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Endoxylanases active under extreme conditions of temperature and alkalinity can replace the use of highly pollutant chemicals in the pulp and paper industry. Searching for enzymes with these properties, we carried out a comprehensive bioinformatics study of the GH10 family. The phylogenetic analysis allowed the construction of a radial cladogram in which protein sequences putatively ascribed as thermophilic and alkaliphilic appeared grouped in a well- defined region of the cladogram, designated TAK Cluster. One among five TAK sequences selected for experimental analysis (Xyn11) showed extraordinary xylanolytic activity under simultaneous conditions of high temperature (90 °C) and alkalinity (pH 10.5). Addition of a carbohydrate binding domain (CBM2) at the C-terminus of the protein sequence further improved the activity of the enzyme at high pH. Xyn11 structure, which has been solved at 1.8 Å resolution by X-ray crystallography, reveals an unusually high number of hydrophobic, ionic and hydrogen bond atomic interactions that could account for the enzyme’s extremophilic nature.
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Phylogenetic, functional and structural characterization of a GH10
xylanase active at extreme conditions of temperature and alkalinity
David Talens-Perales
, Elena Jiménez-Ortega
, Paloma Sánchez-Torres
, Julia Sanz-Aparicio
Julio Polaina
Institute of Agrochemistry and Food Technology, Spanish National Research Council (CSIC), Paterna, Valencia, Spain
Institute of Physical-Chemistry Rocasolano, Spanish National Research Council (CSIC), Madrid, Spain
article info
Article history:
Received 25 February 2021
Received in revised form 29 April 2021
Accepted 1 May 2021
Available online 03 May 2021
Crystal structure
Glycoside hydrolase
Pulp and paper
Endoxylanases active under extreme conditions of temperature and alkalinity can replace the use of
highly pollutant chemicals in the pulp and paper industry. Searching for enzymes with these properties,
we carried out a comprehensive bioinformatics study of the GH10 family. The phylogenetic analysis
allowed the construction of a radial cladogram in which protein sequences putatively ascribed as ther-
mophilic and alkaliphilic appeared grouped in a well-defined region of the cladogram, designated TAK
Cluster. One among five TAK sequences selected for experimental analysis (Xyn11) showed extraordinary
xylanolytic activity under simultaneous conditions of high temperature (90 °C) and alkalinity (pH 10.5).
Addition of a carbohydrate binding domain (CBM2) at the C-terminus of the protein sequence further
improved the activity of the enzyme at high pH. Xyn11 structure, which has been solved at 1.8 Å resolu-
tion by X-ray crystallography, reveals an unusually high number of hydrophobic, ionic and hydrogen
bond atomic interactions that could account for the enzyme’s extremophilic nature.
Ó2021 The Authors. Published by Elsevier B.V. on behalf of Research Network of Computational and
Structural Biotechnology. This is an open access article under the CC BY-NC-ND license (http://creative-
1. Introduction
Enzymes play a fundamental role in the transformation of the
traditional chemical industry into a future, more sustainable, green
alternative. The great diversity of enzymes existing in Nature can
supply suitable catalysts to any type of biochemical reaction.
Although most enzymes are labile material, unable to stand the
harsh physicochemical conditions used in industry, life shows a
surprising plasticity, being able to generate organisms that thrive
under harsh conditions of temperature, pH, pressure, salinity,
etc., which a priori would be unimaginable. Extremophilic organ-
isms, mainly archaea and bacteria, adapted to such extreme condi-
tions become a source of enzymes that widen the range of
processes and operational conditions in which enzymes can be
applied [1,2].
Use of xylanases by the paper industry, for pulp bleaching, offer
a challenging case for stretching the enzyme capability to the limit.
Xylanases facilitate the removal of residual lignin that causes the
dark color of the pulp and can be a total or partial replacement
to the use of hazardous chemical agents [3,4]. The predominant
method used by the paper industry, the kraft process, requires
harsh conditions. Therefore, the availability of xylanases active
under extreme environments of temperature and alkalinity repre-
sents an economic bonus.
Enzymes with xylanolytic activity appear in at least nine fami-
lies of the CAZY database [5]. However, most true xylanases (EC, considered as such because of their high substrate speci-
ficity, are classified in families GH10 and GH11 [6,7]. Family
GH10 enzymes are characterized by the presence of a catalytic
domain of ca. 40 kD, with (
-barrel structure. Family GH11
enzymes contain a smaller (ca. 30 k Da) catalytic domain with b-
jelly roll structure. There is abundant published information about
enzymes from both families. Up to the present the number of
annotated protein sequences and resolved atomic structures is
about 5000 and 50 for GH10, respectively and 2000 and 30 for
GH11 (CAZY database, accessed on January 2021). Although only
a relatively small fraction of the sequences corresponds to
enzymes whose activity has been characterized experimentally,
the available data provide very useful information to predict the
enzymatic properties of an uncharacterized sequence. Several
bioinformatic approaches to this end have been reported.
2001-0370/Ó2021 The Authors. Published by Elsevier B.V. on behalf of Research Network of Computational and Structural Biotechnology.
This is an open access article under the CC BY-NC-ND license (
Corresponding authors.
E-mail addresses: (J. Sanz-Aparicio),
(J. Polaina).
Computational and Structural Biotechnology Journal 19 (2021) 2676–2686
journal homepage:
Conventional strategies used to identify enzymes with extre-
mophilic properties relied on their isolation from microorganisms
with such properties, for instance, Thermotoga and Thermoascus [8].
However, this approach is too limited to yield enzymes with opti-
mal physicochemical and catalytic properties that fulfill industry.
Protein engineering techniques provide a complementary tool for
obtaining enzymes with enhanced thermostability or alkalinity
[9–11].In silico screening of protein sequences from databases,
often derived from genomic and metagenomic analysis, has been
shown to be a powerful methodology that has been successfully
applied to identify extremophilic xylanases in GH11 family [12]
and for discovery of hidden properties of carbohydrate active
enzymes [13–15].
In this communication we describe an in silico analysis of GH10
family, searching for xylanases with extreme thermophilic and
alkalophilic properties, and the functional and structural charac-
terization of the enzyme with best characteristics. Additionally,
we show that the modification of the protein structure by the addi-
tion of carbohydrate binding domains improves the enzyme per-
formance, which is relevant for biotechnological applications.
2. Materials and methods
2.1. Phylogenetic analysis of GH10 sequences
Accession numbers of GH10 protein sequences were obtained
from the CAZy database [5]. The amino acid sequences were
obtained from the NCBI database, using the Batch Entrez Tool
( Pfam [16] pro-
vided protein domain composition and coordinates for each
The linear composition of domains of a given sequence in N-
terminal to C-terminal order, defined what has been called domain
architecture (DA). The assignment of specific DA from the
sequences of the GH10 family was performed following the
methodology previously described for the GH2 family [13]. Only
sequences containing a catalytic domain Glyco_hydro_10 (GH10)
matching at least 80% with the Pfam consensus were included in
this study.
Sequence alignment of GH10 catalytic domains was performed
with CLC sequence viewer (Quiagen), using ClustalO MSA algo-
rithm [17]. Trees were built using W-IQ-TREE Maximum Likehood
algorithm with JTT matrix [15] and a bootstrap of 1000 replicates.
Results were analyzed on Dendroscope Software [18] and repre-
sented using FigTree (
and MESQUITE software (
2.2. Molecular biology
DNA sequences and amino acid selected for experimental anal-
ysis were edited before cloning. Signal peptide sequences were
detected using the Phobius Tool [19] and removed. The coding
sequences were optimized for E. coli expression by using the Inte-
grated DNA Technologies (IDT) Codon Optimization Tool (www. Native restriction sites were eliminated and SacI
and SalI restriction sites were added in 5
and 3
respectively, to
facilitate the cloning in vector pQE80L (Quiagen).
Synthetic genes (codon-optimized) of sequences encoding five
selected putative xylanases (Xyn10-14) were purchased from IDT
(Supplementary Table S3). The DNA fragments (except Xyn10
and Xyn14) were digested with endonucleases SacI and SalI and
cloned into pQE80L plasmid cut with the same enzymes. Xyn10
and Xyn14 could not be synthesized as a single piece and were
therefore assembled by joining two fragments (F1 and F2).
Xyn10-F1 and Xyn14-F1 were cut with SacI and EcoRI, whereas
Xyn10-F2 and Xyn14-F2 were cut with EcoRI and SalI. The
corresponding fragments were then cloned as one piece in pQE80L.
