Purification and Characterization of a Thermostable Xylanase from Paenibacillus sp. NF1 and its Application in Xylooligosaccharides Production

Abstract

High levels of extracellular xylanase activity (211.79 IU/mg) produced by Paenibacillus sp. NF1 were detected when it was submerged-cultured. After three consecutive purification steps using Octyl-Sepharose, Sephadex G75, and Q-Sepharose columns, a thermostable xylanase (XynNF) was purified to homogeneity and showed a molecular mass of 37 kDa according to SDS-PAGE. The specific activity of the purified XynNF was up to 3,081.05 IU/mg with a 14.55-fold purification. The activity of XynNF was stimulated by $Ca^{2+}$, $Ba^{2+}$, DTT, and ${\beta}$-mercaptoethanol, but was inhibited by $Fe^{2+}$, $Zn^{2+}$, $Fe^{2+}$, $Cu^{2+}$, SDS, and EDTA. The purified XynNF displayed a greater affinity for oat spelt xylan with the maximal enzymatic activity at $60^{\circ}C$ and pH 6.0. XynNF, which was shown to be cellulose-free, with high stability at high temperature ($70^{\circ}C-80^{\circ}C$) and low pH range (pH 4.0-7.0), is potentially valuable for various industrial applications. The enzyme hydrolyzed oat spelt xylan to yield mainly xylooligosaccharides (95.8%) of 2-4 degree of polymerization (DP2-4). Moreover, the majority of the xylooligosacharides (DP2-4) products was xylobiose (61.5%). The thermostable xylanase (XynNF) thus seems potentially usefull in the production of xylooligosaccharides.

Introduction

Endo-1,4-β-D-xylanase (E.C. 3.2.1.8) is an important enzyme for the degradation of the major hemicellulosic polysaccharide of lignocellulosic materials, since they hydrolyze xylopyranosyl linkages of β-1,4-xylan [27]. As the key xylan-degrading enzyme, xylanases have attracted considerable attention because of their widespread application in various industrial processes such as pulp bleaching, animal feeding, baking and brewing, wastetreating, bioenergy converting, textiles, and food industry [1, 12]. Recently, Paenibacillus species is frequently isolated and identified from various sources for their ability to hydrolyze lignocellulosic materials. Many strains of Paenibacillus species, like P. campinasensis G1-1 [27] producing a thermostable xylanase, Paenibacillus terrae HPL-003 producing a new bimodular xylanase [17], and alkalophilic Paenibacillus sp. 12-11 [26] producing an alkaline xylanase, showed various application potential.

Xylan, composed of a linear backbone of 1,4-β-linked D-xylose units, is the major constituent of hemicellulose, which is the second most abundant component of plant biomass [29]. It can be hydrolyzed to xylose by the synergistic effect of endoxylanase, exoxylanase, and β-xylosidase. When it was hydrolyzed by only endoxylanase, the major product was xylooligosaccharides (XOS). XOS are newly developed functional oligosaccharides that exhibit excellent prebiotic effect and health benefits, such as the stimulation of human intestinal Bifidobacteria and Lactobacillus growth [5, 6, 13]. Currently, XOS are produced mainly by enzymatic hydrolysis owing to high specificity, negligible substrate loss, and side product generation [22, 29]. However, the xylanases to be used for XOS production should preferably be free of or have very little exoxylanase and β-xylosidase activities stable at high temperature to withstand xylan extraction conditions and be available at low cost [3, 13]. Therefore, thermophilic endoxyalnases are best suited for XOS production.

In our previous report, a novel strain, Paenibacillus sp. NF1, producing high levels of extracellular thermostable xylanase was isolated from cow dung compost in China [20]. In order to characterize the xylanase from Paenibacillus sp. NF1, it was purified to homogeneity from culture supernatants by three kinds of chromatography. The attractive attributes and special hydrolysis products of this xylanase suggest that it is potentially valuable for various industrial applications, especially for xylooligosaccharides production.

