Sugar Tech
https://doi.org/10.1007/s12355-021-01096-x
S . I . : D I V E R S I F I C A T I O N O F S UG A R C R O P S F O R V A L U E A D D I T I O N
Xylooligosaccharide Production from Sugarcane Bagasse using
Recombinant Endoxylanase of Bacillus Halodurans
Ying-Hsuan Tseng1 • Wen-Chien Lee1 • Kridsana Krisomdee2 • Waranya Natesuntorn2 •
Sunisa Chatsurachai2 • Klanarong Sriroth2
Received: 30 July 2020 / Accepted: 26 December 2021
Ó The Author(s), under exclusive licence to Society for Sugar Research & Promotion 2022
Abstract Xylooligosaccharides (XOSs) can be manufac-
tured from sugarcane bagasse as a value-added product in
the sugar industry. To produce XOS, xylan was extracted
from sugarcane bagasse by using sodium hydroxide, pre-
cipitated in ethanol, and subjected to enzymatic hydrolysis
by using endoxylanase to form xylooligomers. We con-
structed a recombinant strain of Escherichia coli harbour-
ing the recombinant plasmid containing a gene encoding
endoxylanase modified from GH10 xylanase of alkaliphilic
Bacillus halodurans BCRC 910,501. Results from the flask
culture showed that in terms of activity, 80.43 ± 6.36% of
recombinant proteins were secreted to the medium under
the induction of 0.1 mM isopropyl b-D-1-thiogalactopy-
ranoside (IPTG) for 18 h at 30 °C. The activity concen-
tration in the medium was 20.04 ± 4.11 U/mL. When this
strain was fed-batch cultivated to high cell density in a
bench-top fermenter, 89.13 ± 0.66% of the recombinant
enzymes were found in the medium, and the active protein
concentration was 45.9 ± 7.32 U/mL. Thus, the recombi-
nant E. coli has the potential for large-scale production of
endoxylanase. A 24-h enzyme reaction yielded 44.91% (w/
w) of XOS from xylan. Among the total soluble oligomers
in the product, 40.6% (w/w) were xylobiose and xylotriose.
& Wen-Chien Lee
chmwcl@ccu.edu.tw
1
Department of Chemical Engineering, National Chung Cheng
University, 168 University Road, Minhsiung, Chiayi 621,
Taiwan
2 human, XOSs have been used as animal feed additives.
Mitr Phol Sugarcane Research Center Co., Ltd., 399 Moo 1
Chumpae-Phukieo Road, Khoksa-at, Phukieo, Chaiyaphum
36110, Thailand
Keywords Sugarcane bagasse

Xylooligosaccharides (XOS)
Endoxylanase

Recombinant
Bacillus halodurans
Introduction
Sugarcane bagasse is a potential lignocellulose feedstock
for the production of biofuels and bio-based chemicals,
which can be generated through fermentation from the
cellulosic biomass after pretreatment and enzymatic
hydrolysis of cellulose. In addition to cellulose (Uppal
et al. 2011; Bhatia and Johri 2016), hemicellulose is
another primary fraction in this cellulosic biomass that can
be converted into biomass-based chemicals, such as fur-
fural (Uppal and Kaur 2011) and xylitol (Xu et al. 2019),
and high value-added compounds, such as xylooligosac-
charides (XOSs) (Brienzo et al. 2010; Jayapal et al. 2013).
Hemicellulose comprises 22–36% of sugarcane bagasse.
Xylan is the predominant component of sugar in the
hemicellulose of sugarcane bagasse because it con-
tains [ 80% of xylose in the backbone. Thus, xylan
extracted from sugarcane bagasse is ideal for XOS
production.
XOSs are oligomers of the five-carbon sugar xylose and
are currently used as a prebiotic. XOS uptake has several
benefits to human health, including favouring the selective
growth of beneficial Bifidobacterium spp, suppressing the
activity of entero-putrefactive and pathogenic intestinal
bacteria through the production of short-chain fatty acids,
and facilitating nutrient absorption (Alonso et al. 2003). In
addition to their nutritional value as functional foods to
XOSs are chains of xylose molecules linked with b1–4
bonds with a degree of polymerisation (DP) ranging from 2
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to 10 (Makelainen et al. 2009). XOSs can be produced
through xylan hydrolysis by using enzymatic or chemical
methods (hydrothermal treatments) or a combination of
both (Aachary and Prapulla 2011; Mandelli et al. 2014).