Fast Digest enzymes and T4 ligase were purchased from
The xylanase encoding plasmids were used for transforming
E. coli XL1 Blue. Selection of E. coli clones expressing xylanase genes
was performed as described in (Talens-Perales et al., 2020). The
cloned sequences were confirmed by sequencing. Once checked,
the plasmids were transferred to E. coli Rosetta (Stratagene) for
protein production. Hybrid enzymes Xyn11-CBM2 and Xyn11-
CBM9 were constructed from plasmids Xyn5-CBM2 PQE80L and
Xyn5-CBM9, respectively [12], replacing the Xyn5 gene by the
Xyn11 gene.
2.3. Protein purification
Cell crude extracts were obtained from E. coli cultures grown at
37 °C until reaching OD
of 0.6 and then were induced with 1 mM
IPTG, either at 37 °C for 5 h or 16 °C overnight. Buffer A (20 mM
phosphate buffer, pH 7.4, 10 mM imidazole, 500 mM NaCl) was
used to disrupt the cells by sonication. Protein extracts were
obtained by centrifugation at 12,000 g during 25 min. Protein
purification was performed using nickel affinity chromatography
with 1 mL HisTrap FF crude column (GE Healthcare) mounted in
AKTA-Purifier (GE). Buffer B (20 mM phosphate buffer, pH 7.4,
500 mM imidazole, 500 mM NaCl) was used for elution. Eluted
fractions with xylanase activity were dialyzed against buffer C
(20 mM Tris-HCl, pH 7.0, 50 mM NaCl). Protein concentration in
the eluted fractions was determined in a NanoDrop spectropho-
tometer (Thermo Fisher). The purity of the protein recovered was
analyzed by SDS-PAGE, using Blue Safe staining (Nzytech). An
image of the gel was taken with a Proxima AQ-4 gel documentation
system (Isogen) and the amount of protein in the gel bands was
quantified using FIJI software [20].
2.4. Evaluation of xylanase activity at different conditions of
temperature and pH
Xylanase activity of purified enzymes was assayed at range of
temperature and pH. Enzyme reactions for temperature assays
were prepared by mixing 180
L of substrate (1% oat spelt xylan,
in Tris-HCl 50 mM buffer pH 9.0) and 20
L of purified protein
(protein concentrations were adjusted to assure linear enzyme
response) and then incubated at 60 °C, 70 °C, 80 °Cor90°C, for
10 min. The reaction was stopped by putting the tubes on ice.
Activity at different pH was determined using 50 mM buffered
solutions, at the following pH values: 5.0 (acetate), 6.0 and 7.0
(phosphate), 8.0, 9.0 and 10.0 (Tris-HCl). The enzyme reactions
were prepared as above described, using the appropriate buffer.
The reactions were incubated at 65 °C during 10 min and then
stopped on ice. Control reactions, without enzyme, were carried
out for each assay condition.
Measurement of reducing sugars was carried out by adding
L of DNS reactive to the reaction tubes that were then boiled
for 10 min. Next, 900
L of miliQ H
O were added and the tubes
were centrifuged. Measurements were done at OD
in 96-well
plates by transferring 300
L of the supernatant using PowerWave
HT equipment, from BioTek Instruments (Winooski, VT, USA).
Cleavage pattern of xylooligosaccharides was analyzed by using
L of 2 mM of the corresponding substrate, from 2 to 6 units
(Megazyme), dissolved in 50 mM Tris-HCl buffer pH 9.0. To each
tube, 20
L of enzyme solution were added. The amount of enzyme
used was estimated to obtain 1
mol of reducing sugars min
of enzyme. The reactions were carried for 5 h at 90 °C and
stopped on ice. Analysis of reaction products was carried out by
ion exchange chromatography using a Dionex (Thermo Fisher Sci-
D. Talens-Perales, E. Jiménez-Ortega, P. Sánchez-Torres et al. Computational and Structural Biotechnology Journal 19 (2021) 2676–2686
entific) with CarbonPac PA100 column and a pulsed amperometric
2.5. Protein crystallization, data collection and structure
Firstly, different crystallization screens were explored, using the
sitting-drop vapor-diffusion method. A wide variety of crystals
grew in several commercial kits such as Index (Hamptom
Research), JBScreen JCSG++ (Jena Bioscience), JBScreen Classic (Jena
Bioscience). Different manuals grids were used in order to optimize
the quality of the crystals. Finally, the best crystals were obtained
with 10% PEG 3350, 0.2 M Proline, 0.1 M Hepes pH 7.5. Each drop
presented the same proportion of protein (6.47 mg/mL) and reser-
voir solution (250 nl). Mother liquor solution was supplemented
with 25% of glycerol to cryoprotect the crystal during the data col-
lection. Diffraction data were collected at the ALBA synchrotron
station of Barcelona (Spain). The X-ray images were processed with
XDS [21], and merged using Aimless from CCP4 suite package [22].
space group was obtained with cell parameters 91.45
95.27 100.53 Å
and two molecules in the asymmetric unit.
The structure was solved by molecular replacement using MOL-
REP from CCP4 [23]. The xylanase CbXyn10C from Caldicellulosirup-
tor bescii [24], with the Protein Data Bank code 5OFJ (56.5%
sequence homology) was selected as a template. Restraint refine-
ment was carried out using REFMAC5 from CCP4 with local non-
crystallographic symmetry (NCS) [25]. This was combined with
several rounds of model building in COOT [26]. Figures were pre-
pared with PyMOL [27]. Final crystallography results are given in
Supplementary Table 4.
2.6. Analysis of protein stability
Different web servers were employed to analyze the structure
and thermostability of the protein. Protein Blast [28] and END-
script server [29] were used to compare protein sequence and
the tridimensional structures were inspected in Dali server [30].
ProtParam tool (ExPASy) [31] was used to compute amino acid
composition. Intraprotein hydrophobic and ionic interactions,
hydrogen bonds, aromatic and cation-
interactions were deter-
mined by PIC server (Protein Interactions Calculator) [32]. The
accessible surfaced area (ASA) and the exposed charged accessible
area were both calculated with VADAR server [33]. The formula for
compactness was: ASA/number of total residues, and for % exposed
charged accessible surface area was: exposed charged accessible
area /ASA *100.
3. Results and discussion
3.1. In silico screening of GH10 sequences for thermophilic alkaliphilic
The nearly 5000 entries currently available in the CAZy data-
base were expurgated discarding repetitions, incomplete
sequences and those having less than 20% matching with the Pfam
Glyco_Hydro_10 domain (GH10). This yielded a total of 2309
sequences that were further processed (Supplementary material,
Table S1). About 80% of these sequences (1848) corresponded to
bacteria whereas the remaining 20% (461) were from eukaryotes.
No archaeal sequences matched the established requirements.
GH10 protein sequences showed a great variety of domain archi-
tectures. Up to 197 different domain architectures (DA) could be
distinguished (Supplementary material,Table S2), in contrast with
the lesser diversity (69 DA) described for functionally related GH11
family [12]. About one half of the bacterial sequences and one
tenth of the eukaryotic, showed the simplest possible DA, consisted
of just the catalytic GH10 domain. Regarding more complex DA,
the presence of CBM4-9, which binds amorphous cellulose and sol-
uble oligosaccharides [34,35], was ubiquitous and detected in 14%
of the sequences, in some cases in tandem repeats at N-terminal
position. CBM2 which binds cellulose and chitin [36], or Ricin-B-
lectin domains that mediate sugar recognition [37], were quite
abundant, present in more than 5% of the sequences. A small frac-
tion of the sequences (1–2%) contained C-terminal extensions not
corresponding with identified motifs. These tails were labeled by
their length, as Ct1 (50–150), Ct2 (150–200), Ct3 (200–300), Ct4
(300–400) and Ct5 (400–500) (Supplementary material,Table S2).
