Biochemical characterization and low-resolution SAXS structure of two-domain endoglucanase BlCel9 from Bacillus licheniformis

Article abstract of DOI:10.1007/s00253-018-9508-1

Lignocellulose feedstock constitutes the most abundant carbon source in the biosphere; however, its recalcitrance remains a challenge for microbial conversion into biofuel and bioproducts. Bacillus licheniformis is a microbial mesophilic bacterium capable of secreting a large number of glycoside hydrolase (GH) enzymes, including a glycoside hydrolase from GH family 9 (BlCel9). Here, we conducted biochemical and biophysical studies of recombinant BlCel9, and its low-resolution molecular shape was retrieved from small angle X-ray scattering (SAXS) data. BlCel9 is an endoglucanase exhibiting maximum catalytic efficiency at pH?7.0 and 60?°C. Furthermore, it retains 80% of catalytic activity within a broad range of pH values (5.5–8.5) and temperatures (up to 50?°C) for extended periods of time (over 48?h). It exhibits the highest hydrolytic activity against phosphoric acid swollen cellulose (PASC), followed by bacterial cellulose (BC), filter paper (FP), and to a lesser extent carboxymethylcellulose (CMC). The HPAEC-PAD analysis of the hydrolytic products demonstrated that the end product of the enzymatic hydrolysis is primarily cellobiose, and also small amounts of glucose, cellotriose, and cellotetraose are produced. SAXS data analysis revealed that the enzyme adopts a monomeric state in solution and has a molecular mass of 65.8?kDa as estimated from SAXS data. The BlCel9 has an elongated shape composed of an N-terminal family 3 carbohydrate-binding module (CBM3c) and a C-terminal GH9 catalytic domain joined together by 20 amino acid residue long linker peptides. The domains are closely juxtaposed in an extended conformation and form a relatively rigid structure in solution, indicating that the interactions between the CBM3c and GH9 catalytic domains might play a key role in cooperative cellulose biomass recognition and hydrolysis.

Full text of DOI:10.1007/s00253-018-9508-1

Applied Microbiology and Biotechnology
https://doi.org/10.1007/s00253-018-9508-1
BIOTECHNOLOGICALLY RELEVANT ENZYMES AND PROTEINS
Biochemical characterization and low-resolution SAXS structure
of two-domain endoglucanase BlCel9 from Bacillus licheniformis
Evandro Ares de Araújo1 & Mário de Oliveira Neto2 & Igor Polikarpov1
Received: 14 July 2018 /Revised: 3 October 2018 /Accepted: 7 November 2018
#
Springer-Verlag GmbH Germany, part of Springer Nature 2018
Abstract
Lignocellulose feedstock constitutes the most abundant carbon source in the biosphere; however, its recalcitrance remains a
challenge for microbial conversion into biofuel and bioproducts. Bacillus licheniformis is a microbial mesophilic bacterium
capable of secreting a large number of glycoside hydrolase (GH) enzymes, including a glycoside hydrolase from GH family 9
(BlCel9). Here, we conducted biochemical and biophysical studies of recombinant BlCel9, and its low-resolution molecular
shape was retrieved from small angle X-ray scattering (SAXS) data. BlCel9 is an endoglucanase exhibiting maximum catalytic
efficiency at pH 7.0 and 60 °C. Furthermore, it retains 80% of catalytic activity within a broad range of pH values (5.5–8.5) and
temperatures (up to 50 °C) for extended periods of time (over 48 h). It exhibits the highest hydrolytic activity against phosphoric
acid swollen cellulose (PASC), followed by bacterial cellulose (BC), filter paper (FP), and to a lesser extent carboxymethylcel-
lulose (CMC). The HPAEC-PAD analysis of the hydrolytic products demonstrated that the end product of the enzymatic
hydrolysis is primarily cellobiose, and also small amounts of glucose, cellotriose, and cellotetraose are produced. SAXS data
analysis revealed that the enzyme adopts a monomeric state in solution and has a molecular mass of 65.8 kDa as estimated from
SAXS data. The BlCel9 has an elongated shape composed of an N-terminal family 3 carbohydrate-binding module (CBM3c) and
a C-terminal GH9 catalytic domain joined together by 20 amino acid residue long linker peptides. The domains are closely
juxtaposed in an extended conformation and form a relatively rigid structure in solution, indicating that the interactions between
the CBM3c and GH9 catalytic domains might play a key role in cooperative cellulose biomass recognition and hydrolysis.
.
.
.
Keywords GH9family Family3carbohydrate-bindingmodule(CBM3c) Endoglucanase SmallangleX-rayscattering(SAXS)
Introduction
75% of a dry plant biomass (Marriott et al. 2016). Cellulose
is the main polysaccharide of the plant cell walls (Cosgrove
and Jarvis 2012; Marriott et al. 2016) formed by a linear
poly-β(1,4)-D-glucopyranose chains (Somerville et al. 2004;
Gilbert 2010; Cosgrove and Jarvis 2012; Ding et al. 2012).
The high crystallinity and its intrinsic recalcitrance make this
biopolymer insoluble in most solvents, hindering the process-
ing of vegetable biomass (Lynd et al. 2002; Gilbert 2010;
Igarashi 2013). However, pretreated lignocellulosic biomass
can be biochemically converted into soluble sugars using en-
zymatic technology, which has become the preferable biotech-
nological route to upgrade plant biomass into biofuels, green
chemicals, and sustainable materials (Himmel et al. 2007;
Soccol et al. 2010; Klein-Marcuschamer et al. 2012; Gilbert
2014; Donohoe and Resch 2015; Kafle et al. 2015; Kumar and
Sharma 2017).
