Enzyme and Microbial Technology 79–80 (2015) 42–48

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Enzyme and Microbial Technology

journal homepage: www.elsevier.com/locate/emt

Accessory enzymes influence cellulase hydrolysis of the model

substrate and the realistic lignocellulosic biomass
Fubao Fuebiol Sun a,∗ , Jiapeng Hong a , Jinguang Hu b , Jack N Saddler b , Xu Fang c ,
Zhenyu Zhang a , Song Shen a
a
Key Laboratory of Carbohydrate Chemistry and Biotechnology & Key Laboratory of Industrial Biotechnology, Ministry of Education, School of
Biotechnology, Jiangnan University, Wuxi 214122, China
b
Forestry Products Biotechnology/Bioenergy Group, Wood Science Department, University of British Columbia, 2424 Main Mall, Vancouver BC, V6T 1Z4,
Canada
c
State Key Laboratory of Microbial Technology, Shandong University, Jinan 250100, China

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

Article history: The potential of cellulase enzymes in the developing and ongoing “biorefinery” industry has provided a
Received 9 January 2015 great motivation to develop an efficient cellulase mixture. Recent work has shown how important the
Received in revised form 24 June 2015 role that the so-called accessory enzymes can play in an effective enzymatic hydrolysis. In this study,
Accepted 25 June 2015
three newest Novozymes Cellic CTec cellulase preparations (CTec 1/2/3) were compared to hydrolyze
Available online 17 July 2015
steam pretreated lignocellulosic substrates and model substances at an identical FPA loading. These cel-
lulase preparations were found to display significantly different hydrolytic performances irrelevant with
Keywords:
the FPA. And this difference was even observed on the filter paper itself when the FPA based assay was
Accessory enzyme (xylanase and AA9)
Cellulase preparations (Cellic® CTec1/2/3)
revisited. The analysis of specific enzyme activity in cellulase preparations demonstrated that different
Filter paper activity (FPA) accessory enzymes were mainly responsible for the discrepancy of enzymatic hydrolysis between diver-
Enzymatic hydrolysis sified substrates and various cellulases. Such the active role of accessory enzymes present in cellulase
Cellulase mixtures/cocktails preparations was finally verified by supplementation with ␤-glucosidase, xylanase and lytic polysaccha-
ride monooxygenases AA9. This paper provides new insights into the role of accessory enzymes, which
can further provide a useful reference for the rational customization of cellulase cocktails in order to
realize an efficient conversion of natural lignocellulosic substrates.
© 2015 Elsevier Inc. All rights reserved.

1. Introduction achieve an efficient enzymatic co-hydrolysis of the cellulose and

One limiting step in an enzyme based biorefinery process is
the fast, complete hydrolysis of the cellulose to monomeric sug-
ars, which can be subsequently converted to a wide range of fuels,
chemicals and biomaterials [1]. To improve the hydrolytic effi-
ciency of cellulase enzyme preparations, considerable attempts
have traditionally focused on their component cellulases because
cellulose is the most abundant polysaccharide constituent in lig-
nocellulose. However, it is now recognized that it is necessary to
Abbreviations: AA9, auxiliary activity family 9; BG, ␤-glucosidase; CTec1, Cellic®
CTec1; CTec 2, Cellic® CTec2; CTec 3, Cellic® CTec3; EX, endoxylanase; FPA, filter
paper activity; FPU, filter paper unit; Htec, Cellic® Htec; LPMOs, lytic polysaccharide
monooxygenases; SPCS, SO2 steam pretreated corn stalk; SPP, SO2 steam pretreated
poplar; SPSSB, SO2 steam pretreated sweet sorghum bagass.
∗ Corresponding author at: School of Biotechnology, Jiangnan University; 1800
Lihu Road, Wuxi 214122, China.
E-mail addresses: fubaosun@jiangnan.edu.cn, sunfubao@hotmail.com (F.F. Sun).
hemicellulose present in pretreated lignocellulosic substrates to
fermentable sugars, which requires a supplementation of the cel-
lulases with some accessory enzymes [2–4].
Recent work has shown how important the role that the
so-called accessory enzymes can play in the effective enzy-
matic hydrolysis [5–8]. These accessory enzyme activities can
improve the performance of enzyme preparations even when
not directly involved in the hydrolysis of cellulose. For example,
␤-glucosidase activity is of uttermost importance to avoid accu-
mulation of cellobiose and thus severe inhibition of the cellulases
[9–11]. Hemicellulases and pectinases can hydrolyze their respec-
tive non-cellulosic polysaccharide substrates that coat cellulose
fibers, and thus assist in the hydrolysis of cellulose to boost a
release of fermentable sugars from lignocellulosic biomass [4,12].
A xylanase-boosting effect has been observed on a range of pre-
treated lignocellulosic materials, regardless of their xylan content
[7]. Another newly found variety, lytic polysaccharide monooxyge-
nases (LPMOs) Auxiliary Activities Family 9 (AA9, formerly known