 

Materials and Methods

Strain and Chemicals

Paenibacillus sp. NF1, which was isolated, identified, and cultivated for xylanase production as described earlier [20], was preserved in our laboratory. Oat spelt xylan, birchwood xylan, carboxymethyl cellulose (CMC), Avicel, xylose, xylobiose, xylotriose, xylotetraose, sodium dodecyl sulfate (SDS), β-mercaptoethanol, bovine serum albumin (BSA), and 3,5-dinitrosalicylic acid (DNS) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Octyl-Sepharose, Sephadex G75, and Q-Sepharose were purchased from GE Healthcare Life Sciences (Pittsburgh, PA, USA). All other chemicals were of analytical reagent grade purity and obtained from commercial sources.

Production of Xylanase by Paenibacillus sp. NF1

For xylanase production, Paenibacillus sp. NF1 was grown in optimized medium [20] containing corncob 20 g/l, beef extract 28.2 g/l, K2HPO4 5 g/l, MgSO4 0.5 g/l, and NaCl 7.1 g/l, at 28℃ with shaking at 160 rpm. Culture samples were collected at 6 h intervals over the period of 96 h cultivation. The initial pH of the medium was 6.9 and the inoculum size was 2%.

Purification of Extracellular Xylanase

The culture supernatants of Paenibacillus sp. NF1 were collected by centrifugation (8,000 rpm for 10 min at 4℃). The supernatants collected after centrifugation were salted out with ammonium sulfate at 70% saturation. After that, the precipitates were dissolved in sodium phosphate buffer (20 mM, pH 6.0), and solid ammonium sulfate was added to adjust to the final saturation of 40%. The samples were loaded onto an Octyl-Sepharose FF column (2.6 × 20 cm) pre-equilibrated with sodium phosphate buffer (20 mM, pH 6.0) containing 40% saturation of ammonium sulfate. The target protein was eluted with a linear gradient (40%-0% saturation) of ammonium sulfate in sodium phosphate buffer (20 mM, pH 6.0). The active fractions were loaded onto a Sephadex G75 column (1.6 × 60 cm) equilibrated with sodium phosphate buffer (20 mM, pH 8.0). The xylanase active fractions were collected for further purification by a Q-Sepharose HP column (2.6 × 20 cm) using linear gradient elution from 0-1 M NaCl in sodium phosphate buffer (20 mM, pH 8.0). Finally, the purified xylanase was desalted by dialysis against deionized water and concentrated by freeze-drying. The protein concentration of the samples at each step of chromatography was determined spectrophotometrically at 280 nm. The protein concentration of the pooled enzyme solution from each purification step was determined according to the Bradford method [2] using BSA as the standard.

SDS-PAGE and Zymogram Analysis

The purified and concentrated xylanase preparations were analyzed by SDS-PAGE using a 12% polyacrylamide gel containing 0.1% (w/v) oat spelt xylan. After electrophoresis, the gel was divided and the protein bands were visualized by staining with Coomassie Brilliant Blue G250 (Sigma) or detected for xylanase activity [9, 11]. For zymogram analysis, the gel was treated with 25% 2-propanol and incubated at 60℃ for 30 min in 20 mM sodium phosphate buffer (pH 6.0). After incubation, the gel was subsequently incubated under agitation at room temperature in Congo red solution (1 mg/ml) for 10 min, washed with 1 M NaCl, and fixed with 5% (v/v) acetic acid [28]. Clear areas in the zymogram indicated enzyme activity. Molecular weight standards from Fermentas were used as molecular mass markers.

Xylanase Assay

The activity of xylanase was measured by determining the amount of reducing sugars liberated from 1% oat spelt xylan using 3,5-dinitrosalicylic acid at 60℃ and pH 6.0 [10]. The method was described previously by Zheng et al. [28]. The amount of enzyme that liberated 1 μmol of reducing sugars per minute was defined as one unit (IU) of xylanase activity.