Plant biomass constitutes the most abundant, natural, renew-
able, and biodegradable carbon source in the biosphere. Plant
cell walls form a complex matrix composed mainly of cellu-
lose, hemicellulose, and lignin (Keegstra 2010; Cosgrove
2014; Marriott et al. 2016) which comprise approximately
Electronic supplementary material The online version of this article
(https://doi.org/10.1007/s00253-018-9508-1) contains supplementary
material, which is available to authorized users.
* Igor Polikarpov
ipolikarpov@ifsc.usp.br
1
Instituto de Física de São Carlos, Universidade de São Paulo, Av.
Trabalhador Saocarlense 400, São Carlos, SP 13560-970, Brazil
Lignocellulosic biomass enzymatic hydrolysis is car-
ried out by a diversity of enzymes that differ structurally
and mechanistically (Henrissat and Davies 1997) and
2
Departmento de Física e Biofísica, Universidade Estadual Paulista
BJúlio de Mesquita Filho^, R. Prof. Dr. Antonio Celso Wagner Zanin
689, Jardim Sao Jose, Botucatu, SP 18618-970, Brazil

Appl Microbiol Biotechnol
which are cataloged in the CAZy (Carbohydrate-Active
EnZymes) database (Cantarel et al. 2009; Lombard et al.
2014). Enzymatic hydrolysis of cellulose involves a range
of cellulases, including members of glycoside hydrolase
families (GHs) 5, 6, 7, 8, 9, 12, 44, 45, and 48 (Lynd et al.
2002; Wilson 2011; Lombard et al. 2014; Berlemont and
Martiny 2015; Talamantes et al. 2016).
According to the CAZy database (http://www.cazy.org/
GH9), GH9 contains over 2800 known enzymes, of which
approximately 55% are from bacterial organisms. In fact, the
ability to secrete at least two GH9 enzymes is shared by most
of the cellulolytic bacteria in the CaZy database, including
Mucilaginibacter, Pedobacter, Luteibacter, Clostridium
cellulolyticum, Ruminiclostridium thermocellum, and
Caldicellulosiruptor bescii (Gilad et al. 2003; Yi et al. 2013;
Ravachol et al. 2014; López-Mondéjar et al. 2016; Dassa et al.
2017); however, Bacillus licheniformis, unlike most cellulo-
lytic bacteria, has only one GH9 enzyme. B. licheniformis is a
facultative anaerobe bacteria widely used in biotechnological
applications (Rey et al. 2004; Schallmey et al. 2004) for pro-
duction of chemicals (Erickson 1976; Ghani et al. 2013;
Elshaghabee et al. 2017) and important enzymes to biofuel
industry (Liberato et al. 2016; Evangelista et al. 2017).
Here, BlCe9 from B. licheniformis was cloned and
expressed in Escherichia coli. The recombinant enzyme was
purified, and its biochemical properties, including optimal pH
and temperature, thermostability, substrate specificity, and the
pattern of released products, were investigated. Furthermore,
we also determined the SAXS-derived low-resolution molec-
ular envelope of BlCe9 in solution.
GH9 enzymes are an important part of the cellulolytic sys-
tems of many bacteria involved in the decomposition of plant
cell walls (Liu et al. 2004; Davison and Blaxter 2005; El
Kaoutari et al. 2013; Brunecky et al. 2013; Artzi et al. 2016;
Talamantes et al. 2016). These bacterial organisms display a
variety of strategies to secrete these enzymes, sometimes using
free enzyme systems, sometimes presenting the catalytic do-
mains (CDs) associated with non-catalytic accessory domains
(e.g., in cellulosomes), or even as a part of large protein chains
with multiple GH domains (Wilson 2011; Brunecky et al.
2013; Artzi et al. 2016; Yarbrough et al. 2017; Brunecky
et al. 2017). These enzymes exhibit distinct multi-modular ar-
chitectures that, in part, underlie their diversity in substrate
hydrolysis (Urbanowicz et al. 2007a, b; Burstein et al. 2009;
Dassa et al. 2017). A particular two-domain arrangement is
composed of a GH9 CD covalently linked to an N-terminal
carbohydrate-binding domain 3c, CBM3c (Sakon et al.
1997). Although the contribution of CBM3c to the catalytic
activity of the GH9 members remains only partially clear
(Irwin et al. 1998; Burstein et al. 2009; Petkun et al. 2015), this
domain seems to be essential for processivity of the enzyme
during the cellulose hydrolysis (Sakon et al. 1997; Gilad et al.
2003; Li et al. 2007; Oliveira et al. 2009; Batista et al. 2011).
GH9 has known catalytic activities against a large number
of polysaccharides, including crystalline cellulose (Gilad
et al. 2003; Liu et al. 2004; Chen et al. 2007), PASC (Gilad
et al. 2003; Telke et al. 2012), CMC (Master et al. 2004; Liu
et al. 2004), xylan (Urbanowicz et al. 2007a, b),
(1,3)-(1,4)-β-D-glucan (Urbanowicz et al. 2007a, b), and
xyloglucan (Ravachol et al. 2014; Ravachol et al. 2016).
Moreover, some of the GH9 enzymes were shown to signif-
icantly enhance the catalytic activity levels of cellulase mix-
tures on cellulose and other polysaccharides (Vazana et al.
2010; Brunecky et al. 2013; Bhattacharya et al. 2015;
Brunecky et al. 2017), and in some cases outperforming cel-
lulolytic fungi enzyme mixtures in cellulose hydrolysis (Gal
et al. 1997; Brunecky et al. 2013; Brunecky et al. 2017).
