Industrial Crops & Products 176 (2022) 114349

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Industrial Crops & Products

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Improving catalytic efficiency of endoxylanase for degrading corncob xylan

to produce xylooligosaccharides by fusing a β-xylosidase
Na Li a, Huan Xia a, Zifu Ni b, Zewang Guo b, Yang Song a, Wenquan Huang a, Yanbin Jiang a, *,
Wenyong Lou b, *
a
Guangdong Provincial Key Lab of Green Chemical Product Technology, School of Chemistry and Chemical Engineering, South China University of Technology,
Guangzhou 510640, China
b
School of Food Science and Engineering, South China University of Technology, Guangzhou 510640, China

A R T I C L E I N F O A B S T R A C T

Keywords: A robust endoxylanase with high activity has great potential in biomass utilization. Here, the catalytic efficiency
Endoxylanase of the endoxylanase XynF derived from Herbinix hemicellulosilytica was improved by fusing a β-xylosidase xln-DT
Fusion enzyme derived from Dictyoglomus thermophilum with linkers (L0 and L3) and the direction from xln-DT to XynF. The
Catalytic efficiency
biochemical properties and application potential of chimeras towards corncob xylan were explored. The result
Corncob xylan
showed that the chimeras Xln-L0-XynF and Xln-L3-XynF exhibited higher catalytic efficiency (31-fold and 20-fold
Xylooligosaccharides
enhancement, respectively), and maintained high stability in some harsh environments compared with parental
enzymes. This might be attributed to the appropriate linkers and the direction which decrease the adverse in­
teractions between the two modules. These two chimeras could hydrolyze corncob xylan to produce approxi­
mately 30% xylooligosaccharides with polymerization of 2–6. This work represents the promising xylanases for
participating in XOS production from an abundant corncobs and promote us to modify glycoside hydrolase by
fusion technology.

1. Introduction GH5, GH7, GH8, GH10, GH11, GH16, GH26, GH30, GH43, GH52,
GH62. The structure of GH11 endoxylanase contains an α-helix and two
Xylan, as the most abundant component in hemicellulose, is β-sheets. GH11 endoxylanases with low-molecule weight usually have
commonly found in agricultural wastes such as corncobs and bagasse high substrate specificity and adapt to a wide pH range (Basit et al.,
(Pinales-Márquez et al., 2021). The backbone of xylan primarily consists 2018). But in the XOS production or in some other fields such as food
of β-D-xylopyranosyl residues linked by β-1, 4-glycosidic bonds. processing, feed additives, biofuel production and papermaking,
Endoxylanases could act on these bonds to produce xylooligosaccharides endoxylanases owning resistance and tolerance to derivatives of sub­
(XOS) (Carvalho et al., 2017). The XOS with the degrees of polymeri­ strates, ethanol, salts, high temperature and extreme pH are necessary
zation (DP) between 2 and 7 can promote calcium absorption, reduce (Álvarez et al., 2017; Bernardi et al., 2021). This is why some research
cholesterol levels and the risk of colorectal cancer, improve immunity, has been carried out to make GH11 endoxylanases robust, in which the
and have antioxidant activity and be antibacterial, and they occupy a amino acid composition would be changed, resulting in the conforma­
very important position in the market (Jagtap et al., 2017; Milessi et al., tion change and improving their activity or stability (Wu et al., 2018).
2021). However, high production costs hinder the application of XOS. The common method of directed evolution in engineering enzyme are
Significantly, hydrolyzing xylan by endoxylanases to produce XOS is a time-consuming and labor-intensive, or requires enough understanding
very effective method and is environmentally friendly (Jagtap et al., of the structure and mechanism of enzymes, but in many cases, it is
2017; Surek et al., 2021). And compared with hydrothermal process to difficult to know the structure and key sites of enzymes accurately
produce XOS, enzymatic hydrolysis was milder and could obtain high (Wang et al., 2021).
quality XOS with confirmed DP (Ruiz et al., 2020). Alternatively, the fusion enzyme would be a fast method of directed
Endoxylanase is a glycoside hydrolase (GH) and was recorded in evolution in the case of only knowing the overall structure and

* Corresponding authors.
E-mail addresses: cebjiang@scut.edu.cn (Y. Jiang), wylou@scut.edu.cn (W. Lou).

https://doi.org/10.1016/j.indcrop.2021.114349
Received 10 October 2021; Received in revised form 18 November 2021; Accepted 28 November 2021
Available online 6 December 2021
0926-6690/© 2021 Elsevier B.V. All rights reserved.
N. Li et al. Industrial Crops & Products 176 (2022) 114349

biochemical properties of enzymes. Fusion enzyme technology can be six chimeras, and the specific activity and optimum temperature of
classified into end-to-end fusion, insertional fusion and branched fusion chimeras were characterized. Two chimeras with higher activity were
(Rizk et al., 2012; Guntas et al., 2005; Schoffelen and van Hest, 2012). selected to further study their biochemical properties and ability to
The fusion GHs usually adopts an end-to-end fusion, in which another produce XOS from corncob xylan. In addition, the strategy and the trend
enzyme or carbohydrate-binding module (CBM) is linked at the N- or of fusion GH were discussed.
C-terminus of target enzyme directly or through a linker peptide.
Accordingly, fusing an excellent enzyme or domain into the target 2. Materials and methods
enzyme seems to improve activity or stability of target enzyme (Chen
et al., 2019; Wang et al., 2010). However, it was difficult to obtain the 2.1. Strains, materials and chemicals
fusion xylanse with high activity and high stability at the same time,
since the effects of direction and linker (Fan et al., 2009; Wang et al., E. coli DH5α, E. coli BL21 (DE3) and pET24c(+) vector were pur­
2010). Therefore, in order to avoid the adverse interactions between chased from TransGen (TransGen, Beijing, China). Ni2+-NTA agarose
enzymes, the direction and linker need to be considered in the fusion was purchased from Qiagen (Valencia, CA, U.S.A.). BCA Protein Assay
xylanase with linker peptide. Kit was purchased from Sangon Biotech (Shanghai). Sugarcane xylan
In this study, in order to obtain an endoxylanase which could be was purchased from Shanghai Yuanju Bio-Technology Co. Ltd. Corncob
applied in some industries, we adopt the heterologous expression to xylan, p-Nitrophenol (pNP), p-Nitrophenyl-β-D-xylopyranoside (pNPX),
obtain two xylanases of a GH11 endoxylanase XynF from D-xylose (X1), xylobiose (X2), xylotriose (X3), xylotetraose (X4), xylo­
H. hemicellulosilytica (Mechelke et al., 2017) and an exoxylanase pentaose (X5) and xylohexaose (X6) were purchased from Shanghai
(β-xylosidase) xln-DT from D. thermophilum (Li et al., 2018b). They yuanye Bio-Technology Co. Ltd. 2,2-Diphenyl-1-picrylhydrazyl was
showed similar optimal reaction conditions, but xln-DT has some su­ purchased from Aladdin. All other chemicals are analytical grade.
perior performance, e.g. higher NaCl-resistance, NaCl-stability, and
better thermostability. Encouraged by the idea that fusing the protein
with better performance into other proteins could promote chimeras to 2.2. Sequence and structure analysis
perform better, four DNA sequences of different linkers were used to
combine the β-xylosidase gene (xln-DT) and the endoxylanase gene The signal peptides of XynF (accession number: CRZ33495) derived
(XynF) to construct plasmids Xln-Ln-XynF-pET24c(+) and XynF-Ln-Xln- from H. hemicellulosilytica (Mechelke et al., 2017) and GH39 β-xylosi­
pET24c(+), then the plasmids were transformed into E. coli to express dase xln-DT (accession number: KX449145) from D. thermophilum (Li
et al., 2018b) were predicted using the online tool SignalP (http://www.

