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Bioresource Technology 277 (2019) 195–203

Contents lists available at ScienceDirect

Bioresource Technology
journal homepage: www.elsevier.com/locate/biortech

Review

Thermostable xylanases from thermophilic fungi and bacteria: Current T


perspective

B.S. Chadhaa, , Baljit Kaura, Neha Basotraa, Adrian Tsangb, Ashok Pandeyc
a
Department of Microbiology, Guru Nanak Dev University, Amritsar 143 005, India
b
Center for Structural and Functional Genomics, Concordia University, Sherbrooke Street West, Montreal, Quebec H4B 1R6, Canada
c
Centre for Innovation and Translational Research, CSIR-Indian Institute of Toxicology Research, Lucknow 226 001, India

A R T I C LE I N FO A B S T R A C T

Keywords: Thermostable xylanases from thermophilic fungi and bacteria have a wide commercial acceptability in feed,
Thermophilic fungi and bacteria food, paper and pulp and bioconversion of lignocellulosics with an estimated annual market of USD 500 Million.
Thermostable xylanases The genome wide analysis of thermophilic fungi clearly shows the presence of elaborate genetic information
Glycoside hydrolases coding for multiple xylanases primarily coding for GH10, GH11 in addition to GH7 and GH30 xylanases. The
Genomics and metagenomics
transcriptomics and proteome profiling has given insight into the differential expression of these xylanases in
Enzyme production
some of the thermophilic fungi. Bioprospecting has resulted in identification of novel thermophilic xylanases
that have been endorsed by the industrial houses for heterologous over- expression and formulations. The future
use of xylanases is expected to increase exponentially for their role in biorefineries. The discovery of new and
improvement of existing xylanases using molecular tools such as directed evolution is expected to be the
mainstay to meet increasing demand of thermostable xylanases.

1. Introduction conversion efficiency (Collins et al., 2005; Nagar et al., 2012). In


comparison to other operational costs, the expenses of animal feed in
Xylanase represents a key component of hemicellulases that cata- production of poultry and livestock are very high. Thus, to improve the
lyzes the breakdown of β 1–4 linkage present in the xylan backbone of feed digestivity of livestock and cut down the expenses with profit gain,
hemicelluloses for subsequent conversion to xylose moieties. Xylanases enzymes are the best and safe option to be employed in feed industry. In
have found beneficial role in feed and food by liberating essential nu- addition to animal feed industry, the paper and pulp industry also aids
trients through hydrolysis/cleavage of non-degradable hemicellulose in the growth of global xylanase market. The eco-friendly use of xyla-
fibers (Leisola et al., 2002). Commercially, xylanase as an important nase enzyme over the harsh chemical usage further fuels the global
ingredient has attained a good market proportion in paper and pulp xylanase market. On the basis of grade, global xylanase market is seg-
industry (Kumar et al., 2016). Besides, some of the potential applica- mented into: feed grade and food grade whereas on the basis of their
tions of microbial xylanase includes, as a food additive ingredient for application, the global xylanase market is dissected according to their
poultry, in baked products, coffee extractions, agriculture silage, as well use in different sectors such as feed and livestock, bleaching of pulp, as
as functional foods, etc. In baking industry, xylanases are being em- an additive (in poultry), bakery, and bioconversion of agro-wastes. The
ployed for enhancing the quality of dough such as stability, flexibility global xylanase market is geographically divided in to five key regions
and extensibility by acting on soluble as well as non- soluble pentosans including North America, Latin America, Europe, Asia-Pacific and
in flour (Butt et al., 2008; Ahmad et al., 2014). The xylanase market is Middle East Africa. Among these, Asia Pacific, especially China, In-
expected to be robust during the forecast period due to increase re- donesia holds maximum share of xylanase market due to major poultry
quirement in animal feed industry. The estimated market share of this production, followed by Asia Pacific is North America, Europe, Latin
enzyme is between 200 and 300 million dollars and slated to reach 500 America and MEA. In Europe, the presence of poultry companies such
million dollars by 2023. In animal feed, nutritional additives are a as PHW-Gruppe Lohmann & CO. AGxylanase, Plukon Food Group, LDC
prime need for poultry industry as they are involved in improving di- Group momentous the growth of xylanase market.
gestibility of broiler chickens by lowering the viscosity level in their Xylanases are produced by diverse variety of micro-organisms in-
intestines thus leading to the improvement in the weight gain and feed cluding extremophilic fungi, bacteria, yeast etc. which are classified in


Corresponding author at: Department of Microbiology, Guru Nanak Dev University, Amritsar, Punjab, India.
E-mail addresses: chadhabs@yahoo.com (B.S. Chadha), adrian.tsang@concordia.ca (A. Tsang), ashok.pandey1@iitr.res.in (A. Pandey).

https://doi.org/10.1016/j.biortech.2019.01.044
Received 28 November 2018; Received in revised form 6 January 2019; Accepted 8 January 2019
Available online 11 January 2019
0960-8524/ © 2019 Elsevier Ltd. All rights reserved.
B.S. Chadha et al. Bioresource Technology 277 (2019) 195–203