Both chemical and enzymatic methods have been used for
xylan hydrolysis from sugarcane bagasse to yield XOS
(Brienzo et al. 2010, 2016; Bian et al. 2013; Bragatto et al.
2013; Mandelli et al. 2014; Kaur et al. 2019). Enzymatic
hydrolysis is advantageous in preventing the formation of
toxic by-products due to low temperature and high speci-
ficity. For use as a food ingredient, XOS must be produced
through enzymatic reaction. For XOS production, xyla-
nases must mainly have the function of endo-1,4-xylanases.
Many studies have explored the microbial sources of
enzymes for XOS production, including Aspergillus foeti-
dus MTCC 4898 (Chapla et al. 2012), A. oryzae MTCC
5154 (Aachary and Prapulla 2009), Bacillus amylolique-
faciens NRRL B-14393 (Rashad et al. 2016), B. mega-
terium (Sun et al. 2015), B. pumilus MK001 (Kapoor and
Kuhad 2007), B. subtilis KCX006 (Reddy and Krishnan
2016), B. subtilis (Bragatto et al. 2013), Clostridium ther-
mocellum (Mandelli et al. 2014), Eupenicillium javanicum
(Tanaka et al. 2009), Paenibacillus sp. NF1 (Zheng et al.
2014), Paecilomyces themophila J18 (Teng et al. 2010),
Pichia stipites (Yang et al. 2011), Streptomyces oli-
vaceoviridis E-86 (Ai et al. 2005), S. thermovulgaris
TISTR1948 (Boonchuay, et al. 2014), S. violascens BF
3.10 (Salupia et al. 2015), Talaromyces thermophilus
(Maalej-Achouri et al. 2009), Thermoascus aurantiacus
ATCC 204,492 (Brienzo et al. 2010), Thermobifida fusca
(Yang et al. 2007), and Trichoderma longibrachiatum
(Saleh et al. 2016). We previously found that endoxy-
lanases in B. halodurans were effective in producing XOS
from xylan (Lin et al. 2011). Two extracellular endo-1,4-b-
xylanases (E.C. 3.2.1.8), which are encoded by xyn45 and
xyn23 genes and belong to the glycosyl hydrolase (GH)
family 10 and 11, respectively, can be found in B. halo-
durans (Tseng et al. 2002; Chang et al. 2004). According to
Hu and Saddler (2018), GH10 xylanase performs better
than GH11 xylanase for deconstructing pretreated biomass.
In this study, the recombinant GH10 xylanase of B. halo-
durans (encoded by xyn45 gene) was thus constructed for
producing XOS from sugarcane bagasse. GH10 genes from
B. halodurans BCRC 910,501 (Chang et al. 2004) and B.
halodurans C-125 (Takami et al. 2000) were combined for
the recombinant enzyme.
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Sugar Tech
Materials and Methods
Composition Assay of Sugarcane Bagasse
The composition of bagasse samples was analysed
according to the following protocol: 1 g of the milled
sample was extracted using deionised water and 95%
ethanol in Soxhlet apparatus for 6 h each. Then, 300 mg of
the extracted sample was hydrolysed with 72% sulphuric
acid at 45 °C for 10 min. The resultant mixture was diluted
to a final concentration of 4% and then heated at 121 °C in
an autoclave for 30 min. After filtration, glucose, xylose,
and arabinose in the filtrates were determined through
high-performance liquid chromatography (HPLC). The
chromatographic separation was performed in a Biorad
Aminex HPX-87H column (300 9 7.8 mm i.d.) at 65 °C,
and the flow rate was set as 0.6 mL min-1 by using 5 mM
sulphuric acid as the mobile phase. The residual solid from
the filtration was dried to a constant weight at 60 °C and
weighted as acid-insoluble lignin (AIL). For determining
acid-soluble lignin (ASL), the filtrate was assayed through
ultraviolet–visible spectroscopy to determine the absor-
bance of the sample at a wavelength of 240 nm, as sug-
gested by Sluiter et al. (2012). For determination of the ash
content, the sample was burned in a high-temperature
furnace. After placing the sample in the furnace, the tem-
perature was increased from room temperature to 105 °C
and held for 12 min, and then, the temperature was
increased to 575 °C at a rate of 20 °C/min. After burning at
575 °C for 3 h, the temperature dropped to 105 °C and
then to room temperature (Sluiter et al. 2012).