Scheme 1. Construction of parental enzymes and chimeras. T: template; p: primer; PCR: polymerase chain reaction; +Plasmids: the obtained recombinant plasmids
(listed in Table S2) and the genes produced by PCR were simultaneously treated with restriction enzymes.

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N. Li et al. Industrial Crops & Products 176 (2022) 114349

cbs.dtu.dk/services/SignalP/). The structure homology model of XynF 2.6.2. Impact of metal ions and organic reagents
was accomplished by SwissModel (http://swissmodel.expasy.org/). NaCl resistance of endoxylanase and β-xylosidase was measured with
QMEAN Z-score, GMQE and Local Quality plot assisted to estimate 0.5% xylan and 2 mM pNPX as substrates that contained 3%, 5%, 10%,
model quality. Tertiary structures of enzymes were visualized using 15%, 20%, 25%, 30% (w/v) NaCl, respectively. The reaction was carried
PyMOLWin. out under optimal pH and temperature, where the control group was
conducted in the same manner but NaCl was replaced by distilled water.
2.3. Gene cloning and vector construction To investigate NaCl-tolerance, the enzyme was treated in 3%, 5%, 10%,
15%, 20%, 25%, 30% (w/v) NaCl solution for 1 h, and then the residual
Scheme 1 illustrates the construction of parental enzymes and chi­ activity was detected under optimal conditions. Similarly, 0.5% xylan or
meras. Using the gene sequences of XynF and xln-DT (Xln) synthesized 2 mM pNPX containing different concentrations (0%− 30% (v/v)) of
in General Biosystems (Anhui) Co. Ltd as templates, and the primers ethanol was prepared to characterize the ethanol-resistance of the
(Table S1) with restriction sites (GGATCC and CTCGAG) were designed enzyme; the enzyme was treated at different concentrations (0%− 30%
to amplify the target fragments. The amplified genes were recovered and (v/v)) of ethanol for 1 h to determine ethanol stability. Metal ions and
purified by nucleic acid electrophoresis, then the recovered genes and β-Mercaptoethanol (β-ME) (final concentration of 5 mM) were added
pET24c(+) vector were simultaneously treated with restriction enzymes into the reaction system to explore their impact on the enzyme. The final
BamHI (recognizing GGATCC) and XhoI (recognizing CTCGAG), fol­ concentration of Triton X-100 (Triton) and Tween 80 (Tween) was 2%
lowed by T4 ligase connecting to obtain the recombinant plasmids Xln- (v/v).
pET24c(+) and XynF-pET24c(+). Xln-pET24c(+) and XynF-pET24c(+)
were used as templates to construct chimeras. Templates, recombinant 2.6.3. Determination of kinetic parameters
plasmids (Table S2) and primers (Table S1) containing the DNA se­ The catalytic rate of the enzyme for different concentrations
quences of linker peptides (Ln) and restriction sites were used for (0.05%− 1.00% (w/v)) of sugarcane xylan was determined within the
cloning to form recombinant plasmid XynF-Ln-Xln-pET24c(+) and Xln- first-order reaction time (7 min), and the kinetic parameters (Km, Vmax
Ln-XynF-pET24c(+) (Scheme 1). and kcat) of the enzyme were obtained from the Michaelis-Menten
equation (Xia et al., 2019).

2.4. Expression and purification of enzyme Vmax [S]

V= (1)
Km + [S]
The above plasmids XynF-pET24c(+), Xln-pET24c(+), XynF-Ln-Xln-
pET24c(+) and Xln-Ln-XynF-pET24c(+) were verified by digestion with kcat =
Vmax
(2)
restriction enzymes and transformed into E. coli. DNA sequencing was Et
conducted for further confirmation of the recombinant enzymes. Puri­
fication was accomplished by the affinity between Ni2+-NTA agarose where V (μmol/min/mg) represents the catalytic rate; Km (mg/mL) is
and His tag. The target protein was identified by SDS-PAGE and protein the Michaelis-Menten constant; Vmax (μmol/min/mg) represents the
quantification was measured using a BCA protein quantification kit maximum enzyme velocity in the same units as V; [S] (mg/mL) repre­
(Bradford method) (Ni et al., 2019). sents the initial concentration of substrate; kcat (s− 1) is the turnover
number, the frequency of substrate conversion by each enzyme site at
per second; Et (mol) is the molar concentration of enzyme catalytic sites.
2.5. Enzymatic assays
In order to demonstrate the structural changes of fusion on enzymes,
the synergism of XynF and xln-DT in equal moles was also investigated
The β-xylosidase activity of xln-DT was tested using the pNP method
in the same condition. The synergy rate was defined as the ratio of xylose
(Li et al., 2018a). The reaction system contained 450 μL of 2 mM pNPX
released by mixed enzymes to the sum of xylose released by each
and 50 μL enzyme, and was stopped by 2 mL of 1 M Na2CO3 solution
enzyme respectively.
after 10 min. Under particular conditions， the enzyme activity is
defined as the amount of enzyme necessary to decompose pNPX to
produce 1 μmol pNP per minute. The endoxylanase activity of XynF was 2.7. XOS production from corncob xylan by XynF and chimeras
detected by the DNS method (Bailey et al., 1992). The reaction system
contained 450 μL of 0.5% (w/v) xylan and 50 μL enzyme. Subsequently, 20.93 U/mg XynF (0.458 nM), 89.25 U/mg Xln-L0-XynF (0.216 nM)
the reaction was stopped by adding 750 μL DNS after 10 min and placing and 68.69 U/mg Xln-L3-XynF (0.270 nM) were added to the reactions
it in a boiling water bath for 5 min. The endoxylanase activity is defined containing 1% (w/v) corncob xylan (pH 6.5). The reactions were incu­
as the amount of endoxylanase which catalyze the formation of 1 μmol bated at 65 ◦ C for various time (1 h, 3 h, 6 h, and 12 h) and terminated
reducing sugar at per minute under standard conditions. The above re­ in a boiling water bath for 5 min to investigate the effect of reaction time
actions were both accomplished in 100 mM McIlvaine buffer with 3 on the products. The samples were centrifuged (10,000 rpm for 2 min),
parallels for each reaction. and a 0.22 µm membrane was used to filter the supernatant. XOS were
further purified by ultrafiltration (UF) with 3 kDa MWCO membrane.
2.6. Biochemical properties of enzymes Thin layer chromatography (TLC) was used to detect the production and
DP of XOS, and high performance liquid chromatography (HPLC) was
2.6.1. Effect of pH and temperature used to quantify XOS. Briefly, the samples were spotted on Silica gel
The optimum pH of enzymes was determined at 75 ◦ C and pH GF254 and developed with the mixture of water, n-butanol and acetic
4.0–8.0 (McIlvaine buffer). The enzymes were treated in McIlvaine acid (1:2:1, v/v/v). The mixture of acetone (50 mL), aniline (1 mL),
buffer (pH 2.0–8.0) and Glycine-NaOH buffer (pH 9.0–12.0) at 45 ◦ C for diphenylamine (1 g), and phosphoric acid (5 mL) was used to stain. The
1 h to determine their pH stability. The catalytic reaction of enzyme was HPLC system was equipped with a Aminex HPX-87 H Column and an
carried out at various temperatures (50–90 ◦ C) and optimal pH condi­ RID index detector, and the column was maintained at 55 ◦ C and eluted
tions to determine the optimal temperature. Enzymes were treated at with 5 mM sulphuric acid at a flow rate of 0.2 mL/min. The yield (w/w)
45 ◦ C, 55 ◦ C, 65 ◦ C and 75 ◦ C for various time (0 min, 30 min, 60 min, of XOS or xylose is determined as follows (Liu et al., 2018):
90 min and 120 min), and then the residual activity (the activity relative MXOS + MX1
to the untreated control sample or treated for 0 min) was detected to Yield (%) = (3)
Mxylan
identify their thermal stability.