Noteworthy, some of the enzymes, such as BlCel9A from
Bacillus licheniformis (Liu et al. 2004), TfCel9A from
Thermobifida fusca (Kostylev and Wilson 2014), CbCel9A
from Caldicellulosiruptor bescii (Yi et al. 2013), and
CcCel9B from Clostridium cellulosi (Yang et al. 2016), are
capable of synergistically enhancing the activities of the
GH48enzymesduringthehydrolysisofinsolublecrystalline
cellulose(Liuet al. 2004; Brunecky et al. 2013;Kostylevand
Wilson 2014; Yang et al. 2016; Brunecky et al. 2017).
Materials and methods
Gene cloning, overexpression, and purification
The BlCel9 gene cloning (GenBank access number
AAU40775 and Uniprot access number Q65JI9) was per-
formed from the genomic DNA (gDNA) of Bacillus
licheniformis (ATCC 14580) using PCR primers containing
the Ligase Independent Cloning (LIC) site region: 5′-CAGG
GCGCCATGATGTCAGATTACAACTATGTAGAGGTGC-
3′ (forward) and 5′-GACCCGACGCGGTTATTATT
TATTCGGAAGCGTACCAA-3′ (reverse). Predicted signal
peptide sequence (amino acid residue 1 to 33) was omitted.
The PCR fragment was inserted into pETTrx-1a/LIC vector
using the previously described method (Camilo and
Polikarpov 2014). This plasmid with the target gene was
transformed and overexpressed using E. coli BL21
Rosetta (DE3). The positive transformants were confirmed
by DNA sequencing using a 3500 Genetic Analyzer (Life
Technologies: California, USA).
The pETTrx-1a/LIC-BlCel9 was cultivated in Luria-
Bertani liquid medium (LB) with 50 μg/mL kanamycin,
34 μg/mL chloramphenicol, at 37 °C and 150 rpm agitation.
When the culture OD600nm reached 0.6, BlCel9 expression
was induced by adding 1 mM of isopropyl-β-D-
thiogalactoside (IPTG); this expression was performed for
20 h at 20 °C. The cells were harvested by centrifugation at
6000×g for 30 min, afterward resuspended in 20 mM Tris-

Appl Microbiol Biotechnol
HCl pH 7.0 containing 300 mM NaCl, disrupted by sonica-
tion, and centrifuged again at 17,000×g for 30 min at 4 °C.
The supernatant was loaded onto a Ni-NTA agarose resin (GE
Healthcare; Massachusetts, USA), previously equilibrated
with the same buffer following the steps: the resin was washed
with ten column volumes (CV) of 20 mM Tris-HCl pH 7.0,
300 mM NaCl, 10 mM imidazole, and the protein eluted with
2CVof 20 mM Tris-HCl pH 7.0, 300 mM NaCl, 300 mM of
imidazole. After extensive dialysis in 20 mM Tris-HCl
pH 7.0, 300 mM NaCl buffer, the pETTrx-1a/LIC-BlCel9
was incubated in the presence of TEV protease (Parks et al.
1994) for 20 h to cleave the His-Trx tag and then re-loaded
onto buffer-equilibrated Ni-NTA agarose resin.
stability was assessed by incubation of the purified enzyme
at temperatures of 40, 50, and 60 °C for 0, 1, 3, 6, 12, 24, 48 h,
and subsequent measurement of its residual activities.
Thereafter, the enzymatic activity was assessed using several
ions and chemical compounds adding them to the reaction at
the concentration of 5 mM. These assays were performed after
the pre-incubation of BlCel9 (at 3.0 μM in 50 mM citrate-
phosphate, pH 7.0) with PASC (at 10 mg/mL) at 50 °C for
1 h. Finally, the enzyme specific activity was measured using
enzyme mass of 0.01 mg and substrates at 10 mg/mL (w/v) for
15 min at 50 °C. The glucose-equivalent yield was measured
according to the 3,5-dinitrosalicylic acid (DNS) method
(Ghose 1987).
The resulting cleaved BlCel9 was further purified by size
exclusion chromatography (SEC) using a Superdex 75 col-
umn (GE Healthcare; Massachusetts, USA), pre-equilibrated
with 20 mM Tris-HCl pH 7.0, 200 mM NaCl. The protein
concentration was determined by extinction coefficient ( =
185,655 M−1 cm−1) at 280 nm using a Nanodrop 1000 spec-
trophotometer (Thermo Scientific; Massachusetts, USA) and
its purity monitored by SDS-PAGE 12% (Laemmli 1970).
Enzymatic cleavage pattern
The soluble oligosaccharides derived from the enzymatic hy-
drolysis of PASC were evaluated using HPAEC-PAD. The
assays were performed by pre-incubating the enzyme
(20 nM) with PASC (2.5 mg/mL) at 50 °C and under an
agitation speed of 200 rpm. Aliquots of 200 μL were taken
after 0, 1, and 48 h, boiled at 95 °C for 5 min and centrifuged
at 17,000×g at 4 °C for 10 min. The soluble sugars were
analyzed in an ICS-5000 HPAEC system (Dionex; CA,
USA) equipped with a pulsed amperometric detector (PAD).
The samples were applied onto the CarboPAC1 analytical
column 250 mm × 2 mm (Dionex; CA, USA) following the
previously established method (de Araújo et al. 2017). As a
control, soluble cello-oligosaccharides (C1 to C6) were used.
Thermal stability analysis
The effects of pH on structural stability of BlCel9 were per-
formed using differential scanning fluorimetry (Thermofluor).