Xylan Extraction
Sugarcane bagasse obtained from Mitr Phol sugarcane mill
(Phukiao factory: PK sample) was milled into powders (40
mesh) and then soaked in 4% NaOH with a solid–liquid
ratio of 1:15 (w/v) at 30 °C for 24 h. The incubated mix-
ture was then centrifuged, and the resulting alkali-soluble
fraction was neutralised with hydrochloric acid solution to
a final pH of 7.0. Thrice the volume of 95% ethanol was
then added, and the resultant mixture was incubated at 4 °C
for 24 h. Xylan was obtained from the recovered precipi-
tate as the substrate for XOS production.
Construction of the Endoxylanase Recombinant
Strain
The amino acid sequence of recombinant endoxylanase is
shown in Fig. 1. Corresponding DNA, named as xynM
(1191 bp), was chemically synthesised and inserted into a
plasmid pET-15b. Codons for this hybrid endoxylanase,
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200 and 1000 rpm. The cultivation pH was controlled at

Fig. 1 Amino acid sequence of endoxylanase modified from the
endoxylanase GH10 of B. halodurans BCRC 910,501
6.8 by using 25% ammonia solution and a feeding solution,
respectively, as alkaline- and acid-adjusting agents. The
feeding solution consisted of 500 g/L of glycerol, 15 g/L of
MgSO4
7H2O, and 50 g/L of yeast extract. When OD600nm
of 35 was achieved, IPTG with the concentration of
0.1 mM/OD600nm was used to induce the expression of
recombinant protein for 18 h at 25 °C.

Characterisation of Protein Expression

including the original signal peptide (first 23 amino acids),
were optimised for expression in Escherichia coli. To
The broth culture in the flask or fermenter was harvested at
construct the recombinant plasmid, the target gene (xynM)
postinduction and centrifuged. The collected supernatant
was first amplified using polymerase chain reaction (PCR)
contained extracellular proteins secreted into the culture
with the forward primer 5’-CGC CAT ATG ATT ACC
medium. Cell pellets were suspended in 100 mM Tris–HCl
CTG TTC AA-3’ and the reverse primer 5’-GCG GGA
buffer (pH 7.5) supplemented with 0.1 mM phenyl-
TCC TTA ATC AAT GCG CCA-3’. The PCR-amplified
methylsulphonyl fluoride. The cells were disrupted by
product was confirmed through agarose gel electrophoresis,
passing them through a French press at 1000–1500 psi. The
purified, and double digested with the restriction endonu-
lysate was centrifuged at 7537 9 g for 10 min at 4 °C, and
cleases NdeI and BamHI. Furthermore, plasmid DNA pET-
the supernatant and precipitate were collected. The pre-
15b (5708 bp) was purified and double digested using the
cipitate was suspended in 100 mM Tris-base (pH 7.5).
same restriction endonucleases. Ligation was performed
Proteins in the intracellular soluble (supernatant) and
through a combination of 50 ng of vector and threefold
insoluble (precipitate) fractions as well as extracellular
molar excess of insert. After adjusting the volume to 10 lL
proteins were analysed through sodium dodecyl sulphate
with nuclease-free water and adding 10 lL of 2 9 Quick
polyacrylamide gel electrophoresis (SDS-PAGE) by using
Ligation Buffer and 1 lL of Quick T4 DNA Ligase, the
a 10% acrylamide gel. The resolved protein bands were
mixture was mixed thoroughly and incubated at room
visualised through Coomassie brilliant blue staining. Fur-
temperature (25 °C) for 6 h. After cooling on ice, the
thermore, extracellular and intracellular protein fractions
recombinant plasmid was transformed into E. coli DH5a.
were assayed for endoxylanase activity.
