicellulose degradation and lignin modification
is inducible in vitro.
7. Future perspectives
Termites can digest 74–99% lignocelluloses (Prins and Kreulen,
1991). Hence, lignocellulose-degrading enzymes produced by ter-
mites and their symbiotic microorganisms should permit efficient uti-
lization of lignocellulosic materials. Multiple genes explored through
“omics” studies and successful recombinant techniques with such
enzyme genes have provided initial tools for further research. How-
ever, our knowledge is limited with respect to the potential for indus-
trial applications of termite lignocellulolytic enzymes. Studies on
hemicellulose degradation are few and effects of lignin modifications
on cellulose and hemicellulose digestion are not fully understood. Be-
cause little heterologous production of termite-derived enzymes has
been reported, further studies on more diverse enzyme overexpression
techniques using different systems, especially overexpression of CBHs,
are needed. Also, novel enzymes with high lignocellulose degradation
activity may reside in termite genes, but the difficulty of identifying
functions of “hypothetical proteins” may have caused investigators to
overlook new enzymes. Thus, a comprehensive quantitative proteomic
analyses combined with enzyme activity screenings are needed. Com-
parative secretome analysis may also identify novel enzymes involved
in lignocellulosic biomass decomposition (Adav et al., 2012). Addition-
ally, optimal enzyme “cocktails” for efficient lignocellulose degradation
(based on the termite enzymatic system) could provide novel ap-
proaches for industrial applications.
Acknowledgments
We thank Prof. Y. Shen at Shandong University for reading the man-
uscript, and for his helpful comments. J.N is grateful to Prof. B.G. Kim
and the Korea Foundation for Advanced Studies for support to conduct
research at Seoul National University. This work was supported by
grants from the National Basic Research Program of China (973 pro-
gram: 2011CB707402) and the National Natural Science Foundation of
China (31272370, 30870085). Part of this work was supported by the
Program for Promotion of Basic and Applied Researches for Innovations
in Bio-oriented Industry (PROBRAIN) and by a grant for the 21st Centu-
ry COE program of University of the Ryukyus and Grants-in-aid for Sci-
entific Research No. 20380037, 17405025, 16780037, and 13760043
from the Japan Society for the Promotion of Science.
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