several glycoside hydrolase (GH) families (5, 7, 8, 10, 11, 26, 30 and on recent understanding on the mode of action and structure of the
43). Xylanases from these sources have been characterized and possess thermophilic xylanases belonging to different families (Table 2), the
different characteristics such as pH and temperatures to retain their GH10 family xylanase from Thermoascus aurantiacus revealed the pre-
functionality. The industrial application of xylanase demands that the sence of (α/β)8 Tim barrel fold comprising of eight major parallel β-
enzymes must be able to withstand the harsh conditions such as acid/ strands and eight major α helices. In addition to this, six short helices
alkaline environment and elevated temperature, where alkaline xyla- are present along the polypeptide chains. Side view of the molecule
nase is beneficial for pulp and kraft bleaching while the preferred xy- resembles a “Salad bowl” as the upper face of the molecule on the β-
lanases in poultry feed must be active under acidic to neutral pH, the barrel side possess a greater radius due to the extended loops of β-α
prevalent conditions in the gut. Therefore, bioprospecting for thermo- loops. The bottom face has a narrow radius with α-β turns (Pollet et al.,
stable xylanases keeping in mind the pH and temperature optima has 2010). The catalytic site of enzyme comprises of two glutamate residues
been considered as two major criteria for their application besides where one acts as an acid/base catalyst and the other behaves as a
being resistant to proteases, metal ion, etc. This review keeping in view nucleophile. The catalytic domain comprising of 250 to 450 amino
the immense potential of xylanases from thermophilic fungi and bac- acids are highly conserved in xylanases but the affinity differences
teria primarily focuses on the research and development that has taken between these sub-sites significantly affect their modes of action, sub-
place in last 5–8 years and explore the methodologies being used for strate preference and product preference (Bar et al., 2004). In order to
discovery of novel xylanases. decipher the mechanism underlying their heat stability, the crystal
structure of many thermostable xylanases have been resolved. The
stability at high temperature is thought to result from intermolecular
1.1. Xylanase coding genes in thermophilic fungi
and intra-molecular interactions (like hydrogen and disulfide bonds),
interactions with carbohydrate binding units and stabilized C- and N-
It is well recognized that thermophilic fungi produce multiple xy-
terminal ends. Through these interactions, the N-terminal region in-
lanases (Hinz et al., 2009). The current advances in genome sequen-
creases the conformational stability and prevents the destabilization of
cing, annotation and analysis of thermophilic fungi (www. fungal
proteins at higher temperatures (Cheng et al., 2014). Since the terminal
genomics.ca) shows the presence of multiple genes coding for xylanases
regions of the proteins are generally more flexible than other parts of
(Table 1). The majority of the xylanases from thermophilic fungi harbor
the structure, an increased terminal flexibility may enhance the process
either GH10 or GH11 xylanase except for Myceliophthora sepedonium,
of denaturation and promote irreversible aggregation. Thus, the for-
Pseudocercosporella, Thermomyces stellatus, Scytalidium thermophilum
mation of a disulfide bond increases the surface pKa and hydrogen
that also code for functionally distinct GH 30 xylanases (Table 1). GH30
bonds for stabilizing the N-terminal random structure, which are the
xylanases specifically active against methyl-glucouronoxylans have
key determinants for the catalytic activity under conditions of high
been studied in thermophilic Clostridium thermocellum (st John et al.,
temperature and pH (Boonyapakron et al., 2017).
2016) but have not been functionally purified from thermophilic fungi.
The structural elucidation of GH11 xylanase exhibits the presence of
Of the different thermophilic fungi genome sequenced, Myceliophthora
catalytic domain that displays a β-jelly-roll architecture comprising of
sepedonium, codes for maximum xylanase genes (seventeen) including
two anti-parallel β sheets and one single major α-helix forming fingers
eight that belongs to GH10 and six categorized to GH11. The genome of
and palm. The presence of loops in between the structure depicts thumb
other strains Pseudocerco sporella and Myceliophthora thermophila also
and cord. The structure is compact and closely packed and mimics
coded for 14 and 13 xylanase genes, respectively. Whereas, Thermo-
partially closed right hand. However, a unique structure for a GH11
myces lanuginosus, considered as one of the most prolific xylanase pro-
xylanase (XynCDBFV) has been reported in ruminant fungus
ducer and Thermoascus aurantiacus coded for single GH11 and GH10
Neocallimastix patriciarum. It harbors an extended N-terminal region
xylanase genes (Singh et al., 2003; Winger et al., 2014). Thermophilic
comprising 11 amino acids that adheres to several β strands and
bacterial genome, however, do not contain such elaborate diversity and
spanned the convex side of the palm β-sheet. The N-terminal region is
multiplicity of xylanases possibly owing to smaller genome size. Most of
attached to the catalytic core by hydrogen bonds with stacking forces
thermophilic bacterial genome has genetic information for GH 10/GH
along a disulfide bond between Cys-4 and Cys- 172. Through these
11 xylanases (Chakdar et al., 2016).
interactions, the N-terminal region clumps the catalytic core into a
more restrained structure, which confers to its stabilization at elevated
1.2. Mode of action of thermophilic xylanases temperatures (Cheng et al., 2014). The active site of thermophilic GH11
xylanase is a deep (∼9 Å), narrow (∼4 Å) and long (∼25 Å) cleft with
The mode of action of the xylanases has been elucidated in excellent the catalytic dyad located in the middle, which supports the endo
reviews previously (Collins et al., 2005; Dodd and Cann, 2009). Based mechanism of GH11 xylanases. The active site residues are two mole-
cules of glutamic acid acting as a nucleophile and an acid/base catalyst
Table 1 that actively participates in a double-displacement catalytic mechanism
Genome wide analysis of thermophilic fungi coding for diverse xylanases. and the existence of a covalent glycosyl-enzyme intermediate complex.
Fungal strains Number of genes GH 7 GH 10 GH 11 GH 30 Xylanases from the GH11 family have low molecular weight and high pI
values (Alvarez-cervantes et al., 2016).
Chaetomium thermophilic 8 3 4 1
Endoxylanases, belonging to GH family30 (GH30), are classified
Myceliophothora sepedonium 17
Myceliophothora fergussi 9 4 5 0
into 8 different subfamilies of which subfamily 7 (GH30-7) is derived
Malbranchea cinnamomea 4 3 1 from fungi and subfamily 8 (GH30-8) belongs to bacterial strains and
Rasamsonia byssochlamys 6 1 2 1 1 are single -domain enzymes that act on xylans that are decorated with
Pseudocercosporella 14 6 6 2 4-O-methylglucuronic acid (MeGlcA) moieties to yield glucouronic acid
Rhizomucus pusilus 7
(GlcA) containing xylooligosaccharides or aldouronates (Valenzuela
Scytalidium thermophilium 10 7 3 1
Thermoascus aurantiacus 1 1 et al., 2012; Padhila et al., 2014). However, unlike well characterized
Thermomyces lanuginosus 1 1 GH30 glucurono-xylanases from Bacillus subtilis and Erwinia chry-
Thermomyces stellatus 10 4 4 2 santhemi (st John et al., 2016), xyn30D reported from the secretome of
Thielavia australiensis 7 3 3 1 Paenibacillus barcinonensis is a modular enzyme comprising of carbo-
Thielavia terrrestaris 9 5 4
Myceliophthora themophilum 13 5 8
hydrate binding module belonging to family CBM35 (Sainz-polo et al.,
2014). Moreover it has been reported that recent GH30-8 xylanase from
www.fungalgenomics.ca (Prof Adrian Tsang, Principal Invest. thermophilic anaerobic bacterium Clostridium thermocellum (CtXyn30A)