Fig. 1A shows a schematic representation of the cladogram
resulting from the phylogenetic analysis of the Glyco_Hydro_10
domain of the 2309 selected sequences. A detailed version of the
cladogram is presented as supplementary material (Fig. S1). In
Fig. 1 and S1 and in Table S2, different DA with common domains
are represented by the same color code. In the cladogram, eukary-
otic sequences appear in three groups, generated by different evo-
lutionary events, at a late stage in the evolutionary process. The
simplest DA, a single GH10 domain, appears in some instances
mingled with composed DA, but most sequences with additional,
non-catalytic domains, emerge from separate nodes. Sequences
putatively identified as thermoresistant, are marked by a gray label
in Fig. 1A. Among these, a cluster (hereinafter designated as TAK)
marked by a triangle, corresponds to sequences that putatively
are thermostable and alkaliphilic. The sequences included in the
TAK cluster, belong to genera the Caldicellulosiruptor, Thermotoga,
Pseudothermotoga, Petrotoga and Defluviitoga, and are listed in
Fig. 1B. Most of these sequences contain a multidomain DA, with
CBM4-9 present in ca. 70% of them. Another domains present are
CBM9, CBM3 and the catalytic domain GH48 with cellulase activity
[38]. CBM9 and CBM3 have been described to have xylan-binding
activity [38,39]. Five of these sequences, representing different
DA, were selected for experimental analysis: Xyn10 from Dyc-
tioglomus turgidum, Xyn11 from Pseudothermotoga thermarum,
Xyn12 and Xyn13 from Caldicellulosiruptor bescii and Caldicellu-
losiruptor owensensis, respectively and Xyn14 from Thermotoga sp.
3.2. Production, purification and functional characterization of
selected enzymes
Synthetic, codon-optimized gene sequences encoding the
selected putative xylanases were cloned and expressed in E. coli.
Synthetic DNA coding sequences could be obtained as a single frag-
ment, except for Xyn10 and Xyn14, which had to be reconstructed
from two fragments (Xyn10-F1, Xyn14-F1 and Xyn10-F2, Xyn14-
F2). Proteins were purified from bacterial crude cell extracts by
thermal treatment and nickel affinity chromatography. Xyn12
was discarded at this point because no substantial amount of pro-
tein could be recovered from the recombinant E. coli cultures.
Semiquantitative evaluation of Xyn14 showed very low level of
xylanase activity (results not shown) and therefore was also dis-
carded. The other three enzymes were purified and analyzed by
SDS-PAGE (Fig. 2). In all three cases, electrophoretical mobility
was in accordance with the expected molecular mass of the
polypeptides, predicted by ProtParam [40].
Oat spelt xylan hydrolysis by Xyn10, Xyn11, and Xyn13 was
measured at different pH and temperature. Assays at different val-
ues of pH were carried out at 90 °C(Fig. 3A). Xyn10 and Xyn11
showed the expected profile for alkaliphilic enzymes, with optimal
activity at pH between 8.0 and 9.0. Xyn13 showed maximal activ-
ity at pH between 6.0 and 7.0, but retained high activity at pH 9.0.
In buffered solution at pH 9.0, the three xylanases showed a ther-
mophilic profile, with optimal temperature 70 °C and 90 °C. The
highest activity measured corresponded to Xyn11 at 90 °C
D. Talens-Perales, E. Jiménez-Ortega, P. Sánchez-Torres et al. Computational and Structural Biotechnology Journal 19 (2021) 2676–2686
(Fig. 3B). These results are remarkably different from a report by
Shi et al., [41], describing the properties of a xylanase encoded
by the Xyn10B gene of T. thermarum (now Pseudothermotoga ther-
marum), same sequence as Xyn11, but optimal activity at 80 °C,
and weak activity at pH > 8.0. A different xylanase from the
GH10 family from P. thermarum has also been described. This
one displays a complex DA, which three tandem CBM4-9 at N-
terminal position and two CBM9 at C-terminal position. Despite
of the differences in DA, the catalytic domain of this enzyme,
named Xyn10A, is very similar to Xyn11 being located very close
in the phylogenetic tree (Figs. 1 and S1). Xyn10A, was reported
to be optimally active at 95 °C and pH 7.0 [8]. Other thermostable
xylanases (from GH10 and GH11 families) have been characterized
but their optimal temperatures are around 70 °C and their opti-
mum pH ranged from 6.5 to 9.0. Only Bacillus halodurans TSEV
xylanase combines thermostability and alkaliphility, with optimal
values of pH of 9.0 and temperature 80 °C[42], but still below the
score of Xyn11. Xylanases from Geobacillus, Caldicellusiruptor [43]
and Thermotoga, showed high optimum temperatures. Remarkably,
Thermotoga naphthophila RKU-10 and Thermotoga petrophila RKU-1
(95 °C) or Thermotoga maritima MSB8, (90 °C), but optimum pH val-
ues for these enzymes were between 5.0 and 6.0 [44–46]. Xyn11
represented an extraordinary performance under simultaneous
conditions of high temperature and alkalinity, showing at pH
Fig. 1. (A) Cladogram of the GH10 family. The gray triangle marks the TAK cluster. (B) Phylogenetic tree of the TAK cluster sequences. Asterisks mark the sequences that were
selected for experimental analysis.
Fig. 2. SDS-PAGE analysis and domain architecture of selected enzymes. Protein ladder mass in kDa.
D. Talens-Perales, E. Jiménez-Ortega, P. Sánchez-Torres et al. Computational and Structural Biotechnology Journal 19 (2021) 2676–2686
10.5 and 90 °C, an activity value of 600 mmol min
A previous study carried out with GH11 xylanases showed that
in some instances, the addition of a Carbohydrate Binding Domain
(CBM2, CBM9) to the protein structure has a positive effect on
enzyme performance [12]. Therefore, we undertook the construc-
tion of hybrid enzymes derived from Xyn11, by fusing this enzyme
with CBM2 and CBM9. The SDS-PAGE profile and DA of the hybrids
are shown in Fig. 4A. Addition of CBM9 caused a decrease of Xyn11
activity at different pH (Fig. 4B). This result was unexpected as it is
the opposite to what was observed by the addition of CBM9 to a
GH11 xylanase [12]. However, the addition of CBM2 improved
the activity of the enzyme at high pH. Hybrid Xyn11-CBM2 showed
an activity two times higher than Xyn11 at 90 °C and pH 10.5
(Fig. 4B).
The cleavage pattern of xylanases was analyzed using as sub-
strate oligoxylosides from 2 to 6 units. Enzyme reactions were car-
ried out for 5 h at pH 9.0 and 90 °C. The products released from the
reactions were analyzed chromatographically. As expected, the
enzymes showed characteristic behavior of endoxylanases, but
with some interesting differences. Xyn10 and Xyn13 were unable
to hydrolyze xylobiose and xylotriose. Xyn10 hydrolyzed xylote-
traose, xylopentaose and xylohexaose, yielding mainly xylobiose
and xylotriose (Fig. 5A). Xyn13 was inactive towards xylotetraose,
but excised xylopentaose and xylohexose, yielding mainly xylo-
biose and xylotriose (Fig. 5C). Xyn11, as well as its hybrids
Xyn11-CBM9 and Xyn11-CBM2, were unable to cut xylobiose,
but were active against xylotriose, xylotetraose, xylopentaose
and xylohexaose yielding mainly xylose and xylobiose (Fig. 5B).
These results agree with previous studies on GH10 xylanases
[42]. The observed differences in the cleavage pattern of Xyn10,
Xyn11 and Xyn13 is relevant from a biotechnological point of view,
as it can be used to produce short-chain oligoxylosides from xylan.
These oligosaccharides are compounds with recognized value as
prebiotics, whose administration confers a proven heath benefit
3.3. Crystallographic structure of Xyn11
The structure of Xyn11 was solved at 1.8 Å resolution by X-ray
crystallography. The protein folds into an (b/
barrel (TIM-barrel)
architecture (Fig. 6A), typical of GH10 xylanases. From the loops
linking the C-terminal of each b-strand to the succeeding
the long L4, L7 and L8 loops protrude markedly and define an
extended groove to accommodate the substrate. An additional b-
hairpin motif is present in the loop L2, linking b2to
According to its primary structure, the closest homologues of
Xyn11 are thermoresistant multidomain proteins from Caldicellu-
losiruptor species (N-terminal domain of CbXyn10C-Cel48B [24],
PDB code 5OFJ, and GH10 domain of WP_045175321 [49], PDB
code 6D5C, showing 55% sequence identity, both presenting the
Fig. 3. Activity of selected enzymes at different values of pH (A) and temperatures
(B). Activity values correspond to
mol of reducing sugars min
of enzyme.
Fig. 4. (A) Physical characterization (DA and SDS-PAGE analysis) of hybrid xylanases. (B) Activity of hybrid xylanases assayed at 90 °C and different pH. Displayed values
correspond to activity expressed as
mol of reducing sugars min
of enzyme. (C) Activity of hybrid xylanases at pH 9.0 and different temperatures.