Each measurement was carried out using 10 μM of protein at
different pH values in 50 mM citrate-phosphate-glycine buffer
system covering pH range from 2.0 to 10.0 using SYPRO
Orange (Invitrogen; California, USA) as a fluorescent dye at
final concentration of 1/2.000. The assays were conducted in
an iCycler iQ Real-Time PCR Detection System (Bio-Rad;
California, USA) with programmed temperature set-up rang-
ing from 25 to 90 °C and stepwise increments of 1 °C per
minute separated by 10-s hold step. Fluorescence intensity
was monitored with excitation/emission wavelengths of 490/
530 nm, respectively.
Chromatography-coupled small-angle X-ray
scattering data and structural analyses
Small-angle X-ray scattering (SAXS) measurements coupled
to the size-exclusion chromatographic system were conducted
at the B21 beamline on Diamond Light Source Synchrotron
(Didcot, Oxfordshire, UK). The protein sample was centri-
fuged at 17,000×g for 2 h at 4 °C, then loaded onto a
4.8-mL KW-403 SEC column (Shodex; New York, USA)
equilibrated with a buffer solution (50 mM Tris-HCl pH 7.0,
150 mM NaCl, 5% w/v glycerol) coupled to an Agilent 1200
HPLC system with flow rate of 0.16 mL min−1 at a controlled
temperature of 25 °C followed by the X-ray scattering data
collection in in-line mode.
X-ray scattering data were measured using Pilatus 2 M
detector (Dectris; Baden-Daettwil, Switzerland) set to cover
momentum transfer within the range: 4.014E-03 Å−1 < q <
4.07E-01 Å−1 (q = 4πsinθ/λ, where 2θ is the scattering angle).
The SAXS data frames were collected with exposure intervals
of 3 s, and the buffer frames used for the background-
subtracted SAXS were collected after 1.5 column volume.
The scattering image frames were spherically averaged,
Enzymatic assays
Evaluation of the enzymatic activity was conducted using
several substrates: carboxymethylcellulose (CMC), Avicel
PH-101, Whatman® qualitative filter paper-grade 1 (FP),
and xylan (all from Sigma-Aldrich; St. Louis, USA); phos-
phoric acid swollen cellulose (PASC), and bacterial cellulose
(BC). PASC and BC were prepared as described previously
(Zhang et al. 2006). Reaction mixtures containing 3.0 μM of
BlCel9 and 10 mg/mL (w/v) of each substrate were incubated
at 50 °C for 3 h in 50 mM citrate-phosphate buffer at pH 7.0.
The optimal pH was evaluated at 50 °C in a 50 mM citrate-
phosphate-glycine buffer system with pH ranging from 3.0 to
10.0, and the temperature-activity evaluation was carried out
at temperatures ranging from 30 to 70 °C; and the thermal

Appl Microbiol Biotechnol
scaled, and merged using in-house software and ScÅtter pro-
gram version 3.0 (http://www.bioisis.net/tutorial/9).
the data set, resulting in 25 complete sequences used to
construct the phylogenetic tree. The enzymes are sepa-
rated into two distinct clusters (branch 1 and branch 2,
Fig. S1). The branch 1, which contains the enzymes
most distantly related to BlCel9, contains ten enzymes,
including the cellobiohydrolase CtCbhA (Uniprot num-
ber A3DCH2) from Clostridium thermocellum (Kataeva
et al. 2005) and the endoglucanase AaCel9A (Uniprot
number C8WSJ6) from Alicyclobacillus acidocaldarius.
Both of these enzymes display a modular architecture
composed of accessory domains connected to a catalytic
domain. CtCbhA and AaCel9A show sequence identity
of 22% and 26%, respectively, with BlCel9. The branch
2 of the phylogenetic tree contains 15 enzymes, includ-
ing BlCel9. This branch englobes enzymes from
mesophilic as well as thermophilic microorganisms,
such as Clostridia and Myxobacter species, including
the CaCel9 (Uniprot number Q97KK3) from
Clostridium acetobutylicum (Lopez-Contreras et al.
2003), the CcCel9R (Uniprot number B8I2K6) from
Clostridium cellulolyticum (Badalato et al. 2017), the
TfCel9A (Uniprot number P26221) from T. fusca
(Sakon et al. 1997), and the MspCel9 (Uniprot number
Q9RAI8) from Myxobacter sp. AL-1 (Ramírez-Ramírez
et al. 2008). The endoglucanase CcCel9R (Uniprot num-
ber B8I2K6) from Clostridium cellulolyticum (Badalato
et al. 2017) has 34% of sequence identity with BlCel9.
This enzyme exhibits a modular architecture composed
of an N-terminal catalytic domain from GH9 followed
by two carbohydrate-binding module family 3 (CBM3)
and a dockerin module at its C-terminal end. Similarly,
the full-length TfCel9A is a processive endoglucanase
containing four distinct domains (sequence identity of
54% as compared to BlCel9) while the MspCel9 (se-
quence identity of 81% with BlCel9) has a module ar-
chitecture composed of an N-terminal CD from GH9
followed by C-terminal CBM3.