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B.S. Chadha et al. Bioresource Technology 277 (2019) 195–203

Table 2
Structure and folding topologies of xylanases of different GH families.
GH families Organism Folding topology Structure Mechanism References

8 Paenibacillus barcinonensis (α/α)6 Inverting Valenzuela et al.,2016

10 Thermoascus auranticus (β/α)8Tim barrel Retaining Pollet et al., 2010

11 Neocallimastix patriciarum β Jelly roll Retaining Chenget al., 2014

30 Clostridium thermocellum (β/α)8Tim barrel Retaining Freire et al., 2016

43 Butyrivibrio proteoclasticus β-propeller Inverting Tillet al., 2014

show preference for GlcA containing substrates as well as possess a 2014).


significantly high activity on wheat arabinoxylan (WAX) and several
other biomass derived polysaccharides that do not contain GlcA (Verma 2. Characteristics, properties and applications of xylanases from
et al., 2016). The three dimensional structure of endo 1–4 xylanase thermophilic fungi
(CtXyn30A) showed (β/α)8 TIM domain which is specific characteristic
of clan A. This domain comprises of Val11–Pro295 residues, with a core 2.1. Myceliophthora strains
of eight β strands and is surrounded by eight α helices. Whereas, the
strongly attached side β-domain contains His3 to Gln10 and Gly296 to M. thermophila, a thermophilic fungus (optimum growth tempera-
Val386 residues. The structure analysis has revealed that Glu 136 and ture of 45 ˚C) previously identified as Chrysosporium lucknowense has
Glu 225 are absolutely conserved within the subfamily and are the key been found to be an efficient source of hemicellulolytic enzymes in-
constituents of the catalytic residues. The distance between these car- cluding xylanases (Hinzet al., 2009). The genome of C. lucknowense
boxyl residues is less than 5Ǻ thus support retention mechanism for strain was annotated to contain 11 xylanases genes for GH10 and seven
hydrolysis. CtXyn30A is active over a broad range of pH and displays an for GH11 (Hinz et al., 2009). Whereas, Myceliophthora thermophila
optimum temperature of 70 °C. The thermostability of the enzyme may genome was later found to contain 12 xylanase genes (Karnaouriet al.,
be attributed to the presence of higher number of salt bridges and 2014). A thermophilic strain identified as Myceliophthora sp (IMI,
strong hydrogen bonding between the side chain residues (Freire et al., 38079) by IMI Kew, UK and as C. lucknowense (MTCC) by Microbial
2016). Type Culture Collection, Chandigarh, India, had also been reported
GH family 8 alongwith endo-1,4-β-xylanases also contain cellulases, previously to produce functionally distinct multiple xylanases
chitosanases, lichenases . The cold-adapted xylanase act on xylan to get (Badhanet al., 2004). Ten different functionally diverse xylanases from
converted to primarily xylotriose and xylotetraose and was found to be Myceliophthora sp. were resolved electrophoretically using PAGE/IEF
most active on long chain xylo-oligosaccharides. Modeling of the three- and were found to show characteristically different activity against
dimensional (3D) structure of Rex8A from Paenibacillus barcinonensis unsubstituted xylans, arabinoxylans and methyl-glucouroxylan
BP-23 shows an (α/α)6 barrel fold topology where the loops connecting (Badhan et al., 2004). Ustinov and co-workers (2008) purified six xy-
the α-helices contour the active site. Moreover Rex8A enzyme has been lanases from C. lucknowense (3 each of GH10 and GH11 xylanases) and
reported for the first time that acts on branched xylo-oligosaccharides found that GH11 in comparison to GH10 xylanases displayed high
(Valenzuela et al., 2016). It has been proposed that like GH11 family substrate specificity when subjected to catalysis against different xylans
xylanases, GH 8 xylanases contain the catalytic site in the middle (Table 3). Structurally two of the GH10 xylanases (Xyl 10B and Xyl
alongwith the large substrate binding cleft comprising of at least six 10C) were found to be distinct with modular configuration and were
xylose binding residues. But they differ from GH10 and GH11 by their tethered by CBM1 at N and C terminal, respectively. Further they found
inverting single displacement reaction mechanism (Pollet et al., 2010). these xylanases to be catalytically versatile and exhibited high ther-
According to CAZy database, GH 43 family includes an array of en- mostability. Myceliophothora thermophila is also known to produce low
zymes such as β-xylosidase, α-L-arabinofuranosidase, arabinase, xyla- molecular weight xylanase (14 kDa) that is active under extreme alka-
nase and galactan 1,3-β-galactosidase (Lombard et al., 2013). The line condition (Boonrung et al., 2018). Myceliophthora is now known to
structural elucidation of GH 43 family shows that proteins of this family contain four species namely M. theromophila, M. heterothallica, M. Hi-
contain five bladed β-propeller domains and possess inverting me- nulea and M. fergusis (van de Brink et al., 2013), where M. thermophila is
chanism where three residues are involved in catalysis (Till et al., better producer of xylanases when compared to M. heterothallica. The

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B.S. Chadha et al. Bioresource Technology 277 (2019) 195–203

Table 3
Production and properties of purified xylanases from thermophilic and thermotolerant fungi.
Fungal strain, (xylanase activity) MW (kDa) /pI Optimum pH/Temp Km (mg/ml) /Vmax (U/mg/ GH Family Reference
min)

