Novel substrates for the measurement of endo-1,4-β-glucanase (endo-cellulase)

Article abstract of DOI:10.1016/j.carres.2013.12.001

A specific and sensitive substrate for the assay of endo-1,4-β- glucanase (cellulase) has been prepared. The substrate mixture comprises benzylidene end-blocked 2-chloro-4-nitrophenyl-β-cellotrioside (BzCNPG3) in the presence of thermostable β-glucosidase. Hydrolysis by exo-acting enzymes such as β-glucosidase and exo-β-glucanase is prevented by the presence of the benzylidene group on the non-reducing end d-glucosyl residue. On hydrolysis by cellulase, the 2-chloro-4-nitrophenyl-β-glycoside is immediately hydrolysed to 2-chloro-4-nitrophenol and free d-glucose by the β-glucosidase in the substrate mixture. The reaction is terminated and colour developed by the addition of a weak alkaline solution. The assay procedure is simple to use, specific, accurate, robust and readily adapted to automation. This procedure should find widespread applications in biomass enzymology and in the specific assay of endo-1,4-β-glucanase in general.

Full text of DOI:10.1016/j.carres.2013.12.001

Carbohydrate Research 385 (2014) 9–17
Contents lists available at ScienceDirect
Carbohydrate Research
journal homepage: www.elsevier.com/locate/carres
Novel substrates for the measurement of endo-1,4-b-glucanase
(endo-cellulase)
a
b
a
a
Barry V. McCleary a, David Mangan a, , Robin Daly , Sébastien Fort , Ruth Ivory , Niall McCormack
⇑
a Megazyme International Ireland, Bray Business Park, Southern Cross Road, Bray, County Wicklow, Ireland
b Centre de Recherches sur les Macromolécules Végétales (CERMAV, UPR-CNRS 5301), Affiliated with Grenoble Alpes University, Member of Institut de
Chimie Moléculaire de Grenoble (ICMG, FR-CNRS 2607) and Institut Carnot PolyNat, BP53, 38041 Grenoble Cedex 9, France
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 30 October 2013
Received in revised form 2 December 2013
Accepted 3 December 2013
Available online 8 December 2013
A specific and sensitive substrate for the assay of endo-1,4-b-glucanase (cellulase) has been prepared. The
substrate mixture comprises benzylidene end-blocked 2-chloro-4-nitrophenyl-b-cellotrioside (BzCNPG3)
in the presence of thermostable b-glucosidase. Hydrolysis by exo-acting enzymes such as b-glucosidase
and exo-b-glucanase is prevented by the presence of the benzylidene group on the non-reducing end
D-glucosyl residue. On hydrolysis by cellulase, the 2-chloro-4-nitrophenyl-b-glycoside is immediately
hydrolysed to 2-chloro-4-nitrophenol and free -glucose by the b-glucosidase in the substrate mixture.
D
Keywords:
endo-1,4-b-Glucanase
Cellulase
Assay procedure
2-Chloro-4-nitrophenyl
Colourimetric
The reaction is terminated and colour developed by the addition of a weak alkaline solution. The assay
procedure is simple to use, specific, accurate, robust and readily adapted to automation. This procedure
should find widespread applications in biomass enzymology and in the specific assay of endo-1,4-b-glu-
canase in general.
Ó 2013 Elsevier Ltd. All rights reserved.
Oligosaccharides
1. Introduction
ase enzymes can be characterised by studying the rates of
hydrolysis of cello-oligosaccharides, borohydride reduced
endo-1,4-b-Glucanase (EC 3.2.1.4) (cellulase) is widely used in
the poultry industry to degrade b-glucan and thus increase the
digestibility of feeds rich in barley.1 In the malt industry it is used
to hydrolyse b-glucan, increase the rate of wort filtration and re-
duce the possibility of b-glucan precipitation in beer. In the textile
industry endo-1,4-b-glucanase is used for denim finishing and cot-
ton softening, in the detergent industry it is used for cleaning and
colour care and in the paper industry it modifies fibre and im-
proves drainage.2 It is generally accepted that the market for this
enzyme will increase dramatically when the second generation
biofuel industry becomes economic.3
cello-oligosaccharides4 or nitrophenyl-cello-oligosaccharides.6–9
However, these substrates cannot be used to measure endo-1,4-
b-glucanase in the presence of other cellulose degrading enzymes
such as b-glucosidase and exo-1,4-b-glucanases which occur in fer-
mentation broths and industrial enzyme preparations.
The aim of this study was to develop a specific substrate and a
robust assay procedure for the measurement of endo-1,4-b-glucan-
ase activity in complex biological materials.
2. Results and discussion
endo-1,4-b-Glucanase in biological materials and microbial fer-
mentation broths can be specifically measured by the decrease in
the viscosity of water soluble, chemically modified cellulose deriv-
atives such as CM-cellulose 7 M.4 Cellulose derivatives either dyed
or dyed and crosslinked also give specific measurement of endo-
1,4-b-glucanase. With soluble dyed substrates, hydrolysis by
endo-1,4-b-glucanase gives an increase in dyed fragments soluble
in an aqueous–organic solvent, whereas with insoluble, dyed and
crosslinked cellulose substrates, cellulase hydrolysis gives an in-
crease in water soluble, dyed fragments.5 Pure endo-1,4-b-glucan-
4,6-O-Benzylidene end-blocked 4-nitrophenyl-a-maltoheptao-
side10 has been commercially available for 25 years (from
Megazyme) and is used, in combination with the ancillary enzymes
a
-glucosidase and/or amyloglucosidase (AMG), for the measure-
ment of
-amylase.10–12 The length of the maltodextrin chain
ensures maximal rate of hydrolysis by fungal and pancreatic
-amylase, and near maximal rate of hydrolysis by bacterial and
cereal -amylase (which require a longer maltodextrin chain
a
a
a
length for binding and hydrolysis). Synthesis of 4,6-O-benzylidene
end-blocked 2-chloro-4-nitrophenyl cello-oligosaccharides poses a
number of challenges. Because cellulose is insoluble, production in
good yield of oligosaccharides of the required degree of polymeri-
sation (DP) of 3–6 is challenging. Cello-oligosaccharides of DP 6
⇑
Corresponding author. Tel.: +353 12861220.