The radius of gyration (Rg) of the enzyme was estimated
through the Guinier approximation—I(q) = I(0).exp[(−q2.Rg )/
2
3], q.Rg < 1.3 (Guinier 1939)—and by using ScÅtter; the same
was done with the pair distribution of the particle, P(r), and the
maximum dimension Dmax, which also were computed using
ScÅtter. Nine independent Bdummy atom^ models (DAMs)
were obtained using DAMMIF (Franke and Svergun 2009)
and the DAMSTART model was improved with the software
DAMMIN (Svergun 1999). Alternatively, the reconstruction of
nine independent Bdummy residues^ models by the chain-like
ensemble of dummy residues was conducted using GASBOR
(Svergun et al. 2001). The models generated in GASBOR or
DAMMIN were averaged using DAMAVER (Volkov and
Svergun 2003) and the best model was superimposed with
the crystallographic structure of TfCel9A (PDB id 1JS4;
(Sakon et al. 1997)) using the SUPCOMB software (Kozin
and Svergun 2001). Based on the reported crystallographic
structures, the X-ray scattering profiles were computed and
the discrepancies between experimental and theoretical SAXS
curves were quantified using the minimized χ-parameter using
FoXS web-server (Schneidman-Duhovny et al. 2010). Finally,
SAXSMoW 2.0 web-server was used to evaluate the molecular
weight of BlCel9 in solution (http://saxs.ifsc.usp.br/; (Fischer
et al. 2010; Piiadov et al. 2018)).
Results
Gene cloning and recombinant protein production
The ORF of gene AAU40775.1 from Bacillus
licheniformis encodes a protein with 654 amino acid res-
idues, including a secretion signal peptide composed of
the first 33 amino acid residues, as based on the predic-
tion by SignalP v4.0 (Petersen et al. 2011). The full-
length BlCel9 enzyme is composed of GH9 CD (residues
34 to 496) connected to CBM3c module (residues 496 to
654) by a Thr/Ser-rich linker. The cloned enzyme com-
prised amino acid residues 34 to 654 with predicted mo-
lecular weight of 69.9 kDa; after purification, the obtain-
ed protein shows a single band with an apparent molec-
ular mass of approximately 70 kDa (Fig. S1).
These enzymes are involved in the catalytic hydrolysis of a
wide range of polysaccharides, acting as cellulases, lichenases,
laminarinases, β-glucosidases, and xyloglucanases (http://
www.cazy.org/GH9.html).
BlCel9 activity was tested against a range of insoluble cel-
lulosic substrates, including PASC, FP, Avicel PH-101, and
BC. The catalytic activity of BlCel9 was highest against
PASC, followed by BC, FP, CMC and quite low against
Avicel (Fig. 1a). The BlCel9-specific activities against these
substrates were 2.01 μmol/min/mg, 1.73 μmol/min/mg,
0.76 μmol/min/mg, 0.42 μmol/min/mg, 0.30 μmol/min/mg,
and 0.04 μmol/min/mg for PASC, BC, FP, Sigmacell CMC,
and Avicel, respectively. The GH9 enzymes showed a large
range of specific activities against cellulosic substrates, such
as PASC varying considerably from 0 to 736 μmol/min/mg
(Ravachol et al. 2014). For example, CtCel9 (CelK; Kataeva
Phylogenetic analysis, enzyme characterization,
substrate specificity, and effects of chemicals
on the enzymatic activity
The amino acid sequences of previously characterized
GH9 enzymes were compared to BlCel9, and a phylo-
genetic tree was assembled (Fig. S2). Entries with only
partial or highly identical sequences were removed from

Appl Microbiol Biotechnol
Fig. 1 Biochemical characterizations and substrate specificity. a The
substrate specificity of BlCel9 was tested against several cellulosic
substrates, including microcrystalline cellulose (Avicel PH-101),
carboxymethylcellulose (CMC); Sigmacell cellulose type 20
(Sigmacell), Whatman grade 1 filter paper (Filter paper), bacterial cellu-
lose (BC), and phosphoric acid-swollen cellulose (PASC). Furthermore,
the influences of pH (b) and temperature (c) on the enzymatic activity
were evaluated as well as the effects of metal ions and chemical agents (d)
et al. 1999) and CtCel9 (CbhA; Schubot et al. 2004), from
Clostridium thermocellum, have specific activities of
2.0 μmol/min/mg and 1.7 μmol/min/mg, respectively, while
the TfCel9A from Thermobifida fusca exhibits specific activ-
ity of 0.018 μmol/min/mg (Rudsander et al. 2008).
Furthermore, the effects of pH and temperature on the en-
zymatic activity of BlCel9 were evaluated. The dependence of
enzymatic activity on pH was studied using PASC as a sub-
strate at 50 °C, covering pH range from 3.0 to 10.0. BlCel9
exhibited activity levels of more than 80% within a broad pH
range (from 6.0 to 8.5), with optimal pH at 7.0 (Fig. 1b).
However, the enzymatic activity decreased to less than 60%
at pH values lower than 4.0 or higher than 8.5.
Temperature dependence of BlCel9 activity was measured
between 30 and 70 °C with the highest activity exhibited at
60 °C (Fig. 1c). At 40 and 50 °C, BlCel9 retained more than
80% of the highest activity levels, whereas at 70 and 30 °C,
the enzymatic activity decreased to less than 60% (Fig. 1c).
To understand the impact of diverse chemical com-
pounds on the activity of BlCel9, the influence of sev-
eral metallic ions and chemical agents was investigated
(Fig. 1d). For most chemicals and additives tested, the
improvement of BlCel9 enzymatic activity was not sig-
nificant. Moreover, in the presence of DMSO, CuCl2,
and FeCl2, the catalytic activity of BlCel9 was strongly
inhibited.
To further analyze the structural stability of BlCel9 in
different pH values, we applied Thermoflour technique.
The analysis of thermal transition curves revealed melt-
ing temperatures (Tm) above 40 °C for pH values rang-
ing from 5.0 to 9.0 (Fig. 2a). The melting temperature
of 77 °C was reached for close to neutral pH values
between 6.6 and 7.0. However, in pH ranges from 5.0
to 6.5, as well as 7.5 to 9.0, the melting temperatures
fell within the 68–76 °C range. Finally, for pH values
above 9.0 and lower than 4.5, the melting temperature
was strongly reduced to approximately 55 °C.