http://dx.doi.org/10.1016/j.enzmictec.2015.06.020
0141-0229/© 2015 Elsevier Inc. All rights reserved.
 F.F. Sun et al. / Enzyme and Microbial Technology 79–80 (2015) 42–48
as GH 61) can greatly enhance cellulose accessibility by disrupting
the crystalline cellulose structure [1,13,14], resulting in significant
improvements to the hydrolytic performance of cellulase enzymes.
These accessory enzymes can contribute significantly to the effec-
tiveness of cellulase preparations, and improve sugar release to
varying degrees depending on the type of substrate and pretreat-
ment [2,15]. Therefore, it is of interest to make more efforts to the
cellulase supplementation with accessory enzymes.
Very recently, some researchers have also suggested a non-
neglectable effect of accessory enzymes made in measuring the
enzyme activity with the model substrate [2,16,17]. And the exis-
tence of accessory enzymes resulted in a significant deviation
at real hydrolytic efficiency of cellulase preparations from the
standardized assay. Kabel et al. [16] showed that the activity
analyzed in the standard cellulase/xylanase test did not present
a high correlation with the corresponding components degra-
dation of wheat bran and grass. Several commercial cellulase
preparations exhibited different degradations on wheat bran/grass
cellulose as they had different accessory enzyme activities. Like-
wise, it was concluded by Pryor and Nahar [17] that the accessory
enzyme (i.e., inherent xylanase and pectinase) activities were at
least one of the main reasons that standardized protocols for
measuring enzyme activity are not adequate for assessing activ-
ity using pretreated lignocellulosic substrates. Notably, Berlin
et al. evidenced the active role of accessory enzymes in cellulase
hydrolysis of natural lignocellulosic substrates, arguing that the
enzyme preparations with similar cellulase activity would show
differences in performance on lignocellulose if they differ in acces-
sory enzyme composition [2,15]. Considering that the commonly
used filter paper activity defines a combination of the endoglu-
canase, the cellubiohydrolase and at most the ␤-glucosidase
activities, some activities of accessory enzymes/proteins can-
not be measured by the standardized FPA assay. It is logic
to infer that these accessory enzymes should have exerted an
active role in the cellulase activity assay, which is neglected by
FPA.
To elucidate these, in this study, three leading Novozymes
cellulase preparations, Cellic® CTec1/2/3, were compared in the
practical hydrolysis of natural lignocellulosic biomass. To ascertain
the uttermost effect of accessory enzymes, the hydrolytic per-
formance of these cellulase preparations was carefully assessed
by revisit of the FPA assay protocol. Eventually, the role
of accessory enzymes was checked out by supplementation
of the preparation with some essential accessory enzymes
such as xylanase and lytic polysaccharide monooxygenases
(LPMOs).
2. Materials and methods
2.1. Lignocellulosic biomass and cellulase enzymes preparation
Poplar, corn stover and sweet sorghum bagasse were steam pre-
treated at different conditions (200 ◦ C, 5 min; 200 ◦ C, 5 min; 180 ◦ C,
5 min), respectively, as described by Bura et al. [18]. Prior to steam
explosion, the dry substrate chips were impregnated with SO2 in
the plastic bags at room temperature overnight. SO2 was absorbed,
in which the absorption of SO2 was app. 3% weight of dried sub-
strate. Commercial Cellulase preparations, Cellic® CTec1 (CTec1),
Cellic® CTec2 (CTec2) and Cellic® CTec3 (CTec3), and endoxylanase
preparation, Cellic® Htec (Htec) were supplied by Novozymes
(Franklington, NC). Beta-glucosidase preparation, Novozyme 188,