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N. Li et al. Industrial Crops & Products 176 (2022) 114349

where MXOS and MX1 (mg) represent the amount of XOS and X1 obtained sequencing, in particular, DNA sequencing results of Xln-L0-XynF and
by enzymatic hydrolysis, respectively; Mxylan (mg) represents the Xln-L3-XynF are shown in Fig. S4.
amount of xylan. X1-X6 were used as standards. Table 2 lists the specific activity (towards sugarcane xylan) of XynF
and fusion enzymes (chimeras), which shows that the specific activity of
six chimeras constructed with various linkers was different. Many
2.8. Antioxidant activities assay
studies on fusion GH have explored the effects of length and amino acid
compositions of linker peptides on the chimeras (Lu and Feng, 2008). Lu
A scavenging rate of 2, 2-diphenyl-1-picrylhydrazyl (DPPH) radicals
and Feng (2008) used linkers of different lengths (5–15 amino acids) to
was used to measure the antioxidant activities of XOS produced in the connect the glucanase from B. amyloliquefaciens and the xylanase from
above way. In brief, 400 μL of 0.1 mM DPPH dissolved in ethanol was
B. subtilis, and tried to find a linker peptide that could make the parental
added to an equal volume of XOS. The reaction was mixed well and kept enzymes interact favorably. Their results showed that (GGGGS)2 and
for 2 h in the dark. The absorbance of DPPH radicals was measured at (EAAAK)3 could enhance the activity of glucanase and xylanase. And the
517 nm using a Perkin Elmer Lambda 365 spectrophotometer (USA). Xln-L0-XynF constructed with L0 ((GGGGS)2) as a linker exhibited the
XOS were replaced by ddH2O in the control, while 50% ethanol was used highest activity, but XynF-L1-Xln and Xln-L2-XynF constructed by
as the blank. The DPPH radical scavenging ability of XOS was evaluated referring to other fusion protein experiences showed lower activity
by the following equation (Bian et al., 2013): Antioxidant activity (Wang et al., 2010; Fan et al., 2009). Fusion protein technology with
( )
ODS linker could be used in some fields such as food, biomedicine, paper­
Antioxidant activity (%) = 1 − × 100% (4) making, textile and biological detection (Aalbers and Fraaije, 2019).
ODC
Hence, to get the insight of linker design is important.
where ODS (Optical Density) and ODC represent the absorbance of Similar to the diversity of protein structure and function, the variety,
sample and control, respectively. quantity, order of amino acids and length of peptides result in the di­
versity of the structure and properties of linker peptides. According to
3. Results and discussion their properties, they could be roughly classified into flexible linker
peptides, rigid linker peptides and anti-protease linker peptides (Chen
3.1. Analysis of sequence and structure et al., 2013). A linker library containing a series of flexible to rigid
peptides was established by a simulation method and the accuracy of the
The prediction of signal peptides is shown in Fig. S1, which shows simulation was verified by Forster Resonance Energy Transfer (FRET)
that xln-DT has no signal peptide (Fig. S1a), thus its gene sequence (Li et al., 2016). Their work provides a guidance for controlling the
(1509 bp) was completely synthesized, while the gene (552 bp) of XynF flexibility of linker peptides. van Rosmalen et al. (2017) explored the
was synthesized after the signal peptide (Fig. S1b) was removed. The effects of glycine ratio and length on flexibility of linker peptides and
endoxylanase rXynA (PDB ID: 1xxn) derived from Bacillus subtilis 1A1 found that a reduced glycine ratio would lead to low frequency FRET,
(Murakami et al., 2005) was used as a template to establish a protein indicating that the interaction between the enzymes was weakened.
model of XynF. The sequence identity between rXynA and XynF is Through computational simulation, linker libraries and reported expe­
80.11%, and the GMQE value (0.96), QMEAN value (0.59) and the Local rience, linker peptide seems to be reasonably designed. However, the
Quality Estimate (Fig. S2) indicate that the model is highly reliable. performance of the six chimeras reminds us that linker peptide is not
Fig. S3 shows the models of XynF and xln-DT. The visual analysis with only a simple connector, but also affects the microenvironment and
PyMOLWin shows that XynF has a similar structure containing two function of fusion enzymes, which is difficult to control in practical
β-sheets, one α-helix and the N-terminal to other GH11 endoxylanases application. Even now, it is difficult to design a perfect linker for two
(Fig. S3a). The three-dimensional structure of xln-DT is represented by B proteins. In this study, XynF-L0-Xln and Xln-L0-XynF were constructed
chain of DtXyl (PDB ID: 6yyi), which has three domains composed of a with (GGGGS)2 as the linker peptide, but the specific xylanase activity of
β-sandwich, a (β/α)8-barrel fold and a C-terminal loop that interacts the former was decreased, thus the direction also plays an important role
with another monomer (Fig. S3b) (Bretagne et al., 2020). in the fusion enzymes.
Moreover, the specific activity of XynF-L0-Xln, XynF-L1-Xln and
3.2. Enzymes preparation and their special activity XynF-L3-Xln was decreased sharply after purification, while XynF, Xln-
L0-XynF, Xln-L2-XynF and Xln-L3-XynF retained high activity after pu­
XynF-pET24c(+), Xln-pET24c(+), XynF-Ln-Xln-pET24c(+) and Xln- rification (Table 2). This suggested that the fusion direction affected the
Ln-XynF pET24c(+) were constructed and transformed into E. coli suc­ structure and expression level of the fusion enzymes. When xln-DT was
cessfully. The linkers, plasmids and names of the six chimeras are connected to the N-terminus of XynF, the His tags at the C-terminus of
showed in Table 1. These recombinant enzymes were confirmed by DNA the chimeras were exposed so that the chimeras could be purified
through affinity chromatography with Ni2+-NTA agarose; when xln-DT

Table 1
The linkers and fusion enzymes studied in this work. Table 2
Specific activity of XynF and fusion enzymes.
Linker Amino acid sequence Plasmid Fusion Source
enzyme Enzyme Specific activity (U/mg)

L0 GGGGSGGGGS Xln-L0-XynF- Xln-L0- Lu and Crude enzyme Pure enzyme

pET24c(+), XynF, Feng
XynF 0.20 ± 0.00 16.35 ± 1.09
XynF-L0-Xln- XynF-L0- (2008)
XynF-L0-Xln 0.15 ± 0.01 1.11 ± 0.19*
pET24c(+) Xln
Xln-L0-XynF 0.99 ± 0.02** 120.39 ± 2.42**
L1 GGGGGADQLAIGPM XynF-L1-Xln- XynF-L1- Fan et al.
XynF-L1-Xln 0.11 ± 0.01* 0.99 ± 0.02*
YNQVVYQYPN pET24c(+) Xln (2009)
Xln-L2-XynF 0.14 ± 0.01* 13.77 ± 2.03
L2 SAGSSAAGSGSG Xln-L2-XynF- Xln-L2- Wang
XynF-L3-Xln 0.23 ± 0.02 4.25 ± 0.04*
pET24c(+) XynF et al.
Xln-L3-XynF 0.84 ± 0.06* 76.72 ± 1.45**
(2010)
L3 KAKLKEEEERKQREEEE XynF-L3-Xln- XynF-L3- Chen The specific activity (U/mg) was determined for corncob xylan. The values
RIKRLEELAKRKEEERK pET24c(+), Xln- Xln, Xln-L3- et al. represent the means ± SD (n = 3).
L3-XynF-pET24c XynF (2016) *
P < 0.05 versus XynF (Control) (Student’s t-test).
(+) **
P < 0.01 versus XynF (Control) (Student’s t-test).