D. Talens-Perales, E. Jiménez-Ortega, P. Sánchez-Torres et al. Computational and Structural Biotechnology Journal 19 (2021) 2676–2686
same b-hairpin observed in Xyn11. Next homologues are two
intracellular xylanases, IXT6 from Geobacillus stearothermophilus
[50], PDB code 1 N82, with 50% identity, and the mesophilic
Xyn10B from Paenibacillus barcinonensis [51], PDB code 3EMC,
showing 44% identity. These enzymes show very similar topology,
with equivalent L4, L7 and L8 loops both in length and position.
Despite fairly conserved topology, loops L7 and L8 are highly
variable in sequence (Fig. 7). Therefore, these loops shape very dif-
ferent active site crevices. To fully depict the Xyn11 active site, the
binding mode of an heptaxylose has been inferred from structural
superimposition of its complex from Caldicellulosiruptor bescii
Xyn10C [43], PDB code 5OFK, onto the Xyn11 coordinates
(Fig. 6B–D). As it is observed in Fig. 6, Xyn11 seems able to accom-
modate at least seven units of xylose spanning its catalytic tunnel.
The most peculiar feature of Xyn11 is the presence at the non-
reducing end of three consecutive Phe residues (Phe312, Phe313
and Phe314) at L8 (Fig. 7) that define a narrow entrance to the cat-
alytic channel. Only the central Phe313 is conserved in homo-
logues, which have Ser/Thr and Pro at the other two positions.
This feature places Phe313 in a more prominent position within
a narrower cavity and suggests a potential subsite 3, by
hydrophobic interaction to Phe312, not seen in the Xyn11 ana-
logues. Apart from this hydrophobic wall, subsite 2 is well-
characterized by the presence of a great amount of hydrogen bonds
at the opposite wall with a number of very conserved residues.
Thus, and according to the complex model, O2 for the xylose would
interact with Trp301 and Glu50, O3 with Glu50 and Asn51, O4
would be stabilized by Trp301 and Lys54, which also form a hydro-
gen bond with O5. Furthermore, the xylose bound at subsite 1
would also present polar interactions to conserved residues at
the same wall of the active site, i.e. to Lys54 (O3), His91 (O2 and
O3), and Asn143 and Gln220, (O2).
The xylose bound at subsite + 1 is mainly fixed by stacking to
Tyr189 and hydrophobic interaction to Trp309 and Phe313, all con-
served residues, and lacking direct polar interactions as in other
xylanases. However, in Xyn11, the presence of the pair Phe313-
Phe314 narrow the cavity at this subsite + 1 and, more important,
fixes the conformation of the Arg260 side-chain in a very
restrained position pointing to the inner part of the cavity, in
which it could be hydrogen linked to O2 and O3 of xylose bound
at subsite + 2. As a consequence, Xyn11 does not present at this
subsite + 2 the pocket observed in its homologues that seems able
to allocate a putative xylan decoration [51]. Furthermore, Ser259, a
position in which most analogues present an aromatic residue with
a protruding side-chain that makes a more restricted subsite + 3,
precedes Arg260, unique to Xyn11 at loop L7 (Fig. 7). Thus, the
reducing-end of the Xyn11 tunnel exhibit rather open subsites
from + 3 that are markedly different to that observed in its homo-
logues. Only the bifurcated polar link from Asn190 to the xyloses
bound at subsite + 2 (O5) and + 3 (O3) is conserved.
A last interesting feature of the channel is the presence of a
small pocket created by a cluster of three aromatic residues located
at the end of the tunnel, Phe192, Phe193 and Trp231, only Phe193
being conserved (Fig. 6D).Interestingly, a glycerol molecule from
Fig. 5. Cromatographic profile of reaction products obtained from the action of xylanases on xylooligosaccharides of different degree of polymerization.
D. Talens-Perales, E. Jiménez-Ortega, P. Sánchez-Torres et al. Computational and Structural Biotechnology Journal 19 (2021) 2676–2686
the cryoprotectant has been trapped within this pocket in our crys-
tals, suggesting a putative role in accommodating a potential O3
substituent at the succeeding xylose unit, which would represent
a putative subsite + 5. As it is shown in Fig. 6D, an additional glyc-
erol molecule has been trapped in a groove close to the free O2 and
O3 hydroxyls of xylose at subsite + 3. The particular shape of both
cavities might reflect a specific pattern of xylan decorations that
would be recognized and bound at subsite + 3 and + 5.
In summary, Xyn11, as its closest thermoresistant homologues, is
able to degrade xylan, and presents 6–7 subsites (from 2 to + 4/+5)
for binding this polysaccharide. However, inspection of its active site
crevice reveals that substitutions are not allowed at 2 and 1, nor
at + 2 and + 4. Therefore, two consecutive unsubstituted xylose units
are required to bind at positions 2, 1, for hydrolysis to occur, and a
subsequent sequence of alternate substituted/unsubstituted
xyloses is envisaged from subsite + 1. This result is in accordance
with the cleavage pattern observed in Fig. 5. The especial shape of
the cavities described above may match the chemical structure of
its natural substrate.
3.4. Structural basis of Xyn11 extremophilic properties
A great effort has been directed to disclose the molecular basis
of protein adaptability to extreme conditions [52–55]. The general
agreement is that this ability results from the cumulative effect of
multiple stabilizing factors that may be present to a different
degree in each extremophilic protein. Nevertheless, despite the
ample variation observed, some trends can be observed between
the sequences and structures of extremophilic vs their mesophilic
In order to explain the extremophilic properties of Xyn11, its
primary and tertiary structure was compared to other family
GH10 xylanases, including thermoresistant CbXyn10C (PDB code
5OFJ) [49] and three mesophiles: PbXynB from Paenibacillus barci-
nonensis; (3EMC) [51], CmXyn10B from Cellvibrio mixtus; (2CNC)
[53] and SoXyn10A from Streptomyces olivaceoviridis (1V6Y) [56].
Several features analyzed in Xyn11 and its homologues are sum-
marized in Tables 1 and 2.
As a general rule, extremophilic proteins contain low number of
residues that decompose easily, such as hydroxy amino acids: Ser
and Thr [57], amino acids with an amide terminal group: Asn,
Gln [58] or sulfur containing amino acids: Cys, Met [57]. Accord-
ingly, Xyn11 complies this rule (Table 1). On the other hand, the
relative abundance of Pro observed in Xyn11, compared with its
homologues, can been related to increased stabilization provided
by a higher chain rigidity that reduces fluctuation of the secondary
structure and helps to keep the proper folding at high temperature
[59]. Finally, an increased number of charged residues, meaning an
increment in the ion pair interactions, derive in thermostability
[58,60]. Thus, Xyn11 presents the highest value of Lys, Arg and
Glu compared with the homologues.
Considering the tertiary structure (Table 2), hydrophobic interac-
tions have been reported to play a key role in protein folding [53].
Thus, the buried hydrophobic interactions in the core of the protein
increase the van der Waals contacts, reducing the exposure of
hydrophobicresiduestosolventand promoting rigidity [53]. In addi-
tion, aromatic clusters have also been commonly considered to con-
tribute to the extreme stability of the proteins, resulting in internal
stacking interactions that enhance the stability of the protein [61].
In this respect, Xyn11 presents the highest number of both,
hydrophobic and aromatic interactions. Whereas Xyn11 compact-
ness is similar to its mesophilic analogues, its accessible surface area
(ASA) is higher. Hydrogen bonding is correlated with an increase in
Fig. 6. Crystal structure of Xyn11. (A) Overall folding as an (b/
(TIM-barrel) architecture, represented in green (helices) and violet (strands). The
most relevant loops that participate in the substrate binding and the catalytic
residues are colored in orange. (B) The active site tunnel with a heptaxylose
molecule, modelled from structural superimposition with its complex from
Caldicellulosiruptor bescii Xyn10C (PDB code 5OFK). (C) Proposed atomic interac-
tions of heptaxylose at Xyn11 active site. The catalytic residues are colored in
orange, while the non-conserved residues are colored in yellow. Polar interac-
tions are marked in dashed lines. (D) Detail of the reducing-end moiety of the
active site, showing two glycerol molecules (blue) trapped in the crystals, and
the three aromatic residues shaping a pocket in the tunnel. (For interpretation of
the references to color in this figure legend, the reader is referred to the web
version of this article.)