Colony PCR was used for analysing the recombinant
plasmid that was transformed into E. coli DH5a. The
Enzymatic Activity Assay
bacterial colony harbouring the recombinant plasmid was
confirmed through DNA sequencing. This recombinant
Enzymatic activity was determined based on the release of
plasmid was named as pET-15b-xynM and was finally
reducing sugar from xylan by using the dinitrosalicylic acid
transformed into E. coli BL21 (DE3) for endoxylanase
(DNS) method. A mixture of crude enzyme and 1% (w/v)
production.
birchwood xylan dissolved in 10 mM sodium phosphate
buffer, pH 7.0, was incubated at 60 °C for 5 min. The
Xylanase Production
reaction was stopped with the addition of DNS reagent.
One unit of the xylanase activity was defined as the amount
Batch culture was conducted at 37 °C in an Erlenmeyer
of enzyme released in 1 lmol of reducing sugar equivalent
flask containing 50 mL of LB medium and 50 mg mL-1
to xylose per minute under the assay conditions.
ampicillin. When OD600nm achieved 0.8–1.0, IPTG was
added to induce recombinant protein expression for 18 h at
XOS Production Through Enzymatic Hydrolysis
30 °C.
For fed-batch cultivation, a flask culture of 50 mL of LB
Crude enzyme xylanase, with a total activity of 0.225
medium was inoculated to a bench-top fermenter contain-
U/mL, was added to a well-mixed solution of 2.0% (w/v)
ing 1 L of PM medium composed of the following (per
xylan in 1 L 100 mM Tris–HCl buffer (pH 7.0). The
litre): 20 g of glycerol, 3 g of (NH4)2HPO4, 7 g of KH2-
mixture was incubated at 60 °C with mild agitation
PO4, 0.8 g of citric acid, 1 g of MgSO4
7H2O, 2 g of yeast
(200 rpm) and air input (around 2 vvm) to prevent pre-
extract, and 3 mL of trace metal solution. Fermentation
cipitation at the bottom of the reactor. The reaction was
was conducted at 37 °C with an aeration rate of 2 vvm. The
performed with thorough mixing. A sample of 100 lL was
dissolved oxygen concentration was maintained at 20% of
obtained at different reaction times and diluted with ddH2O
air saturation through varying the agitation rate between
to 10% before filtration. The filtrate was analysed through

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HPLC by using the HPX-87H column to determine the
concentrations of xylose, xylobiose, xylotriose, and
xylotetraose. Details of the HPLC conditions were the
same as those used for the composition assay.
Results and Discussion
Xylan Preparation
For xylan extraction, sugarcane bagasse was pretreated
with sodium hydroxide solution. As shown in Table 1, the
raw material sugarcane bagasse contains 31.17% w/w
glucan, 26.58% w/w lignin, and 15.35% w/w xylan in the
dry biomass. The raw material also contains 1.11% of
extractive, which is soluble in water or ethanol during the
Soxhlet extraction. The ethanol-soluble fraction in sugar-
cane bagasse may be waxy materials, low molar mass
aromatics, and oligosaccharides (Masarin et al. 2011). For
xylan extraction, sugarcane bagasse is commonly pre-
treated with NaOH and KOH. An increasing alkaline
concentration can increase the dissolution and removal of
hemicellulose and lignin in pretreated bagasse (Safirzadeh
et al. 2017). On the basis of the best conditions obtained by
Nascimento et al. (2016), 4% NaOH was used here.
However, we used 30 °C for 24 h instead of their harsh
conditions (121 °C for 60 min) for saving energy. Pre-
treatment for large-scale production under harsh conditions
(121 °C) requires special reactors and well-managed con-
trol systems, which will increase capital and operating
costs. In addition, pretreatment under harsh conditions will
cause partial hemicellulose degradation and produce toxic
by-products, which will inhibit enzymes in subsequent
steps. We are trying to optimise the alkaline pretreatment
by changing the parameters in the range of low temperature
(40–60 °C) and low sodium hydroxide concentration
(2–6%). Basically, increasing the temperature or increasing
the NaOH concentration can shorten the incubation time.