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N. Li et al. Industrial Crops & Products 176 (2022) 114349

was connected to the C-terminus of XynF, the His tags were buried and xylanase would affect the thermal stability (Ventorim et al., 2018).
could not bind with Ni2+-NTA agarose. Ultimately, the concentration of Accordingly, it is likely that the N-terminus of the XynF module in
xln-DT, XynF, Xln-L0-XynF and Xln-L3-XynF after being purified was Xln-L0-XynF and Xln-L3-XynF are connected and extended by the L0 and
1.22 mg/mL, 0.20 mg/mL, 0.35 mg/mL and 0.45 mg/mL, respectively. L3, respectively, which could not change the compact structure of XynF
And the SDS-PAGE of purified enzymes is shown in Fig. 1. Xln-L0-XynF module.
and Xln-L3-XynF were selected for further studies, since they showed In recent years, there have many studies on improving thermosta­
higher specific activity (Table 2) and retained high temperature activity bility of enzymes through fusion enzyme, all trying to fuse thermostable
of XynF among six chimeras (Fig. S5). proteins or domains into the N- or C-terminus of the enzymes owning
poor thermostability (Chen et al., 2019; Xu et al., 2018; Yang et al.,
2018; Olson et al., 2012). Different from this work, for the sake of
3.3. Biochemical properties of enzymes
improving the thermostability of a Xyn (GH11 xylanase) derived from
Aspergillus niger, a carbohydrate binding module (C2) of a GH10 xylanase
3.3.1. Impact of pH and temperature
derived from Thermotaga maritime was connected to the N-terminus and
The activity and stability of XynF, Xln-L0-XynF and Xln-L3-XynF in
C-terminus of Xyn concurrently to form C2-Xyn-C2 like a sandwich
various condition were determined with sugarcane xylan as substrate.
structure; two C2 protect Xyn like arms, causing the half-life of
Fig. 2 shows the impact of pH and temperature on enzymes. The
C2-Xyn-C2 at 50 ◦ C to be 24.8 times than that of Xyn (Yang et al., 2018).
maximum activity of XynF, Xln-L0-XynF and Xln-L3-XynF was detected
This also showed the diversity of fusion design. Referring to the strategy
at pH 6.0 (Fig. 2a), and they were stable in pH 2.0–12.0 (Fig. 2c); the
of using fusion enzyme technology to improve the thermostability of
optimum temperature of XynF and Xln-L0-XynF was 75 ◦ C, while the
GHs, we would enhance their stability in other extreme environments.
optimum temperature of Xln-L3-XynF was 70–75 ◦ C (Fig. 2b). Their
general trend of temperature stability at 55 ◦ C, 65 ◦ C and 75 ◦ C was
3.3.2. Resistance and stability of NaCl and ethanol
similar (Fig. 2d and e). But the residual activities of XynF and Xln-L0-
Fig. 4 shows the effect of NaCl and ethanol on enzymes. Compared
XynF after the treatment at 75 ◦ C for 30 min were 6.7% and 43.7%,
with the parents XynF and xln-DT, the activity and stability of Xln-L0-
respectively, and their residual activities after the treatment at 65 ◦ C for
XynF and Xln-L3-XynF in the solution containing high concentration
6 h were 57.1% and 71.2%, respectively. In addition, xln-DT showed the
of NaCl were improved slightly (Fig. 4a and b). The activity and stability
same optimum pH and temperature, and still having 100% relative ac­
of the enzyme in NaCl solution were affected by its surface polarity and
tivity after being treated at 75 ◦ C for 2 h (Li et al., 2018b). These results
structural stability, which depended on the type, arrangement and
not only proved that when two enzymes with similar optimal pH and
interaction of amino acids, but sometimes the structure amplitude
temperature are fused, the fusion enzymes tend to keep the same
caused by structure flexibility could compensate for the hydrophobicity
optimal condition (Rizk et al., 2012), but also implied that an appro­
effect to make the enzyme maintain high activity in a high concentration
priate strategy would improve the stability of enzymes in the extreme
of NaCl solution (Zhang et al., 2019). The xln-DT maintained the highest
pH and temperature. This improvement seems to be attributed to the
activity in the reaction system containing 30% (w/v) NaCl. It is specu­
xln-DT module with large-molecule weight and better performance.
lated that the xln-DT domains of Xln-L0-XynF and Xln-L3-XynF
The variety of pH will cause different charges in enzymes and sub­
contribute the most to the better performance of two chimeras in a
strates, thereby affecting the interaction between enzymes and sub­
high-concentration NaCl solution.
strates, and extreme pH would cause denaturation and inactivation of
As shown in Fig. 4c, compared with the parents XynF and xln-DT, the
enzymes. For Xln-L0-XynF and Xln-L3-XynF, it’s speculated that acidic
activity of Xln-L0-XynF and Xln-L3-XynF in ethanol had no significant
amino acids (Asp8 and Asp117) and basic amino acid Arg110 near the
increase, but they had higher activity than XynF after incubation in
active sites (Glu75 and Glu169) could keep the pKa in the neutral range
lower concentrations of ethanol (Fig. 4d). It is speculated that the flex­
(Fig. 3a) (Sundd et al., 2002). Thus the optimal pH (pH 6.0) of the two
ibility of chimeras increased by L0 or L3 could balance electrostatic
chimeras was consistent with that of XynF, indicating that the modifi­
interaction resulted by ethanol (Li et al., 2018a). Compared with XynF,
cation had no significant impact on these key amino acids. But the
xln-DT also had higher stability in low-concentration ethanol, hence it is
mechanism that the enzymes could resist extreme pH in this work needs
very likely that the xln-DT modules of Xln-L0-XynF and Xln-L3-XynF
to be revealed.
were charged with a large number of ethanol molecules in
The β-xylosidase with low temperature activity usually possesses a
low-concentration ethanol, which reduced the effect of ethanol on the
flexible structure caused by the lack of salt bridges and hydrogen bonds,
XynF modules.
or having more small amino acids and random coils in the structure
(Zhang et al., 2019; Li et al., 2018a). On the contrary, xylanases with
3.3.3. The influence of metal ions and organic reagents on enzymes
high temperature activity and stability depend on a stable structure,
The influence of metal ions and organic reagent on enzymes is shown
while local flexibility such as the free peptide at the N-terminus of GH11
in Fig. 5. Hg2+ had a strong inhibition on XynF, Xln-L0-XynF and Xln-L3-
XynF. Ag+ had a strong inhibition on XynF, Xln-L0-XynF, but Xln-L3-
XynF still having about 62% relative activity. Cu2+ had a slight inhibi­
tion on XynF and Xln-L0-XynF, but only about 38% relative activity for
Xln-L3-XynF. Similar to XynF, β-ME could increase the activity of Xln-
L0-XynF and Xln-L3-XynF, and most metal ions had no obvious effect
on them. Some previous studies reported that Cd2+ and Ba2+ could
inactivate xylanases (Basit et al., 2018), but XynF, Xln-L0-XynF and
Xln-L3-XynF in this study were not sensitive to them.