and LPMOs preparation, AA9, were kind gifts from Novozymes A/S
(Bagsværd, Denmark).
43
2.2. Enzymatic hydrolysis
Batch enzymatic hydrolysis experiments were conducted in
10 mL total volume in a 50 mL glass bottle with a rubber plug-in
and round aluminum cap sealed at 2% substrate consistency [(g dry
substrate) × (g dry substrate + g water)−1 ] in 50 mmol L−1 acetate
buffer at pH4.8. Bottles and substrates were pre-incubated at 50 ◦ C
in a shaker (MAXQ 4000, Barnstead Line-lab) for 20 min before
the enzyme addition. For the hydrolysis of different lignocellulosic
substrates (SO2 steam pretreated poplar (SPP) and sweet sorghum
bagasse (SPSSB)), the cellulases were added at 14.2 FPU and 20 mg
protein per gram cellulose. For the complement of cellulase with
some accessory enzyme, different amounts of hemicellulase (1.0,
2.0, 4.0 and 8.0 mg protein g−1 cellulose), ␤-glucosidase (1.0, 2.0,
4.0 and 8.0 mg protein g−1 cellulose) and LPMOs (0.2, 0.5, 1.0 and
2.0 mg protein g−1 cellulose) were added, respectively. The cellu-
lase hydrolysis was performed at 50 ◦ C at a shaking speed of 150
rotations per minute. At an interval time, 500 ␮L hydrolytic slurry
was withdrawn and deactivated on a hot plate at 100 ◦ C for 10 min,
followed by centrifugation at 13,000 × g for 10 min, and subse-
quently the supernatant was stored at −20 ◦ C until sugar analysis
was made. A prolonged hydrolysis of filter paper was made com-
pletely in terms of the standard protocol of NREL, in which each
sampling was taken for a set of test tubes alone for the sugar
analysis by 3,5-dinitrosalicylic acid method [19]. For a hydroly-
sis of SO2 catalyzed steam pretreated corn stalk (SPCS), it was
devised only with the SPCS (50.0 mg dried substrate) instead of fil-
ter paper based on the standard protocol, in which each cellulase
loading (mg protein/g cellulose) was the same to that on the filter
paper.
2.3. Analytical method
The substrates were analyzed for acid insoluble lignin and car-
bohydrates using the Tappi-T-22 om-88 as previously described
[20]. The hydrolysate from this analysis was retained and ana-
lyzed for soluble lignin by reading the absorbance at 205 nm.
Sugars were measured on Dionex (Sunnyvale, CA) HPLC (ICS-3
000) equipped with an AS 50 auto sampler, ED50 electrochem-
ical detector, GP 50 gradient pump and anion exchange column
(Dionex-CarboPac PA1). Acetone soluble extractives were esti-
mated using Tappi T 204 om-88 with the following modifications.
Briefly, 10 g of air-dried sample was extracted for 8 h with ace-
tone with 6 cycles/h. The acetone in the round-bottomed flask is
then evaporated in the fume hood and then dried in the oven at
100 ◦ C overnight to determine the weight of extractives present in
the sample flasks. The filter paper activity was determined accord-
ing to the NREL laboratory analytical procedure [19]. Xylanase and
CMCase activities were determined as described elsewhere [21].
Cellobiohydrolase I, ␤-xylosidase and ␤-glucosidase activities were
determined using p-nitrophenyl-␤-d-cellobioside, p-nitrophenyl-
␤-d-xylopyranoside, and p-nitrophenyl-␤-d-glucopyranoside as
substrates, respectively, as described previously [21]. Protein
concentration was measured using the ninhydrin assay using
BSA as the protein standard [22]. In addition to the hydrol-
ysis based on FPA assay using the 3,5-dinitrosalicylic acid to
detect reducing sugar contents, other enzymatic hydrolysis (%)
was calculated based on the glucose content detected with YSI
Biochemistry Analyzer (2700 Select). All the FPA based assays
were made for 6 to 8 times. All hydrolysis experiments were
performed at least in triplicate, and mean values are pre-
sented.
44 F.F. Sun et al. / Enzyme and Microbial Technology 79–80 (2015) 42–48