E-mail address: david@megazyme.com (D. Mangan).
0008-6215/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.carres.2013.12.001

10
B. V. McCleary et al. / Carbohydrate Research 385 (2014) 9–17
Figure 1. CNPG3 hydrolysis by A. niger endo-1,4-b-glucanase (A = 0 U, B = 0.64 U and C = 6.4 U) and CNPG4 hydrolysis by A. niger endo-1,4-b-glucanase (D = 0 U, E = 0.064 U
and F = 0.64 U). All incubations were carried out at 40 °C, pH 4.5 for 10 min with 6.66 mg/mL substrate concentration.
and greater are sparingly soluble in water and thus are very diffi-
cult to purify in quantity. The current work has thus focused on
the modification of cello-oligosaccharides of DP 3–5, which could
be obtained in quantity from Megazyme.
The synthesis of colourimetric cello-oligosaccharide substrates
has been described by a number of groups using a variety of glyco-
sylation methods. Initial approaches employed peracetylated
strictly anhydrous conditions.15 It was later discovered that the
use of a biphasic CHCl3/NaOH solvent system for the same glyco-
sylation reactions afforded higher yields.16,17 This methodology
has been successfully employed here to synthesise a series (DP
2–DP 4) of colourimetric cello-oligosaccharides. Extensive spectro-
scopic data on these compounds not previously reported are also
included here in the Supporting information.
a
-bromo glycosyl donors in mono-phasic acetone/aq. NaOH glyco-
These colourimetric substrates have been further functionalised
with the incorporation of a 4,6-O-benzylidene group on the non-
reducing terminus. Benzylidene acetals have been routinely used
for the selective protection of the 4 and 6 positions of glucosyl
sylation reactions with a variety of phenol acceptors.13,14 This reac-
tion was subsequently improved by replacing the aqueous base
with 2,6-lutidine and carrying out the transformation under

B. V. McCleary et al. / Carbohydrate Research 385 (2014) 9–17
11
Figure 2. CNPG3 hydrolysis by T. longibrachiatum endo-1,4-b-glucanase (A = 0 U, B = 0.027 U and C = 0.27 U) and CNPG4 hydrolysis by T. longibrachiatum endo-1,4-b-glucanase
(D = 0 U, E = 0.027 U and F = 0.27 U). All incubations were carried out at 40 °C, pH 4.5 for 10 min with 6.66 mg/mL substrate concentration.
residues in a wide range of applications.18–20 This synthetic modi-
fication has been shown to block the action of exo-acting glucosi-
dase enzymes in the analogous malto-oligosaccharide series.9
Difficulty in the chemical modification of the cello-oligomers
increased in moving from cellotriose to cellopentaose, and this
was reflected in lower final yields of the modified tetramer
(10%), and particularly the modified pentamer (not isolated, esti-
mated 5%). This can be attributed to the fact that a greater number
of undesired benzylidene acetal derivatives are obtained during
the benzylidene acetal formation step with the higher DP oligosac-
charides. In addition, the higher DP oligosaccharides are obviously
less amenable to purification and isolation by crystallisation/pre-
cipitation methods. Consequently, the work described here has
focused on the production and evaluation of 4,6-O-benzylidene
end-blocked 2-chloro-4-nitrophenyl cellotrioside (BzCNPG3) and
4-6-O-benzylidene end-blocked 2-chloro-4-nitrophenyl cellotet-
raoside (BzCNPG4). 4-6-O-Benzylidene end-blocked 2-chloro-
4-nitrophenyl cellobioside (BzCNPG2) was also prepared for
comparative studies.
It is well known that the rates of hydrolysis of cello-oligosaccha-
rides by endo-1,4-b-glucanase is dependent both on the DP of the
cello-oligosaccharide (or reduced cello-oligosaccharide) and on
the specific endo-1,4-b-glucanase being studied. In a comparison
of the rates of hydrolysis of borohydride reduced cello-oligomers
of DP 2–6 by endo-1,4-b-glucanase,4 it was shown that Trichoderma
longibrachiatum endo-1,4-b-glucanase hydrolysed cellotriitol at a
rate similar to that for cellohexaitol, whereas Aspergillus niger
endo-1,4-b-glucanase hydrolysed cellotetraitol at just 24% the rate
of cellohexaitol, and the hydrolysis of cellotriitol was just 6% the
rate for cellohexaitol. The susceptibility to the hydrolysis of
2-chloro-4-nitrophenyl-cellotrioside (CNPG3) and 2-chloro-4-
nitrophenyl-cellotetraoside (CNPG4) by A. niger and T. longibrachia-
tum endo-1,4-b-glucanase is shown in Figures 1 and 2. Complete
hydrolysis of both CNPG3 and CNPG4 is obtained with just 0.27 U
of T. longibrachiatum endo-1,4-b-glucanase in 10 min at 40 °C. Near
complete hydrolysis of CNPG4 by A. niger endo-1,4-b-glucanase
requires 0.64 U/assay, and of CNPG3 requires 6.4 U/assay. Of the en-
zymes studied by McCleary et al.4 these two endo-1,4-b-glucanases

12
B. V. McCleary et al. / Carbohydrate Research 385 (2014) 9–17
Figure 3. Relative rates of hydrolysis of NPG2, CNPG2, BzNPG2 and BzCNPG2 by
Trichoderma endo-1,4-b-glucanase. Incubation of each substrate (0.1 mL, 3 mM in
33% DMSO/0.1 M KCl) in the presence of b-glucosidase (0.82 U) with endo-1,4-b-
glucanase (1.42 U, 0.1 mL, pH 4.5 100 mM sodium acetate) at 40 °C.