Humicola insolens (16.4 units/ml) Xyn W (44.0)w/NR 6.0/70 °C 2.8 / 311.8 GH10 Du et al.,2013
Xyn A (41.5)a/NR 6.0/80 °C 1.6 / 974.4 GH10
Xyn B (42.0)a/NR 7.0/70 °C 1.1 / 306.8 GH10
Xyn C (44.0)a/NR 6.0/70 °C 2.1 / 287.7 GH10
T. lanuginosus DSM 10,635 (1180 units/ml) (25.5)/N.R 6.5/70 °C 3.85/ N.R NR Xiong et al.,2004Le and Wang, 2014
T. lanuginosusa (36 units/ml) (44.0)f/N.R 6.0/65 °C N.R/ N.R GH 11
Thermoascus aurianticus (575.9 units/ml) (33.0)/N.R 4.0–4.5/70–75 °C N.R/ N.R GH10 Kalogeris et al., 2001
Chaetomium thermophile (61.1 units/ml) Xyl I (26.0)/N.R 5.4–6.0/ 0.55/58.8 NR Ganju et al., 1989Katapodis et al.,
Xyl II (7.0)/N.R 70 °C4.8–6.4/ 60 °C 0.1/5.26 2007
Melanocarpus albomyces (415 units/ml) Xyl I (38.0)/N.R 6.6/65 °C 0.30/311 NR Biswas et al., 2010a,b
Xyl II (24.0)/N.R 5.5/65 °C 1.69/500
Myceliophthora sp. IMI 387,099 (16.2 units/ Xyl Ia (53.0)/5.2 6.0/75 °C 1.63/55.5 GH10 Chadha et al., 2004
ml) Xyl IIa (53.0)/4.8 6.0/75 °C 1.69/33.3 GH10
Malbranchea flava MTCC 4889 (164 units/ml) MFX I (25.2)/4.5 9.0/70 °C 1.25/1666 GH11 Sharma et al., 2010
MFX II (30.0)/3.7 9.0/70 °C 3.7/1923 GH10
Chrysosporiium lucknowenese (96.6 units/ml) Xyn 10A (47.8)/4.7 5.5–7.0/65–70 °C N.D/65d GH10 Ustinov et al., 2008
Xyn 10a (31.0)/8.9 5.5–7.0/65–70 °C N.D/83d GH10
Xyn 10B (57.0)/4.4 5.5–7.0/80–85 °C N.D/39d GH10
Xyn 10b (46.0)/4.3 5.5–7.0/80–85 °C N.D/85d GH10
Xyn 10C(40.0)/4.8 5.0/80 °C N.D/32d GH10
Xyn 11A(24.0)/7.7 5.5–7.0/70 °C N.D/395d GH11
Xyn 11B (23.0)/8.4 5.5–7.0/65–70 °C N.D/169d GH11
Xyn 11C (22.0)/6.7 4.5/65 °C N.D/300d GH11

Sporotrichum thermophile StXyn I (24.0)/8.7 5.0/60 °C NR GH11 Vafiadi et al., 2010


StXyn II (48.0)/8.0 5.0/60 °C GH10
Remersonia thermophila rtXylI (42.0)/N.R 6.5/60 °C 2.48 /5.79 GH 10 McPhillips et al., 2014

a
recombinant xylanases;
f
fusion protein xylanase;
w
wild;
d
(units/mg).

transcriptome profiling of M. thermophilia showed that the expression of NC38 in P. pastoris (261. 7 ± 0.61Uml) was 545-folds higher than ob-
GH 1 xylanases was up-regulated when cultured in presence of monocot served in E. coli (Mchunu et al., 2009). Engineering of an N-terminal
cereals, whereas, the expression of GH10 showed no selective up-reg- disulfide bridge resulted in improved thermal performance of re-
ulation in presence of either monocots/dicots as substrate and its level combinant T. lanuginosus GH11 xylanase as evidenced by apparent
remained unchanged (Kolbusz et al., 2014). M. thermophila JCP 1–4 upward temperature optima shift upwards at pH 6.5 by about 10 °C to
strain from Brazil (Pereiera et al., 2015) was found to produce 931.1 75 °C (Wang et al., 2012). T. lanuginosus produces low molecular weight
(units/g) of xylanase on sugarcane bagasse, whereas, Myceliophthora (25.0 kDa) and highly thermostable xylanase that is active between pH
sp., isolated from composting soils of India was reported to produce 5.0 and 9.0 and has been classified as GH 11 family member. The se-
2366 (units/g) of xylanase when cultured on rice straw employing solid cretome based analysis of T. lanuginosus grown on corn-cob based
state fermentation (Badhan et al., 2007). Recently two GH11 xylanase medium showed high proportion of protein as xylanase (22–30%)
genes MYCTH_49824 and MYCTH_56237 from M. thermophila have (Winger et al. 2014) and may be the reason for its purification using
been cloned and expressed in Pichia pastoris. The multiple template simple two-step process involving weak anion exchanger and gel fil-
alignment for modelling further revealed their catalytic domains (Basit tration (Bennet et al., 1998). Recent studies have shown that the xy-
et al., 2018). lanase purification was also possible by partitioning using PEG1000/
NaCit based two phase aqueous system. The separation based on par-
titioning is best because of high Kp value of 17.7 ± 0.3 thus making it
2.2. Thermomyces lanuginosus
applicable for industrial purification of xylanase (Loureiro et al., 2017).
Xylanase from T. lanuginosus is commercially produced as recombinant
Thermomyces lanuginosus with optimum growth temperature of 50˚C
enzyme using Fusarium venentatum as host organism and is marketed as
is considered as one of the most prolific producer of alkaline active
Novozyme 899 as processing aid in baking industry to improve dough
virtually cellulase free thermostable xylanases that exhibited 3500
stability and crumb structure. Similarly, Pentopan Mono BG, a re-
(units/ml) and 48,000 (units/g substrate) of activity under shake flask
combinant xylanase from T. Lanuginosus expressed in A. oryzae, is
and solid substrate fermentation, respectively (Sonia et al., 2005;
known to improve bread loaf volume by 41% (Butt et al., 2008). Ro-
Kumar et al., 2009; Winger et al., 2014). However, T. lanuginosus strains
nozyme WX CT, an endoxylanase derived from T. Lanuginosus em-
isolated from different geographical regions vary in their xylanase
ploying submerged fermentation by Novozymes A/S (Denmark), and
production capacity. A T. lanuginosus strain VAPS isolated from de-
marketed by DSM Nutritional Products (Switzerland) is authorized in
caying wood sample produced as low as (61 units/ml) that was im-
the EU (Commission Regulations (EC) No 1332/2004 and No 2036/
proved to 132.5 (units/ml) using combination of optimization ap-
2005) as feed enzyme. The xylanase product was found to positively
proaches such as Genetic Algorithm-Response Surface Methodology
influence the feed conversion efficiency in non-ruminants (Nielsen
(GA-RSM), Artificial Neural Network (ANN), Genetic Algorithm-Artifi-
et al., 2007). The treatment of Kraft pulp during milling with T. lanu-
cial Neural Network (GAANN) (Kumar et al., 2016). Heterologous
ginosus xylanase was found to partially hydrolyze xylan associated with
production of cloned T. lanuginosus gene using E. coli and Pichia pastoris
cellulose fibres thus resulting in subtle changes in the pulp structure
as host has also been reported (Mchunu et al., 2009). Upon comparison,
and enhanced susceptibility of the pulp to refining (refining energy was
it was found that expression of xylanase by alkaline stable variant