100 enzymes at the same FPA would present a consistent hydroly-

sis performance on the same substrate. Similar observations have
80 also been made by other researchers [15,16]. For example, Kabel
Glucan hydrolysis (%)

et al. [16] tested fourteen commercial enzymes for their cellu-

60 lase/ xylanase activity, claiming that these enzyme preparations
presented a big difference between standardized activity and
40
the actual hydrolytic performance on grass cellulose and wheat
bran xylan. Since these CTec preparations are all derived from
CTec1 CTec2 Novozymes, it is evident that the product upgrade was not only
20
CTec3 CTec1- achieved by improving the protein expression, but also by mixing
and matching various enzymes as accessory enzymes to strengthen
0
their synergistic cooperation on the lignocellulosic hydrolysis.
0 8 16 24 32 40 48
a) Enzymac hydrolysis me (h) 3.2. Characterization on specific enzyme activity of cellulase
preparations

100
To confirm it, three cellulase preparations, CTec1/2/3, were ana-
CTec1 CTec2 lyzed at FPA, protein content and specific enzyme activities as
80 CTec3 CTec1- shown in Table 1. As expected, these enzyme preparations dis-
Glucan hydrolysis (%)

played a considerable difference at the FPA and protein content.

60
The FPA of CTec3 was 165 FPU per gram of enzyme preparation,
which was almost 7 times that of CTec1 (23 FPU g−1 ) and 1.2 times
that of CTec2. Meantime, the protein content of CTec3 was 230 mg
40 protein per gram of enzyme preparation, while CTec1 and CTec2
were 160 mg g−1 and 190 mg g−1 , respectively. With specific activ-
20 ity of the preparation calculated on the protein content, there were
high specific enzyme activities in both CTec2 and CTec3 prepara-
tions as compared to the CTec1. The CTec1 was at 0.14 FPU mg−1
0
0 4 8 12 16 20 24 28 32 36 40 44 48 52 protein, while CTec2 reached almost an equivalent level as CTec3,
which were both at 0.71 FPU mg−1 protein. Notably, the CTec2 had
b) Enzymac hydrolysis me (h) a highest specific activity of the xylanase (21.2 U mg−1 ) and CMCase
(2.3 U mg−1 ). And the CTec3 contained a highest ␤-glucosidase
Fig. 1. Enzymatic hydrolysis (pH4.8, 150 rpm, 50 ◦ C) of 2% (w/w) SPP 200 and SPSSB
with various cellulase enzymes, respectively. The ‘CTec1’ means an enzyme load- (10.2 U mg−1 ), Cellobiohydrolase I (3.1 U mg−1 ) and ␤-xylosidase
ing of 14.2 FPU/g substrate; the ‘CTec2’ and ‘CTec3’ both mean enzyme loadings of (1.2 U mg−1 ) activity. That is, compared with the CTec2, there was
14.2 FPU/g substrate and 20 mg protein g substrate; the ‘CTec1-’ means an enzyme a relatively low xylanase and CMCase activity in the CTec3 prepa-
loading of 20 mg protein/g substrate. ration, decreasing 30% and 45%, respectively. Remarkably, a large
number of ␤-glucosidase, cellobiohydrolase I and especially ␤-
3. Results and discussion xylosidase were added in CTec3 preparation, resulting in a big
increase at 150%, 300% and 450%, respectively.
3.1. Hydrolysis of various lignocellulosic substrate with cellulase These results have showed that the cellulase preparation was
preparations tailored gradually in the variety and proportion of enzyme proteins
in order to achieve a better hydrolysis performance on lignocellu-
Three cellulase preparations were assessed on steam pre- losic biomass. In the current cellulase preparation, there exists large
treated poplar and sweet sorghum bagasse during time course of accessory non-cellulase enzymes (␤-glucosidase, xylanase and ␤-
hydrolysis, as shown in Fig. 1. With the same enzyme loading, xylosidase), whose activity cannot be detected with the common
CTec3 resulted in 94% glucan hydrolysis of pretreated poplar at FPA that is used at most to predict hydrolytic potential of the
24 h, which was higher than those reached by CTec1 (86%) and canonical cellulase enzymes such as CBH and EG. Accordingly, it
CTec2 (80%). When acting on the steam pretreated sweet sorghum is evident that some accessory enzymes and/or disrupting proteins
bagasse, CTec3 reached 66% of glucan hydrolysis (24 h) while only which have significant effects on lignocellulosic hydrolysis might
47% for CTec1 and 55% for CTec2, respectively. With an addition be neglected by the common FPA assay. These accessory enzymes
of CTec1 at the same protein level as CTec2 and CTec3, the glu- which might not be reflected by FPA can synergistically cooperate
can hydrolysis of pretreated poplar and sweet sorghum bagasse with canonical cellulase enzymes for hydrolysis of lignocellulosic
was only up to 38% and 20%, respectively. The result has indicated substrates [1]. This also helps to explain our and other’s findings
that the older cellulase preparation, CTec1, has a relatively lower that the FPA of cellulase enzymes did not reflect well their actual
hydrolytic efficacy than the new products, CTec2 and CTec3 on hydrolytic potential to different lignocellulosic substrates [16,17].
pretreated lignocellulosic substrate. In addition, the CTec3 is more
efficient than CTec2 whatever at the same specific enzyme loading 3.3. Prolonged hydrolysis of filter paper based on FPA assay
and enzyme activity.
The result showed that, at a fixed enzyme loading, the three Since the existence of accessory enzymes in the cellulase prepa-
cellulase preparations presented surprisingly different hydrolytic ration can greatly affect the enzymatic hydrolysis of lignocellulosic
profiles on the different lignocellulosic substrates. Here, the fixed substrates, it is of interest to study whether these accessory
enzyme loading represented as below: firstly, one cellulase prepa- enzymes have some influence on the FPA assay based hydrolysis of
ration had different hydrolytic potential on different biomass filter paper. Accordingly, in this experiment, the standardized FPA
substrates; secondly, different cellulase preparations also exhib- protocol was revisited. To follow the analytical procedure of FPA,
ited different hydrolytic performance on the same substrate [17]. 0.5 mL diluted cellulase enzyme preparations, namely 0.1850 FPU,
The result seems contradictory to the expectation that cellulase was used for hydrolyzing 50 mg filter paper (Fig. 2). As expected,
 F.F. Sun et al. / Enzyme and Microbial Technology 79–80 (2015) 42–48 45

