Endo-1, 4-β-D-xylanohydrolase (EC3.2.1.8), referred to as
endoxylanase, is the most critical enzyme in the xylan degrading enzyme system
(Collins et al. 2005). Endoxylanases
are widely used in the paper industry (Basu
et al. 2018). During the pretreatment of raw materials, the endoxylanase
can effectively degrade the xylan re-adsorbed or deposited on the fiber surface
during the cooking process, while loosening the paper fiber structure and
making it easier to separate (Ashwani et
al. 2009; Weerachavangkul et al.
2012). During paper bleaching and fiber modification, xylanase can act on
hemicellulose in pulp connected with residual lignin, thus swelling and
loosening the fibers (Sandrim et al.
2005; Nagar et al. 2013). Xylanase
removes part of the hemicellulose and refine fibers on the surface of straw
pulp fibers, which can be used to improve the performance of straw pulp and
increase production efficiency (Buzała et al. 2016; Sridevi et al. 2017). Because of the
characteristics of the paper industry process, xylanases are required to be
alkali-resistant and have high thermal stability (Kumar et al. 2018).
Endoxylanase-producing microorganisms including
bacteria, molds, actinomyces, and yeasts were widely distributed in natural
environments (Juturu and Wu 2012). Fungi often produce acid xylanase which has
low thermal stability (Liu et al.
2013). Alkaline xylanase was usually derived from bacteria and actinomycetes
(Chakdar et al. 2016). Alkaline
xylanase was first isolated and purified from alkalophiles in 1973 (Horikoshi
1973). Sanjivkumar et al. (2017)
screened a xylanase-producing strain Streptomyces
olivaceus MSU3 from mangroves, the optimal reaction pH of the endoxylanase
produced was 8.0. Bagewadi et al.
(2016) isolated a thermostable GH-10 xylanase from Penicillium citrinum HZN13. The endoxylanase gene xynB was
cloned using a pair of degenerate primers designed according to the conservative
amino acid sequences of xylanases. The gene was expressed in E. coli, the molecular weight of the recombinant
endoxylanase is about 66 kDa.
Endoxylanase produced by Actinomycete is similar
to that produced by Bacterium, both have a wide range of pH tolerance, better
thermal stability, etc., and therefore have attracted more and more attention
from researchers (Beg et al. 2001).
An endoglucanase-producing strain Streptomyces spp. H31 was isolated from
soil in the early stage. In this paper, an alkali-resistant endoxylanase xynh31
was isolated and purified from the fermentation broth of this strain, and its
gene was cloned and expressed in E. coli.
This enzyme shown excellent properties such as alkali
resistance and thermal stability, and shows good industrial application
prospects. This research describes the isolation and purification of the
enzyme, the cloning and expression of the enzyme gene, and its enzymatic
properties.
Materials and Methods
SP-sepharose Fast Flow column and HisTrapTM
column was purchased from GE Healthcare (General Electric Co., U.S.A.). Vector
pET-28a (+) and E. coli BL21 (DE3)
were used for the expression of endoxylanase. Vector pMD19-T and E. coli
top10F´were used for plasmid construction and propagation. Xylan (Birchwood)
and other reagents were all of analytical grade.
Isolation and purification of natural endoxylanase Xynh31
The culture broth of Streptomyces sp. H31 was centrifuged at 12000 r/min and
the supernatant was loaded onto an SP-Sepharose Fast Flow column with PBS
buffer pH 6.0 at a flow rate of 1 mL/min. Gradient elution was performed using
NaCl-containing PBS buffer (pH 6.0), and the elution peaks were collected to
determine the enzyme activity. The enzyme activity peak was dialyzed and
concentrated, and then analyzed by SDS-PAGE electrophoresis (Brunelle and Green 2014).
Cloning and plasmid construction
Extraction protocols of Genomic DNA and plasmid were used as described (Sambrook et al. 1989). Endoxylanases mostly
belong to the glycoside hydrolase (GH) family 10/11. The Pfam 26.0 (Sanger
institute) platform (Finn et al.