D. Talens-Perales, E. Jiménez-Ortega, P. Sánchez-Torres et al. Computational and Structural Biotechnology Journal 19 (2021) 2676–2686
rigidity in the core of the protein [62] and is considered one of the
major contributors to extreme-stability, especially when consider-
ing the main chain-main chain interactions [53,63]. Theoretical
studies show the stabilizing effect of electrostatic interactions and
salt-bridges at high temperature [64]. Again, Xyn11 presents a high
number of hydrogen bonds and ionic interactions.
It is worth noting that, even when Xyn11 is first-ranked in only
5 out of 11 stabilizing parameters that we have analyzed (Tables 1
and 2), it is one of the two best proteins in all cases, revealing that
its molecular structure is well suited to stand denaturing condi-
tions. All the atomic interactions considered in this work have
been previously identified as molecular mechanism underlying
resistance in both, thermophilic and also alkaliphilic proteins
[53], which explains not only Xyn11 thermal resistance but also
its activity at high pH. Furthermore, the increased number of
atomic interactions observed in Xyn11 must derive in protein
rigidity, which is the key to high-temperature adaptation by pre-
venting unfolding. In this context, Xyn11 presents a markedly
low chain flexibility, as deduced from the analysis of the atomic
B factors values along the polypeptide chain that is shown in
Fig. 8A.
Lastly, an interesting feature in Xyn11 structure is the long loop
L2 (Fig. 6A), connecting b2to
2, also present in close homologues
from Caldicellulosiruptor species (xylanases from C. bescii, PDB code
5OFK and C. danielli, PDB code 6D5C). This loop is making a
b-hairpin which is protruding from the (
barrel that,
Fig. 7. Sequence variability of loops L7 and L8. Sequences are of xylanases CbXynb10C from C. bescii (5OFJ), GH10 module from C. danielli (6D5C), IXT6 from G.
stearothermophilus (1N82), and Xyn10B from P. barcinonensis (3EMC). (ESPript -
Table 2
Analysis of the atomic interactions and packing. Number of hydrophobic and aromatic interactions, main chain-main chain hydrogen bonds and ionic interactions in Xyn11,
CbXyn10C from Caldicellulosiruptor bescii (5OFJ), XynB from Paenibacillus barcinonensi (3EMC), CmXyn10B from Cellvibrio mixtus (2CNC) and SoXyn10A from Streptomyces
olivaceoviridis (1V6Y). Compactness and percentage of exposed residues in the accessible surface area are also computed and shown. In each column, the two first-ranked proteins
are shown in bold type, underlined number indicate the most favorable value.
Atomic Interactions and Packing
Xyn11 381 32 449 47 42.4 21.8
CbXyn10C 361 21 497 39 40.6 19.1
PbXynB 323 24 428 38 42.7 25.1
CmXyn10B 347 27 419 56 43.1 21.4
SoXyn10A 274 18 439 34 37.9 16.2
Table 1
Analysis of amino acid composition. Percentage of Asn, Gln, Ser, Thr, Pro, Met, Cys, Glu, Arg and Lys in Xyn11, CbXyn10C from Caldicellulosiruptor bescii (5OFJ), XynB from
Paenibacillus barcinonensi (3EMC), CmXyn10B from Cellvibrio mixtus (2CNC) and SoXyn10A from Streptomyces olivaceoviridis (1V6Y). In each column, the two first-ranked proteins
are shown in bold type, underlined number indicate the most favorable value.
AA Composition (%)
Asn + Gln Ser + Thr Pro Met + Cys Glu + Arg + Lys
Xyn11 7.4 10.0 4.9 3.0 19.0
CbXyn10C 10.0 13.0 4.1 3.3 17.1
PbXynB 9.0 10.8 3.3 3.0 18.4
CmXyn10B 7.2 9.6 3.4 3.1 18.6
SoXyn10A 10.8 11.8 2.8 4 13.9
D. Talens-Perales, E. Jiménez-Ortega, P. Sánchez-Torres et al. Computational and Structural Biotechnology Journal 19 (2021) 2676–2686
nevertheless is stabilized by two cation-
interactions keeping a
close packing of L2 (Fig. 8B). Thus, Arg61 is stacking to Trp102,
located at L3, while Arg131, at the end of helix
3, is stacking to
Tyr69. Only the last interaction is conserved in the other thermore-
sistant homologues. Cation-
interactions have been described as
another form of electrostatic interaction that make an important
contribution to protein stability, which appears to increase at
higher temperatures [65]. Thus, Xyn11 might have developed addi-
tional local molecular mechanisms that combined to the above
parameters make a very resistant protein.
4. Conclusions
The comprehensive bioinformatics screening of the GH10 fam-
ily reported in this study represents a powerful methodological
approach that allowed the identification and characterization of
xylanases active at extreme conditions of pH and temperature.
The enzyme with best performance, Xyn11, corresponds to a xyla-
nase from the bacterium Pseudothermotoga thermarum. The
enzyme shows an exceptional level of activity at 90
C and pH
10.5. Fusion of a carbohydrate binding module (CBM2) to Xyn11
further increased its activity at extreme conditions. In addition to
Xyn11, two other xylanases, Xyn10 and Xyn13 showed activity
high at extreme conditions. Interestingly, these enzymes differ in
their cleavage pattern on xylan derived substrates, yielding differ-
ent proportions of xylose, xylobiose and xylotriose as final prod-
ucts, which may have practical consequences from a
biotechnological point of view. The crystallographic resolution at
1.8 Å of Xyn11 structure provides an explanation of its function
at extreme conditions. Not surprisingly, qualitatively, the atomic
interactions responsible for Xyn11 resistance are the same known
to sustains protein stability, namely hydrogen bonds, ion pairs,
hydrophobic and aromatic interactions. However, the number
and distribution in which these interactions appear and its extre-
mophilic enzyme properties makes Xyn11 an outstanding case
for study.
5. Accesion code
CRediT authorship contribution statement
David Talens-Perales: Formal analysis, Investigation. Elena
Jiménez-Ortega: Investigation. Paloma Sánchez-Torres: Investi-
gation. Julia Sanz-Aparicio: Supervision, Funding acquisition. Julio
Polaina: Supervision, Funding acquisition.
Declaration of Competing Interest
The authors declare that they have no known competing finan-
cial interests or personal relationships that could have appeared
to influence the work reported in this paper.
This work was funded as part of the European Project WOOD-
ZYMES, by the Bio Based Industries Joint Undertaking, under the
European Union’s Horizon 2020 research and innovation program
(Grant Agreement H2020-BBI-JU-792070) and by grants
BIO2016-76601-C3-3-R and PID2019-105838RB-C33 from the
Spanish Government.
Appendix A. Supplementary data
Supplementary data to this article can be found online at
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... This is particularly relevant for the kraft process, given the harsh physicochemical conditions that characterise this pulping method, which is used predominantly by the paper industry to obtain pulp from wood. Most of the commercial enzymes are unable to withstand the conditions of high temperatures and alkaline pH used in this particular industry [11]. ...
... In particular, extremophilic bacteria or archaea adapted to extreme temperatures, pH, pressure or salinity become valuable sources of enzymes to be applied as biocatalysts at the harsh operational conditions of the industrial processes [12]. In silico screening of protein sequences from databases, often derived from genomic and metagenomic analysis, has been successfully applied to identify extremophilic enzymes [11,13,14]. ...
... A bioinformatic screening of the glycosyl hydrolase family GH10 allowed the identification and characterisation of xylanases active at high pH and temperature. One enzyme in particular, Xyn11, a xylanase from Pseudothermotoga thermarum, showed an exceptional xylanolytic activity at 90 • C and pH 10.5 [11]. Here, this extremophilic enzyme (extremozyme) is integrated in an industrial ECF bleaching sequence of eucalyptus kraft pulp with the aim of increasing pulp bleachability and reducing the need for ClO 2 as bleaching agent. ...