Table 1
Chemical composition of pretreated lignocellulosic substrates.

Substrate Composition of pretreated feedstock (%)

Glucan Xyl Ara Gal Man AIL ASL Moisture

FP 99.9% None None None None None None 6.5 ± 0.4

SPP200 52.4 ± 1.6 6.3 ± 0.4 0.10 ± 0.01 0.40 ± 0.03 1.20 ± 0.07 31.6 ± 2.2 2.3 ± 0.9 61.1 ± 0.6
SPSSB 55.3 ± 1.7 4.4 ± 0.3 0.30 ± 0.02 0.10 ± 0.01 0.50 ± 0.04 35.5 ± 2.6 1.6 ± 1.1 76.9 ± 0.8
SPSC180 46.3 ± 1.4 19.8 ± 1.2 1.64 ± 0.11 0.65 ± 0.05 1.05 ± 0.06 24.1 ± 1.7 2.9 ± 0.9 71.7 ± 0.6

80

70

60
Glucan hydrolysis (%)

50

40

30

20 CTec1
CTec2
10 CTec3

0
0 12 24 36 48 60 72 84 96 108 Fig. 3. FPA based assay of three cellulase preparations with 50.0 mg filter paper at
50 ◦ C in a pH4.8 sodium citrate buffer solution at an shaking speed of 150 rpm.
Enzymac hydrolysis me (h)

Fig. 2. FPA based enzymatic hydrolysis of 50.0 mg filter paper with CTec1/2/3 at
enzyme loadings of 0.185 FPU in the standard FPA assay condition. The hydrolysis
was performed at 50 ◦ C in a pH4.8 sodium citrate buffer solution in a static state.
three cellulase enzyme stocks, CTec1/2/3, all produced 2.0 mg of
reducing sugars from 50 mg filter paper and gave the same hydrol-
ysis extent (3.6%) at the rigid 1 h hydrolysis. With the hydrolysis
time extending, however, the hydrolysis of filter paper showed
a different profile. The CTec1/2/3 presented significantly different
hydrolysis performances, resulting in 40%, 55% and 70% conversion
of filter paper after 96 h, respectively. Results showed that the FPA
of three preparations acting on a standardized assay condition did
not predict the long-time hydrolysis. To our knowledge, there is no
literature that has yet described this phenomenon. These results
indicate that, with the extra introduction of accessory enzymes in
the cellulase preparation, the FPA can only formulate the hydrolytic
activity of some enzyme preparation with different dilution. The
accessory enzymes present in the enzyme preparation also affected
the FPA itself.
data showed that the shaking activity of three cellulase prepa-
rations increased to a different degree. The oscillation enhanced
the activity of CTec3 greatly, thus a low enzyme loading for the
production of equal reducing sugars. The result indicates that the
accessory enzymes can contribute to a high enzymatic hydroly-
sis of filter paper at the shaking condition. Also, the contribution
can be made to a different degree with the different variety and
concentration of every accessory enzyme in cellulase preparations.
Given that the practical enzymatic hydrolysis of ‘real life’ lignocel-
lulosic substrate is engineered at the shaking condition to facilitate
enzyme catalytic reaction, it is within the expectation that vari-
ous cellulase preparations at an identical dosage would probably
release different fermentative sugars from the substrate, owing to
the different variety or/and concentration of accessory enzymes.
This at least can partly explain why the above hydrolysis of various
lignocellulosic substrates was different with cellulase preparations
at the same loading.
3.5. Hydrolysis of SPCS 180 similar to FPA assay