Effects of Temperature and pH on Xylanase Activity and Stability

The optimum temperature of the xylanase was measured by incubating the reaction mixtures at different temperatures (40-80℃) and pH 6.0 for 10 min. The relative enzymatic activity was calculated as a percentage of the maximal activity. Its thermal stability was determined by incubating the purified xylanase in sodium phosphate buffer (20 mM, pH 6.0) at various temperatures (40-80℃) for 180 min. The residual enzyme activity was calculated as a percentage of the starting activity. The optimum pH of xylanase activity was measured at 60℃ using acetic acid sodium buffer (pH 3.0-5.0), sodium phosphate (pH 6.0-7.0), Tris-HCl (pH 8.0-9.0), and glycine-NaOH (pH10.0), respectively, at the same concentration of 20 mM. The relative enzymatic activity was calculated as a percentage of the maximal activity. To assess its pH stability, the purified xylanase was preincubated in buffers at various pH values (pH 3.0-10.0) at 60℃ for 180 min and the residual activities were determined at 30 min intervals.

Effects of Various Metal Ions and Other Additives on Xylanase Activity

The effects of various metal ions (KCl, CaCl2, BaCl2, MgCl2, MnCl2, FeCl2, FeCl3, ZnCl2, and CuSO4), metal chelators (EDTA) and surfactants (DTT, β-mercaptoethanol, and SDS) on the purified xylanase activity were evaluated by including it in the reaction mixtures at the concentration of 1 mM and 10 mM. Enzyme solutions were incubated at 60℃ for 60 min before the residual activity was measured. The activity of the enzyme assayed under the same condition without additives was defined as 100%.

Substrate Specificity and Kinetic Studies

The specificities and kinetic constants (Km and Vmax) of xylanase were determined following the method of Zheng et al. [28]. For specificities determination, the activity of the purified xylanase against oat spelt xylan, birchwood xylan, rice hull xylan, bran xylan, CMC, and Avicel at concentrations of 1% (w/v) at 60℃ and pH 6.0 were assayed. The enzyme preparations were incubated with oat spelt xylan and birchwood xylan at concentrations between 0.1% (w/v) and 1% (w/v) to estimate the kinetic parameters (Km and Vmax) of the purified xylanase using a Lineweaver-Burk double reciprocal plot.

Analysis of Xylooligosaccharides in Xylanase Hydrolysates by High Performance Liquid Chromatography (HPLC)

One hundred fifty micrograms of oat spelt xylan in 1.5 ml sodium phosphate buffer (pH 6.0) was treated with 30 IU of the diluted purified xylanase at 60℃ for 10 min, 1 h, and 2 h, respectively. The xylanase hydrolysates were analyzed by an HPLC (Agilent). HPLC analysis was done by using a chromatographic column (Shodex SUGAR KS-801, 8.0 × 300 mm) at 65℃ with water as the mobile phase at 0.6 ml/min.

 

Results and Discussion

Production of Extracellular Xylanase by Paenibacillus sp. NF1

When Paenibacillus sp. NF1 was cultured by the submerged method, the xylanase was secreted to the culture liquid. The production of extracellular xylanases by Paenibacillus sp. NF1 was investigated during 96 h of cultivation in the optimized production medium (detailed in Materials and Methods). As shown in Fig. 1, exponential growth of Paenibacillus sp. NF1 was observed until 42 h, and then the stationary phase was ended after 30 h. Moreover, Paenibacillus sp. NF1 secreted extracellular xylanases at a low rate in the exponential phase and achieved the maximal xylanase production of up to 165.2 IU/ml at the end of the stationary phase (72 h, Fig. 1). After the stationary phase (72 h), the xylanase activity was gradully decreased because of hydrolysis by autologous protease in the decline phase of strain NF1. This course of xylanase degradation was also observed from Fomitopsis pinicola KMJ812 [15] and Paenibacillus campinasensis G1-1 [27] reported previously. Paenibacillus sp. NF1 producing relatively high levels of xylanases compared with those reported previously [21, 24, 25] could be an efficient and economical microorganism with potential applications in industrial production.

Fig. 1.Growth and extracellular xylanase production of Paenibacillus sp. NF1. The culture growth was monitored by measuring the optical density at 600 nm with a 6 h interval, and the xylanase activity was also assayed using the DNS method. Data are presented as means ± SD (n = 3).