Figure 5. Effect of BzCNPG3 concentration on reaction kinetics over 12 min in the
presence of T. longibrachiatum endo-1,4-b-glucanase (170 mU/assay) and b-gluco-
sidase (0.82 U/assay) at 40 °C in pH 4.5 100 mM sodium acetate at 40 °C.
and the reaction terminated at 0–12 min by the addition of 3 mL
of 2% Tris solution (pH 9). b-Glucosidase at a level of 0.5 U/assay
(5 U/mL of substrate) was sufficient to give maximal colour release
on hydrolysis of the substrate (Fig. 4), but a level of 0.66 U/assay
was routinely used to allow for the possibility of loss of enzyme
activity on longer term storage of the substrate.
Under the assay conditions employed, the effect of substrate
concentration was determined by incubating 0.1 mL of substrate
mixture containing from 0.66 to 3.0 mM BzCNPG3 plus b-glucosi-
dase (at 0.82 U/assay) with 0.1 mL of buffered solution containing
T. longibrachiatum endo-1,4-b-glucanase (170 mU on this sub-
strate) and the reaction was terminated at 0, 3, 6, 9 and 12 min
by the addition of 3 mL of 2% Tris solution (pH 9) (Fig. 5). Near
maximum rate of hydrolysis was obtained at a final substrate con-
centration of 1.2 mM in the reaction mixture, so a concentration of
1.5 mM was routinely used. At this substrate concentration, the
reaction was linear up to an absorbance value of 1.0 (corresponds
to <65% substrate hydrolysis). If absorbance values above 1.0 are
obtained, the enzyme should be diluted and the assay re-run.
In the hydrolysis of BzCNP-cello-oligosaccharides, the endo-1,4-
b-glucanase can potentially cleave within the oligosaccharide
Figure 4. Effect of b-glucosidase concentration on reaction kinetics over 12 min in
the presence of T. longibrachiatum endo-1,4-b-glucanase (170 mU/assay) and
BzCNPG3 (1.5 mM) in pH 4.5 100 mM sodium acetate at 40 °C.
occur at either end of a spectrum in terms of their ability to
hydrolyse low DP cello-oligosaccharides as well as their ability to
hydrolyse tamarind seed xyloglucan (containing a highly substi-
tuted 1,4-b-D-glucan backbone) or konjac glucomannan in which
just 40% of the 1,4-b-linked hexoses in the linear polysaccharide
chain or between the terminal
D-glucosyl residue and the CNP
chain are
residues).
D-glucosyl residues (the remainder being D-mannosyl
group (Fig. 6). The effect of saturating levels of b-glucosidase on
the rates of hydrolysis of BzCNPG2 and BzCNPG3 by T. longibrachi-
atum and A. niger endo-1,4-b-glucanase is shown in Figure 7. With
A. niger endo-1,4-b-glucanase, addition of b-glucosidase has no
effect on the rate of hydrolysis of either BzCNPG2 or BzCNPG3,
showing that hydrolysis occurs exclusively between the terminal
BzNPG2, BzCNPG2, BzCNPG3 and BzCNPG4 are sparingly soluble
in water. They are best handled by dissolving in dimethyl sulphox-
ide (DMSO) at a concentration of 9.15 mM. Before use, 3 mL of this
substrate solution is diluted with 6 mL of 100 mM KCl solution,
mixed, and 50 lL of thermostable b-glucosidase is added to a
D-glucosyl residue and the CNP group. The same is true for the
3 mL aliquot of the result 33% DMSO solution to make the assay
reagent mixture.
hydrolysis of BzCNPG2 by T. longibrachiatum endo-1,4-b-glucanase,
but with BzCNPG3, the rate of release of CNP in the presence of
b-glucosidase is approximately fivefold higher than without added
b-glucosidase. This shows that the preferred site of hydrolysis is
within the oligosaccharide chain, most probably between the
The relative rates of hydrolysis of NPG2, CNPG2, BzNPG2 and
BzCNPG2 by T. longibrachiatum endo-1,4-b-glucanase is shown in
Figure 3. These incubations were performed in the absence of
b-glucosidase and the release of 4-nitrophenol (NP) or 2-chloro-4-
nitrophenol (CNP) was measured. b-Glucosidase was not required
in these incubations because with the modified disaccharide, the
reducing-end terminal and the penultimate-D-glucosyl residues.
The relative rates of hydrolysis of BzCNPG2, BzCNPG3 and
BzCNPG4 by A. niger and T. longibrachiatum endo-1,4-b-glucanase
areshownin Figure8. WithT. longibrachiatum endo-1,4-b-glucanase,
BzCNPG3 is hydrolysed at the same rate as BzCNPG4. This is consis-
tent with the relative rates of hydrolysis of cellotetraitol and cello-
pentaitol by this enzyme.4 BzCNPG2 is hydrolysed, but very slowly.
With A. niger endo-1,4-b-glucanase, BzCNPG4 is hydrolysed approx-
imately five times faster than BzCNPG3, again consistent with the
rate of hydrolysis of cellopentaitol and cellotetraitol by this enzyme.
The determined activity of different endo-1,4-b-glucanase on
BzCNPG3 relative to CM-cellulose 4 M varies significantly as shown
in Table 1. This can be used to characterise the particular endo-1,4-
b-glucanase. From a quality control standpoint, this is of no conse-
quence as protocols and activity relationships can be established
cellulase cleaves between the terminal D-glucosyl unit and NP or
CNP. The CNP derivatives are hydrolysed at 2–3 times the rate for
the NP derivatives. This effect was to be expected given the differ-
ence in leaving group ability between CNP (pKa = 5.45) and NP
(pKa = 7.18) and has been studied in great detail by Tull and With-
ers.14 All subsequent research has focused on the CNP derivatives.
The concentration of b-glucosidase required to optimise the
sensitivity of hydrolysis of BzCNPG3 by T. longibrachiatum endo-
1,4-b-glucanase was determined using substrate solutions contain-
ing 3 mM BzCNPG3 plus 3.2–8.2 U/mL of b-glucosidase. An aliquot
of these solutions (0.1 mL) was incubated with 0.1 mL of Tricho-
derma endo-1,4-b-glucanase (170 mU on this substrate) at 40 °C

B. V. McCleary et al. / Carbohydrate Research 385 (2014) 9–17
13
Figure 6. Observed sites for hydrolysis of BzCNPG2 and BzCNPG3 by endo-1,4-b-glucanase.