2016) was used to analyze the conserved sequences of glycoside hydrolase
families 10 and 11. Degenerate primers that were based on the most highly
conserved amino acid residues are then designed to amplify the conserved
sequence of endoxylanase gene (Staheli et al.
2011). The upstream and downstream sequences were amplified by TAIL-PCR as
described (Liu and Whittier 1995). The cloned endoxylanase gene xynh31 was ligated into the vector
pET-28a (+) and then transformed into E. coli
BL21 (DE3).
Expression and purification of the recombinant
enzyme
The selected recombinant was induced by IPTG and the cultures were
harvested by centrifugation. The bacteria deposit was suspended in Tris-HCl
buffer (50 mM, pH 8.0) and lysis by ultrasonic for 20 min. The cell-free
extract was dialyzed and then was loaded onto a HisTrapTM column
with Equilibration buffer (Tris-HCl buffer, 50 mM; NaCl, 0.2 M, pH 8.0). A linear gradient (i.e.,
0.02, 0.05, 0.1, 0.2 and 0.5 M) of
imidazole containing 0.2 M NaCl (flow rate: 1 mL/min) was used for the elution
of adsorbed proteins. Fraction with endoxylanase activity were collected and
tested for hydrolysis of 1% xylan at 45°C for 20 min. The Recombinant rXynh31
was subsequently examined by SDS-PAGE.
Biochemical characterization of endoxylanase
Xylan (Birchwood) was used as the substrate for the assay of the
endoxylanases activity in different pH values and temperatures. The amount of
enzyme required to produce 1.0 mg of xylose per hour is defined as one enzyme
activity unit (U/mL). The method was used as described previously (Costa-Ferreira
et al. 1994).
Results
Purification of natural endoxylanase Xynh31
The strain Streptomyces spp. H31
was inoculated in liquid culture at 37°C, 200 r/min
for 5 days and the fermentation broth was centrifuged at 4°C, 12000 r/min for
10 min.
The SP-Sepharose Fast Flow column was equilibrated
to baseline using pH 6.0 PBS buffer. Then apply the crude enzyme solution at a
flow rate of 1 mL/min. Gradient elution was performed with PBS solutions
containing NaCl (0.1 M/0.2 M/1 M)
and the eluate is collected and measured for enzyme activity. The purified fraction which can be detected
enzyme activity was further dialyzed and concentrated and analyzed by SDS-PAGE
protein electrophoresis (Fig. 1). A single band was visible, which proved that
the endoxylanase xynh31 was successfully separated and purified by
one-step cation exchange chromatography. Protein electrophoresis showed that
the molecular weight of the enzyme was about 42 kDa. The specific activity of
the enzyme was calculated to be 671.63 U/mg.
Cloning and sequence analysis of gene xynh31
The conservative sequence of the xyn31
gene was amplified by conservative primers. TAIL-PCR was used to clone the
full-length endoxylanases gene from the genome of Streptomyces spp. H31. The gene has an open reading frame (ORF)
with 1380 bp, which starts with ATG, and TGA as the stop codon. The gene
encoded a protein consisting of 459 amino acids with a calculated molecular
mass of 42 kDa.
BLAST analysis showed that the amino acid sequence
of this gene had the highest homology with the family 10 xylanase from Streptomyces Halstedii Jm8 but had only 82% similarity. Analysis
of the conserved domain of Xynh31 revealed that it belongs to the glycoside
hydrolase 10 family (GH10) and contains a second family carbohydrate-binding
domain (CBM_2) at the C-terminal (Fig. 2).
Expression and purification of recombinant endoglucanases rXynh31
The endoxylanase gene xynh31 was ligated into the expression vector pET-28a (+) and
introduced into E. coli BL21 (DE3).
The optimal enzyme activity was up to 186.3 U/mL.