3.3.4. Kinetic studies

The kinetic analysis of XynF, Xln-L0-XynF and Xln-L3-XynF for
degrading xylan is shown in Table 3 and Fig. 6. Both Xln-L0-XynF and
Xln-L3-XynF exhibited enhanced affinity (Km is 3.61 mg/mL and
Fig. 1. SDS-PAGE analysis of the purified enzymes. M, molecular weight 4.50 mg/mL for Xln-L0-XynF and Xln-L3-XynF, respectively) for sugar­
standard. Lane 1, xln-DT(~58.7 kDa). Lane 2, XynF(~21.4 kDa). Lane 3, Xln- cane xylan, and their catalytic efficiency was 31.15 times and 18.07
L0-XynF(~80.6 kDa). Lane 4, Xln-L3-XynF(~84.3 kDa). times than that of XynF, respectively. Meanwhile, the synergism of XynF

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N. Li et al. Industrial Crops & Products 176 (2022) 114349

Fig. 2. The impact of pH and temperature on

XynF and two chimeras. The optimal pH (a) and
temperature (b) of XynF and two chimeras; (c)
the residual activity of enzymes after being
treated in pH 2.0–12.0; the residual activity of
XynF and Xln-L0-XynF (d) and the residual ac­
tivity of XynF and Xln-L3-XynF (e) after being
treated in different temperature for various
time. The reaction was carried out in the mix­
tures containing 0.5% sugarcane xylan and
detected by DNS method with 3 parallels for
each reaction. The data show the means ± SD
(n = 3) relative to the control samples.

Fig. 3. Some key amino acids in the catalytic

cleft of XynF module. (a) Glu75 and Glu169 are
catalytic acid/base and catalytic nucleophile;
Asp8 and Asp117 are acidic amino acids;
Arg110 is basic amino acid. (b) Tyr2, Gln4,
Tyr5, Trp6, Thr7, Asp8 at N-terminus partici­
pate in formation of catalytic pocket, which are
pulled by the xln-DT module. The amino acids
were numbered according to the sequence of
endoxylanase XynF but removed the signal
peptide. The stick circle indicates the space
occupied by substrates.

and xln-DT in equal moles was also investigated, and the synergy rate each other in the process of expression and catalysis, resulting in a
was only 1.3. This implied that xln-DT and XynF modules in Xln-L0- conformation change and making xylan easier to interact with active
XynF and Xln-L3-XynF are not completely separate but interact with sites. This might be interpreted as that the six amino acids (Tyr2, Gln4,

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N. Li et al. Industrial Crops & Products 176 (2022) 114349

Fig. 4. The impact of NaCl and ethanol on enzymes. The activity (a) and stability (b) of parental and fusion enzymes in 0%− 30% (w/v) NaCl; the activity (c) and
stability (d) of parental and fusion enzymes in 0%− 30% (v/v) ethanol. The data show the means ± SD (n = 3) relative to the control samples.

Tyr5, Trp6, Thr7, Asp8) at N-terminus and near the catalytic site of XynF
are pulled by the xln-DT module to make the catalytic pocket wider
(Fig. 3b). There is also a hypothesis that the xln-DT module could bind
and immobilize xylan, so that the catalytic sites in the XynF modules
could contact more xylan. However, the two chimeras had no activity
toward pNPX, since the dimers or tetramers formation of the xln-DT
module was disturbed (Bretagne et al., 2020).
In industry, improving the activity, stability and reuse rate of en­
zymes would reduce the cost of enzyme production. Consequently, the
purpose or advantages of constructing fusion GHs could be divided into:
improving expression level of enzymes, improving catalytic efficiency,
improving the stability and repeatability, and realizing the one-step
immobilization and purification of the enzyme (Chang et al., 2018).
Table 4 shows some fusion GHs and their properties. Compared with
some chimeras of xylanase-glucanase and xylanase-arabinosidase, the
xylanase activity (towards xylan) of the Xln-L0-XynF and Xln-L3-XynF
needs to be improved, but they were more stable in extreme pH and
high temperature. Particularly, their activity and stability have been
greatly improved compared with the xylosidase-xylanase obtained by
Fig. 5. The influence of metal ions and organic reagents on enzymes. The data Xue et al. (2009) and Wang et al. (2010) (Table 4).
show the means ± SD (n = 3) relative to the control sample.

Table 3
Kinetic parameters of XynF, xln-DT, Xln-L0-XynF and Xln-L3-XynF.
Enzyme Km (mg/mL) Vmax (μmol/min/mg) kcat (s− 1) kcat/Km (mL/mg/s)

XynF 7.91 ± 1.68 45.71 ± 5.78 16.30 ± 3.31 2.06 ± 0.22

xln-DT 0.08 ± 0.01 0.20 ± 0.00 0.20 ± 0.00 2.55 ± 0.36
Xln-L0-XynF 3.61 ± 0.29 172.65 ± 5.62* 231.06 ± 3.51*** 64.01 ± 4.51*
Xln-L3-XynF 4.50 ± 0.35 131.27 ± 4.65* 184.53 ± 0.79** 41.00 ± 0.73**

Note: reactions were performed in triplicate with 0.05%− 1.00% (w/v) xylan, except that kinetic parameters of xln-DT were determined with 0.01–0.68 mg/mL pNPX
as substrates. The values represent the means ± SD (n = 3).
*
P < 0.05 versus XynF (Control), (Student’s t-test).
**
P < 0.01 versus XynF (Control), (Student’s t-test).
***
P < 0.001 versus XynF (Control) (Student’s t-test).

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N. Li et al. Industrial Crops & Products 176 (2022) 114349

obtained by enzymes degrading corncob xylan. It can be seen from the

TLC that the XOS produced by three enzymes were mainly X3, X4 and X5
(Fig. S7a), but the amount of XOS produced by XynF was significantly
lower than that of two chimeras, and XynF could not produce X2 even if
the reaction time was extended to 12 h (Fig. S7b). While the DP of XOS
obtained by two chimeras was 2–6, hence these XOS would be used as
feed additives and ingredients in food (Rahmani et al., 2019; Yegin,
2017).
The products changes caused by reaction time were also explored.
With the extension of reaction time, the product category did not
change, and the product content gradually increased in time of 1–6 h but
remained unchanged in 6–12 h, indicating that the amount of XOS ob­
tained from hydrolysis after 6 h had reached the maximum under the
defined conditions (pH 6.5 and 65 ◦ C). However, X1 was not detected in
the products, therefore the X1 inhibition was avoided (Rahmani et al.,
2019). A quantity of XOS obtained after the hydrolysis of 12 h was
analyzed with HPLC (Fig. S7b). The conversion rate of XynF,
Xln-L0-XynF and Xln-L3-XynF towards corncob xylan was approxi­
mately 22.1%, 32.8% and 25.5%, respectively. The conversion rate of
Fig. 6. The kinetic analysis of XynF, Xln-L0-XynF, Xln-L3-XynF. The catalytic
Xln-L0-XynF was close to the maximum yield of 31.8% and 26.4% ob­
rate was characterized with various concentrations (0.05%− 1.00% (w/v)) of
sugarcane xylan as substrate. The values represent the means ± SD (n = 3). tained by Bian et al. (2013) and Wu et al. (2019) respectively. In the
absence of chemical treatment, this value was also higher than the yield
(23.2%) obtained by Liu et al. (2018).
3.4. Production of XOS by XynF and chimeras degrading corncob xylan

The reaction was conducted in the conditions of pH 6.5 and 65 ◦ C to 3.5. Antioxidant activity of XOS obtained by XynF and chimeras
obtain XOS. XOS were purified by filtration and ultrafiltration, and the
absorption of crude XOS and purified XOS liquids from 260 to 500 nm The antioxidant activity of XOS released by the enzymatic hydrolysis
wavelengths are shown in the Supplementary Data (Fig. S6). The for 6 h is shown in Fig. 7. DPPH radicals were significantly decreased in
amount of XOS was monitored at UV 280 nm. The amount of XOS the reaction systems containing XOS (Fig. 7a). The XOS obtained by
produced by XynF was decreased sharply compared with Xln-L0-XynF XynF, Xln-L0-XynF and Xln-L3-XynF degrading corncob xylan exhibited
and Xln-L3-XynF, and the amount of XOS after ultrafiltration was 49.5%, 67.9% and 61.4% antioxidant activity, respectively (Fig. 7b).
reduced a little. Fig. S7 shows the TLC and HPLC analysis of XOS The amount of XOS produced by two chimeras was increased, therefore
this result was consistent with the result obtained by Bian et al. (2013),