Figure 7. Hydrolysis of BzCNPG2 and BzCNPG3 by endo-1,4-b-glucanase in the presence and absence of saturating levels of b-glucosidase (0.82 U/assay); (a) BzCNPG2 with T.
longibrachiatum endo-1,4-b-glucanase (600 mU/assay), (b) BzCNPG3 with T. longibrachiatum endo-1,4-b-glucanase (30 mU/assay), (c) BzCNPG2 with A. niger endo-1,4-b-
glucanase (17.5 U/assay), (d) BzCNPG3 with A. niger endo-1,4-b-glucanase (700 mU/assay). All incubations were carried out at 40 °C in pH 4.5 100 mM sodium acetate.
for the particular endo-1,4-b-glucanase and then the determined
activity value is the sole consideration.
at 20 °C there is a significant increase in the blank absorbance va-
lue, but only after extended periods. The increase in the reaction
absorbance value parallels the increase in the blank value, such
that on subtracting the blank value from the reaction value, the
determined absorbance value is very similar to those obtained on
storage of the reagent at À20 and 4 °C. This demonstrates that
the b-glucosidase in the reagent mixture is stable, even on storage
at 20 °C for 100 days. Since on storage of the substrate at 4 or
À20 °C there is essentially no increase in blank absorbance value
and the determined activity of the cellulase control is the same,
these are the recommended storage temperatures. These studies
would indicate that the prepared substrate should be stable for
>2–3 years at À20 °C.
The thermostable b-glucosidase used in combination with
BzCNPG3 is stable up to 90 °C and has activity over the pH range
of 4.5–7.5, meaning that the reagent can be used over these ranges
of temperature and pH. A standard curve relating absorbance in-
crease at 400 nm to level of Thermotoga maritima endo-1,4-b-glu-
canase at 80 °C is shown in Figure 9. BzCNPG3 substrate mixture
is stable at 80 °C for up to 30 min.
The stability of the reagent mixture containing BzCNPG3 and
thermostable b-glucosidase (400 U/mL) at À20, 4 and 20 °C is
shown in Figure 10. On storage of the reagent for up to 100 days,
0.1 mL aliquots were removed and the blank absorbance was
determined as well as the absorbance on incubation of the sub-
strate with a set amount of T. longibrachiatum endo-1,4-b-glucan-
ase (20 mU on BzCNPG3) at 40 °C for 10 min. With reagent stored
It can thus be concluded that BzCNPG3 is a useful and versatile
substrate for the measurement of endo-1,4-b-glucanase from
microbial sources. The assay procedure employing this substrate

14
B. V. McCleary et al. / Carbohydrate Research 385 (2014) 9–17
Figure 9. Standard curve relating absorbance increase at 400 nm observed with
BzCNPG3 to level of T. maritima endo-1,4-b-glucanase activity on CM-cellulose-4 M
at 80 °C in pH 6 100 mM sodium phosphate.
Figure 8. Relative rates of hydrolysis of BzCNPG2, BzCNPG3 and BzCNPG4 by (a) A.
niger endo-1,4-b-glucanase and (b) T. longibrachiatum endo-1,4-b-glucanase at 40 °C
in pH 4.5 100 mM sodium acetate.
Table 1
Relative rates of hydrolysis of CellG3 mixture [containing 1.5 mM BzCNPG3 (concen-
tration in the assay) and b-glucosidase] and CM-cellulose 4 M (10 mg/mL) by several
endo-1,4-b-glucanases at 40 °C and optimal pH
Source of
pH of
CMC-4 M
CellG3
endo-cellulase
reaction
mixture
Specific Relative
activity activity (%) activity activity
Specific Relative
(U/mg)
(U/mg)
(%)
Figure 10. Stability study on BzCNPG3/b-glucosidase assay reagent in 33% DMSO/
100 mM KCl at À20, 4 and 20 °C over 50 days.
T. longibrachiatum
B. amyloliquefaciens 6.0
T. maritima
T. emersonii
A. niger
4.5
68
82
53
64
82
100
100
100
100
100
47.6
52.2
27.3
2.6
70.0
63.6
51.6
4.0
6.0
4.5
4.5
were purchased from Sigma Aldrich, Lennox Laboratory Supplies
or Lab unlimited (Carl Stuart Group) and were analytical reagent
grade. A Brüker Avance 400 was employed for 1H (400.13 MHz)
and 13C (100.61 MHz) NMR spectra. Resonances d, are in ppm units
downfield from an internal reference in C2D6SO (dH = 2.50). Mass
spectrometry analysis was performed with a Q-Tof Premier Waters
Maldi-quadrupole time-of-flight (Q-Tof) mass spectrometer
equipped with Z-spray electrospray ionisation (ESI) and matrix as-
sisted laser desorption ionisation (MALDI) sources. Silica gel Flori-
sil (200 mesh; Aldrich) was used for column chromatography.
Analytical thin-layer chromatography was performed using Merck
60 F254 silica gel (pre-coated sheets, 0.2 mm thick, 20 cm  20 cm)
and visualised by UV irradiation or 5% H2SO4/EtOH staining.
2.1
2.5
is versatile, accurate and very reproducible. Activity on BzCNPG3
compared to that on CM-cellulose 4 M varies from one endo-cellu-
lase to the next, but this is of no concern if the aim is to monitor and
control a particular fermentation broth when the nature of the ma-
jor endo-cellulase component is known. The principle of the assay
procedure is summarised in Figure 11. On hydrolysis of BzCNPG3
by endo-1,4-b-glucanase, the b-glucosidase in the substrate mixture
immediately hydrolyses the non-blocked CNP-gluco-oligosaccha-
rides releasing D-glucose and free 2-chloro-4-nitrophenol. The reac-
tion is terminated and colour developed by the addition of an
alkaline solution. The substrate is absolutely specific for endo-1,4-
b-glucanase. The benzylidene blocking group prevents hydrolysis
by b-glucosidase, cellobiohydrolase or exo-glucanases.