Additionally, the residual activity of BlCel9 was eval-
uated by pre-incubations of this enzyme at 40, 50, and
60 °C for 48 h (Fig. 2b) and subsequent measurements
of the residual activity against PASC. Our results show
that BlCel9 was very stable at temperatures ranging
from 40 to 50 °C, for at least 48 h. At 50 °C, this
enzyme was able to retain more than 80% of its

Appl Microbiol Biotechnol
Fig. 2 Protein stability assays. a Structural stability of BlCel9 was
evaluated at different pH values. b Effect of temperature on the stability
of BlCel9 and activity was evaluated at temperature values of 40 °C,
50 °C, and 60 °C. Furthermore, the profile of the product of BlCel9 on
PASC was analyzed through HPAEC-PAD chromatography for 1 and
48 h (c), where the released sugar amounts were normalized to relative
values (%) (d)
hydrolytic activity after 24 h. However, at 60 °C, its
enzymatic activity decreased significantly to less than
50% after an incubation period of 3 h.
C2 was the dominant hydrolytic product of BlCel9 re-
leased from PASC (Fig. 2c, d). These results are con-
sistent with a notion that BlCel9 is an endoglucanase.
Product profile analyses
SAXS data collection and analysis
The hydrolytic products formed by BlCel9 acting on
PASC were analyzed by HPAEC-PAD (Fig. 2c and
Fig. S3). Identification of the product generated during
the enzymatic hydrolysis using the retention times of
standard mono and oligosaccharides (Fig. S3) reveals
that BlCel9 acting on PASC produces cellotetraose
(C4), cellotriose (C3), cellobiose (C2), and glucose
(C1). After 60 min of hydrolysis, BlCel9 produces
mostly C2 (~ 46%), as well as a small amount of C3
(~ 23%) and C4 (~ 17%), Fig. 2d. After 48 h of hydro-
lysis, the main product was again C2, which amounted
to approximately 59% of all released soluble oligosac-
charides. C4 present at the initial stages of PASC hy-
drolysis (after 1 h of reaction) was reduced fourfold
after 48 h (Fig. 2d). This indicates a slow, but consis-
tent enzymatic hydrolysis of C4 into C2. Furthermore,
We applied small angle X-ray scattering (SAXS) technique to
retrieve the molecular shape of bacterial endoglucanase
BlCel9 in solution. SAXS measurements were carried out
using the SEC-SAXS approach. The experimental and the
overall SAXS-derived parameters of BlCel9 are shown in
Table 1.
According to the data analysis of the SAXS measurements,
the aggregation-dependent or interparticle effects were not
observed in the measured scattering intensities. Rg variation
in the low q-region was also not detected. In the Guinier ap-
proximation, the initial q-region of the scattering curves ex-
hibited a linear behavior characteristic to monodisperse pro-
tein species. Additionally, the radius of gyration (Rg), with the
Guinier limit q.Rg ≥ 1.3 for each scattered frame, remained
constant within the experimental errors and the range of uti-
lized protein concentration. The radius of gyration calculated

Appl Microbiol Biotechnol
Table 1 Overall SAXS parameters for BlCel9. The numbers in the parentheses are the obtained discrepancy from expected values for the expected
protein molecular weights calculated using SAXSMoW 2.0 software (http://saxs.ifsc.usp.br/; (Fischer et al. 2010 ; Piiadov et al. 2018))
Sample
Rg, Å (Guinier)
Rg, Å (P(r)
Dmax, Å
SAXS_MoW MW, kDa
χ2-
Resolution, Å
References
parameter
BlCel9a
29.1 0.7
29.6
30.2 0.3
30.1
101
65.8 0.1
ND
0.7
1.3
1.5
2.1
3.7
4.1
5.2
30 2 Å
NR
This work
CtCel9I
106.2
104.7
102.6
79.9
79.3
76.1
(Petkun et al. 2015)
(Mandelman et al. 2003)
(Sakon et al. 1997)
(Eckert et al. 2009)
(Schubot et al. 2004)
(Honda et al. 2016)
CcCel9G
TfCel9A
AaCel9A
CtCbhA
PpGlcNase
29.3
29.8
ND
NR
28.9
29.1
ND
NR
22.9
23.3
ND
NR
23.3
23.8
ND
NR
23.1
23.7
ND
NR
a Ensembled resolution computed by SASRES (Tuukkanen et al. 2016)
ND not determined, NR not reported
with basis on the Guinier plot was 29.3 Å. These findings are
also supported by molecular mass estimations determined
from the SAXS data, which was approximately 65.8 kDa
(Table 1) with a discrepancy value of only 5.4% from the
theoretical molecular weight of the enzyme calculated on the
basis of its amino acid sequence (69.6 kDa). The close agree-
ment of the molecular weight experimentally determined from
the SAXS experiments and the theoretically expected molec-
ular weight indicates that BlCel9 exists as monomers in
solution.
The pair-distance distribution function, P(r), calculated
from the scattering data was characteristic of an elongated
particle with a maximum distance of 101 Å (Fig. 3). The Rg
determined on the basis of P(r) was approximately 29.3 Å,
which was very close to Rg obtained from the Guinier analysis
(Table 1). The distribution of interatomic function is
characteristic of particles that have two spatially well-
separated subunits, displaying two maxima corresponding to
the separation between them. This molecule has a tadpole-like
shape, quite similar to the already-known crystallographic
structures of bimodular CcCel9G from C. cellulolyticum
(Mandelman et al. 2003), TfCel9A from T. fusca (Sakon
et al. 1997), and CtCel9I from C. thermocellum (Petkun
et al. 2015). The computed P(r) from CcCel9G (Mandelman
et al. 2003), TfCel9A (Sakon et al. 1997), CtCel9I (Petkun
et al. 2015)) are very similar to P(r) of BlCel9 (Fig. 3). In all
cases, the overall SAXS-derived parameters extracted from
Guinier and P(r) analyses were in close agreement.
