NC State
Sun, J., Shi, S., Wu, J., Xie, R., Geng, A., and Zhu, D. C. (2016). "Characterization of a salt-tolerant and cold-adapted xylanase from Bacillus cellulosilyticus," BioRes. 11(4), 8875-8889.


A xylanase (Xyn10A) gene from the saline-alkali-tolerant microorganism Bacillus cellulosilyticus DSM 2522 was cloned and expressed in Escherichia coli BL21 (DE3). The open reading frame was composed of 1008 base pairs, and it encoded 335 amino acid residues belonging to glycosyl hydrolase family 10. The optimal temperature and pH of the purified Xyn10A were 40 °C and 8.0, respectively. The Xyn10A was sensitive to heat and showed obvious cold-adapted activity, retaining 38.3%, 55.7%, and 82.9% of the optimal activity at 4, 20, and 30 °C, respectively. Xyn10A also showed a high level of NaCl tolerance. The highest activity was observed with 1.5 M NaCl. The specific enzyme activity of Xyn10A was as much as 163.8 U/mg. Kinetic assays showed that Km, Vmax, and Kcat were 2.56 mg/mL, 202.5 μM/min/mg, and 132.6 /s, respectively. Additionally, the main hydrolysis products using birchwood xylan as substrate were xylobiose, xylotriose, and xylotetraose, as determined by thin layer chromatography analysis. As a cold-adapted and salt-tolerant enzyme, Xyn10A is an ideal candidate for further research and biotechnological applications.

Download PDF

Full Article

Characterization of a Salt-Tolerant and Cold-Adapted Xylanase from Bacillus cellulosilyticus

Jianzhong Sun, Sailing Shi, Jian Wu,* Rongrong Xie, Alei Geng, and Dao Chen Zhu

A xylanase (Xyn10A) gene from the saline-alkali-tolerant microorganism Bacillus cellulosilyticus DSM 2522 was cloned and expressed in Escherichia coli BL21 (DE3). The open reading frame was composed of 1008 base pairs, and it encoded 335 amino acid residues belonging to glycosyl hydrolase family 10. The optimal temperature and pH of the purified Xyn10A were 40 °C and 8.0, respectively. The Xyn10A was sensitive to heat and showed obvious cold-adapted activity, retaining 38.3%, 55.7%, and 82.9% of the optimal activity at 4, 20, and 30 °C, respectively. Xyn10A also showed a high level of NaCl tolerance. The highest activity was observed with 1.5 M NaCl. The specific enzyme activity of Xyn10A was as much as 163.8 U/mg. Kinetic assays showed that KmVmax, and Kcat were 2.56 mg/mL, 202.5 μM/min/mg, and 132.6 /s, respectively. Additionally, the main hydrolysis products using birchwood xylan as substrate were xylobiose, xylotriose, and xylotetraose, as determined by thin layer chromatography analysis. As a cold-adapted and salt-tolerant enzyme, Xyn10A is an ideal candidate for further research and biotechnological applications.

Keywords: Xylanases; Bacillus cellulosilyticus; Cold adapted; Salt tolerant

Contact information: Biofuels Institute, School of the Environment and Safety Engineering, Jiangsu University, Zhenjiang 212013, Jiangsu, China; *Corresponding author:


Endo-1,4-β-xylanases (EC3.2.1.8), which randomly hydrolyze the β-1,4-xylosidic bonds of xylan, have gained much attention in biotechnological fields because of their potential value. They have been used widely in the conversion of lignocelluloses into fermentable sugars and prebiotic oligosaccharides (Juturu and Wu 2011; Shi et al. 2013; Amel et al. 2016). They can also be used to improve the quality of breads and beverages, enhance the digestibility of animal feeds, and improve the bleaching and deinking of paper (Huang et al. 2006; Dhimanet al. 2008; Garg et al. 2009; Maity et al. 2012; Singh et al. 2012).

Many xylanases have been reported from various microbes, such as bacteria, fungi, and yeasts (Polizeli et al. 2005; Dhiman et al. 2008; Rao et al. 2008; Khandeparker et al. 2011; Tenkanen et al. 2013). Xylanases originating from the Bacillus strains are intrinsically interesting because of their characteristics, such as alkali and salt tolerance, cold adaptation, or thermostability; they are widely employed in industrial applications (Chang et al. 2004; Huang et al. 2006; Mamo et al. 2006; Ruller et al. 2008; Khandeparker et al. 2011; Haddar et al. 2012; Thomas et al. 2014).

The genome for Bacillus cellulosilyticus, a soil microorganism that is both alkaliphilic and halophilic, has recently been revealed (GenBank accession number: GCA_000177235.2) (Mead et al. 2013). B. cellulosilyticus possesses rich biomass-degrading enzymes besides those for crystalline cellulose. However, research on the enzymes from B. cellulosilyticus is still limited. Among the enzymes researched, three predicted xylanases have been denoted as Bcell_0541, Bcell_0537, and Bcell_0547, respectively. Bcell_0541 was predicted to belong to the GH8 family. Bcell_0537 and Bcell_0547 were classified in the GH10 family, with the former composed of a catalytic domain and carbohydrate-binding module (CBM), and the latter lacking the CBM domain. In this study, Bcell_0547 was studied and designated as Xyn10A.

Salt-tolerant xylanases can be applied in marine products, wastewater processing, and bioethanol production from seaweeds (Bai et al. 2012; Liu et al. 2013, 2014). The cold-adapted enzymes are most active at low and medium temperatures, which has obvious advantages in the bioprocess, such as saving energy, reducing production costs, and improving food flavor (Cavicchioli et al. 2002; Vester et al. 2014).