3.2. Colourimetric substrate synthesis
3.2.1. Nitrophenyl-cello-oligosaccharide synthesis
NPG2, CNPG2, CNPG3 and CNPG4 were synthesised without dif-
ficulty according to the general methods employed by Planas
et al.16 The compounds reported here have been previously syn-
thesised by Claeyssens and Henrissat.9
3. Experimental
3.1. Materials
Cello-oligosaccharides, thermostable b-glucosidase (T. mariti-
ma; accession number Q08638, Cat. No. E-BGOSTM) and highly
purified endo-1,4-b-glucanases4 from T. longibrachiatum (accession
number Q12714, Cat. No. E-CELTR), Bacillus amyloliquefaciens
(accession number Q8RPQ6, Cat. No. E-CELBA), Thermatoga mariti-
ma (accession number Q9X273, Cat. No. E-CELTM), Talaromyces
emersonii (accession number Q8NIB5, Cat. No. E-CELTE) and A. niger
(O74705, Cat. No. E-CELAN), were obtained from Megazyme Inter-
national, Ireland. All other chemicals used in organic synthesis
3.2.1.1. 4-Nitrophenyl-cellobioside (NPG2).
Yield: 41%; mp
(H2O) 244–247 °C (dec); 1H NMR (400 MHz C2D6SO) d 2.94–3.57
(m, 8H), 3.58–3.79 (m, 4H), 4.30 (d, J = 7.53 Hz, 1H(anom)), 4.62–
4.76 (m, 2H), 4.87 (br s, 1H), 4.99–5.12 (m, 2H), 5.18 (d,
J = 7.53 Hz, 1H(anom)), 5.28 (br s, 1H), 5.65 (br s, 1H), 7.24 (d,
J = 7.53 Hz, 2H), 8.22, (d, J = 8.03 Hz, 2H); 13C NMR (100 MHz,
C2D6SO) d 60.3, 61.5, 70.5, 73.3, 73.8, 75.2, 75.6, 76.9, 77.3, 80.0,
99.8, 103.6, 117.0, 117.0, 126.2, 126.2, 142.2, 162.7; HRMS ESÀ
[MÀH]À Calcd 462.1248, Found 462.1250.

B. V. McCleary et al. / Carbohydrate Research 385 (2014) 9–17
15
Figure 11. Overview of endo-1,4-b-glucanase assay format.
3.2.1.2. 2-Chloro-4-nitrophenyl-cellobioside (CNPG2). Yield:
37%; mp (H2O) 200–203 °C (dec); 1H NMR (400 MHz C2D6SO) d
2.94–3.08 (m, 2H), 3.09–3.24 (m, 2H), 3.24–3.51 (m, 4H), 3.58–3.78
(m, 4H), 4.28 (d, J = 7.78 Hz, 1H (anom)), 4.62 (t, J = 5.14 Hz, 1H),
4.66 (t, J = 5.53 Hz, 1H), 4.85 (d, J = 2.01 Hz, 1H), 5.00 (d, J = 5.53 Hz,
1H), 5.04 (d, J = 4.77 Hz, 1H), 5.25 (d, J = 5.02 Hz, 1H), 5.32 (d,
J = 7.78 Hz, 1H (anom)), 5.64 (d, J = 5.14 Hz, 1H), 7.47 (d,
J = 9.09 Hz, 1H), 8.20 (dd, J = 9.09, 2.79 Hz, 1H), 8.32 (d,
J = 2.79 Hz, 1H); 13C NMR (100 MHz, C2D6SO) d 60.2, 61.6, 70.5,
73.2, 73.8, 75.4, 75.7, 76.9, 77.3, 79.9, 99.9, 103.6, 116.1, 122.7,
124.7, 126.1, 142.0, 158.1; HRMS ESÀ [MÀH]À Calcd 496.0858,
Found 496.0858.
(d, J = 5.27 Hz, 1H), 7.46 (d, J = 9.17 Hz, 1H), 8.19 (dd, J = 9.17,
2.38 Hz, 1H), 8.32 (d, J = 2.38 Hz, 1H); 13C NMR (100 MHz, C2D6SO)
d 60.2, 60.8, 60.8, 61.5, 70.5, 73.2, 73.5, 73.5, 73.7, 75.2, 75.3, 75.3,
75.3, 75.4, 75.7, 76.9, 77.3, 79.9, 80.9, 80.9, 99.9, 103.2, 103.3,
103.7, 116.1, 122.7, 124.7, 126.1, 142.1, 158.1; HRMS ESÀ [MÀH]À
Calcd 820.1914, Found 820.1906.
3.2.2. Benzylidene blocked nitrophenyl-cello-oligosaccharide
synthesis
BzNPG2, BzCNPG2, BzCNPG3 and BzCNPG4 were synthesised
from the corresponding unprotected substrates according to the
following general procedure. To a solution of 2-chloro-4-nitro-
phenyl-cellotrioside (1 g, 1.515 mmol) and p-toluenesulfonic acid
monohydrate (86 mg, 0.45 mmol) under an argon atmosphere in
anhydrous dimethylformamide (10 mL) containing activated 4A
molecular sieves (200 mg) was added benzaldehyde dimethylace-
3.2.1.3. 2-Chloro-4-nitrophenyl-cellotrioside (CNPG3). Yield:
35%; mp (H2O) 228–231 °C (dec); 1H NMR (400 MHz C2D6SO) d
2.92–3.87 (m, 18H), 4.23 (d, J = 7.78 Hz, 1H (anom)), 4.35 (d,
J = 7.78 Hz, 1H (anom)), 4.43–4.88 (m, 6H), 4.99 (br s, 1H), 5.23 (br
s, 1H), 5.31 (d, J = 7.78 Hz, 1H (anom)), 5.41 (br s, 1H), 5.64 (br s,
1H), 7.46 (d, J = 9.23 Hz, 1H), 8.19 (dd, J = 9.23, 2.64 Hz, 1H), 8.32
(d, J = 2.64 Hz, 1H); 13C NMR (100 MHz, C2D6SO) d 60.1, 60.8, 61.5,
70.5, 73.2, 73.5, 73.7, 75.3, 75.3, 75.4, 75.8, 76.9, 77.3, 79.8, 80.9,
99.9, 103.2, 103.8, 116.1, 122.7, 124.7, 126.1, 142.1, 158.1; HRMS
ESÀ [MÀH]À Calcd 658.1386, Found 658.1392.