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B.S. Chadha et al. Bioresource Technology 277 (2019) 195–203

significantly reduced) and improved the static strength properties of approved by FDA and is used commercially for application in food
paper (Buzala et al., 2016). (brewery, bread, starch extraction from wheat) and feed industry. The
xylanases from H. Insolens are capable of improving the starch extrac-
2.3. Malbranchea strains as source of xylanase tion from wheat and is sold commercially as Shearzyme by Novozymes.
The optimum growth temperature of H. insolens is 45 ˚C (Basotra et al.,
Malbranchea strains produce multiple forms of thermostable alka- 2016). Four GH10 xylanases from H. insolens have been purified and
line active xylanases (Sharma et al., 2010). The culture produces high characterized to be optimally active between 70 and 80 °C and at pH
levels of xylanases (27193 units/g substrate) at 45˚C on solidified cul- 6.0–7.0 (Du et al., 2013). Later Shi et al., (2015) cloned and expressed
ture medium containing rice straw as carbon source (Mahajan et al., multi-modular GH11 xylanases form H. insolensY1that was found to be
2014). Employing transcriptomics profiling it was observed that the alkaline tolerant.
expression of M. cinnamomea GH11 xylanase gene was up-regulated by Besides the strains cited above, different thermophilic fungus like
301.8 times when cultured on xylan and 22.79 times on wheat bran as Thielavia terrestaris (Berka et al., 2011; Garcia-Haunte et al., 2017),
compared to glucose (Huttner et al., 2017). The xylanases MEX-I(GH11) Corynascus thermophilus (van de Brink et al., 2013), Rhizomucor pusillus
and MEX-II(GH10) from a thermophilic isolate Malbranchea flava were (Huttner et al., 2018) have also been reported as source of xylanase. T.
purified and characterized and found to be optimally active at pH 9.0 terrestris was found to produce hyper-thermophilic active xylanase (Tt
and 70OC (Sharma et al., 2010). The secretome profiling of the M. Xyn A) with molecular weight of 82 kDa and was found to be optimally
Cinnamomea indicated it to be a source of thermostable metal depen- active at pH 5.5 and at 85 °C. The xylanase exhibited half life of
dent glycosyl hydrolases (Mahajan et al., 2016) including xylanases 23.1 days at 65 °C. The thermostability was found to be associated with
that are involved in boosting the hydrolytic potential of commercial gain in secondary structures at high temperature (Garcia-Haunte et al.,
cellulase Cellic Ctec 2 during saccharification of rice straw, carrot grass 2017). C. thermophilus previously known as Myceliophthora fergusii, re-
and corn stover (Sharma et al.,. 2016). The cloning of 50 kDa thermo- ported to produce low levels of xylanases (van de Brink et al., 2013),
stable GH10 xylanase MpXyn10A and its expression in Aspergillus ni- was taken up for expression of GH11 xylanase for heterologous over
dulans was carried out and the expressed enzyme was optimally active expression in P. pastoris. To obtain perfect expression, the 870 bp gene
at pH 5.8 and 80 °C and found to be 16% glycosylated and thermo- sequence was codon optimized and synthesized. The recombinant
stable, preserving 85% activity after 24 h at 65 °C. Circular dichroism Xyn11A was found to be optimally active at pH 7.4 and 70 °C. The
showed high alpha-helical content consistent with the canonical GH10 enzyme was resistant to metal ions and protease such as pepsin which
family (β/α)8 barrel fold observed in molecular modeling. The xylanase makes it suitable for biotechnological applications (Yang and Zhang,
resulted in effective hydrolysis of native and pretreated sugarcane ba- 2017). Paecilomyces thermophile, a thermotolerant fungus that had been
gasse (Rebeiro et al., 2014). In a recent study, two xylanase genes reported to produce high levels of thermostable xylanases was cloned
(GH10 and GH11) designated as XYN10A_MALCI and XYN11A_MALCI and expressed in P. pastoris. The recombinant xylanase produced 8.1 (g/
from Malbranchea cinnamomea, respectively, were cloned and expressed L) of protein and 2940 (U/ml) of activity in 5 L fermenter fed se-
in P. pastoris X33. The resultant clones of GH11 and GH10 produced quentially with methanol. The recombinant enzyme was active at 80 °C