3.4. Shaking hydrolysis of filter paper based on FPA assay
The FPA assay based experiment was further carried out at a
shaking condition to assess the effect of accessory enzymes on the
filter paper hydrolysis. In terms of the standardized analytical pro-
cedure of FPA, properly diluted cellulase preparations of 0.5 mL
were added to 50.0 mg filter paper for an accurate 60 min hydroly-
sis. Then the two data points that were very close to release 2.0 mg
reducing sugars would be considered, by which a straight line was
drawn then to interpolate the enzyme dilution. As shown in Fig. 3,
with just the release of 2.0 mg reducing sugars at 60 min, the dosage
of three 0.5 mL properly diluted cellulase preparations, CTec1/2/3,
was 0.0985 FPU, 0.1195 FPU and 0.0817 FPU, respectively, which
was far lower than that of 0.1850 FPU determined in the stan-
dardized FPA assay. Correspondingly, the protein loading of each
enzyme dilution reduced 48%, 35% and 56% compared with that
(1321 ␮g, 261 ␮g and 261 ␮g) used in the original FPA assay. These
To further elucidate the effect of accessory enzymes on FPA
assay, the hydrolytic performance of cellulase preparations on the
natural lignocellulosic biomass was characterized in terms of the
standardized analytical procedure of FPA. Fifty milligrams of SPCS
were used as the substrate instead of filter paper strip for hydrolysis
with 0.5 mL diluted cellulase enzyme preparation (0.1850 FPU). The
hydrolysis profile is shown at Fig. 4. At an exact 60 min hydrolysis,
the three cellulase enzymes, CTec1/2/3, released 1.6 mg, 2.6 mg and
2.8 mg reducing sugar, resulting in 4.3%, 6.9% and 7.3% hydrolysis
of SPCS cellulose, respectively. At 96 h, three cellulase preparations
performed different hydrolysis profiles on SPCS, reaching 33%, 40%
and 44% conversion of cellulose, respectively. Results showed that
the hydrolytic ability of cellulase preparations on the natural ligno-
cellulosic substrates was not consistent with that on the filter paper,
even at the standardized FPA assay condition. Unlike the filter
paper, the composition of natural lignocellulosic biomass is com-
plex, in which exist the components that the accessory enzymes
46
60
50
Glucan hydroysis (%)
40
30
20
10
0
0 12 24 36 48 60 72 84
Enzymac hydrolysis me (h)
Fig. 4. FPA based assay of three cellulase preparations with 50.0 mg SPCS 180 alter-
native to the filter paper in the standard assay condition. The total enzyme loading
was at 0.185 FPU (8.4 FPU/g glucan).
hydrolyze selectively as the corresponding substrates. Such the
selective hydrolysis can strengthen the action of cellulase prepa-
ration, and thus a different hydrolytic performance. Therefore, the
different variety and its content of accessory enzymes is at least
partly responsible for the different hydrolysis of lignocellulosic sub-
strate alternative to filter paper during the revisit of FPA assay. In
addition, it is inferred that the attribute of substrate multiplicity
in the composition of lignocellulosic biomass should enable more
than one accessory enzyme to concertedly strengthen the cellulase
hydrolysis of the lignocellulosic biomass to a different degree.
3.6. Composition of the lignocellulosic substrate
Since the complexity of components in lignocellulosic biomass
is at least one of the reasons that accessory enzymes resulted in
the above hydrolytic difference of cellulase preparation, the main
composition of these substrates was analyzed in the ensuing exper-
iment as shown in Table 2. For the filter paper substrate, the
composition was pretty simple, which contained almost pure cellu-
lose. However, in the natural lignocellulosic feedstock, there were
some other components such as hemicellulose and lignin [15,23].
Furthermore, the content of various components was unequal. The
SPSSB had a high glucan (55%) and lignin content (37%), together
with a low hemicellulose content (5%). The SPCS180 contained an
obviously high hemicellulose (23%). Therefore, the composition of
pretreated lignocellulosic feedstock was quite complex, very differ-
ent from the model substrate as such filter paper [12]. Actually, such
the difference in the variety and content of components is depen-
dent on the biomass nature and the pretreatment strategy [24,25].
The composition complexity of substrates intensified by various
pretreatment often results in their different hydrolytic profile even