After alkaline pretreatment and ethanol precipitation, crude
xylan was obtained. Xylan composition was determined
using the same method used for determining the bagasse
sample composition. The purity of xylan was determined
based on the measurement of xylose released after
Table 1 Composition of sugarcane bagasse as the raw material*
Moisture (%) %Ts %Tfinal %Extractives
0.11 99.89 99.84 1.11
*
%Ts and %Tfinal are percentages of dried mass before and after Soxhlet extraction, respectively. The weight of the air-dried sample after
extraction is subtracted from the weight of the sample before extraction, and the obtained value is divided by the oven dry weight of the extracted
sample to obtain the percentage of extractive (%Extractive)
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hydrolysis. The purity of xylan was 51.91%, suggesting
that the xylan extracted after NaOH pretreatment and
ethanol precipitation contains not only xylan but also lig-
nin, glucan, arabinosyl, and uronic acid groups. The xylan
yield could be briefly increased to 61.7% with an increased
concentration (40%) of NaOH (w/w) (Sporck et al. 2017).
We have tried to reduce the amount of ethanol used in
xylan precipitation step. After the sugarcane bagasse was
pretreated with 4% NaOH at 60 °C for 24 h and the pH
value of the obtained alkali-soluble fraction was neu-
tralised to 7.0, then 95% ethanol was added in different
ratios of the alkali-soluble fraction to ethanol for further
incubation at 4 °C for 24 h. When the ratios of alkali-
soluble solution to ethanol were 1:0.5, 1:1, 1:2, and 1:3, the
yields of crude xylan were 0.077 ± 0.008, 0.096 ± 0.019,
0.138 ± 0.010, and 0.135 ± 0.019 g/g-sugarcane bagasse,
respectively. The results show that the best condition for
ethanol precipitation is that the ratio of alkali-soluble
fraction to ethanol is 1:2. When the ratio is less than 1:2,
the yield of crude xylan decreased significantly.
Endoxylanase Production
The protein overexpressed by recombinant E. coli BL21
(DE3) harbouring pET-15b-xynM was endoxylanase
modified from the GH10 xylanase of alkaliphilic B. halo-
durans BCRC 910,501, which was previously named as B.
firmus (Chang et al. 2004). The amino acid sequence is
identical to the one obtained from B. halodurans BCRC
910,501 except for two amino acid mutations: 364D ? G
and 393G ? R. These changes are based on the amino
acid sequence of endoxylanase from B. halodurans C-125.
According to the calculation performed using the ExPASy
protein structure homology-modelling server Swiss-Model
(Waterhouse et al. 2018), the changes in the two amino
acid residues of this enzyme had little effect on the protein
confirmation. However, this inconspicuous change may
cause the structure at the C terminus to become tight. The
endoxylanase GH10 obtained from B. halodurans BCRC
910,501 showed enzymatic activities over a broad pH
range of 4–11 at 37 °C, and it was optimally active at
70 °C when assayed at either pH 7 or 9. Furthermore, this
enzyme could be fully active and retained 80% of its
%ASL %AIL %Ash Glucan (%) Xylan (%) Arabinan (%)
0.63 25.95 0.12 31.17 15.35 0.18
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activities after 14 and 16 h, respectively, of preincubation
at 62 °C (Chang et al. 2004). Because of its high ther-
mostability, the enzyme reaction for XOS production can
be performed at a high temperature.