573.3 and 24.3 (units/ml) of xylanase, respectively, when fed with for 30 min (Fanet al., 2012). A highly catalytically efficient thermo-
methanol under shake flask cultures (Basotra et al., 2018) were purified philic xylanase belonging to family GH10 from Achaetomium Sp X2-8
and the recombinant rXYN11A MALCI was found to be stable at 70 °C was stable at 75 °C and at broad range of pH 4.0 to 10.0 with Kcat/ Km
and catalytically active against a variety of substituted (arabinoxylans) of 3710/m/s/mg (Zhao et al., 2013). The xylanase was comparable to
as well as unsubstituted xylans. The recombinant xylanases were found Ultraflo, commercially produced from H. insolens by Novozyme and was
to act synergistically with commercial cellulases and resulted in 1.54 used for filtration of brewing mash and was found to perform much
and 1.58 folds improved hydrolysis of acid and alkali treated rice straw better in combination with β-glucanase when compared to Ultraflo.
(Basotra et al., 2018). Surprisingly, the genome of thermophilic fungus Rhizomucor pusillus
which do not harbor any gene for xylanase (Huttner et al., 2018) was
2.4. Thermoascus aurantiacus as source of xylanase reported to produce 6 (Units/ml) of xylanase when cultured on
beechwood xylan (Huttner et al., 2018). R. pusillus, isolate from maize
T.aurantiacus (with optimal growth temperature at 50˚C), a well silage, has also previously been shown to produce 824 (U/g) of xylanase
known source of GH10 xylanase, has been reported to produce xylanase which was found to be stable at 75 °C (Robeldo et al., 2016). The ob-
both under solid state fermentation where wheat bran supported 6543 served xylanase activity in the secretome can be ascribed to the broad
(units/g substrate) (Jainet al., 2015) and shake flask culture (130 units/ specificity of some of other glycosyl hydrolases like GH5 and even
ml) using corn cob as substrate (Olivirea et al., 2010). T.aurantiacus has auxiliary activity proteins in the secretome against the xylan as sub-
also been found to express xylanase constitutively using xylose as in- strate (Fromhaggen et al., 2016). Talaromyces emersonii now renamed as
ducer where, Schuerg et al. (2017) showed that feeding xylose con- Rasmasonia emersonii is an important source of xylanases that is com-
tinuously in fed batch mode resulted in production of 80 (units/ml) of mercially produced by Novozymes DSM and is in the list of food en-
xylanase. The crystal structure of T. aurantiacus GH10 xylanases had zymes approved for use in mashing process in breweries as well as
been shown to complex with xylobiose substitute with arabinofuranosyl biorefineries. A thermotolerant strain T. Leycettanus, reported as a novel
feraloyl ester side chain and the enzyme. The enzyme showed fourfold source of the thermostable GH10 xylanase, was cloned and expressed in
higher substrate specificity for xylotriose which is linked to arabinose at P. pastoris. The purified recombinant TlXyn10A was acidic and hyper-
non reducing xylan moiety compared to xylose alone (Vardakou et al., thermophilic and retained stability over the pH range of 2.0–6.0 and at
2005). Thermoascus aurantiacus M-2, producing novel acidophilic and 90 °C (Wang et al., 2017a,b). Later Wang and co-workers (2017) pre-
thermostable xylanase 39.07 (U/mL) was purified. The purified xyla- pared three mutants of GH10 xylanases by swapping gene of a xylanase
nase (31.0 kDa) was characterized to be active at 75 °C and pH 5.0 and of another fungal strain Bispora sp., MEY-1 reported to be source of
maintained stability at 80 °C (Ping et al., 2018). thermostable xylanases and showed site directed mutation of E229I,
F232E and G145D resulted in weakened substrate specificity and im-
2.5. Scytalidium thermophilium and other thermophilic fungal strains as proved catalytic efficiency. Melanocarpus albomyces, a thermophilic and
source of xylanases non-sporulating fungus that is considered as proficient strain for pro-
duction of alkaline active xylanase with application in paper and pulp
Xylanases produced from Humicola insolens (syn. S. thermophilum industry was subjected to strain development. The mutant M. albomyces
and now Mycothermus thermophilus) being GRAS organism has been IITD3A produced 415 (IU/mL) xylanase on alkaline lignocellulosic