tal (684
l
L, 4.545 mmol) via syringe. The reaction was heated to
L, 0.62 mmol) was
50 °C and stirred for 14 h. Triethylamine (86
l
added and the reaction cooled to room temperature. The crude
reaction mixture was absorbed onto silica gel and semi-purified
by flash chromatography. The fractions obtained containing the de-
sired product were combined and further purified by recrystallisa-
tion from ACN/H2O.
3.2.1.4.
2-Chloro-4-nitrophenyl-cellotetraoside
(CNPG4).
3.2.2.1.
4,6-O-Benzylidene-4-nitrophenyl-cellobioside
Yield: 32%; mp (H2O) 243–245 °C (dec); 1H NMR (400 MHz
C2D6SO) d 2.89–3.33 (m 6H), 3.24–13.86 (m, 18H), 4.22 (d,
J = 7.78 Hz, 1H(anom)), 4.31 (d, J = 8.03 Hz, 1H(anom)), 4.36 (d,
J = 7.78 Hz, 1H(anom)), 4.59 (t, J = 5.27 Hz, 1H), 4.62–4.71 (m, 4H),
4.73 (br s, 1H), 4.78 (br s, 1H), 4.98 (d, J = 5.02 Hz, 1H), 5.02 (d,
J = 4.77 Hz, 1H), 5.22 (d, J = 4.77 Hz, 1H), 5.31 (d, J = 7.53 Hz,
1H(anom)), 5.41 (d, J = 3.77 Hz, 1H), 5.42 (d, J = 3.76 Hz, 1H), 5.64
(BzNPG2). Yield: 58%; mp (H2O) 266–267 °C (dec); 1H NMR
(400 MHz C2D6SO) d 3.07–3.23 (m, 1H), 3.27–3.84 (m, 10H), 4.13–
4.28 (m, 1H), 4.55 (d, J = 6.53 Hz, 1H(anom)), 4.67 (br s, 1H), 4.76
(br s, 1H), 5.17 (d, J = 7.03 Hz, 1H(anom)), 5.44 (br s, 1H), 5.60 (app
br s, 2H), 5.67 (br s, 1H), 7.24 (d, J = 7.53 Hz, 2H), 7.31-7.52 (m, 5H),
8.23 (d, J = 7.53 Hz, 2H); 13C NMR (100 MHz, C2D6SO) d 60.0, 66.5,
68.2, 73.3, 73.5, 74.8, 74.8, 75.7, 78.6, 80.8, 99.9, 101.2, 103.5,

16
B. V. McCleary et al. / Carbohydrate Research 385 (2014) 9–17
117.0, 117.0, 126.2, 126.2, 126.8, 126.8, 128.5, 128.5, 129.4, 138.1,
10 min. The reaction was terminated by adding 3 mL of 2% triso-
dium phosphate (pH 11.0) or 3 mL of 2% Tris (pH 9). The tube con-
tents were well mixed and the absorbance measured at 400 nm
against a reagent blank. One Unit of activity is the amount of en-
zyme required to release one micromole of 2-chloro-4-nitrophen-
olate per minute at 40 °C and the pH used in the particular assay.
142.2, 162.7.; HRMS ESÀ [MÀH]À Calcd 550.1561, Found 550.1556.
3.2.2.2. 4,6-O-Benzylidene-2-chloro-4-nitrophenyl-cellobioside
(BzCNPG2).
Yield: 57%; mp (H2O) 236–237 °C (dec); 1H NMR
(400 MHz C2D6SO) d 3.07–3.24 (m, 1H), 3.25–3.85 (m, 10H),
4.11–4.30 (m, 1H), 4.56 (d, J = 7.78 Hz, 1H (anom)), 4.71 (d,
J = 3.01 Hz, 1H), 4.79 (t, J = 5.51 Hz, 1H), 5.32 (d, J = 7.52 Hz, 1H
(anom)), 5.46 (d, J = 3.95 Hz, 1H), 5.53–5.65, (m, 2H), 5.71 (d,
J = 4.96 Hz, 1H), 7.27–7.60 (m, 6H), 8.22 (dd, J = 9.31, 2.78 Hz,
1H), 8.35 (d, J = 2.78 Hz, 1H); 13C NMR (100 MHz, C2D6SO) d 59.6,
66.1, 67.9, 73.0, 73.1, 74.5, 74.6, 75.5, 78.1, 80.5, 99.7, 100.8,
103.2, 115.8, 122.4, 124.4, 125.7, 126.5, 126.5, 128.2, 128.2,
129.1, 137.8, 141.7, 157.8; HRMS ESÀ [MÀH]À Calcd 584.1171,
Found 584.1168.
3.5. Standard assay for endo-1,4-b-glucananse using CM-
cellulose 4 M (10 mg/mL) by reducing sugar determination
To a pre-equilibrated aliquot of CM-cellulose 4 M (0.5 mL,
10 mg/mL) in 100 mM sodium acetate buffer (pH 4.5) or sodium
phosphate, pH 6.0, a pre-equilibrated aliquot of endo-1,4-b-glucan-
ase was added. The resulting mixture was agitated on a vortex stir-
rer and incubated at 40 °C. Incubations were terminated by the
addition of Nelson–Somogyi solution C4,21 (0.5 mL) at 0, 3, 6, 9
and 12 min. The colour was developed according to the Nelson–
Somogyi procedure4,21 and incubation tubes centrifuged at
1500 g for 10 min to remove insoluble CM-cellulose 4 M. The
absorbance was measured at 520 nm against a substrate/reagent
3.2.2.3. 4,6-O-Benzylidene-2-chloro-4-nitrophenyl-cellotrioside
(BzCNPG3).