The recombinant E. coli BL21 (DE3) harbouring pET-
15b-xynM was cultured in batch (in flask) and fed-batch (in
bench-top fermenter) modes separately for producing
endoxylanase. When bacterial cells were cultivated in a
flask containing LB medium, the induction for recombinant
protein expression with IPTG began when OD600nm
reached 0.8–1.0. When bacterial cells were cultivated in a
fermenter and fed with PM medium, a high cell density up
to OD600nm of 35 could be achieved before adding IPTG
with a concentration of 0.1 mM/OD600nm to induce
recombinant protein expression. Figure 2 shows the SDS-
PAGE of proteins expressed through recombinant E. coli
BL21 (DE3) harbouring pET-15b-xynM. The target protein
is shown as a dark band on the SDS-PAGE gel of proteins
in the culture medium. The first 23-amino-acid sequence
shown in Fig. 1 is the signal peptide. When a His-tag was
included in front of the signal peptide at N terminus, the
estimated molecular mass of this fused protein containing
416 amino acids was theoretically 47,490.79 Da. If the
signal peptide is removed from the synthesised protein
during the secretory process, the resultant protein could
have an estimated molecular mass of 42,999.52 Da. The
location of the dark band of target protein in Fig. 2 is closer
to 43 kDa than to 47.5 kDa, suggesting that the signal
peptide works well in E. coli.
Results shown in Table 2 indicate that the recombinant
E. coli could produce recombinant endoxylanase through
Fig. 2 SDS-PAGE analysis of proteins expressed by recombinant
E. coli BL21 (DE3) harbouring pET-15b-xynM in a high cell density
culture after IPTG induction at 25[Error hu2103] for 18 h. Lane M,
marker; Lane 1, extracellular proteins in the culture medium; Lane 2,
soluble fraction of intracellular proteins; and Lane 3, insoluble
fraction of intracellular proteins. The arrow indicates the band for
target protein
enzymatic activity mainly in the culture medium. When the
batch-grown bacteria were induced to express recombinant
proteins at 30 °C for 18 h, extracellular protein activity
accounted for 80.43 ± 6.36% of the total enzymatic
activity. The concentrations of extracellular proteins in
terms of amount and activity were 2.67 ± 0.05 mg/mL and
20.04 ± 4.11 U/mL, respectively. Compared with enzyme
production from B. halodurans S7, 0.574 mg/mL amount
and 5.1 U/mL activity were observed in the culture
supernatant of a 36-h-old culture (Mamo et al. 2006).
Table 2 shows also the results of recombinant endoxy-
lanase expressed by recombinant E. coli with fed-batch
cultivation to produce high cell density in a bench-top
fermenter. The concentrations of extracellular proteins in
terms of amount and activity were 10.84 ± 1.07 mg/mL
and 45.9 ± 7.32 U/mL, respectively. Furthermore, the
majority of overexpressed proteins were found in the cul-
ture medium. The extracellular protein activity accounts
for 89.13 ± 0.66 of the total enzymatic activity. In com-
parison with the batch culture in a flask, the recombinant
bacteria cultivated in fed-batch mode could produce more
amount of endoxylanase because of high cell density and
secretion of almost all the overexpressed proteins out of the
cell and into the culture. The fed-batch culture was induced
to produce recombinant protein expression at 25 °C for
18 h. Moreover, induction at 30 °C could increase the
activity concentration to 53.53 U/mL.