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extract in a 14 L bioreactor with volumetric productivity of 11,530 (IU/ cellulose besides xylan and barley β-glucan (Xue et al., 2015), thus
L/h),which was 8-fold higher than that of the wild-type strain (Biswas making it useful candidate for bioconversion. C. lactoaceticus is also
et al., 2010, Gupta et al., 2013). The cyclic maintenance of pH of fer- known to express GH10 xylanase (47 kDa) that exhibited good stability
mentation medium between 7.8 and 8.2 increased the productivity to at 80 °C at pH4.5. The enzyme was shown to liberate branched XOS
16, 670 (IU/L/h), it was found that change in the fungal morphology to with methyl-glucouronic acid sub-chains (Jia et al., 2014). Recently, C.
a pellet form resulted in very high xylanase productivity of 22,000 IU/ owensensis has been shown to produce highly thermostable xylanase
L/h (Biswas et al., 2010a,b). which is optimally active at 90 °C. The recombinant CoXynA was shown
to exhibit half life of 1 h at 80 °C. The authors further solved crystal
3. Thermostable xylanases from thermophilic bacteria structure of the recombinant xylanase. The relative high thermo-
stability of the enzyme was proposed to be due to increased overall
3.1. Geobacillus strains as source of thermostable xylanase protein rigidity resulting from reduced length and fluctuation of loop 7
(Liu et al., 2017). The hemicellulase from C. owensensis was capable of
Geobacillus perhaps is the most widely reported thermophilic bac- degrading hemicellulose of native corn stover and cob efficiently (Peng
teria for its ability to produce thermostable xylanase. Geobacillus sp. et al., 2015). Herbivorax saccincola A7 isolated through enrichment
WSUCEF1 (grows optimally at 60˚C) isolated from soil (Bhalla et al., culture using untreated corn stover as carbon source was found to grow
2015) and strain DUSELR 13 isolated from deep gold mines of South optimally at 55 °C and at alkaline pH (9.0). The draft genome of the
Dakota (Bibra et al., 2018) have been reported for producing highly culture revealed the presence of GH11 xylanase and essential genes for
thermostable enzyme whereas xylanase from the former strain showed xylose metabolism, xylose isomerase, xylose transporter etc. (Aikawa
half life of 18 and 12 days at 60 °C and 70 °C, respectively, while DU- et al., 2018). A high xylanase producing Caldicoprobacter algeriensis
SELR 13 produced 31 (U/ml) after optimization and exhibited t ½ of isolated from hydrothermal hot spring of Guelma produced 250 (U/ml)
13 days at 60 °C and 70 °C. The enzymes also showed better hydrolysis of xylanase in a medium comprising birchwood and oat spelt xylan at
of beechwood xylan when compared to commercial xylanases Cellic 70 °C in 24 h (Amel et al., 2016). Modular trifunctional xylanolytic
HTEC 2 and Accelarase XY. A GH10 xylanase from Geobacillus sp Axy43A has also been reported from Paenibacillus curdlanolyticus strain
WSUCFI cloned and expressed in E. coli exhibited 461 (units/mg) of B-6. This GH43 hydrolase with family 6 carbohydrate binding module
specific activity and exhibited t ½ at 60 °C after 60 h (Bhalla et al., exhibited endoxylanase, β-xylosidase and arabinoxylan hydrolyzing
2015). G. stearothermophilus KIBGE-IB29, a soil isolate was found to activities (Teeravivattanaki et al., 2016). Modular family 8 xylanase
produce xylanase optimally at 60 °C and at pH 6.0 after 24 h of in- from marine bacteria Glaciecola mesophila was found to have new type
cubation period (Bibi et al.,2014).The GH10 xylanase from G. stear- of CBM with eight β-strands (Chen et al., 2018). Improved expression of
othermophilus and Rhodothermus marinus (Mathew et al., 2018) pro- Thermoanaerobacterium aotearoense SCUT27 GH10 xylanase harbouring
duced xylo-oligosaccharides (XOS) and arabino-xylooligosaccharides CBM modules and three surface layer homology domains (Huang et al.,
(AXOS) such as A3X, A2XX and A2 + 3XX. The strain produced a single 2015) expressed in B. subtilis was optimally active at 80 °C and at pH
45 kDa xylanase; however unlike G. Stearothermophilus, xylose was not 6.5. The hydrolysis of beechwood xylan resulted in xylobiose and me-
an inducer of the resultant xylanase (Gerasimova & Kuisiene, 2012). thylglucourono-xylotriose as the main products.
The xylanase expression in G. stearothermophilus was found to be con-
trolled by multiple regulatory mechanisms including XylR as the master 3.3. Thermopolyspora flexuosa as source of industrial xylanase
negative regulator in addition to carbon catabolite repression. The
genes for xylanase expression were positively modulated in presence of A GH10 xylanase from Thermopolyspora flexuosa (Nonomuraea flex-
xylose as inducer which negates the effect of XylR, in addition quorum uosa) DSM43186 which is an actinomycete known to produce ther-
sensing played an important role as evidenced from 50 fold increase in mophilic xylanase exhibited temperature optima of 65–70 °C against
expression of xylanase during exponential growth phase (Shulami et al., insoluble xylan and 75–80 °C in presence of 3% xylan was found to be
2014). G. galactosidasius BS61 isolated from geothermal resource in stabilized in presence of ionic liquid [EMIM]OAc at inactivating tem-
Turkey producing (15 U/ml) xylanase was purified and applied for peratures (80–90 °C). The increased stability of TfXYN10A indicated to
clarification of juices of orange and pomegranate as evident by reduced the binding of IL molecules that stabilized the protein structure.
turbidity (Sari et al., 2018). Geobacillus thermodenitrificans JK1 is an- Furthermore, the xylanase exhibited low Km (∼1 mg/mL) demon-
other well studied strain for the production of xylanase that was shown strated higher tolerance to ILs than xylanases with higher
to produce two xylanase (XynA1 and XynA2) isoforms that synergisti- Km (Anbarasan et al., 2017). The GH10 xylanase from T. flexuosawas
cally act with β-xylosidases and arabinofuranosidase from efficient was taken up for commercial production. The gene was cloned and
hydrolysis of birchwood xylan (Huang et al., 2017). The xylanase gene expressed in Trichoderma reesei by AB enzymes. The enzyme produced
GtxynA1 of G. thermodenitrificans was found to be associated with HUS in GRAS organism has been cleared by Canadian government for ap-
(hemicellulose utilization) locus. This GH10 family protein was opti- plication in baking, brewery, potable alcohol and grain processing
mally active at 60 °C in pH range of 3.0–9.0 and its stability was shown (GRAS Notice No. GRN000628). The recombinant xylanase produced
to be improved by applying protein engineering where mutants of G. by T. Reesei at industrial scale was subjected to ultrafiltration and spray
thermodenitrificans C5 with enhanced temperature by 11 °C substituting drying and is marketed in powdered form that has an activity of
histidine, tryptophan and aspartic acid with glutamic acid and proline 2,28,200 (units/g).
(H82E/ W185P/ D186E) was reported (Irfan et al., 2018). In addition, some of the novel thermophilic bacterial strains capable
of producing xylanase including, Caldoprocter, Herbinix, Herbivorax
3.2. Caldicellulosiruptor as source of thermostable xylanase saccinola from bovine manure as well as biogas digester beside
Rhodothermus marinus from marine environment have also been re-
C. bescii. C. lactoaceticus and C. owensensis have been reported for ported (Amel et al., 2016; Mechelke et al., 2017; Aikawa et al., 2018;
the secretion of thermostable xylanases in recent past (An et al., 2015, Mathew et al., 2018).
Jia et al., 2014; Liu et al., 2017). The GH10 xylanase from C. bescii was
over-expressed in E. coli and found to exhibit high catalytic activity 4. Metagenomic DNA as source of thermostable xylanases
with half life of 7.7 h at 60 °C and the enzyme was optimally active at
70 °C at pH 7.0 with 85% activity at pH 4.0 and 12.0 (Anet al., 2014). Owing to the search for novel enzymes for industrial exploitation,
The N-terminal domain of GH10 xylanase from C. bescii was found to be metagenomic approaches can overcome the limitations of culture-de-
multidomain cbxyn10C/cel48B capable of degrading crystalline pendent methods and can facilitate the discovery of enzymes from