Table 4
Lists of fusion GHs and their properties.
Chimera Linker Host kcat/Km (s− 1mg− 1mL); stable conditiona Reference

Glucanase→Xylanase (GGGGS)2, (EAAAK)3 E. coli 31.3; pH 8–10, ≦ 50 C◦

Lu and Feng (2008)
Glucanase→Xylanase EFPDLGTGGGSGS Yarrowia lipolytica 646.6; ≦ 70 ◦ C Chen et al. (2018)
Xylanase→Glucanase EFPDLGTGGGSGS Y. lipolytica 0
Xylanase→Glucanase AGSGSGSGS Y. lipolytica 0
Xylanase→Glucanase (GGGGS)2 Pichia pastoris 1360.4; ≦ 60 ◦ C Kim et al. (2015)
Xylanase→xylosidase GGGGGADQLAIGPMYNQVVYQYPN E. coli NR Fan et al. (2009)
Xylanase→arabinosidase GGGGGADQLAIGPMYNQVVYQYPN E. coli 274.9 Fan et al. (2009)
Xylosidase→Xylanase SAGSSAAGSGSG E. coli 17.2; pH 4.6–10, ≦ 60 ◦ C Wang et al. (2010)
Xylosidase→Xylanase (GGGGS)2 E. coli 64.0; pH 2.0–12, ≦ 65 ◦ C This work
Xylosidase→Xylanase KAKLKEEEERKQREEEERIKRLEELAKRKEEERK E. coli 41.0; pH 2.0–12, ≦ 65 ◦ C This work

"→" indicates the fusion order that the C-terminus of the former is combined with the N-terminus of the latter; NR: not reported.
a
The substrate is xylan.

Fig. 7. The antioxidant activity of XOS obtained by XynF, Xln-L0-XynF, Xln-L3-XynF hydrolyzing corncob xylan. (a) UV–vis absorption spectra of DPPH in different
reaction systems. (b) Quantitative determination of antioxidant activity.

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N. Li et al. Industrial Crops & Products 176 (2022) 114349

which exhibited a concentration dependent scavenging effect. In fact, Declaration of Competing Interest
the antioxidant activity could be higher, since the XOS mixed with DPPH
were diluted twice in this study. This suggests that the chimeras has We have no competing financial interests or personal relationships
potential in converting waste corncobs to produce XOS with low cost. that could have appeared to influence the work reported in this paper.
Generally, the performance of Xln-L0-XynF and Xln-L3-XynF was
better than that of XynF, XynF-L0-Xln and XynF-L3-Xln, since when the Acknowledgement
fusion direction was xln-DT to XynF, the C-terminal loop of xln-DT and
linkers could extend the N-terminus of XynF, which reduced the adverse Financial support from the National Natural Science Foundation of
interactions between parental enzymes. It can be concluded that the China (No. 21776102) is greatly appreciated.
fusion strategy plays an important role in the construction of fusion GH.
The structure and function of the fusion enzyme are affected by linker Appendix A. Supporting information
and direction. This influence is actually caused by the interaction be­
tween the parental enzymes. Whether the interaction is beneficial to the Supplementary data associated with this article can be found in the
fusion enzyme depends on the conformation and biochemical properties online version at doi:10.1016/j.indcrop.2021.114349.
of parental enzymes.
Some previous research also proved that parental enzymes would References
make a difference on fusion enzyme. Kim, Jung, Lee, Song and Bae
(2015) used an identical linker to fuse a xylosidase or arabinosidase into Aalbers, F.S., Fraaije, M.W., 2019. Enzyme fusions in biocatalysis: coupling reactions by
the C-terminus of the xylanase (derived from Clostridium thermocellum). pairing enzymes. Chembiochem 20, 20–28. https://doi.org/10.1002/
cbic.201800394.
It was found that the chimera xylanase-arabinosidase had enhanced Álvarez, C., González, A., Negro, M.J., Ballesteros, I., Oliva, J.M., Sáez, F., 2017.
activity towards arabinoxylan, and the released reducing sugar was Optimized use of hemicellulose within a biorefinery for processing high value-added
increased by 30% compared with the mixed parental enzymes with the xylooligosaccharides. Ind. Crop. Prod. 99, 41–48. https://doi.org/10.1016/j.
indcrop.2017.01.034.
same moles, while the xylanase-xylosidase had no such improvement. In
Bailey, M.J., Biely, P., Poutanen, K., 1992. Interlaboratory testing of methods for assay of
the work of Chen et al. (2018), the N-terminus and C-terminus of the xylanase activity. J. Biotechnol. 23, 257–270. https://doi.org/10.1016/0168- 1656
glucanase (derived from Teleogryllus emma) were connected to the same (92)90074-J.
Basit, A., Liu, J., Rahim, K., Jiang, W., Lou, H., 2018. Thermophilic xylanases: from
xylanase (derived from Thermomyces lanuginosus) with the same linker,
bench to bottle. Crit. Rev. Biotechnol. 38, 989–1002. https://doi.org/10.1080/
and obtained glucanase-xylanase and xylanase-glucanase, respectively. 07388551.2018.1425662.
The temperature stability and activity of the former were improved, Bernardi, A.V., Gerolamo, L.E., Uyemura, S.A., Dinamarco, T.M., 2021. A thermophilic,
while the activity of the latter was decreased; the activity of the pH-tolerant, and highly active GH10 xylanase from Aspergillus fumigatus boosted pre-
treated sugarcane bagasse saccharification by cellulases. Ind. Crop. Prod. 170,
xylanase-glucanase linked by another linker was also decreased. 113697 https://doi.org/10.1016/j.indcrop.2021.113697.
What’s more, the GHs chimeras listed in Table 4 were expressed in Bian, J., Peng, F., Peng, X.P., Peng, P., Xu, F., Sun, R.C., 2013. Structural features and