Yield: 49%; mp (H2O) 230–232 °C (dec); 1H NMR
(400 MHz C2D6SO) d 3.00–3.18 (m, 2H), 3.21–3.87 (m, 15H), 4.09–
4.25 (m, 1H), 4.36 (d, J = 7.78 Hz, 1H(anom)), 4.49 (d, J = 7.78 Hz,
1H (anom)), 4.51 (d, J = 1.75 Hz, 1H), 4.67 (t, J = 5.35 Hz, 1H), 4.73
(t, J = 5.32 Hz, 1H), 4.80 (br s, 1H), 5.32 (d, J = 7.53 Hz, 1H(anom)),
5.35–5.48 (m, 2H), 5.53 (d, J = 4.52 Hz, 1H), 5.57 (s, 1H), 5.64 (d,
J = 4.52 Hz, 1H), 7.29–7.53 (m, 6H), 8.20 (dd, J = 9.16, 2.64 Hz, 1H),
8.32 (d, J = 2.51 Hz, 1H); 13C NMR (100 MHz, C2D6SO) d 60.2, 60.6,
66.4, 68.1, 73.2, 73.3, 73.6, 74.7, 74.7, 75.4, 75.5, 75.8, 79.4, 79.8,
80.8, 99.9, 101.1, 103.3, 103.6, 116.1, 122.7, 124.7, 126.1, 126.8,
126.8, 128.5, 128.5, 129.4, 138.1, 142.0, 158.1; HRMS ES+ [M+Na]+
Calcd 770.1675, Found 770.1679.
blank and a glucose standard solution (50 lg) was included. One
unit of activity is the amount of enzyme required to release one
micromole of glucose reducing sugar equivalents per minute at
40 °C and the pH used in the particular assay.
3.6. Assay of endo-1,4-b-glucananse on CNP-cello-oligomers and
BzCNP-cello-oligomers
The mode of action of A. niger and T. longibrachiatum endo-1,4-b-
glucanase on CNPG3 and CNPG4 was studied by incubating 0.1 mL
of the endo-1,4-b-glucanase (0–6.4 mU; A. niger; Fig. 1) (0–0.27 U;
T. longibrachiatum; Fig. 2) with 0.2 mL of CNPG3 or CNPG4 (10 mg/
mL) in 10 mM sodium acetate buffer (pH 4.5) for 10 min at 40 °C.
The reaction was terminated by heating the reaction tubes at
ꢀ100 °C for 3 min and the tube contents were transferred to a
microfuge tube and centrifuged at 12,000 rpm for 6 min. An aliquot
3.2.2.4. 4,6-O-Benzylidene-2-chloro-4-nitrophenyl-cellotetrao-
side (BzCNPG4).
Yield: 31%; mp (H2O) 235–238 °C (dec); 1H
NMR (400 MHz C2D6SO) d 3.00–3.17 (m, 3H), 3.20–3.87 (m, 20H),
4.12–4.21 (m, 1H), 4.31 (d, J = 7.38 Hz, 1H (anom)), 4.36 (d,
J = 7.01 Hz, 1H (anom)), 4.48 (d, J = 7.47 Hz, 1H (anom)), 4.52 (br
s, 1H), 4.63–4.78 (m, 4H), 4.81 (br s, 1H), 5.31 (d, J = 7.46 Hz, 1H
(anom)), 5.37–5.49 (m, 3H), 5.51–5.56, (m, 2H), 5.66 (br s, 1H),
7.23–7.54, (m, 6H), 8.20, (d, J = 8.84 Hz, 1H), 8.32 (br s, 1H); 13C
NMR (100 MHz, C2D6SO) d 60.2, 60.5, 60.8, 66.4, 68.1, 73.2, 73.3,
73.5, 73.6, 74.7, 74.7, 75.2, 75.3, 75.4, 75.4, 75.7, 79.4, 79.8, 80.7,
80.7, 99.9, 101.1, 103.2, 103.4, 103.5, 115.7, 122.2, 124.2, 125.6,
126.3, 126.3, 128.0, 128.0, 128.8, 137.7, 141.6, 157.6; HRMS ESÀ
[MÀH]À Calcd 908.2227, Found 908.2227.
(50 lL) was analysed directly by HPLC using a Waters Sugar-Pac
Column (6 Â 300 mM), 90 °C, with distilled water containing dical-
cium EDTA as mobile phase at 0.5 mL/min. A Breeze HPLC system
was used incorporating a Waters 2410 RI detector and Empower
2 software.
The relative rates of hydrolysis of NPG2, CNPG2, BzNPG2 and
BzCNPG2 by T. longibrachiatum endo-1,4-b-glucanase (Fig. 3) in-
volved the incubation of 0.1 mL of the substrates at 3 mM in the
presence of b-glucosidase (0.66 U) with 0.1 mL of endo-1,4-b-glu-
canase at 40 °C. Reaction was terminated at 0, 2, 4, 6, 8 and
10 min by adding 3 mL of 2% Tris solution (pH 10) with mixing.
The absorbance values were read at 400 nm against the zero time
reading for the respective substrate. Activity was calculated using
3.3. Dissolution of oligosaccharide substrates
NPG2, CNPG2, CNPG3 and CNPG4 were dissolved directly in
100 mM KCl solution to give a concentration of 3 mM. BzNPG2,
BzCNPG2, BzCNPG3 and BzCNPG4 were dissolved at a concentration
of 9.15 mM in dimethylsulfoxide (DMSO) and stored at À20 °C be-
tween use. In this form they are stable for >4 years. For use as sub-
strates, 3 mL of these solutions were added to 6 mL of 100 mM KCl
the extinction coefficient of CNP of 16,600 MÀ1 cmÀ1
.