with using the same enzyme, much less with different cellulase
preparations. The result has supported the above inference and
explained at least partly why the certain cellulase preparation
showed different hydrolysis profile on the different lignocellulosic
substrates.
3.7. Active role of some accessory enzymes on enzymatic
hydrolysis of CTec1
Since the accessory enzyme that cannot be addressed with the
FPA assay had such a positive role on lignocellulosic hydrolysis,
it is possible to verify it by supplementation of some accessory
enzymes during cellulase hydrolysis of pretreated lignocellulosic
F.F. Sun et al. / Enzyme and Microbial Technology 79–80 (2015) 42–48
CTec1
CTec2
CTec3
96 108 120 132
Fig. 5. Direct addition of accessory enzymes with CTec1 to hydrolyze SPSSB. The
hydrolysis was performed at 50 ◦ C in a pH4.8 sodium citrate buffer solution at an
shaking speed of 150 rpm with an enzyme loading of 20 mg protein/g substrate.
The figure (a) means single additions of the endoxylanase (EX) preparation Htec,
␤-glucosidase (BG) preparation Novozyme 188 and LPMOs preparation AA9; the
figure (b) means a simultaneous addition of EX, BG and AA9.
substrate [7,8,26]. Therefore, some leading accessory enzymes such
as endoxylanase, ␤-xylosidase and AA9 were directly added into
the CTec1 cellulase hydrolysis of SPSSB. As shown in Fig. 5a, only
with the CTec1 at 20 mg protein/g substrate, the glucan hydrolysis
of SPSSB was 21% at 24 h and 33% at 48 h. The hydrolysis increased
significantly with addition of some accessory enzymes. With an
addition of the endoxylanase Htec preparation, the 48 h enzymatic
hydrolysis reached a maximum of 43% at 4.0 mg/g glucan, increas-
ing 30% as compared to that of the CTec1 alone. The data show that
the endoxylanase activity of CTec1 is deficient. And an extra sup-
ply would be necessary for an efficient hydrolysis of SPSSB, which
is also testified by the above analysis of specific activity of cel-
lulase preparations. However, an addition of the Novozyme 188
(␤-glucosidase preparation) at 1.0–8.0 mg/g glucan nearly had no
obvious help for the hydrolysis, suggesting that the ␤-glucosidase
activity of CTec1 itself was sufficient for the hydrolysis of SPSSB. The
result did not accord with the above analysis of specific activity.
To the present knowledge, it may be due to the physicochemi-
cal complexity of SPSSB, and the detailed information has yet to
explore.
The AA9, formerly classified as GH61, is a preparation of copper-
dependent LPMOs. As reported extensively, the LPMOs is helpful
for cellulase hydrolysis of lignocellulose by oxidative cleavage of
crystalline cellulose region [1,13,14]. In the experiment, the AA9
was added with the CTec1 to hydrolyze the SPSSB. At a low addi-
 F.F. Sun et al. / Enzyme and Microbial Technology 79–80 (2015) 42–48
Table 2
Filter paper activity, protein content and specific activities of the cellulase preparations on model substrates.
/g stock /mg protein
FPA (FPU) Protein (mg) Xylanase (U)
CTec1 23.1 ± 2.6 160.4 ± 7.3 0.77 ± 0.21
CTec2 137.0 ± 4.3 193.5 ± 7.6 21.17 ± 5.72
CTec3 165.2 ± 5.1 233.7 ± 8.2 14.93 ± 4.82
CMCase: carboxymethyl cellulase; CBH1: cellobiohydrolase 1; BG: ␤-glucosidase; BX: ␤-xylosidase; FPA: filter paper activity; FPU: filter paper unit.
tion of 0.20 mg AA9, the hydrolysis had a slight increase. With the
more AA9 addition, the hydrolytic performance of CTec1 improved
greatly. At an addition of 2.0 mg, the enzymatic hydrolysis was up
to 26% for 24 h and at 39% for 48 h, which was almost as equiva-
lent as that with the Htec addition at 2.0 mg. Results indicated that
the AA9 addition exerted a positive role in improving the CTec1
activity. The LPMOs greatly strengthen the enzymatic hydrolysis of
lignocellulosic substrate, the ability of which is comparable with
the xylanase Htec.
Finally, the combination of endoxylanase, ␤-glucosidase and
LPMOs greatly improved the CTec1 hydrolysis of SPSSB, as shown
in Fig. 5b. With the simultaneous addition of 1.0 mg Htec, 1.0 mg
Novozyme 188 and 0.20 mg AA9, the enzymatic hydrolysis reached
42% at 48 h, which was 30% higher than the contrast. The increase
was bigger than the sum of that with their single addition of Htec
(20%), Novozyme 188 (0%) and AA9 (6%). Actually the observation
was more clear at 24 h. The effect boosted by the simultaneous addi-
tion of accessory enzymes is far better than the overall that of their