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Xylanases can boost pulp bleachability in Elemental Chlorine Free (ECF) processes, but their industrial implementation for producing bleached kraft pulps is not straightforward. It requires enzymes to be active and stable at the extreme conditions of alkalinity and high temperature typical of this industrial process; most commercial enzymes are unable to withstand these conditions. In this work, a novel highly thermo and alkaline-tolerant xylanase from Pseudothermotoga thermarum was overproduced in E. coli and tested as a bleaching booster of hardwood kraft pulps to save chlorine dioxide (ClO2) during ECF bleaching. The extremozyme-stage (EXZ) was carried out at 90 °C and pH 10.5 and optimised at lab scale on an industrial oxygen-delignified eucalyptus pulp, enabling us to save 15% ClO2 to reach the mill brightness, and with no detrimental effect on paper properties. Then, the EXZ-assisted bleaching sequence was validated at pilot scale under industrial conditions, achieving 25% ClO2 savings and reducing the generation of organochlorinated compounds (AOX) by 18%, while maintaining pulp quality and papermaking properties. Technology reproducibility was confirmed with another industrial kraft pulp from a mix of hardwoods. The new enzymatic technology constitutes a realistic step towards environmentally friendly production of kraft pulps through industrial integration of biotechnology.
... The use of xylanases in industrial processes is often carried out under extreme pH and temperature conditions that conventional enzymes cannot withstand. Therefore, enzymes active under such extreme conditions have been identified and isolated 24,25 . Extremophilic xylanases are particularly useful for XOS production as their operational conditions greatly favor substrate and product solubility and prevent contamination in a sugar-rich environment, otherwise prone to microbial contamination 26 . ...
... Since xylanases are in-demand enzymes for several industrial applications, particularly the digestion of xylan for producing XOS, in this work we analyzed whether the production of one of these enzymes in N. benthamiana using a TMV-derived vector and an export-to-apoplast strategy to facilitate purification is a good alternative to the classic E. coli host. We focused on the extremophilic xylanase Xyn11 from Pseudothermotoga thermarum DSM 5069 25 , an enzyme active under high temperature (90 °C) and alkaline pH (10.5), which are the most convenient conditions in an industrial setting. ...
... Comparative analysis of Xyn11 produced in E. coli and N. benthamiana. Xylanase Xyn11 is produced in E. coli with a high yield: up to 20 mg of protein per liter of culture can be recovered 25 . The thermoresistant nature of the protein makes purification easy to a high degree by subjecting the bacterial cell extract to heating for several minutes. ...
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A gene construct encoding a xylanase, which is active in extreme conditions of temperature and alkaline pH (90 °C, pH 10.5), has been transitorily expressed with high efficiency in Nicotiana benthamiana using a viral vector. The enzyme, targeted to the apoplast, accumulates in large amounts in plant tissues in as little as 7 days after inoculation, without detrimental effects on plant growth. The properties of the protein produced by the plant, in terms of resistance to temperature, pH, and enzymatic activity, are equivalent to those observed when Escherichia coli is used as a host. Purification of the plant-produced recombinant xylanase is facilitated by exporting the protein to the apoplastic space. The production of this xylanase by N. benthamiana, which avoids the hindrances derived from the use of E. coli, namely, intracellular production requiring subsequent purification, represents an important step for potential applications in the food industry in which more sustainable and green products are continuously demanded. As an example, the use of the enzyme producing prebiotic xylooligosdaccharides from xylan is here reported.
... thermostability of its mutant XynAF1-AC was increased by 6-fold through semi-rational design (Li et al., 2021). Xyn11 from Pseudothermotoga thermarum showed an excellent activity at 90°C and pH 10.5, and the fusion of CBM2 to Xyn11 enhanced the enzymatic activity under extreme conditions (Talens-Perales et al., 2021). Therefore, the catalytic efficiency and thermostability of TfXyl10A could be further improved by protein engineering. ...
... The removal of the linker had a great influence on both enzyme activity and thermal stability ( Figures 2C, 3A). The linker region contains 9 Pro and 8 polar amino acids (2 Asp + 3 Glu + 2 Ser + 1 Thr), more than half of the total number of amino acids, which play an important role in structural stability (Talens-Perales et al., 2021). The deletion of the linker region may allow to deconstruct more easily the catalytic domain from the C-terminal under elevated temperatures. ...
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Xylanases have the potential to be used as bio-deinking and bio-bleaching materials and their application will decrease the consumption of the chlorine-based chemicals currently used for this purpose. However, xylanases with specific properties could act effectively, such as having significant thermostability and alkali resistance, etc. In this study, we found that Tf Xyl10A, a xylanase from Thermobifida fusca , was greatly induced to transcript by microcrystalline cellulose (MCC) substrate. Biochemical characterization showed that Tf Xyl10A is optimally effective at temperature of 80 °C and pH of 9.0. After removing the carbohydrate-binding module (CBM) and linker regions, the optimum temperature of Tf Xyl10A-CD was reduced by 10°C (to 70°C), at which the enzyme’s temperature tolerance was also weakened. While truncating only the CBM domain ( Tf Xyl10AdC) had no significant effect on its thermostability. Importantly, polysaccharide-binding experiment showed that the auxiliary domain CBM2 could specifically bind to cellulose substrates, which endowed xylanase Tf Xyl10A with the ability to degrade xylan surrounding cellulose. These results indicated that Tf Xyl10A might be an excellent candidate in bio-bleaching processes of paper industry. In addition, the features of active-site architecture of Tf Xyl10A in GH10 family were further analyzed. By mutating each residue at the -2 and -1 subsites to alanine, the binding force and enzyme activity of mutants were observably decreased. Interestingly, the mutant E51A, locating at the distal -3 subsite, exhibited 90% increase in relative activity compared with wild-type (WT) enzyme Tf Xyl10A-CD (the catalytic domain of Tf Xyl110A). This study explored the function of a GH10 xylanase containing a CBM2 domain and the contribution of amino acids in active-site architecture to catalytic activity. The results obtained provide guidance for the rational design of xylanases for industrial applications under high heat and alkali-based operating conditions, such as paper bleaching.
... New bacterial xylanases with remarkable extremophilic properties were discovered through in silico analyses (2,3). One in particular, Xyn11, presented outstanding activity on xylan at pH 10.5 and 90ºC (3,4). The enzyme was produced at 400 L pilot scale. ...
Conference Paper
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Enzymes can substitute harsh and energy-demanding chemical treatments for production of bio-based building blocks and products from wood processing. However, their properties need to be adapted to the extreme operation conditions (such as high T and pH) commonly used by these industries. Here, we summarize the main results obtained during the WoodZymes European Project (, which aimed to provide tailor-made extremozymes and extremozyme-based processes never assayed before in wood biorefineries. Novel extremophilic enzymes active on kraft lignin (laccases) and xylan (xylanases) were developed and produced at pilot or industrial scales. The enzymatic fractionation of kraft lignins using the METNIN(TM) lignin refining technology, and the extremozyme-aided delignification and bleaching of kraft pulps were demonstrated at pilot scale. The resulting lignin and hemicellulose derived compounds were chemically characterized and applied as components of phenol-(lignin)-formaldehyde resins for wood panels and of polyurethane foams, or as papermaking additives. The new extremozymes were also applied to improve some of the latter applications. The techno-economic and environmental assessment of the new materials and processes, developed in WoodZymes project, showed that extremozyme-based processes led to clear benefits in energy savings during the refining of pulp or wood fibres, enabled lower addition of harsh chemicals (e.g. ClO2 during pulp bleaching), and resulted in a lower carbon footprint of the new bio-based products by substitution of fossil-derived components.
... The research discovered that superhydrophobic coatings may effectively inhibit biofouling development and enhance surface antifouling performance. Talens-Perales et al. (2021) examined the corrosion prevention capability of superhydrophobic coatings [34]. The research discovered that superhydrophobic coatings have successfully protected surfaces against corrosion and might increase the durability of materials used in severe conditions. ...
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In the oil and gas construction industry, the adoption of superhydrophobic coatings is still in the early adoption phase. Due to the lack of research and the importance of hydrophobic coatings in the oil and gas construction business, this study examined the success determinants of superhydrophobic coatings in Malaysia. This quantitative study included a pilot survey to assess questionnaire validity and Exploratory Factor Analysis (EFA) to reduce success variables discovered through a literature review. A structural equation modeling (SEM) approach was used to develop a model involving success factors of superhydrophobic coatings in the oil and gas construction industry of Malaysia. Four constructs in total were found in SEM, namely, performance success, sustainability construct, oil spill management, and safety and economic success. In total, five items were excluded from the model because their loading factors were less than 0.6. All Cronbach Alpha reliability constants were greater than 0.7, the composite reliability indicators were greater than 0.8, and the AVE was greater than 0.6 for all of the constructs, confirming acceptable reliability and validity statistics. Both convergent and discriminant validity confirmed the relationships between all constructs and the latent variable. The observed path coefficients between the constructs and the latent variable were 0.476 for performance success, 0.461 for sustainability success, 0.322 for oil spill management, and 0.242 for safety and economic success. The significance value for all of the constructs was less than 0.05, confirming the strong relationship between the constructs and the critical success of superhydrophobic coatings in the oil and gas industry.