The Ni-column affinity chromatography was used to purify the recombinant
enzyme rXynh31. The purified protein was analyzed by SDS-PAGE (Fig. 3). The
results were shown in Fig. 3, indicating that the enzyme has
Fig. 1: SDS-PAGE of purified Xynh31
M:
Premixed Protein Marker; 1: Crude enzyme; 2: SP-Sepharose
Fast Flow Enzyme activity fraction
Fig. 2: Conserved domain analysis of
Xynh31
The green is the glycosylhydrolase 10 family (GH10)
catalytic domain, red is the binding domain CBM_2
Fig. 3: SDS-PAGE of the expression and purified endoxylanase
M: Premixed Protein Marker (High);
1: Without IPTG; 2: Induction with 0.2 mM
IPTG; 3: Purified protein
been successfully isolated and purified
with a molecular weight of approximately 60 kDa. The theoretical value of the
recombinant protein is about 55 kDa, which may be related to the basic amino
acids (such as His tags at both ends) that affect the SDS-PAGE mobility,
resulting in a difference between the apparent molecular weight and the
theoretical molecular weight. The specific activity of the recombinant
endoxylanase was 628.4 U/mg.
Enzymatic properties of endoxylanase
Xynh31 and rXynh31
Effect of temperature on enzyme activity
Analysis of the optimal reaction temperature of the native endoxylanase
Xynh31and the recombinant rXynh31, the results can be seen from Fig. 4-A. The
optimal reaction temperature range of the endoxylanases
is 45–50℃. When the temperature exceeds 60℃, the enzyme activity
decreases rapidly. The residual enzyme activity of rXynh31 is slightly higher
than that of Xynh31 at a lower temperature (≤ 40℃), and the
residual enzyme activity of the Xynh31 is slightly higher than that of the
recombinant enzyme rXynh31 at higher temperature (≥ 50℃).
Fig. 4: The effect of temperature on the endoxylanase
(A) Optimum reaction temperature of the endoxylanase.
(B) Thermostability
of the endoxylanase
*: P < 0.05;
**: P < 0.01
Fig. 5: The effect of pH on the endoxylanase
(A)
Optimum reaction pH of the endoxylanase. (B) The pH stability of the endoxylanase
*: P < 0.05;
**: P < 0.01
Fig. 6: Effect of
metal ions,
surfactant and metal chelate on endoxylanase activity
The control group
enzyme activity is defined as 100%, Data are expressed as mean ± SD (n = 3)
relative to control samples
Thermal stability of the endoxylanases
The endoxylanase Xynh31 and rXynh31 were analyzed
for thermal stability. It can be seen from Fig. 4-B that the enzyme has a
constant activity after incubation for 60 min at a temperature lower than
50℃. However, when the temperature was higher than 55°C, the temperature
stability of the enzymes decreased rapidly. At 55°C, the relative enzyme
activity was closer to 80%. When the temperature was raised above 70°C, the
relative enzyme activity dropped below 20%.
Effect of pH on enzyme activity
From Fig. 5-A, it can be seen that the relative residual enzyme activity
of the recombinant enzyme under acidic conditions (pH 3–5) is higher than the
native enzyme. The relative enzyme activity is maintained at a very high level
between pH 7–9, and the relative enzyme activity is close to 100% at pH 7 and
pH 9, which indicates that the enzyme Xynh31 is alkaline resistant endoxylanase, its optimum reaction pH is 7–9. The relative
enzyme activity exceeds 50% at a pH of 10, indicating that the enzyme can adapt
to the drastic change from weakly acidic to strong alkaline, and can perform
better under alkaline conditions than under acidic conditions.
pH stability of the
endoxylanases
Endoxylanases Xynh31 and rXynh31 were incubated at different pH for 60
min. The results were shown in Fig. 5-B. At pH 3–4, the relative enzyme
activity of the recombinant enzyme is slightly higher than that of the natural
enzyme; at pH 5–11, the relative enzyme activity of the enzyme is higher than
60%; at pH 11, it can still exceed 50%. It can be seen that the enzyme can
maintain a relatively high relative enzyme activity under the condition of pH 5–11, especially the residual enzyme activity is above 70% in
the entire range of pH 6–10. From the enzymatic nature of the enzyme, its
alkali resistance is better and its stability is high under neutral and
alkaline conditions. This can be reasonably connected with the requirements of
the enzyme used in the paper industry and has a good application prospect.