Structural parameters such as Rg and Dmax, obtained from
measured SAXS intensities, are given in Table 1.
The overall shape of BlCel9 based on P(r) was calculated
using DAMMIN and GASBOR, as shown in Fig. 4. Nine
envelopes were generated using both algorithms. The best fit
envelope in each case was selected based on their exhibiting
lowest χ2 value with respect to the experimental curve (Fig. 5a
and Table 1). The elongated conformation of BlCel9 is con-
sistent with the respective derived P(r) function. Each enve-
lope contains a globular region, one that corresponds to the
GH9 CD, and a second globular region corresponding to the
CBM 3c which are connected by a narrower region
representing the linker region (Figs. 4 and S4).
The crystal structures of the multi-domain TfCel9A (PDB
id 1JS4, (Sakon et al. 1997)) were superimposed within the
best SAXS envelope of BlCel9 (Fig. 4). TfCel9A crystal struc-
ture comfortably fits the molecular envelope (the χ-parameter
of 1.2). In the GASBOR/DAMMIN-calculated ab initio
SAXS model, the separation distance between the CD and
CBM 3c was very short, resembling the conformation adopted
by the crystal structure of the endo-processive TfCel9A.
Since BlCel9 is composed of a CD connected at its N-
terminus to a CBM3c via linker peptide, a significant degree
of flexibility could be expected for this enzyme. Both classic
and dimensionless Kratky plots of SAXS intensity data are
Fig. 3 SAXS data processing and data analysis of BlCel9. P(r) functions
determined from the experimental SAXS scattering data, and from the
computed scattering curve for the crystallographic models of CtCbhA
(PDB id 1UT9 (Schubot et al. 2004)), PpGlcNase (PDB id 5DGQ
(Honda et al. 2016)), AaCel9A (PBD id 3GZK (Eckert et al. 2009)),
CcCel9G (PDB id 1G87 (Mandelman et al. 2003)), TfCel9A (PDB id
1JS4 (Sakon et al. 1997)), and CtCel9I (PDB id 1G87(Petkun et al. 2015))

Appl Microbiol Biotechnol
Fig. 4 Low-resolution
model of BlCel9. The
superimposition of the low-
resolution SAXS envelope of
BlCel9 with the crystallographic
model of the endo-processive
TfCel9A (PDB id: 1JS4; (Sakon
et al. 1997)). Front and side view
of BlCel9 ab initio molecular en-
velope (gray semi-transparent
surfaces) retrieved from SAXS
measurements and the crystallo-
graphic model of TfCel9A are
shown (in ii the molecular enve-
lope was rotated by 180° around
the x-axis). The catalytic domain
was colored in cyan, CBM3c was
colored in blue, and the linker
peptide was colored in red
useful for the qualitative assessment of flexibility and disorder
of a protein in solution, showing a plateau along the baseline
at high q value for a compact and well-folded protein (Glatter
and Kratky 1982; Putnam et al. 2007; Rambo and Tainer
2011; Receveur-Brechot and Durand 2012; Hammel 2012).
Kratky analyses of the SAXS data suggest that BlCel9 is a
compact particle exhibiting a well-defined bell-shaped form
(Fig. 5b). However, the dimensionless Kratky plot exhibited a
bell-shaped peak with a distinct maximum at 2.1, which is
slightly dislocated from the ideal value of q.Rg ≈ √3 ≈ 1.73
(Fig. 5b), which might indicate certain degree of flexibility
in solution.
BlCel9 exhibits its optimal activity at neutral pH (pH 7.0)
and 60 °C. The optimal pH and temperature values obtained
for BlCel9 are similar to other previously characterized GH9
enzymes (Kataeva et al. 2005; Zhang et al. 2011; Ravachol
et al. 2014; Okano et al. 2015; Younesi et al. 2016; Leis et al.
2017). The optimal activity of other family members was
mainly registered within a pH range of 6.0 to 7.5 and temper-
ature of 50 °C. Indeed, BlCel9 shows similarities to some
previously studied GH9 enzymes, such as multimodular
cellobiohydrolase CtCbhA from Clostridium thermocellum
(Kataeva et al. 2005), cellulosomal GH9 enzymes from
Clostridium thermocellum (Leis et al. 2017) and Clostridium
cellulolyticum (Ravachol et al. 2014), endoglucanase
AaCel9A from Alicyclobacillus acidocaldarius (Younesi
et al. 2016), endoglucanases ThCel9A from Thermobifida
halotolerans YIM (Zhang et al. 2011), and metagenome-
derived endoglucanases (Okano et al. 2015), which displayed
optimum activity levels at temperatures ranging from 50 to
70 °C. Additionally, some of these enzymes, such as
endoglucanase CtCel9I from C. thermocellum (Burstein
et al. 2009; Petkun et al. 2015), cellobiohydrolase CtCbhA
(Schubot et al. 2004; Kataeva et al. 2005), and endoglucanase
TfCel9A (Sakon et al. 1997; Kostylev et al. 2012) have their
highest levels of activity at 60–80 °C.
Figures 4 and S4 show the molecular shape comparison
between BlCel9 and the crystallographic structures of TfCel9A
(PDB id 1JS4) and AaCel9A (PDB id 3GZK), suggesting that
CD and CBM domains of BlCel9 are in a close proximity.