... For example, the catalytic activity of Dictyoglomus thermophilum xylanase was increased by 4-fold after fusing the CBM9 to its N-terminus [16]. Also, the fusion of Pyrococcus furiosus-derived CBM2 to Pseudothermotoga thermarum-derived xyn11 resulted in a 2-fold increase in activity compared to xyn11 [30]. Meanwhile, the location of the binding domain also affects the catalytic activity of the fusion protein. ...
In this study, the catalytic activity of xynAm1 (a thermophilic xylanase family 11 variant) was significantly enhanced by fusing a carbohydrate-binding module (CBM) 9-2. First, CBM9-2 was fused to the N- and C-termini of xynAm1, generating the fusion enzymes C-X and X-C, respectively. The specific activity of C-X was 19.0 U/μM, which was 2.3-fold and 1.1-fold higher than that of xynAm1 and X-C, respectively. Then, the flexible linker (GGGGS)2 was used to fuse CBM9-2 at the N-terminus of xynAm1, yielding C-F2-X with an additional 15.91-fold increase in the specific activity. By analyzing their modeled structures, the hydrogen bonds between xynAm1 and CBM in C-F2-X made the distance between the catalytic active site E379 and binding site W176 shorter by 8 Å than that in C-X. Meanwhile, the sugarcane xylan hydrolysis with C-F2-X produced 5.42 times higher amount of the xylose than that with xynAm1. When C-F2-X (200 U) and cellulase (200 U) were added to 30 mL suspension of wheat bran (1.5 g), the reducing sugar content and dry weight loss rate reached 143.23 nM and 82.7% after 24 h reaction, respectively. Therefore, the fusion enzyme C-F2-X could be a robust candidate enzyme for xylose production and lignocellulose degradation.
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Xylanase, a glycoside hydrolase, is widely used in the food, papermaking, and textile industries; however, most xylanases are inactive at high temperatures. In this study, a xylanase gene, CFXyl3, was cloned from Cellulomonas flavigena and expressed in Escherichia coli BL21 (DE3). To improve the thermostability of xylanase, four hybrid xylanases with enhanced thermostability (designated EcsXyl1–4) were engineered from CFXyl3, guided by primary and 3D structure analyses. The optimal temperature of CFXyl3 was improved by replacing its N-terminus with the corresponding area of SyXyn11P, a xylanase that belongs to the hyperthermostable GH11 family. The optimal temperatures of the hybrid xylanases EcsXyl1–4 were 60, 60, 65, and 85°C, respectively. The optimal temperature of EcsXyl4 was 30 C higher than that of CFXyl3 (55°C) and its melting temperature was 34.5°C higher than that of CFXyl3. After the hydrolysis of beechwood xylan, the main hydrolysates were xylotetraose, xylotriose, and xylobiose; thus, these hybrid xylanases could be applied to prebiotic xylooligosaccharide manufacturing.
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The majority of lignocellulosic biomass on the planet originates from plant cell walls, which are complex structures build up mainly by cellulose, hemicellulose and lignin. The largest part of hemicellulose, xylan, is a polymer with a β-(1→4)-linked xylose residues backbone decorated with α-D-glucopyranosyl uronic acids and/or L-arabinofuranose residues. Xylan is the second most abundant biopolymer in nature, which can be sustainably and efficiently degraded into decorated and undecorated xylooligosaccharides (XOS) using combinations of thermochemical pretreatments and enzymatic hydrolyses, that have broad applications in the food, feed, pharmaceutical and cosmetic industries. Endo-xylanases from different complex carbohydrate-active enzyme (CAZyme) families can be used to cleave the backbone of arabino(glucurono)xylans and xylooligosaccharides and degrade them into short XOS. It has been shown that XOS with a low degree of polymerization have enhanced prebiotic effects conferring health benefits to humans and animals. In this review we describe recent advances in the enzymatic production of XOS from lignocellulosic biomass arabino- and glucuronoxylans and their applications as food and feed additives and health-promoting ingredients. Comparative advantages of xylanases from different CAZy families in XOS production are discussed and potential health benefits of different XOS are presented.
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Background Xylanases are one of the most extensively used enzymes for biomass digestion. However, in many instances, their use is limited by poor performance under the conditions of pH and temperature required by the industry. Therefore, the search for xylanases able to function efficiently at alkaline pH and high temperature is an important objective for different processes that use lignocellulosic substrates, such as the production of paper pulp and biofuels. Results A comprehensive in silico analysis of family GH11 sequences from the CAZY database allowed their phylogenetic classification in a radial cladogram in which sequences of known or presumptive thermophilic and alkalophilic xylanases appeared in three clusters. Eight sequences from these clusters were selected for experimental analysis. The coding DNA was synthesized, cloned and the enzymes were produced in E. coli. Some of these showed high xylanolytic activity at pH values > 8.0 and temperature > 80 °C. The best enzymes corresponding to sequences from Dictyoglomus thermophilum (Xyn5) and Thermobifida fusca (Xyn8). The addition of a carbohydrate-binding module (CBM9) to Xyn5 increased 4 times its activity at 90 °C and pH > 9.0. The combination of Xyn5 and Xyn8 was proved to be efficient for the saccharification of alkali pretreated rice straw, yielding xylose and xylooligosaccharides. Conclusions This study provides a fruitful approach for the selection of enzymes with suitable properties from the information contained in extensive databases. We have characterized two xylanases able to hydrolyze xylan with high efficiency at pH > 8.0 and temperature > 80 °C.
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Several organisms, specifically microorganisms survive in a wide range of harsh environments including extreme temperature, pH, and salt concentration. We analyzed systematically a large number of protein sequences with their structures to understand their stability and to discriminate extremophilic proteins from their non-extremophilic orthologs. Our results highlighted that the strategy for the packing of the protein core was influenced by the environmental stresses through substitutive structural events through better ionic interaction. Statistical analysis showed that a significant difference in number and composition of amino acid exist among them. The negative correlation of pairwise sequence alignments and structural alignments indicated that most of the extremophile and non-extremophile proteins didn’t contain any association for maintaining their functional stability. A significant numbers of salt bridges were noticed on the surface of the extremostable proteins. The Ramachandran plot data represented more occurrences of amino acids being present in helix and sheet regions of extremostable proteins. We also found that a significant number of small nonpolar amino acids and moderate number of charged amino acids like Arginine and Aspartic acid represented more nonplanar Omega angles in their peptide bond. Thus, extreme conditions may predispose amino acid composition including geometric variability for molecular adaptation of extremostable proteins against atmospheric variations and associated changes under natural selection pressure. The variation of amino acid composition and structural diversifications in proteins play a major role in evolutionary adaptation to mitigate climate change.
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Background: Regarding plant cell wall polysaccharides degradation, multimodular glycoside hydrolases (GHs) with two catalytic domains separated by one or multiple carbohydrate-binding domains are rare in nature. This special mode of domain organization endows the Caldicellulosiruptor bescii CelA (GH9-CBM3c-CBM3b-CBM3b-GH48) remarkably high efficiency in hydrolyzing cellulose. CbXyn10C/Cel48B from the same bacterium is also such an enzyme which has, however, evolved to target both xylan and cellulose. Intriguingly, the GH10 endoxylanase and GH48 cellobiohydrolase domains are both dual functional, raising the question if they can act synergistically in hydrolyzing cellulose and xylan, the two major components of plant cell wall. Results: In this study, we discovered that CbXyn10C and CbCel48B, which stood for the N- and C-terminal catalytic domains, respectively, cooperatively released much more cellobiose and cellotriose from cellulose. In addition, they displayed intramolecular synergy but only at the early stage of xylan hydrolysis by generating higher amounts of xylooligosaccharides including xylotriose, xylotetraose, and xylobiose. When complex lignocellulose corn straw was used as the substrate, the synergy was found only for cellulose but not xylan hydrolysis. Conclusion: This is the first report to reveal the synergy between a GH10 and a GH48 domain. The synergy discovered in this study is helpful for understanding how C. bescii captures energy from these recalcitrant plant cell wall polysaccharides. The insight also sheds light on designing robust and multi-functional enzymes for plant cell wall polysaccharides degradation.