However, in the past, cold-adapted and salt-tolerant xylanases have rarely been published. In this study, the Bcell_0547 gene was heterologously expressed and characterized. The Xyn10A displayed high salt tolerance and cold-adapted activity. Thus, Xyn10A has widespread application prospects in biotechnology fields and especially in the food industry.



Birchwood xylan, Avicel, carboxymethylcellulose (CMC), and cellobiose were purchased from Sigma Chemical Company (St. Louis, USA). Xylo-oligosaccharides (XOs) were purchased from ADHOC International Technologies Co., Ltd. (Beijing, China). HisTrap HP and HiTrap Q HP columns were obtained from GE Life Sciences (Piscataway, NJ, USA). The primers were synthesized by Sangon (Shanghai, China), and the pEASY-E2 expression kit and the protein assay kit were provided by TransGen Biotech (Beijing, China). All other chemicals were of analytical grade commercially available unless otherwise specified.

The B. cellulosilyticus DSM 2522 was purchased from DSMZ (Braunschweig, Germany). The pEASY-E2 Expression Vector (Transgen Biotech, China) and E. coli BL21 (DE3) (Novagen, Darmstadt, Germany) were employed as the expression vector and host cells, respectively.


Strain culture

The B. cellulosilyticus DSM 2522 strain was maintained in ATCC medium 661 (alkaline Bacillus medium, peptone 10.0 g, yeast extract 5.0 g, glucose 10.0 g, K2HPO4 1.0 g, NaCl 5.0 g, agar 15.0 g, distilled water 900.0 mL, autoclaved at 115 °C for 15 min, cooled to 50 °C, and 100.0 mL of filter-sterilized 10% Na2CO3 solution added). E. coli BL21 (DE3) was grown at 37 °C in a Luria-Bertani medium (LB) and supplemented with ampicillin (100 μg/mL).

DNA manipulation

DNA was extracted from B. cellulosilyticus with the extraction kit (Takara, Dalian, China), and the Xyn10A gene was amplified by PCR using forward primer 5’-AAGCAAAAGCTAGAAGAAAC-3’ and reverse primer 5’-AAAACGAGTAATG-TTCCA-3’. Direct ligations of the PCR products to the pEASY-E2 expression vector were performed according to the manufacturer’s instructions. The recombinant plasmid was named pEASY-E2-Xyn10A, and the sequence was confirmed by Sangon Biotechnology Inc. (Shanghai, China).

Expression and purification of the Xyn10A

Plasmid pEASY-E2-Xyn10A was transformed into E. coli BL21 (DE3) cells. The transformed strains were grown in an LB medium containing 100 μg/mL of ampicillin at 37 °C until the OD600 reached 0.8. Expression was induced by adding isopropyl-β-D-thiogalactopyranoside (IPTG) to a final concentration of 0.5 mM and incubation at 25 °C for 12 h. The cells were harvested by centrifugation at 8,000 × g for 15 min at 4 °C, washed with sterile distilled H2O, and resuspended in buffer A (20 mM sodium phosphate, 0.5 M NaCl, pH 7.4). The cells were disrupted by sonication on ice and centrifuged at 10,000 × g for 10 min at 4 °C. The supernatant was loaded onto a pre-equilibrated 5-mL Histrap HP Ni2+-NTA affinity column for purification. The proteins were eluted by a buffer solution containing stepwise increases of imidazole (50, 100, 200, and 300 mM), desalted, and further purified by Histrap Q HP. The purified recombinant proteins were confirmed by sodium dodecyl sulfate-polycrymide gel electrophoresis (SDS-PAGE). Protein concentrations were determined by the BCA method using bovine serum albumin as the standard.

Determination of enzyme activities

To measure Xyn10A activity, the reaction mixture containing 450 μL of birchwood xylan (1%, w/v) in 50 mM sodium phosphate buffer (pH 8.0) and 50 μL of enzyme was incubated for 10 min at 40 °C. The reaction was stopped by the addition of 1.0 mL of 3,5-dinitrosalicylic acid (DNS), followed by boiling for 5 min. The amount of reducing sugar released was quantified by the 3,5-dinitrosalicylicacid (DNS) method using xylose as a standard. One unit (U) of enzyme activity was defined as the amount of enzyme required to release 1 μmol of reducing sugars per min under the assay conditions. Specific activities were expressed as units per mg of protein.

Enzyme characterization

To determine the optimal pH, the activity of Xyn10A was measured at 40 °C in the following buffers (50 mM) of different pH: citrate acetate buffer (pH 4.0 to 6.0), sodium phosphate buffer (pH 6.0 to 8.0), Tris-HCl buffer (pH 8.0 to 9.0), and NaOH-glycine buffer (pH 9.0 to 11.0). To determine the optimal temperature, the activity of Xyn10A was measured at different temperatures (4 to 60 °C) in the presence of 50 mM phosphate buffer (pH 8.0). The value obtained at the optimum temperature or pH was defined as 100%, and the results were expressed as relative activity.

The pH stability was determined by pre-incubating the enzyme at 30 °C for 1 h at various pH levels from 4.0 to 11.0. The residual activity was assayed by the standard assay methods. Thermal stability was measured by assaying the residual activity after incubation of the enzyme at 30, 40, and 50 °C for various time periods (0, 10, 20, 40, and 60 min) in 50 mM phosphate buffer (pH 8.0). The value from the sample without pre-incubation was defined as 100%.