Discussion
Our biochemical analysis of BlCel9 shows that the enzyme
is active against insoluble substrates (PASC, BC) and its
activity is inhibited by Fe2+ and Cu2+ ions. According to
previous studies of other GH9 enzymes, Ca2+ plays an im-
portant role in their thermostability and substrate binding
affinity (Zhang et al. 2015; Kim et al. 2016; Wang et al.
2016). However, the BlCe9 enzymatic activity was practi-
cally not influenced by Ca2+ ion and molecular reasons for
such behavior are currently unknown.
During PASC hydrolysis, BlCel9 mostly releases C2. For
comparison, TfCel9A (Kostylev and Wilson 2014) and
CcCel9A from C. cellulosi (Yang et al. 2016) also released
small quantities of glucose (C1) and cellotetraose (C4) as their
final product. Based on the analysis of these products, it was

Appl Microbiol Biotechnol
high q value for a compact and well-folded protein (Glatter
and Kratky 1982; Putnam et al. 2007; Rambo and Tainer
2011; Receveur-Brechot and Durand 2012; Hammel 2012).
The dimensionless Kratky plot is independent of the size
and molecular weight of the macromolecule and in this case,
for a well-folded molecule with a well-defined molecular
shape, the q.Rg limit tends to √3, and (qRg)2I(q)/I(0) limit
tends to 1.104 (Receveur-Brechot and Durand 2012; Burger
et al. 2016). The deviations from these limits indicate protein
flexibility or conformational instability (Receveur-Brechot
and Durand 2012; Burger et al. 2016). Although Kratky anal-
yses are consistent with a notion of BlCel9 being a compact
particle, the dimensionless Kratky plot exhibited a bell-shaped
peak with a distinct maximum at 2.1, which is slightly
dislocated from the ideal value of q.Rg ≈ √3 ≈ 1.73 (Fig. 5b).
Thus, taking into account that the linker segment of BlCel9 is
relatively long (∼ 20 residues), BlCel9 might have a certain
degree of flexibility in solution, imprinted by the dynamics of
inter-domain linker between its CD and CBM3c. This ob-
served flexibility could facilitate substrate recognition during
catalytic events.
Comparison of BlCel9 molecular shape with the available
GH9 X-ray structures further confirms that CD and CBM
domains of the enzyme are in a close proximity. This archi-
tecture has been proposed to play an important role in coop-
erative action on crystalline cellulose hydrolysis of some GH9
enzymes (Oliveira et al. 2009; Batista et al. 2011; Petkun et al.
2015). The closely juxtaposed CBM3c module and GH9 cat-
alytic domain of TfCel9A and CcCel9G are believed to form a
single functional entity (Burstein et al. 2009) where a flat
surface formed between their domains should guide the cellu-
lose chain toward the active site of the catalytic domain of
GH9 (Li et al. 2007; Oliveira et al. 2009). Collectively, our
SAXS analyses support the notion that BlCel9 has a well-
defined, elongated shape in solution, which resembles other
GH9 enzymes.
Fig. 5 SAXS data processing and data analysis. Experimental SAXS
intensity (a) from BlCel9 in solution (red line) was fitted to the
theoretical scattering curves calculated from the restored low-resolution
model retrieved from DAMMIN and GASBOR model. The dimension-
less Kratky plot (b) was applied to experimental SAXS data for evaluat-
ing the inter-domain flexibility between the catalytic and accessory do-
main of BlCel9
proposed that TfCel9A initially produces mainly cellotetraose
(C4), which later is converted into cellotriose (C3), cellobiose
(C2), and glucose (C1) (Kostylev and Wilson 2014).
However, BlCel9 produces much more C2 than C3 (which
could be indicative of its processivity), whereas TfCel9A pro-
duces larger quantities of C3 than C2 (Kostylev and Wilson
2014). A similar product pattern has been observed for
CcCel9A from Clostridium cellulosi (Yang et al. 2016),
CbCel9A-TM3 from Caldicellulosiruptor (Yi et al. 2013),
and the processive TfCel9A from Thermobifida fusca (Li
et al. 2007; Kostylev et al. 2012; Kostylev and Wilson 2014).
SAXS-derived low resolution molecular envelope of
BlCel9 shows two domains in a close proximity to each other.
Since BlCel9 is composed of a CD connected at its N-terminus
to a CBM3c via linker peptide, a significant degree of flexi-
bility could be expected for this enzyme. Both classic and
dimensionless Kratky plots of SAXS intensity data are useful
for the qualitative assessment of flexibility and disorder of a
protein in solution, showing a plateau along the baseline at
Acknowledgments The authors acknowledge the Diamond Light Source
for Synchrotron beam time and staff scientists of the B21 beamline for
their assistance. They are also grateful to Maria Auxiliadora Morim
Santos, Ph.D., who conducted gene cloning, and Marco Antonio Seiki
Kadowaki, Ph.D., for HPAEC-PAD data collection.
Authors’ contributions E.A.A. and I.P. conceived and designed the study.
M.O.N. collected SAXS data. E.A.A. performed enzyme production,
enzyme characterization, and SAXS data processing; also carried out data
analyses. E.A.A. and I.P wrote the manuscript with the assistance of all
authors. I.P conducted the overall supervision. All the authors approved
the final version.
Funding This research was supported by Fundação de Amparo à
Pesquisa do Estado de São Paulo (FAPESP) via grant 2015/13684-0,
and by Conselho Nacional de Desenvolvimento Científico e
Tecnológico (CNPq) via grants 405191/2015-4, 140667/2015-6,
158752/2015-5, 303988/2016-9, and 440977/2016-9.

Appl Microbiol Biotechnol
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