The effect of the presence of saturating levels of b-glucosidase,
and its absence, on the rate of increase in absorbance at 400 nm
on hydrolysis of BzCNPG2 and BzCNPG3 by endo-1,4-b-glucanase
was studied by incubating 0.1 mL of oligosaccharide substrate
(3 mM; containing 0 or 0.66 U b-glucosidase per 0.1 mL substrate)
with 0.1 mL of T. longibrachiatum (Fig. 7a and b) or A. niger (Fig. 7c
and d) endo-1,4-b-glucanase in 100 mM sodium acetate buffer (pH
4.5) at 40 °C. Reaction was terminated at 0, 3, 6, 9 and 12 min by
adding 3 mL of 2% Tris solution (pH 9).
To determine the effect of substrate concentration (Fig. 6) on
the rate of hydrolysis of BzCNPG3 by T. longibrachiatum endo-1,4-
b-glucanase, 0.1 mL of BzCNPG3 (0.66–3.0 mM) in the presence of
b-glucosidase (0.66 U) was incubated with 0.1 mL of T. longibrachi-
atum endo-1,4-b-glucanase (170 mU on this substrate) in 100 mM
sodium acetate buffer (pH 4.5) at 40 °C. Reaction was terminated
at 0, 3, 6, 9 and 12 min by adding 3 mL of 2% Tris solution (pH
and mixed thoroughly. Thermostable b-glucosidase (50 lL, 400 U/
mL) was then added to 3 mL aliquots of the resulting 33% DMSO
solution, mixed by swirling and stored at À20 °C between use. In
this form, they were stable for >2 years.
3.4. Standard assay of endo-1,4-b-glucanase using 3 mM
BzCNPG3 in the presence of b-glucosidase (8.2 U/mL) (CellG3
assay)
To a pre-equilibrated aliquot of substrate solution (CellG3)
(0.1 mL, 3 mM) containing thermostable b-glucosidase (0.66 U), a
pre-equilibrated aliquot of endo-1,4-b-glucanase in 100 mM
sodium acetate buffer, pH 4.5 (0.1 mL) or sodium phosphate, pH
6.0 was added and the mixture incubated at 40 °C for exactly

B. V. McCleary et al. / Carbohydrate Research 385 (2014) 9–17
17
10) and absorbance measured at 400 nm. The effect of b-glucosi-
dase concentration on the rate of increase in absorbance at
400 nm was determined by preparing solutions containing 3 mM
BzCNPG3, plus b-glucosidase (3.2–8.2 U/mL) and was determined
as follows (Fig. 4): aliquots (0.1 mL) of the solutions were incu-
bated with 0.1 mL of T. longibrachiatum endo-1,4-b-glucanase
(170 mU/mL on Cell G3) at 40 °C and reaction terminated at 0, 3,
6, 9 and 12 min by adding 3 mL of 2% Tris solution (pH 9). The rel-
ative rates of hydrolysis of BzCNPG2, BzCNPG3 and BzCNPG4 by A.
niger and T. longibrachiatum endo-1,4-b-glucanase was determined
by incubating 0.1 mL of substrate (3 mM, containing b-glucosidase
at 6.6 U/mL) with 0.1 mL of A. niger endo-1,4-b-glucanase
(0–180 mU/assay on CM-cellulose 4 M) or T. longibrachiatum
endo-1,4-b-glucanase (0–57 mU/assay on CM-cellulose 4 M)
(Fig. 8).
in the assay increased by just 0.013 and 0.025, respectively over
the same period. In the assay, the substrate behaved exactly as in
the freshly prepared reagent solution.
Acknowledgements
The authors thank Dr. William York, Complex Carbohydrate Re-
search Centre, The University of Georgia, Athens, Georgia, USA and
Dr. Hugues Driguez, Centre de Recherche sur les Macromolecules
Vegetales, CERMAV, Grenoble, France for valuable discussion and
suggestion. We also thank Stephanie Pradeau Centre de Recherche
sur les Macromolecules Vegetales, CERMAV, Grenoble, France for
her technical assistance.
Supplementary data
Action of T. maritima thermostable endo-1,4-b-glucanase on
BzCNPG3 was determined by incubating 0.1 mL of BzCPNG3
(3 mM, containing b-glucosidase at 6.6 U/mL) with 0.1 mL of T.
maritima endo-1,4-b-glucanase (0–50 mU/assay on CMC4 M in pH
6 100 mM sodium phosphate) at 80 °C for 10 min. The reaction
was terminated by the addition of 3 mL of tri-sodium phosphate
solution (pH 11.0) and the tubes immediately placed into an ice
water bath. They were warmed to ꢀ20 °C before measuring absor-
bance at 400 nm (Fig. 9).
Longer term stability of BzCNPG3 in the presence of b-glucosi-
dase (CellG3 substrate mixture) was determined by storing the
substrate mixture at À20, 4 and 20 °C for up to 100 days. After
the various time intervals, stability was determined by running a
standard assay by mixing 0.1 mL of the substrate mixture with
0.1 mL of T. longibrachiatum endo-1,4-b-glucanase (20.8 mU on
CellG3) for 10 min at 40 °C and stopping the reaction with 3 mL
of 2% Tris solution (pH 10). Changes in blank absorbance values
were determined by adding 3 mL of 2% Tris solution (pH 9) to
0.1 mL of the substrate solution and 0.1 mL of the relevant buffer
solution, mixing well and measuring the absorbance at 400 nm
(Fig. 10). Clearly, the substrate mixture is very stable under each
of the storage conditions. On storage at 20 °C, the absorbance of
the blank solution in the assay (no enzyme) increased from 0.013
to 0.078 over 45 days (i.e., sixfold), but the assay absorbance in-
creased by the same amount such that the determined enzyme
activity was the same. This increase in absorbance value represents
a degradation of approximately 4% of the substrate in the assay
mixture. At À20 °C and 4 °C, the absorbance of the blank solution
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.carres.2013.12.
001.
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