Substrate specificities were studied by measuring enzyme activity using various polysaccharides as substrates in place of the xylan. The enzyme reactions were carried out in the 50 mM phosphate buffer (pH 8.0) at 40 °C.

The effects of metal ions and chemicals on Xyn10A activity were investigated. To study the effects, 1 mM metal salts (NaCl, KCl, CaCl2, FeCl2, NiCl2, CoCl2, MnCl2, CuCl2, MgCl2, and ZnCl2) and 5 mM chemicals (Na2EDTA, SDS, and Tween-20) were added to the reaction mixture, respectively. The residual activities were measured at 40 °C in 50 mM phosphate buffer (pH 8.0), and the activity without the addition of metal ions and chemicals was set as 100%. To assess the effect of sodium chloride on xylanase activity, various concentrations of NaCl (0.5 to 3.0 M) were added to the reaction mixture. Then, the activity was measured under standard assay conditions, and the residual activity (%) was calculated. The enzyme activity without the addition of NaCl was set as 100%.

To estimate the kinetic parameters, the enzyme reaction was carried out at 40 °C for 10 min using different substrate concentrations (0% to 1.0% birchwood xylan) in 50 mM phosphate buffer (pH 8.0). The Michaelis-Menten constant (Km) and maximal reaction velocity (Vmax) of Xyn10A were determined using the Lineweaver–Burk plots.

Hydrolysis products analysis by thin layer chromatography (TLC)

A 500-μL reaction solution containing 50 μL of Xyn10A in 50 mM phosphate buffer (pH 8.0) and 450 μL of birchwood xylan (1%) was incubated at 30 °C for 90 min. After incubation, the solution was boiled for 5 min and then centrifuged at 10,000 × g for 10 min. The supernatant was filtrated through a 0.22-μm cellulose acetate membrane. Analysis of the hydrolytic products (2 μL) was performed on a silica gel-coated glass plate (50 mm × 100 mm) with a mixture consisting of xylose, xylobiose, xylotriose, and xylotetraose as standards. A solvent system composed of n-butanol/acetic acid/H2O (10: 5: 1, v/v/v) was used. After the plate was dried at room temperature, the TLC plate was sprayed with a mixture of diphenylamine/aniline, phosphoric acid/acetone reagents (2 g: 2 mL: 10 mL: 100 mL) and then heated at 85 °C for 10 min for coloration.

Sequence and data statistics

The open reading frame (ORF) was obtained from KEGG ( The signal peptide was predicted using the Signal P 4.0 server ( The amino acid sequence alignment was performed using the DNAMAN program or by using the BLAST program ( The theoretical molecular weight and isoelectric point were predicted by online software ( All of the values shown in the figures were averaged from three replicates. The figures were created with origin 8.0 software.


Expression and Purification of the Recombinant Xyn10A

One of the predicted xylanases (Bcell_0547, Xyn10A) belonging to the GH10 family from B. cellulosilyticus was examined in this study. The ORF of the Xyn10A gene consisted of 1008 base pairs, and it encoded a protein consisting of 335 amino acid residues with a theoretical molecular weight of 39.3 kDa. There was no obvious signal peptide sequence found in Xyn10A using SignalP 4.0 analysis. In addition, the multiple sequence alignment (Fig. 1) revealed two highly conserved residues, Glu48 and Glu138, which are considered to be crucial for the catalytic activity of family 10 glycosyl hydrolases. Xyn10A showed the highest sequence similarity (67%) to the xylanase from Bacillus akibai (Accession number WP_035668525.1) and the highest identity (61%) to the xylanase from Bacillus sp. SN5, among the characterized xylanases (Bai et al. 2012).

Fig. 1. Sequence alignment of Xyn10A to other cold-adapted xylanases belonging to GH10. Sequence alignment was performed using DNAMAN8.0. Stars above residues indicate the conserved catalytic amino acids. Identical residues are shaded. Xyn10A, in this study (CP002394); BS Xyn10A, from Bacillus sp. SN5 (AGA16736); XynA, from G. mesophila KMM241 (FJ715293); ZP: from Z. profunda (WP_013072455).

The Xyn10A gene was cloned from the genomic DNA of B. cellulosilyticus. The amplified gene was transferred into the pEASY-E2 Expression Vector using the pEASY-E2 expression kit. The Xyn10A gene was successfully expressed in E. coli BL21 (DE3) as the his6 tagged fusion protein. The recombinant Xyn10A protein was purified to homogeneity by Ni-NTA affinity and anion exchange chromatography, and the single bands were exhibited by SDS-PAGE analysis (Fig. 2). It showed a molecular weight of about 39 kDa, which was consistent with the theoretical molecular weight.

Fig. 2. SDS–PAGE analysis of purified recombinant Xyn10A. Lanes: M, protein markers; 1, total protein from E. coli without IPTG induction; 2, total protein from E. coli by induction with 0.5 mM IPTG for 12 h; 3, the supernatant from E. coli after cell disruption; 4, purified protein after Ni-affinity; 5, purified protein after anion exchange Q column