E. coli or yeast, and different hosts would affect the folding, secretion antioxidant activity of xylooligosaccharides enzymatically produced from sugarcane
bagasse. Bioresour. Technol. 127, 236–241. https://doi.org/10.1016/j.
and function of fusion enzymes, even if they are closely related and biortech.2012.09.112.
belong to yeast (Xu et al., 2018). This suggest that the same chimera Bretagne, D., Paris, A., de Vaumas, R., Lafite, P., Daniellou, R., 2020. Crystal structure of
would show different properties when it was expressed in different Dictyoglomus thermophilum β-D-xylosidase DtXyl unravels the structural determinants
for efficient notoginsenoside R1 hydrolysis. Biochimie 181, 34–41. https://doi.org/
hosts. In the practical consolidated bioprocessing (CBP) of biomass 10.1016/j.biochi.2020.11.017.
conversion, some hosts would be used to convert the reducing sugars Carvalho, E.A., Dos Santos Goes, L.M., Uetanabaro, A.P.T., da Silva, E.G.P., Rodrigues, L.
produced by the decomposition of biomass into bioethanol or other B., Pirovani, C.P., da Costa, A.M., 2017. Thermoresistant xylanases from Trichoderma
stromaticum: Application in bread making and manufacturing xylo-oligosaccharides.
chemicals through fermentation (Olson et al., 2012; Kumar and Verma,
Food Chem. 221, 1499–1506. https://doi.org/10.1016/j.foodchem.2016.10.144.
2020). For example, Saccharomyces cerevisiae was used to produce Chang, F., Xue, S., Xie, X., Fang, W., Fang, Z., Xiao, Y., 2018. Carbohydrate-binding
ethanol, while Y. lipolytica and Lipomyces starkeyi were used to produce module assisted purification and immobilization of β-glucosidase onto cellulose and
application in hydrolysis of soybean isoflavone glycosides. J. Biosci. Bioeng. 125,
fatty acids or lipids (Tsigie et al., 2012; Lin et al., 2011). The research
185–191. https://doi.org/10.1016/j.jbiosc.2017.09.001.
would prefer these engineering hosts in the future, as they could express Chen, C.C., Gao, G.J., Kao, A.L., Tsai, Z.C., 2018. Bifunctional fusion enzyme EG-M-Xyn
and secrete chimeras that degrade biomass polysaccharides to produce displaying endoglucanase and xylanase activities and its utility in improving
reducing sugars, and switch on their sugar metabolism pathway to lignocellulose degradation. Int. J. Biol. Macromol. 111, 722–729. https://doi.org/
10.1016/j.ijbiomac.2018.01.080.
produce chemicals simultaneously, forming a continuous and recyclable Chen, C.H., Yao, J.Y., Yang, B., Lee, H.L., Yuan, S.F., Hsieh, H.Y., Liang, P.H., 2019.
CPB. Engineer multi-functional cellulase/xylanase/β-glucosidase with improved efficacy
to degrade rice straw. Bioresour. Technol. Rep. 5, 170–177. https://doi.org/
10.1016/j.biteb.2019.01.008.
4. Conclusion Chen, K., Li, K., Deng, J., Zhang, B., Lin, J., Wei, D., 2016. Carbonyl reductase
identification and development of whole-cell biotransformation for highly efficient
Semi-rationally, this study adopted an appropriate strategy of fusion synthesis of (R)-[3,5-bis(trifluoromethyl)phenyl] ethanol. Microb. Cell Factor. 15.
https://doi.org/10.1186/s12934-016-0585-5.
enzyme to modify XynF, two chimeras Xln-L0-XynF and Xln-L3-XynF Chen, X., Zaro, J.L., Shen, W.C., 2013. Fusion protein linkers: property, design and
were obtained and evaluated. The two chimeras not only retained the functionality. Adv. Drug. Deliv. Rev. 65, 1357–1369. https://doi.org/10.1016/j.
superior properties of the parental enzymes, but also had enhanced ac­ addr.2012.09.039.
Fan, Z., Wagschal, K., Lee, C.C., Kong, Q., Shen, K.A., Maiti, I.B., Yuan, L., 2009. The
tivity toward xylan, and they could degrade corncob xylan to produce
construction and characterization of two xylan-degrading chimeric enzymes.
XOS (DP of 2–6) with higher bioactivity efficiently, thus they have great Biotechnol. Bioeng. 102, 684–692. https://doi.org/10.1002/bit.22112.
application prospects in the utilization of waste corncobs. Theoretically, Guntas, G., Mansell, T.J., Kim, J.R., Ostermeier, M., 2005. Directed evolution of protein
switches and their application to the creation of ligand-binding proteins. PNAS 102,
the work implied that the fusion of two GHs with synergism and similar
11224–11229. https://doi.org/10.1073/pnas.0502673102.
biochemical properties could improve the activity and stability of them, Jagtap, S., Deshmukh, R.A., Menon, S., Das, S., 2017. Xylooligosaccharides production
and the parental enzyme with better properties seems to contribute by crude microbial enzymes from agricultural waste without prior treatment and
more favorable factors to chimeras. The application of the fusion their potential application as nutraceuticals. Bioresour. Technol. 245, 283–288.
https://doi.org/10.1016/j.biortech.2017.08.174.
enzyme technology can help in the development of a CPB and achieve Kim, H.M., Jung, S., Lee, K.H., Song, Y., Bae, H.J., 2015. Improving lignocellulose
the “circular bioeconomy” with zero-waste. degradation using xylanase-cellulase fusion protein with a glycine-serine linker. Int.
J. Biol. Macromol. 73, 215–221. https://doi.org/10.1016/j.ijbiomac.2014.11.025.
Kumar, B., Verma, P., 2020. Enzyme mediated multi-product process: a concept of bio-
based refinery. Ind. Crop. Prod. 154, 112607 https://doi.org/10.1016/j.
indcrop.2020.112607.