respective additions, implying a synergistic interaction of multiple
accessory enzymes and cellulase enzymes [1,4,7]. With the simul-
taneous addition of 8.0 mg Htec, 8.0 mg Novozyme 188 and 2.0 mg
AA9, the enzyme hydrolysis at 48 h was 47%, which enhanced
47% as compare to that of the CTec1 loading alone. Relatively, the
enzymatic hydrolysis with the single additions contributed to an
increase of 30%, 0% and 20%, respectively. The data indicated that
the simultaneous addition of accessory enzymes with CTec1 at a
high level boosted the cellulase action. However, the mode of boost-
ing enzymatic hydrolysis is probably different from the synergetic
hydrolysis at the low addition, which tends to a co-hydrolysis, act-
ing as an additive effect [12]. It can be guessed that the coexistence
of various accessory enzymes would present a synergetic interac-
tion with cellulase enzyme at a low loading. And with an increase of
the loading, the interaction would shift from the synergetic coop-
eration to the co-hydrolysis.
For the complex composition of substrate, it is challenging to
degrade one component with the sole or several enzyme activities
[24,25]. Other components in the natural lignocellulosic biomass,
particularly hemicellulose and lignin, exert significant restraints on
cellulose hydrolysis [15,23]. For example, the lignin tends to bind
enzyme components non-productively, thus to reduce hydrolytic
performance of cellulase components. Hemicelluloses and pectin
probably restrict the access of cellulolytic enzymes by coating cel-
lulose fibers. Accordingly, a complete hydrolysis of industrially
relevant lignocellulosic biomass requires relatively large quantities
of cellulase enzymes and also requires the cooperation between
cellulases and several accessory enzymes with different catalytic
targets [2,12,25]. The same cellulase preparation can exhibit dif-
ferences in performance on the different lignocellulosic substrate,
even at an equivalent FPA loading. And various kinds of cellulase
preparations are also within expectation to yield unequal sug-
ars from the same substrate if they contain different varieties or
amounts of accessory enzyme [15–17]. Therefore, it is reasonable
to attach more importance to the accessory enzymes for the rational
design and customization of cellulase cocktails.
47
CMCase (U) BG (U) CBHI (U) BX (U)
0.19 ± 0.05 1.32 ± 0.42 0.36 ± 0.17 0.15 ± 0.03
2.26 ± 0.32 6.04 ± 2.42 0.98 ± 0.32 0.28 ± 0.07
1.24 ± 0.28 10.16 ± 1.92 3.08 ± 0.52 1.15 ± 0.26
4. Conclusion
Accessory enzymes that cannot be addressed by FPA revealed
an extremely important role beyond the expectation in cellulase
hydrolysis of the model substrate (i.e., filter paper) and the realis-
tic lignocellulosic biomass. The active role of accessory enzymes
existing in cellulase preparations contributed to the deviation
of hydrolytic action of cellulases on various lignocellulosic sub-
strates from the standardized FPA. The accessory enzyme boosted
the action of cellulase enzymes on various substrates to different
degree. The co-existence of accessory enzymes at a low level in
the cellulase hydrolysis apparently strengthened the boost by the
synergetic cooperation. In the ongoing lignocellulosic biorefining
industry, more importance should be attached within reason to the
accessory enzymes in rational customization of cellulase cocktails
in order to optimize hydrolysis and to realize an efficient conversion
of natural lignocellulosic substrates.
Competing interests
Authors declare that they have no competing interests.
Acknowledgements
The Natural Sciences and Engineering Research Council of
Canada (NSERC), Natural Resources Canada (NRCan) and Genome
BC are acknowledged for the support of this work. Part of work
was supported by the National Natural Science Foundation of
China (21176106; 31200023) and State Key Laboratory of Micro-
bial Technology (M2013-12), together with the Project Funded by
China Postdoctoral Science Foundation (2015M571666). Authors
also would like to give the thanks to the Priority Academic Pro-
gram Development of Jiangsu Higher Education Institutions, the
111 Project (No. 111-2-06), and the Jiangsu province “Collaborative
Innovation Center for Advanced Industrial Fermentation” industry
development program.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.enzmictec.2015.
06.020
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