The Properties of Xyn10A

Effect of pH and temperature on activity

The results showed the highest enzyme activity of Xyn10A at a pH of 8.0. Almost no activity was found at pH 4.0 and 10.6. However, the enzyme retained more than 50.0% of the relative activity in the pH range from 6.0 to 9.0 (Fig. 3a). When Xyn10A was pre-incubated for 1 h at 30 °C with pH values ranging from 4.0 to 11.0, it showed good stability, retaining at least 90.0% of the original activity in the pH range from 6.0 to 9.0. When the pH value was less than 5.0 or above 9.0, however, the pH stability decreased sharply (Fig. 3b). These results indicated that Xyn10A was stable in moderate and slightly alkaline conditions. This was similar to the xylanases from Bacillus mojavensis A21 (Haddar et al. 2012), as its best activity was also found at pH 8.0. Moreover, several alkaliphilic xylanases from the same genus (Bacillus) showed an optimum activity at pH 9.0 to 10.0, such as B. pumilus SV-85S (Nagar et al. 2010), and B. halodurans MTCC 9512 and S7 (Mamo et al. 2006; Mamo et al. 2009; Garg et al. 2009). These xylanases retained more than 50% activity even at pH 12.0 after 1 h incubation. The xylanases from Bacillus sp. SN5(Bai et al. 2012) showed the highest similarity (61%) with Xyn10A among characterized xylanases. Its optimum pH was 7.0 and not especially alkaline, though Bacillus sp. SN5 (Bai et al. 2012) was the alkaliphilic strain. Compared with the other reported cold-adapted xylanases, only Xyn10A displayed the optimum activity at alkaline conditions.

Fig. 3. Optimal pH and temperature and their stabilities for Xyn10A. a, Effect of pH on Xyn10A activity. Activities at various pHs were assayed at 40 °C; b, pH stability of Xyn10A. Residual activities were assayed at pH 8.0, 40 °C after incubation in buffers of different pH at 30 °C for 1 h; c, Effect of temperature on enzyme activity. The assay was performed at pH 8.0 in phosphate buffer. d, Thermostability of Xyn10A with and without NaCl. The enzyme was incubated at 30, 40, and 50 °C for different periods of time (10, 20, 40, 60 min), and then the residual activity was assayed at standard conditions. Each value represents the average of triplicate experiments. Error bars represent the standard deviation.

The results of testing the optimal temperature for Xyn10A showed the highest activity at a temperature of 40 °C. It is noteworthy that Xyn10A showed high relative activity at low temperature, and even retained significant activity at 4 °C. The relative activity was 38.3%, 55.7%, and 82.9% at 4 °C, 20 °C, and 30 °C, respectively (Fig. 3c). When the temperature was above 50 °C, the catalytic activity was lost quickly. Meanwhile, Xyn10A exhibited low thermostability at elevated temperature. The Xyn10A retained nearly 87.2% residual activity after being incubated at 30 °C for 60 min. However, more than 90% of the activity was lost after 10 min of incubation at 50 °C, and all activity was lost at 60 °C (Fig. 3d). According to previous reports, the typical cold-adapted enzyme exhibited the optimum activity at lower temperatures and was more sensitive to heat (Cavicchioli et al. 2002; Siddiqui and Cavicchioli 2006), which was a sharp contrast with the thermophilic xylanases (Petegem et al. 2003; Chang et al. 2004; Shi et al. 2013; Vester et al. 2014). Therefore, Xyn10A can be considered a cold-adapted xylanase. Recently, the research about cold-adapted enzymes has been given much attention, as the use of cold-adapted enzymes has many advantages, such as reducing energy consumption and maintaining the original food flavor (Liu et al. 2014; Vester et al. 2014).

Various kinds of cold-adapted enzymes have been reported, such as esterases (Novototskaya-Vlasova et al. 2012), lipases (Tian et al. 2014), chitinases (Ramli et al. 2011), β-glucosidases (Vester et al. 2014), and proteinases (Kredics et al. 2008). However, there has been only limited study of cold-adapted xylanase. The xylanases from Glaciecola mesophila(Guo et al. 2009), Bacillus sp. SN5 (Bai et al. 2012), Zunongwangia profunda (Liu et al. 2014), and goat rumen contents belonging to the GH10 family showed obvious cold-adapted activity (Wang et al. 2011). Additionally, several xylanases from G. mesophila KMM241 (Guo et al. 2013), Pseudoalteromonas haloplanktis (Petegem et al. 2003), and the environmental DNA library of lagoons (Lee et al. 2006) belonging to the GH8 family also exhibited the cold activity. The temperature characteristics of these xylanases were similar to the Xyn10A results. The molecular mechanism has been explored with an increasing number of primary sequences and three-dimensional structures of cold-adapted enzymes. The relationship between stability, flexibility, and activity in these enzymes may be of crucial importance. It is generally accepted that cold-adapted proteins are more flexible than their mesophilic counterparts, with a reduced number of weak interactions. However, more details still need to be elucidated (Petegem et al. 2003; Papaleo et al. 2011; Zheng et al. 2016).

Effects of metal ions and chemicals on enzyme activity

The effects of metal ions and chemicals on Xyn10A activity were also tested (Fig. 4). The results revealed that the enzyme activity was positively stimulated by Ca2+, Mn2+, Mg2+, K+, and Tween-20. The activity was enhanced by nearly 20% in a solution of 1 mM Mn2+ and Mg2+. Moreover, Xyn10A activity was inhibited by Cu2+, Zn2+, Ni2+, EDTA, and SDS. The most obvious decreases (-25%) were observed with Zn2+ and SDS. The effects of Fe2+, Co2+, and Na+ on the enzyme activity were insignificant. The results showed differences from the other cold-adapted xylanases, such as xylanases from Bacillus sp. SN5 and G. mesophila KMM241 (Guo et al. 2009; Bai et al. 2012), which showed that Mg2+ had no evident effect on activity. On the contrary, the activity of XynB from G. mesophila KMM241 was obviously inhibited by Mn2+ (Guo et al. 2009).