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B.S. Chadha et al. Bioresource Technology 277 (2019) 195–203

uncharacterized microorganisms. The screening of metagenomic li- Anbarasan, S., Wahlström, R., Hummel, M., Ojamo, H., Sixta, H., Turunen, O., 2017. High
braries coding for xylanases has resulted in few novel ORF’s coding for stability and low competitive inhibition of thermophilic Thermopolyspora flexuosa
GH10 xylanase in biomass-dissolving ionic liquids. Appl. Microbiol. Biotechnol. 101,
thermostable xylanases. The environmental DNA isolated from different 1487–1498.
niches such as insect gut, manure, waste water, hot environmental Badhan, A.K., Chadha, B.S., Kaur, J., Saini, H.S., Bhat, M.K., 2007. Production of multiple
samples, chicken cecum, crater of Avachinsky volcano have been re- xylanolytic and cellulolytic enzymes by thermophilic fungus Myceliophthora sp. IMI
387099. Bioresour. Technol. 98, 504–510.
ported as sources of xylanases (Mientus et al., 2013). The xylanases Badhan, A.K., Chadha, B.S., Sonia, K.G., Saini, H.S., Bhat, M.K., 2004. Functionally di-
from the chicken cecum metagenome was found to require high con- verse multiple xylanases of thermophilic fungus Myceliophthora sp. IMI 387099. Enzy.
centration of salt and organic solvents for their activity (Darkazali et al., Microb. Technol. 35, 460–466.
Bar, M., Golan, G., Nechama, M., 2004. A new crystal form of XT6 enables a significant
2017). Furthermore it was proposed that poultry feed is primarily improvement of its diffraction quality and resolution. Acta. Crystallogr. D.60,
composed of high ratio of non-starch polysaccharides that include xy- 545–549.
lans and arabinoxylans so the microorganisms capable of hydrolyzing Basit, A., Liu, J., Miao, T., Zheng, F., Rahim, K., Lou, H., Jiang, W., 2018. Characterization
of two Endo-β-1, 4-Xylanases from Myceliophthora thermophila and their sacchar-
these polysaccharides should be abundant in chicken intestine. A novel
ification efficiencies, Synergistic with Commercial Cellulase. Front. Microbiol. 9, 233.
alkali-stable and thermostable GH11 endoxylanase encoding gene Basotra, N., Joshi, S., Satyanarayana, T., Pati, P.K., Tsang, A., Chadha, B.S., 2018.
(Mxyl) was retrieved by functional screening of a compost soil meta- Expression of catalytically efficient xylanases from thermophilic fungus Malbranchea
genome. The recombinant xylanase (1077 bp) exhibited activity at cinnamomea for synergistically enhancing hydrolysis of lignocellulosics. Int. J. Biol.
Macromol. 108, 185–192.
80 °C and pH 9.0 (Verma et al., 2013). The thermostability of this en- Basotra, N., Kaur, B., Falco, M. Di., Tsang, A., Chadha, B.S., 2016. Mycothermus ther-
zyme was subsequently engineered through the enrichment of surface mophilus (Syn. Scytalidium thermophilum): Repertoire of a diverse array of efficient
β-sheets with arginine residues by substituting serine/threonine by site cellulases and hemicellulases in the secretome revealed. Bioresour. Technol. 222,
413–421.
directed mutagenesis (Verma and Satyanarayana, 2013) whereas xy- Bennett, N.A., Ryan, J., Biely, P., Vrsanska, M., Kremnicky, L., Macris, B.J., Kekos, D.,
lanases from volcano metagenome was found to be maximally active at Christakopoulos, P., Katapodis, P., Claeyssens, M., Nerinckx, W., 1998. Biochemical
95 °C and exhibited half life of 22 h at 85 °C (Mientus et al., 2013). and catalytic properties of an endoxylanase purified from the culture filtrate of
Thermomyces lanuginosus ATCC 46882. Carbohydr. Res. 306, 445–455.
Similarly, the expression of xylanase gene xyl7 derived from termite Bhalla, A., Bischoff, K.M., Sani, R.K., 2015. Highly thermostable xylanase production
gut, in E. coli showed higher thermostability with 10 °C increase in from a thermophilic Geobacillus sp. strain WSUCF1 utilizing lignocellulosic biomass.
optimal temperature and was active under broad pH range of 5.5–10.0 Front. Bioeng. Biotechnol. 3, 84.
Berka, R.M., Grigoriev, I.V., Otillar, R., Salamov, A., Grimwood, J., Reid, I., Ishmael, N.,
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5. Conclusion Bibra, M., Kunreddy, V., Sani, R., 2018. Thermostable Xylanase Production by Geobacillus
sp. Strain DUSELR13, and Its Application in Ethanol Production with Lignocellulosic
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Biswas, R., Sahai, V., Mishra, S., Bisaria, V.S., 2010b. Development of mutants of
genome editing and synthetic biological techniques in future, the en- Melanocarpus albomyces for hyperproduction of xylanase. Biotechnol. Bioproc. Eng.
zyme producing microorganisms are compelling subjects to explore the 15, 800–809.
growing importance of thermophilic xylanases in various industrial Boonrung, S., Katekaew, S., Mongkolthanaruk, W., Aimi, T., Boonlue, S., 2018.
Purification and characterization of low molecular weight extreme alkaline xylanase
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for thermostable and alkaliphilic enhancement of endo-b-1,4-xylanase for applica-
market share by three folds in next ten years will be hotly pursued.
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Butt, M.S., Tahir-Nadeem, M., Ahmad, Z., Sultan, M.T., 2008. Xylanases and their ap-
Acknowledgements plications in baking industry. Food Technol. Biotechnol. 46.
Buzała, K.P., Przybysz, P., Kalinowska, H., Derkowska, M., 2016. Effect of cellulases and
xylanases on refining process and kraft pulp properties. PloS one 11, 0161575.
The financial support from DBT – India (BT/PR4827/PID/6/647/ Chadha, B.S., Badhan, A., Mello, F., Bhat, M.K., 2004. Two endoxylanases active and
2012) and AMAAS (ICAR) – India (NBAIM/AMAAS/2014-20/PF/51) stable at alkaline pH from newly isolated thermophilic fungus, Myceliophthora sp.IMI
for carrying out some of the research being cited in this review is duly 387099. J. Biotechnol. 109, 227–237.
Chakdar, H., Kumar, M., Pandiyan, K., Singh, A., Nanjappan, K., Kashyap, P.L.,
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