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Background Cellulose and hemicellulose are the two largest components in lignocellulosic biomass. Enzymes with activities towards cellulose and xylan have attracted great interest in the bioconversion of lignocellulosic biomass, since they have potential in improving the hydrolytic performance and reducing the enzyme costs. Exploring glycoside hydrolases (GHs) with good thermostability and activities on xylan and cellulose would be beneficial to the industrial production of biofuels and bio-based chemicals. Results A novel GH10 enzyme (XynA) identified from a xylanolytic strain Bacillus sp. KW1 was cloned and expressed. Its optimal pH and temperature were determined to be pH 6.0 and 65 °C. Stability analyses revealed that XynA was stable over a broad pH range (pH 6.0–11.0) after being incubated at 25 °C for 24 h. Moreover, XynA retained over 95% activity after heat treatment at 60 °C for 60 h, and its half-lives at 65 °C and 70 °C were about 12 h and 1.5 h, respectively. More importantly, in terms of substrate specificity, XynA exhibits hydrolytic activities towards xylans, microcrystalline cellulose (filter paper and Avicel), carboxymethyl cellulose (CMC), cellobiose, p-nitrophenyl-β-d-cellobioside (pNPC), and p-nitrophenyl-β-d-glucopyranoside (pNPG). Furthermore, the addition of XynA into commercial cellulase in the hydrolysis of pretreated corn stover resulted in remarkable increases (the relative increases may up to 90%) in the release of reducing sugars. Finally, it is worth mentioning that XynA only shows high amino acid sequence identity (88%) with rXynAHJ14, a GH10 xylanase with no activity on CMC. The similarities with other characterized GH10 enzymes, including xylanases and bifunctional xylanase/cellulase enzymes, are no more than 30%. Conclusions XynA is a novel thermostable GH10 xylanase with a wide substrate spectrum. It displays good stability in a broad range of pH and high temperatures, and exhibits activities towards xylans and a wide variety of cellulosic substrates, which are not found in other GH10 enzymes. The enzyme also has high capacity in saccharification of pretreated corn stover. These characteristics make XynA a good candidate not only for assisting cellulase in lignocellulosic biomass hydrolysis, but also for the research on structure–function relationship of bifunctional xylanase/cellulase.
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The last few years have witnessed significant changes in Pfam ( The number of families has grown substantially to a total of 17,929 in release 32.0. New additions have been coupled with efforts to improve existing families, including refinement of domain boundaries, their classification into Pfam clans, as well as their functional annotation. We recently began to collaborate with the RepeatsDB resource to improve the definition of tandem repeat families within Pfam. We carried out a significant comparison to the structural classification database, namely the Evolutionary Classification of Protein Domains (ECOD) that led to the creation of 825 new families based on their set of uncharacterized families (EUFs). Furthermore, we also connected Pfam entries to the Sequence Ontology (SO) through mapping of the Pfam type definitions to SO terms. Since Pfam has many community contributors, we recently enabled the linking between authorship of all Pfam entries with the corresponding authors' ORCID identifiers. This effectively permits authors to claim credit for their Pfam curation and link them to their ORCID record.
Xylooligosaccharides (XOS), produced from lignocellulosic biomass (LCB), are short-chain polymers with prebiotic activity which, in the last few decades, have gained commercial interest due to their potential application as ingredients for the nutraceutical industry. This article reviews relevant topics to consider when researching XOS productive processes, such as the selection of raw materials and strategies for XOS production, purification, characterisation, quantification and evaluation of the prebiotic effects. With regard to the production approach, this article focuses on LCB pre-treatments and the enzymatic hydrolysis of xylan, exploring the reported alternatives and enzymes. A critical view on the current process reveals that comparative analysis between different studies is difficult due to the lack of consensus on the criteria and parameters used in the evaluation of XOS production processes. However, the most generally recommended XOS production strategy is the two-stage approach through alkaline pre-treatment and enzymatic hydrolysis with further purification through membrane filtration.
The concurrence of microorganisms in niches that are hostile like extremes of temperature, pH, salt concentration and high pressure depends upon novel molecular mechanisms to enhance the stability of their proteins, nucleic acids, lipids and cell membranes. The structural, physiological and genomic features of extremophiles that make them capable of withstanding extremely selective environmental conditions are particularly fascinating. Highly stable enzymes exhibiting several industrial and biotechnological properties are being isolated and purified from these extremophiles. Successful gene cloning of the purified extremozymes in the mesophilic hosts has already been done. Various extremozymes such as amylase, lipase, xylanase, cellulase and protease from thermophiles, halothermophiles and psychrophiles are of industrial interests due to their enhanced stability at forbidding conditions. In this review, we made an attempt to point out the unique features of extremophiles particularly thermophiles and psychrophiles, at the structural, genomic and proteomic levels, which allow for functionality at harsh conditions focusing on the temperature tolerance by them.
The updated definition of prebiotic expands the range of potential applications in which emerging xylooligosaccharides (XOS) can be used. It has been demonstrated that XOS exhibit prebiotic effects at lower amounts compared to others, making them competitively priced prebiotics. As a result, the industry is focused on developing alternative approaches to improve processes efficiency that can meet the increasing demand while reducing costs. Recent advances have been made towards greener and more efficient processes, by applying process integration strategies to produce XOS from costless lignocellulosic residues and using genetic engineering to create microorganisms that convert these residues to XOS. In addition, collecting more in vivo data on their performance will be key to achieve regulatory claims, greatly increasing XOS commercial value. Copyright © 2019 Elsevier Inc. All rights reserved.
Currently, hyperstable endo-1,4-β-xylanase has been the focus of attention as potent biocatalyst as well as utilization in bioconversion process. Therefore, the gene (1,035 bp) of a monomeric glycoside hydrolase family 10 (GH10) endo-1,4-β-xylanase (TnXynB) from a hyperthermophilic eubacterium Thermotoga naphthophila RKU-10T was cloned and overexpressed in a mesophilic host system. The extracellular TnXynB was purified to homogeneity with a molecular mass of 40 kDa, and showed peak activity at pH 6.0 and 95°C temperature. Purified TnXynB has prodigious stability over a broad range of pH (5.5-8.0) and temperature (50-85°C) even after 8 h incubation, and also revealed great tolerance toward different modulators (metal cations, surfactants and organic solvents). TnXynB exhibited great affinity towards various heteroglycans and para-nitrophenyl glycosides substrates. The values of Km, Vmax, kcat, and kcat Km-1 were 2.75 mg mL-1, 3146.7 µmol mg-1 min-1, 40342.3 s-1, 14669.93 mL mg-1 s-1, respectively using birchwood xylan as substrate. Thermodynamic parameters for birchwood xylan hydrolysis at 95°C as ∆S*, ∆H*, and ∆G* were -22.88 J mol-1 K-1, 62.44 kJ mol-1, and 70.86 kJ mol-1 respectively. TnXynB displayed a half-life (t1/2) of 54.15 min at 96°C with ΔS*D, ΔH*D, and ΔG*D values of 1.074 kJ mol-1K-1, 513.23 kJ mol-1 and 116.92 kJ mol-1, respectively. All noteworthy features of TnXynB make this new recombinant enzyme an appropriate candidate for the biodegradation of lignocellulosic substrates as well as various other industrial bioprocesses.
Lectins are a widespread group of sugar-binding proteins occurring in all types of organisms including animals, plants, bacteria, fungi and even viruses. According to a recent report, there are more than 50 lectin scaffolds (∼Pfam), for which three-dimensional structures are known and sugar-binding functions have been confirmed in the literature, which far exceeds our view in the twentieth century (Fujimoto et al. 2014 Methods Mol. Biol.1200, 579-606 (doi:10.1007/978-1-4939-1292-6_46)). This fact suggests that new lectins will be discovered either by a conventional screening approach or just by chance. It is also expected that new lectin domains including those found in enzymes as carbohydrate-binding modules will be generated in the future through evolution, although this has never been attempted on an experimental level. Based on the current state of the art, various methods of lectin engineering are available, by which lectin specificity and/or stability of a known lectin scaffold can be improved. However, the above observation implies that any protein scaffold, including those that have never been described as lectins, may be modified to acquire a sugar-binding function. In this review, possible approaches to confer sugar-binding properties on synthetic proteins and peptides are described.