Fig. 4. Effects of ions and chemicals on the activity of purified Xyn10A. The final concentration of the ions and chemicals was 1 mM and 5 mM, respectively. The activity was measured at pH 8.0 and 40 °C, and activity without adding ions or chemicals was defined as 100%. Each experiment was performed in triplicate.

Effects of NaCl tolerance on enzyme activity

The halophilic bacteria and enzymes are very interesting (Khandeparker et al. 2011). Because the B. cellulosilyticus was isolated from a saline-alkali soil, the effect of NaCl on Xyn10A activity was evaluated. As shown in Fig. 5, the highest enzyme activity for Xyn10A was obtained by adding 0.5 M NaCl, which is approximately equivalent to the salinity of seawater.Compared to the control, the relative activity reached 127.3%. When the salt concentration reached 3 M, the residual activity remaining was still 66.5%. Additionally, compared to the absence of NaCl, the temperature stability was improved by adding the 0.5 M NaCl into the reaction solution (Fig. 5). The results were similar to xylanases from Bacillus sp. SN5 and G. mesophila KMM241 (Guo et al. 2009; Bai et al. 2012). The highest level of NaCl tolerance for a xylanase was found from Z. profunda (Liu et al. 2014), and its optimal activity was found with 3 M NaCl. The activity of XynB from G. mesophila KMM241 was not obviously affected by NaCl, though it showed some extent of salt-tolerant ability (Guo et al. 2013). Notably, several reported cold-adapted xylanases also showed the NaCl tolerance (Guo et al. 2009; Bai et al. 2012; Guo et al. 2013; Liu et al. 2014). Previous molecular dynamics research has shown that the salt-tolerant and cold-adapted properties may have some relevance, as the enzymes exhibit more flexible loop regions at the appropriate concentration of salt (Benrezkallah et al. 2015). In general, NaCl-tolerant enzymes originated from saline-alkaline land and marine environments, and they had to change their molecular structure in order to adapt to the corresponding environments.

Fig. 5. Effect of NaCl on the activity of Xyn10A. The enzyme activity was measured at 40 °C in phosphate buffer (pH 8.0) containing 0 M to 3.0 M NaCl. The activity in 0 M NaCl was set as 100%.

Specific enzyme activity and kinetic parameters

Under the optimal conditions (pH 8.0 and temperature 40 °C), the Xyn10A exhibited the highest specific enzyme activity of 163.8 U/mg of protein. This activity was slightly higher than that of other NaCl-tolerant and cold-adapted xylanases. The specific activity toward beechwood of xylanase from Bacillus sp. SN5 and G. mesophila KMM241 were 105 U/mg and 143 U/mg, respectively (Bai et al 2011; Guo et al. 2013). The specific activity was also lower than that of some xylanases. The specific activity of the WSUCF1 endo-xylanase from Geobacillus sp. WSUCF1 was 461 U/mg (Bhalla et al. 2014), and the XYN-LXY from rumen contents of Hu sheep showed a specific activity of 664.7 U/mg (Wang et al. 2015). Even higher specific activity was reported for xylanases originating from Paenibacillus campinasensis and Neocallimastix patriciarum (Liu et al. 2008; Ko et al. 2010), with activity reaching 2392 U/mg and 1982.8 U/mg, respectively. The commercial xylanases from Trichoderma viride have been reported to have activity of 100 U/mg to 300 U/mg (Shi et al. 2013).

The kinetic parameters Km and Vmax of the Xyn10A were determined at 40 °C using various concentrations of birchwood xylan as the substrate and calculated by Lineweaver-Burk double-reciprocal plots. The KmVmax, and Kcat of Xyn10A were 2.56 mg/mL, 253.1 μM/min/mg, and 165.8 /s, respectively. The Km value of Xyn10A using birchwood xylan as the substrate was lower than that of some other xylanases, such as the xylanases from goat rumen contents, the rumen contents of Hu sheep, and Malbranchea cinnamomea, which had Kmvalues of 3.2, 4.39, and 7.1 mg/mL, respectively (Fan et al. 2014; Wang et al. 2015). Furthermore, the Km of salt-tolerant and cold-adapted xynlases from the Bacillus sp. SN5, Z. profunda, goat rumen contents, and G. mesophila KMM 241 (XynA and XynB) were 0.6, 2.98, 1.8, 0.78, and 1.22 mg/mL, respectively, toward beechwood xylan (Guo et al. 2009; Baiet al. 2012; Guo et al. 2013; Liu et al. 2014). From the literature, the Km for birchwood xylan was higher than that for beechwood as the substrate. It can be concluded that the salt-tolerant and cold-adapted xylanases showed high affinity to various xylan sources.

Substrate specificity and analysis of hydrolytic products

The activity of the purified xylanase towards various substrates was studied. The Xyn10A had almost no activity on CMC-Na, Avicel, CMC, cellobiose, and xylobiose (data not shown). This result indicated that Xyn10A had no cellulase, glucosidase, or xylosidase activity, and is a strict endoxylanase. Furthermore, the birchwood xylan was incubated with purified Xyn10A, and the hydrolytic products were confirmed by TLC methods using xyloligosaccharides (XOs) as standards (Fig. 6). The results showed that xylobiose, xylotriose, and xylotetraose were the main products in the hydrolytic mixture. No xylose was detected.

Fig. 6. TLC analysis of the hydrolysis products of birchwood xylan (1%) by Xyn10A.The reactions were performed at 30 °C and pH 8.0, and the samples were taken for analysis at 0.5, 1, and 1.5 h. S, Standard of XOs: X1, xylose; X2, xylobiose; X3, xylotriose; X4, xylotetraose.