Effect of metal ions, surfactant and metal chelate on endoxylanase
activity
Metal ions with a final concentration of 10 mM and SDS and EDTA
with a final concentration of 0.5% were used to investigate the effects of
metal ions and surfactants on the activity of endoxylanase (Fig. 6). In
general, metal ions had little effect on the original enzyme and recombinase.
Ni2+, Zn2+ has an obvious promotion effect on enzyme
activity, and Fe3+ has a significant inhibitory effect. SDS and EDTA
had little effect on enzyme activity.
Discussion
Endoxylanase can be widely used in
a variety of industrial production processes (Basit et al. 2018). Currently reported endoxylanases generally have
deficiency such as narrow optimal pH range and poor thermal stability (Walia et al. 2017; Singh 2019) and in
industrial applications (especially in the textile and paper industry),
However, often face situations such as a large change in the pH of the reaction
environment and a drastic change in the reaction temperature. It can be seen
that these shortcomings have become constraints to its further application.
Therefore, it is of great interest to discover new types of endoxylanases with
wide pH adaptability suitable for industrial applications.
In this paper, the endoxylanase gene xynh31 from Streptomyces spp. H31 was successfully cloned. Based on the
sequence analysis results and the differences in molecular weight and enzymatic
characteristics between this enzyme and the other existing endoxylanase, it
showed that the gene xyh31 is a newly discovered endoxylanase gene.
After analysis with signalP (Nielsen 2017), the
possible signal peptide sequences were removed. The gene sequence excluding the
predicted signal peptide was expressed in E.
coli, but the activity was found to be extremely low. It is speculated that
the signal peptide sequence inferred by SignalP software may be inaccurate,
resulting in the N segment of the enzyme being excised, thereby affecting the
activity. Therefore, the entire open reading frame of the gene sequence from
ATG was expressed and analyzed in this paper.
Through the study of enzymatic properties, it was
found that the optimal reaction pH of this enzyme is between
7–9, and the optimal reaction temperature is between 45–50°C. The enzyme
has good alkali-resistance, and the pure enzyme still has nearly 70% residual
activity after being stored for 60 min at pH 10.0. It is a typical
alkali-resistant xylanase. This is related to the natural alkaline environment
of the strain Streptomyces sp. H31
(Chen et al. 2017). In addition, the
enzyme also has many excellent properties such as resistance to a variety of
metal ions and resistance to SDS and EDTA. The enzymatic characteristic of the
endoxylanase determine its application potential and application fields.
Generally, it requires high catalytic efficiency, good resistance to stress, a
wide range of acid-base and temperature adaptation, etc. In the paper and pulp
industry, lignin is often removed by high-temperature cooking under alkaline
conditions (Kumar et al. 2016).
However, endoxylanases currently used in industrial processes is generally not
alkali-resistant and has poor thermal stability, which requires temperature and
pH to be adjusted before adding enzymes, which is laborious, time-consuming,
and extremely uneconomical (Sun and Li 2008). The development of new,
high-quality alkaline-tolerant xylanase can not only improve the quality of
pulp and finished paper, but also contribute to environmental protection (Bajaj
and Singh 2016). Based on the comprehensive enzymatic properties, it can be
judged that the endoxylanase Xynh31 is a high-quality alkaline endoxylanase,
which is suitable for industrial applications such as papermaking, waste paper
deinking and recycling.
Conclusion
A novel endoxylanase Xynh31 was isolated and purified from Streptomyces spp. H31, and the complete open reading frame (ORF) of the
enzyme gene was obtained by PCR cloning. The xynh31 gene was
successfully expressed in E. coli using the pET-28a (+) expression
vector. The natural and recombinant endoxylanases studied in this paper have
excellent enzymatic characteristics such as alkali resistance, high temperature
resistance, metal ion resistance, surfactant resistance, and metal chelator
resistance, which meet the basic requirements of industrial enzymes such as
papermaking.
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