9
N. Li et al. Industrial Crops & Products 176 (2022) 114349

Li, G., Huang, Z., Zhang, C., Dong, B.J., Guo, R.H., Yue, H.W., Yan, L.T., Xing, X.H., 2016. continuous operation, scale-up and pilot reactor under biorefinery concept.
Construction of a linker library with widely controllable flexibility for fusion protein Bioresour. Technol. 299, 122685 https://doi.org/10.1016/j.biortech.2019.122685.
design. Appl. Microbiol. Biotechnol. 100, 215–225. https://doi.org/10.1007/ Schoffelen, S., van Hest, J.C.M., 2012. Multi-enzyme systems: bringing enzymes together
s00253-015-6985-3. in vitro. Soft Matter 8, 1736–1746. https://doi.org/10.1039/c1sm06452e.
Li, N., Han, X.W., Xu, S.J., Li, C.Y., Wei, X., Liu, Y., Zhang, R., Tang, X.H., Zhou, J.P., Sundd, M., Iverson, N., Ibarra-Molero, B., Sanchez-Ruiz, J.M., Robertson, A.D., 2002.
Huang, Z.X., 2018a. Glycoside hydrolase family 39 β-xylosidase of Sphingomonas Electrostatic interactions in ubiquitin: stabilization of carboxylates by lysine amino
showing salt/ethanol/trypsin tolerance, low-pH/low-temperature activity, and groups. In: Biochemistry, 41, pp. 7586–7596. https://doi.org/10.1021/
transxylosylation activity. J. Agric. Food Chem. 66, 9465–9472. https://doi.org/ bi025571dCCC:$22.00.
10.1021/acs.jafc.8b03327. Surek, E., Buyukkileci, A.O., Yegin, S., 2021. Processing of hazelnut (Corylus avellana L.)
Li, Q., Wu, T., Qi, Z., Zhao, L., Pei, J., Tang, F., 2018b. Characterization of a novel shell autohydrolysis liquor for production of low molecular weight
thermostable and xylose-tolerant GH 39 β-xylosidase from Dictyoglomus xylooligosaccharides by Aureobasidium pullulans NRRL Y–2311–1 xylanase. Ind.
thermophilum. BMC Biotechnol. 18, 29. https://doi.org/10.1186/s12896-018-0440- Crop. Prod. 161, 113212 https://doi.org/10.1016/j.indcrop.2020.113212.
3. Tsigie, Y.A., Wang, C.Y., Kasim, N.S., Diem, Q.D., Huynh, L.H., Ho, Q.P., Truong, C.T.,
Lin, J., Shen, H., Tan, H., Zhao, X., Wu, S., Hu, C., Zhao, Z.K., 2011. Lipid production by Ju, Y.H., 2012. Oil production from Yarrowia lipolytica Po1g using rice bran
Lipomyces starkeyi cells in glucose solution without auxiliary nutrients. J. Biotechnol. hydrolysate. J. Biomed. Biotechnol. 2012, 378384 https://doi.org/10.1155/2012/
152, 184–188. https://doi.org/10.1016/j.jbiotec.2011.02.010. 378384.
Liu, X., Liu, Y., Jiang, Z., Liu, H., Yang, S., Yan, Q., 2018. Biochemical characterization of van Rosmalen, M., Krom, M., Merkx, M., 2017. Tuning the flexibility of glycine-serine
a novel xylanase from Paenibacillus barengoltzii and its application in linkers to allow rational design of multidomain proteins. Biochemistry 56,
xylooligosaccharides production from corncobs. Food Chem. 264, 310–318. https:// 6565–6574. https://doi.org/10.1021/acs.biochem.7b00902.
doi.org/10.1016/j.foodchem.2018.05.023. Ventorim, R.Z., de Oliveira Mendes, T.A., Trevizano, L.M., Dos Santos Camargos, A.M.,
Lu, P., Feng, M.G., 2008. Bifunctional enhancement of a β-glucanase-xylanase fusion Guimaraes, V.M., 2018. Impact of the removal of N-terminal non-structured amino
enzyme by optimization of peptide linkers. Appl. Microbiol. Biotechnol. 79, acids on activity and stability of xylanases from Orpinomyces sp. PC-2. Int. J. Biol.
579–587. https://doi.org/10.1007/s00253-008-1468-4. Macromol.. 106, 312–319. https://doi.org/10.1016/j.ijbiomac.2017.08.015.
Mechelke, M., Koeck, D.E., Broeker, J., Roessler, B., Krabichler, F., Schwarz, W.H., Wang, R., Xue, Y., Wu, X., Song, X., Peng, J., 2010. Enhancement of engineered
Zverlov, V.V., Liebl, W., 2017. Characterization of the arabinoxylan-degrading trifunctional enzyme by optimizing linker peptides for degradation of agricultural
machinery of the thermophilic bacterium Herbinix hemicellulosilytica-Six new by-products. Enzym. Microb. Technol. 47, 194–199. https://doi.org/10.1016/j.
xylanases, three arabinofuranosidases and one xylosidase. J. Biotechnol. 257, enzmictec.2010.07.008.
122–130. https://doi.org/10.1016/j.jbiotec.2017.04.023. Wang, Y., Xue, P., Cao, M., Yu, T., Lane, S.T., Zhao, H., 2021. Directed evolution:
Milessi, T.S., Corradini, F.A.S., Marçal, J.V.M., Baldez, T.O., Kopp, W., Giordano, R.C., methodologies and applications. Chem. Rev. https://doi.org/10.1021/acs.
Giordano, R.L.C., 2021. Xylooligosaccharides production chain in sugarcane chemrev.1c00260.
biorefineries: From the selection of pretreatment conditions to the evaluation of Wu, Q., Fan, G., Yu, T., Sun, B., Tang, H., Teng, C., Yang, R., Li, X., 2019. Biochemical
nutritional properties. Ind. Crop. Prod. 172, 114056 https://doi.org/10.1016/j. characteristics of the mutant xylanase T-XynC(122)C(166) and production of
indcrop.2021.114056. xylooligosaccharides from corncobs. Ind. Crop. Prod. 142, 111848 https://doi.org/
Murakami, M.T., Arni, R.K., Vieira, D.S., Degreve, L., Ruller, R., Ward, R.J., 2005. 10.1016/j.indcrop.2019.111848.
Correlation of temperature induced conformation change with optimum catalytic Wu, X., Tian, Z., Jiang, X., Zhang, Q., Wang, L., 2018. Enhancement in catalytic activity
activity in the recombinant G/11 xylanase A from Bacillus subtilis strain 168 (1A1). of Aspergillus niger XynB by selective site-directed mutagenesis of active site amino
FEBS Lett. 579, 6505–6510. https://doi.org/10.1016/j.febslet.2005.10.039. acids. Appl. Microbiol. Biotechnol. 102, 249–260. https://doi.org/10.1007/s00253-
Ni, Z.F., Zeng, Y.J., Xu, P., Guo, Z.W., Ou, X.Y., Peng, F., Yang, J.G., Zong, M.H., Lou, W. 017-8607-8.
Y., 2019. Characterization of a novel methylaspartate ammonia lyase from E. coli Xia, H., Li, Z., Zhong, X., Li, B., Jiang, Y., Jiang, Y., 2019. HKUST-1 catalyzed efficient in
O157:H7 for efficient asymmetric synthesis of unnatural amino acids. ACS Sustain. situ regeneration of NAD+ for dehydrogenase mediated oxidation. Chem. Eng. Sci.
Chem. Eng. 8, 329–334. https://doi.org/10.1021/acssuschemeng.9b05424. 203, 43–53. https://doi.org/10.1016/j.ces.2019.03.076.
Olson, D.G., McBride, J.E., Shaw, A.J., Lynd, L.R., 2012. Recent progress in consolidated Xu, Q., Alahuhta, M., Wei, H., Knoshaug, E.P., Wang, W., Baker, J.O., Vander Wall, T.,
bioprocessing. Curr. Opin. Biotechnol. 23, 396–405. https://doi.org/10.1016/j. Himmel, M.E., Zhang, M., 2018. Expression of an endoglucanase-cellobiohydrolase
copbio.2011.11.026. fusion protein in Saccharomyces cerevisiae, Yarrowia lipolytica, and Lipomyces starkeyi.
Pinales-Márquez, C.D., Rodríguez-Jasso, R.M., Araújo, R.G., Loredo-Treviño, A., Biotechnol. Biofuels 11, 322. https://doi.org/10.1186/s13068-018-1301-y.
Nabarlatz, D., Gullón, B., Ruiz, H.A., 2021. Circular bioeconomy and integrated Xue, Y., Peng, J., Wang, R., Song, X., 2009. Construction of the trifunctional enzyme
biorefinery in the production of xylooligosaccharides from lignocellulosic biomass: a associating the Thermoanaerobacter ethanolicus xylosidase-arabinosidase with the
review. Ind. Crop. Prod. 162, 113274 https://doi.org/10.1016/j. Thermomyces lanuginosus xylanase for degradation of arabinoxylan. Enzym. Microb.
indcrop.2021.113274. Technol. 45, 22–27. https://doi.org/10.1016/j.enzmictec.2009.03.010.
Rahmani, N., Kahar, P., Lisdiyanti, P., Lee, J., Yopi, Prasetya, B., Ogino, C., Kondo, A., Yang, A., Cheng, J., Liu, M., Shangguan, Y., Liu, L., 2018. Sandwich fusion of CBM9_2 to
2019. GH-10 and GH-11 Endo-1,4-β-xylanase enzymes from Kitasatospora sp. enhance xylanase thermostability and activity. Int. J. Biol. Macromol. 117, 586–591.
produce xylose and xylooligosaccharides from sugarcane bagasse with no xylose https://doi.org/10.1016/j.ijbiomac.2018.05.199.
inhibition. Bioresour. Technol. 272, 315–325. https://doi.org/10.1016/j. Yegin, S., 2017. Single-step purification and characterization of an extreme halophilic,
biortech.2018.10.007. ethanol tolerant and acidophilic xylanase from Aureobasidium pullulans NRRL Y-
Rizk, M., Antranikian, G., Elleuche, S., 2012. End-to-end gene fusions and their impact 2311-1 with application potential in the food industry. Food Chem. 221, 67–75.
on the production of multifunctional biomass degrading enzymes. Biochem. Biophys. https://doi.org/10.1016/j.foodchem.2016.10.003.
Res. Commun. 428, 1–5. https://doi.org/10.1016/j.bbrc.2012.09.142. Zhang, R., Li, N., Liu, Y., Han, X.W., Tu, T., Shen, J.D., Xu, S.J., Wu, Q., Zhou, J.P.,
Ruiz, H.A., Conrad, M., Sun, S.N., Sanchez, A., Rocha, G.J.M., Romani, A., Castro, E., Huang, Z.X., 2019. Biochemical and structural properties of a low-temperature-
Torres, A., Rodriguez-Jasso, R.M., Andrade, L.P., Smirnova, I., Sun, R.C., Meyer, A. active glycoside hydrolase family 43 β-xylosidase: Activity and instability at high
S., 2020. Engineering aspects of hydrothermal pretreatment: from batch to neutral salt concentrations. Food Chem. 301, 125266 https://doi.org/10.1016/j.
foodchem.2019.125266.

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