The XOs could not be hydrolyzed further to form xylose after prolonged hydrolysis time. This is in accordance with the results of the xylanases from B. mojavensis A21 (Haddar et al. 2012), G. mesophila KMM241 (Guo et al. 2013), Geobacillus thermoleovorans (Verma and Satyanarayana 2012), M. cinnamomea (Fan et al. 2014), etc. While some other xylanases showed xylosidase activity, the end product was xylose, such as originated from Thermoascus thermarum (Shi et al. 2013), Thermoascus aurantiacus (Zhang et al. 2011), the rumen contents of Hu sheep, and Caldicoprobacter algeriensis (Amel et al. 2016). The hydrolysis properties could enable Xyn10A to be used as an effective and powerful enzyme for large-scale production of XOs, which can be used as prebiotics.


  1. A GH10 family xylanase, the Xyn10A from B. cellulosilyticus, was heterologously expressed and biochemically characterized successfully.
  2. Xyn10A displayed obvious cold-adapted characteristics and promising activity in the presence of weak alkalinity and a broad range of NaCl concentrations. Therefore, Xyn10 would be a useful candidate for biotechnology applications at low/room temperature conditions and/or a high salinity environment.
  3. Xyn10A hydrolyzed xylans to yield mainly XOs (xylobiose, xylotriose, and xylotetraose). It can be used as excellent biocatalyst for production of XOs.


The authors are grateful for the support of the Natural Science Fund for Colleges and Universities in Jiangsu Province, China (13KJB530003), the startup Foundation of Jiangsu University (11JDG110), and a project funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD). Rongrong Xie acknowledges the support of the Natural Science Foundation of Jiangsu province (BK20130508). Alei Geng acknowledges the support of the National Natural Science Foundation of China (31201752).


Amel, B. D., Nawel, B., Khelifa, B., Mohammed, G., Manon, J., Salima, K. G., Farida, N., Hocine, H., Bernard, O., Jean-Luc, C., and Marie-Laure, F. (2016)“Characterization of a purified thermostable xylanase from Caldicoprobacter algeriensis sp. nov. strain TH7C1T,” Carbohyd. Res. 419, 60-68. DOI: 10.1016/j.carres.2015.10.013

Bai, W. Q., Xue, Y. F., Zhou, C., and Ma, Y. H. (2012). “Cloning, expression and characterization of a novel salt-tolerant xylanase from Bacillus sp. SN5,” Biotechnol. Lett. 34(11), 2093-2099. DOI 10.1007/s10529-012-1011-7

Benrezkallah, D., Dauchez, M., and Krallafa, A. M. (2015). “Molecular dynamics of the salt dependence of a cold-adapted enzyme: Endonuclease I,” J. Biomol. Struct. Dyn. 33(11), 1-11. DOI: 10.1080/07391102.2014.1002007

Bhalla, A., Bischoff, K. M., Uppugundla, N., Balan, V., and Sani, R. K. (2014). “Novel thermostable endo-xylanase cloned and expressed from bacterium Geobacillus sp. WSUCF1,” Bioresource Technol. 165(8), 314-318. DOI: 10.1016/j.biortech.2014.03.112

Cavicchioli, R., Siddiqui, K. S., Andrews, D., and Sowers, K. (2002). “Low-temperature extremophiles and their application,” Curr. Opin. Biotech. 13(3), 253-261. DOI: 10.1016/S0958-1669(02)00317

Chang, P., Tsai, W. S., Tsai, C. L., and Tseng, M. J. (2004). “Cloning and characterization of two thermostable xylanases from an alkaliphilic Bacillus firmus,” Biochem. Bioph. Res. Co. 319(3), 1017-1025. DOI: 10.1016/j.bbrc.2004.05.078.

Dhiman, S. S., Sharma, J., and Battan, B. (2008). “Industrial application and future prospects of microbial xylanase: A review,” BioResources 3(4), 1377-1402. DOI: 10.15376/biores.3.4.1377-1402

Fan, G., Yang, S., Yan, Q., Guo, Y., Li, Y., and Jiang, Z. Q. (2014). “Characterization of a highly thermostable glycoside hydrolase family 10 xylanase from Malbranchea cinnamomea,” Int. J. Biol. Macromol. 70(8), 482-489. DOI: 10.1016/j.ijbiomac.2014.07.025

Garg, S., Ali, R., and Kumar, A. (2009). “Production of alkaline xylanase by an alkalo-thermophilic bacteria, Bacillus halodurans, MTCC 9512 isolated from dung,” Curr. Trends Biotechnol. Pharm. 3(1), 90-96.

Guo, B., Chen, X. L., Sun, C. Y., Zhou, B. C., and Zhang, Y. Z. (2009). “Gene cloning, expression and characterization of a new cold-active and salt-tolerant endo-β-1,4-xylanase from marine Glaciecola mesophila KMM 241,” Appl. Microbiol. Biot. 84(6), 1107-1115. DOI: 10.1007/s00253-009-2056-y

Guo, B., Li, P. Y., Yue, Y. S., Zhao, H. L., Dong, S., Song, X. Y., Sun, C. Y., Zhang, W. X., Chen, X. L., Zhang, X. Y., et al. (2013). “Gene cloning, expression and characterization of a novel xylanase from the marine bacterium, Glaciecola mesophila KMM241,” Mar. Drugs 11(4), 1173-1187. DOI: 10.3390/md11041173

Haddar, A., Driss, D., Frikhac, F., Ellouz-Chaabouni, S., and Nasria, M. (2012)“Alkaline xylanases from Bacillus mojavensis A21: Production and generation of xylooligosaccharides,” Int. J. Biol. Macromol. 51(4), 647-656. DOI: 10.1016/j.ijbiomac.2012.06.036