XOS Production
To 1 L of reactor, 0.225 U/mL of endoxylanase produced
by recombinant E. coli was used as a biocatalyst. Time
courses of XOS production from sugarcane bagasse xylan
at pH 7.0 and 60 °C are shown in Fig. 3. For mixing xylan
and enzyme evenly in the reactor, the reaction was per-
formed with mild agitation (200 rpm) and air purge (* 2
vvm). Initially, most of the xylan in the solution was
insoluble, but small amounts of soluble xylooligomers
(DP C 4) were produced due to autolysis in alkaline con-
ditions. Furthermore, xylobiose and xylotriose were pro-
duced within 2 h when the recombinant endoxylanase was
used in the xylan solution. Simultaneously, insoluble xylan
was digested to form soluble high DP xylooligomers,
which subsequently degraded into xylobiose and xylo-
triose. As suggested by Bian et al. (2013), xylobiose and
xylotriose accumulate rapidly in the first 2 h, possibly
because the enzyme preferentially acts on the end of the
xylan chain. Figure 3 shows that the concentrations of
xylobiose and xylotriose gradually increased with time,
whereas soluble xylooligomers (DP C 4) accumulated
rapidly after 6 h. Subsequently, all the components
increased slowly over time, and the soluble high DP
xylooligomers continued to degrade into oligomers with
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Table 2 Activity of the crude enzyme induced using 0.1 mM inducer IPTG final concentration for 18 h
Cultivation Inducing Inducer (IPTG) Sample* U/mL Ratio to total Protein conc. Specific activity,
methods temperature (°C) conc activity (%) (mg/mL) U/mg

Batch 30 0.1 mM Medium 20.04 ± 4.11 80.43 ± 6.36 2.67 ± 0.05 7.5 ± 1.41
Supernatant 3.63 ± 0.16 14.81 ± 2.51 4.86 ± 0.3 7.48 ± 0.15
Pellet 1.12 ± 0.81 4.76 ± 3.86 0.65 ± 0.06 16.86 ± 11.07
Fed-batch 25 0.1 mM Medium 45.9 ± 7.32 89.13 ± 0.66 10.84 ± 1.07 4.23 ± 0.26
Supernatant 4.39 ± 0.32 8.67 ± 1.94 12.35 ± 3.58 3.67 ± 0.8
Pellet 1.19 ± 0.83 2.21 ± 1.27 2.5 ± 1.32 6.5 ± 6.72
*
’’Medium’’ is the culture medium from which cells have been removed and contains extracellular proteins. ‘‘Supernatant’’ and ‘‘Pellet’’ are the
supernatant and precipitate of the cell lysate after centrifugation, respectively

8.98/10.38 = 86.5%. This conversion rate was as high as

the production of XOS from corncob xylan using the intact
enzymes of B. halodurans BCRC 910,501 (Lin et al. 2011).

Conclusion

To produce XOS, hemicellulose consisting of xylan was

Fig. 3 Time course of XOS production from xylan of sugarcane
bagasse using recombinant endoxylanase
lower DP. Towards the end of the 24-h reaction, the con-
tent of xylobiose and xylotriose in the total soluble oligo-
mers of the product (8.98 g/L) was 40.6%. The
monosaccharide (xylose) produced was \ 1%, suggesting
the exclusively endoxylanase function of this enzyme.
Similar to enzyme form other microbial sources (Mamo
et al. 2006), the recombinant enzyme degraded xylan in an
endo-fashion.
The XOS yield was calculated based on the mass of total
soluble oligomers (DP C 2) in the product obtained from
one unit mass of xylan. A 24-h enzyme reaction yielded
44.91% (w/w) of XOS from xylan. Thus, the XOS yield
obtained was higher than that (37.1%) obtained through the
hydrolysis of sugarcane bagasse hemicellulose by using the
enzyme from T. aurantiacus at 50 °C. Hemicellulose
containing approximately 80% xylan was extracted from
dewaxed sugarcane bagasse by using alkaline hydrogen
peroxide and further purified through fractional precipita-
tion with ethanol (Brienzo et al. 2010). Because the purity
of xylan obtained in this study was 51.91%, the theoreti-
cally maximal XOS concentration that could be obtained
from 2% xylan is 20 g/L 9 51.91% = 10.38 g/L. The
conversion rates with recombinant endoxylanase use were
extracted from sugarcane bagasse through pretreatment
with sodium hydroxide followed by ethanol precipitation.
Subsequently, it was hydrolysed using an enzyme belong-
ing to family GH10 xylanase. We constructed a recombi-
nant E. coli carrying a plasmid containing a gene encoding
a modified endoxylanase from alkaliphilic B. halodurans
BCRC 910,501 and cultured it to produce enzyme. As the
recombinant endoxylanase was induced in a high-cell-
density culture, approximately 90% of the enzyme activity
was observed in the culture medium. Using the recombi-
nant endoxylanase, a high conversion rate could be
achieved with the enzyme hydrolysis of xylan to form XOS
at pH 7.0 and 60 °C. Thus, this recombinant enzyme
increases the potential of obtaining a high-value product
from sugarcane bagasse.
Declarations
Conflict of interest The authors declare that there is no conflict of
interest.
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