Huang, J., Wang, G., and Xiao, L. (2006). “Cloning, sequencing and expression of the xylanase gene from a Bacillus subtilis strain B10 in Escherichia coli,” Bioresource Technol. 97(6), 802-808. DOI: 10.1016/j.biortech.2005.04.011

Juturu, V., and Wu, J. C. (2011). “Microbial xylanases: Engineering, production and industrial applications,” Biotechnol. Adv. 30(6), 1219-1227. DOI: 10.1016/j.biotechadv.2011.11.006

Khandeparker, R., Verma, P., and Deobagkar, D. (2011). “A novel halotolerant xylanase from marine isolate Bacillus subtilis cho40: Gene cloning and sequencing,” New Biotechnol.28(6), 814-821. DOI: 10.1016/j.nbt.2011.08.001

Ko, C. H., Tsai, C. H., Tu, J., Lee, H. Y., Ku, L. T., Kuo, P. A., and Lai, Y. K. (2010). “Molecular cloning and characterization of a novel thermostable xylanase from Paenibacillus campinasensis BL11,” Process Biochem. 45(10), 1638-1644. DOI: 10.1016/j.procbio.2010.06.015

Kredics, L., Terecskei, K., Antal, Z., Szekeres, A., Hatvani, L., Manczinger, L., and Vagvolgyi, C. (2008). “Purification and preliminary characterization of a cold-adapted extracellular proteinase from Trichoderma atroviride,” Acta. Biol. Hung. 59(2), 259-68. DOI: 10.1556/ABiol.59.2008.2.11

Lee, C. C., Kibblewhite-Accinelli, R. E., Wagschal, K., Robertson, G. H., and Wong, D. W. (2006). “Cloning and characterization of a cold-active xylanase enzyme from an environmental DNA library,” Extremophiles 10(4), 295-300. DOI: 1007/s00792-005-0499-3

Liu, J. R., Duan, C. H., Zhao, X., Tzen, J. T., Cheng, K. J., and Pai, C. K. (2008). “Cloning of a rumen fungal xylanase gene and purification of the recombinant enzyme via artificial oil bodies,” Appl. Microbiol. Biot. 79(2), 225-233. DOI: 10.1007/s00253-008-1418-1

Liu, X. S., Huang, Z., Zhang, X. N., Shao, Z. Z., and Liu, Z. D. (2014). “Cloning, expression and characterization of a novel cold-active and halophilic xylanase from Zunongwangia profunda,” Extremophiles 18(2), 441-450. DOI: 10.1007/s00792-014-0629-x

Liu, Z., Zhao, X., and Bai. F. (2013). “Production of xylanase by an alkaline-tolerant marine-derived Streptomyces viridochromogenes strain and improvement by ribosome engineering,” Appl. Microbiol. Biot. 97(10), 4361-4368. DOI: 10.1007/s00253-012-4290-y

Maity, C., Ghosh, K., Halder, S. K., Jana, A., Adak, A., Mohapatra, P. K. D., and Pati, B. R. (2012). “Xylanase isozymes from the newly isolated Bacillus sp. CKBx1D and optimization of its deinking potentiality,” Appl. Biochem. Biotech. 167(5), 1208-1219. DOI: 10.1007/s12010-012-9556-4

Mamo, G., Hatti-Kaul, R., and Mattiasson, B. (2006). “A thermostable alkaline active endo-β-1-4-xylanase from Bacillus halodurans S7: Purification and characterization,” Enzyme Microb. Tech. 39(7), 1492-1498. DOI: 10.1016/j.enzmictec.

Mamo, G., Thunnissen, M., Hatti-Kaul, R., and Mattiasson, B. (2009). “An alkaline active xylanase: Insights into mechanisms of high pH catalytic adaptation,” Biochimie 91(9), 1187-1196. DOI: 10.1016/j.biochi.2009.06.017

Mead, D., Drinkwater, C., and Brumm, P. J. (2013). “Genomic and enzymatic results show Bacillus cellulosilyticus uses a novel set of LPXTA carbohydrases to hydrolyze polysaccharides,” 8(4), 464-483. DOI: 10.1371/journal.pone.0061131

Nagar, S., Gupta, V. K., Kumar, D., Kumar, L., and Kuhad, R. C. (2010). “Production and optimization of cellulase-free, alkali-stable xylanase by Bacillus pumilus SV-85S in submerged fermentation,” J. Ind. Microbiol. Biot. 37(1), 71-83. DOI: 10.1007/s10295-009-0650-8

Novototskaya-Vlasova, K., Petrovskaya, L., Yakimov, S., and Gilichinsky, D. (2012). “Cloning, purification, and characterization of a cold-adapted esterase produced by Psychrobacter cryohalolentis K5 T from Siberian cryopeg,” FEMS Microbiol. Ecol. 82(2), 367-375. DOI: 10.1111/j.1574-6941.2012.01385.x

Papaleo, E., Pasi, M., Tiberti, M., and Gioia, L. D. (2011). “Molecular dynamics of mesophilic-like mutants of a cold-adapted enzyme: Insights into distal effects induced by the mutations,” PLoS One 6(9), e24214. DOI: 10.1371/journal.pone.0024214

Petegem, F. V., Collins, T., Meuwis, M. A., Gerday, C., Feller, G., and Beeumen, J. V. (2003). “The structure of a cold-adapted family 8 xylanase at 1.3 Å resolution,” J. Biol. Chem.278(9), 7531-7539. DOI: 10.1074/jbc.M206862200

Polizeli, M. L., Rizzatti, A. C., Monti, R., Terenzi, H. F., Jorge, J. A., and Amorim, D. S. (2005). “Xylanases from fungi: Properties and industrial applications,” Appl. Microbiol. Biot. 67(5), 577-591. DOI: 10.1007/s00253-005-1904-7

Ramli, A. N., Mahadi, N. M., Rabu, A., Murad, A. M., Bakar, F. D., and Illias, R. M. (2011). “Molecular cloning, expression and biochemical characterisation of a cold-adapted novel recombinant chitinase from Glaciozyma antarctica PI12,” Microb. Cell Fact. 10(69), 15667-15672. DOI: 10.1186/1475-2859-10-94

Rao, R. S., Singh, P. K., Sarka, P. K., and Shivaji, S. (2008). “Yeasts and yeast-like fungi associated with tree bark: Diversity and identification of yeasts producing extracellular endoxylanases,” Curr. Microbiol. 56(5), 489-494. DOI: 10.1007/s00284-008-9108-x

Ruller, R., Deliberto, L., Ferreira, T. L., and Ward, R. J. (2008). “Thermostable variants of the recombinant xylanase A from Bacillus subtilis produced by directed evolution show reduced heat capacity changes,” Proteins 70(4), 1280-1293. DOI: 10.1002/prot.21617

Shi, H., Zhang, Y., Li, X., Huang, Y. J., Wang, L. L., Wang, Y., Ding, H. H., and Wang, F. (2013). “A novel highly thermostable xylanase stimulated by Ca2+ from Thermotoga thermarum: Cloning, expression and characterization,” Biotechnol. Biofuels 6(1), 1-9. DOI: 10.1186/1754-6834-6-26

Siddiqui, K. S., and Cavicchioli, R. (2006). “Cold-adapted enzymes,” Annu. Rev. Biochem. 75(75), 403-433. DOI: 10.1146/annurev.biochem.75.103004.142723

Singh, A., Yadav, R. D., Kaur, A., and Mahajan, R. (2012). “An ecofriendly cost effective enzymatic methodology for deinking of school waste paper,” Bioresource Technol. 120(3), 322-327. DOI: 10.1016/j.biortech.2012.06.050

Tenkanen, M., Vrsanska, M., Siika-Aho, M., Wong, D. W., and Puchart, V. (2013). “Xylanase XYNIV from Trichoderma reesei showing exo- and endo-xylanase activity,” FEBS J280(1), 285-301. DOI: 10.1111/febs.12069

Thomas, L., Ushasree, M. V., and Pandey, A. (2014). “An alkali-thermostable xylanase from Bacillus pumilus functionally expressed in Kluyveromyces lactis and evaluation of its deinking efficiency,” Bioresource Technol. 165(8), 309-313. DOI: 10.1016/j.biortech.2014.03.037

Tian, J. W., Lei, Z. C., Peng, Q., Wang, L., and Tian, Y. Q. (2014). “Purification and characterization of a cold-adapted lipase from Oceanobacillus strain PT-11,” PLoS One 9(7): e101343. DOI: 10.1371/journal.pone.0101343

Verma, D., and Satyanarayana, T. (2012). “Cloning, expression and applicability of thermo-alkali-stable xylanase of Geobacillus thermoleovorans in generating xylooligosaccharides from agro-residues,” Bioresource Technol. 107(2), 333-338. DOI: 10.1016/j.biortech.2011.12.055

Vester, J. K., Glaring, M. A., and Stougaaed, P. (2014). “Discovery of novel enzymes with industrial potential from a cold and alkaline environment by a combination of functional metagenomics and culturing,” Microb. Cell Fact. 13(3), 1-14. DOI: 10.1186/1475-2859-13-72

Wang, G., Luo, H., Wang, Y., Huang, H., Shi, P., Yang, P., Meng, K., Bai, Y., and Yao, B. (2011). “A novel cold-active xylanase gene from the environmental DNA of goat rumen contents: Direct cloning, expression and enzyme characterization,” Bioresource Technol. 102(3), 3330-3336. DOI: 10.1016/j.biortech.2010.11.004

Wang, Q., Luo, Y., He, B., Jiang, L. S., Liu, J. X., and Wang, J. K. (2015). “Characterization of a novel xylanase gene from rumen content of Hu sheep,” Appl. Biochem. Biotech. 22(1), 33-38. DOI 10.1007/s12010-015-1823-8

Zhang, J., Siika-aho, M., Puranen, T., Tang, M., Tenkanen, M., and Viikari, L. (2011). “Thermostable recombinant xylanases from Nonomuraea flexuosa and Thermoascus aurantiacusshow distinct properties in the hydrolysis of xylans and pretreated wheat straw,” Biotechnol. Biofuels 4(1), 12. DOI: 10.1186/1754-6834-4-12

Zheng, Y. Y., Li, Y. J., Liu, W. D., Chen, C. C., Ko, T. P., He, M., Xu, Z. X., Liu, M. X., Guo, R. T., Yao, B., et al. (2016). “Structural insight into potential cold adaptation mechanism through a psychrophilic glycoside hydrolase family 10 endo-β-1,4-xylanase,” J. Struct. Biol. 193(3), 206-211. DOI: 10.1016/j.jsb.2015.12.010

Article submitted: May 28, 2016; Peer review completed: July 18, 2016; Revised version received and accepted: August 18, 2016; Published: August 31, 2016.

DOI: 10.15376/biores.11.4.8875-8889