Purification of Xylanase XynNF

Under the optimized culture conditions, the specific activity of xylanase produced by Paenibacillus sp. NF1 reached to 211.79 IU/mg (Table 1). The xylanase was purified to electrophoretic homogeneity by the steps listed in Table 1. The specific activity of the purified xylanase was 3,081.05 IU/mg with a 14.55-fold purification, and the final yield for the enzyme was 10.63% (Table 1). As a single band on SDS-PAGE with an apparent molecular mass of about 37 kDa, the purified xylanase from culture supernatants was designated as XynNF (Fig. 2, lane 3), whereas zymogram analysis of the purified XynNF exhibiting a clear activity band on the corresponding zone further confirmed the presence of active xylanase (Fig. 2, lane 4). It also indicates that XynNF from Paenibacillus sp. NF1 was a monomeric protein comparable to those previously reported for other xylanases [16, 21, 23].

Fig. 2.SDS-PAGE and zymogram analysis of the purified xylanase from Paenibacillus sp. NF1. The contents of each lane are as follows: Lane M: molecular weight of marker standard; Lane 1: crude extracellular xylanase from Paenibacillus sp. NF1; Lane 2: XynNF purified by hydrophobic-interaction chromatography; Lane 3: purified XynNF; Lane 4: zymogram analysis of the purified XynNF.

Table 1.Purification of xylanase (XynNF) from Paenibacillus sp. NF1.

Effects of Temperature and pH on Enzyme Activity and Stability

The optimal reaction temperature of XynNF was 60℃, which is higher than that of Paenibacillus sp. 12-11 (55℃) [26], and the enzyme retained 46.88% of its maximum activity at 70℃ (Fig. 3A). The thermal stability of XynNF was evaluated by incubation at temperatures from 40℃ to 80℃ for 180 min. The enzymatic activity retained at least 71.07% of the original activity after incubation at 40℃ to 60℃ for 180 min, and still retained about 70.65% and 42.66% of the original activity after incubation at 70℃ and 80℃ for 60 min, respectively (Fig. 3B). This thermal stability is better than earlier reports on most of the bacterial xylanases [1, 14, 19, 26]. The purified XynNF showed the maximum activity at pH 6.0, which is lower than that of XynG1-1 (7.5) from P. campinasensis G1-1 [28] and retained 59.78% of its maximum activity at pH 3.0 (Fig. 4A). It was stable between pH 4.0 and 7.0 for 180 min, retaining at least 59.11% of its activity at pH 6.0 (Fig. 4B), manifesting much greater pH stability at the condition of lower pH than those of most bacterial xylanases reviewed [1, 19, 26]. Its stability at high temperature (70℃-80℃) and activity at the condition of lower pH (pH 4.0-7.0) (Figs. 3 and 4) make XynNF valuable for various industrial applications, such as animal feeding, bioenergy converting, and food industry.

Fig. 3.Effects of temperature on the activity (A) and stability (B) of the purified XynNF. (A) Relative activities at different temperatures; the maximum activity observed at 60℃ was taken as 100%. (B) Residual activities after incubation for various times at 40℃ to 80℃, pH 6.0. The original activity without incubation was taken as 100%. Data are presented as means ± SD (n = 3).

Fig. 4.Effects of pH on the activity (A) and stability (B) of the purified XynNF. (A) Relative activities at different pH values; the maximum activity observed at pH 6.0 was taken as 100%. (B) Residual activities after incubation for various times at pH 3.0 to 10.0, 60℃. The original activity without incubation was taken as 100%. Data are presented as means ± SD (n = 3).

Effects of Various Metal Ions and Additives on Xylanase Activity

Addition of 1 mM or 10 mM of K+, Mg2+, and Mn2+ had no significant effect on the purified XynNF (Table 2). However, the purified XynNF was activated by Ca2+ and Ba2+ and was inhibited by certain metal ions in the following order: Fe3+ > Zn2+ > Fe2+ > Cu2+ (Table 2). The reducing agents, DTT and β-mercaptoethanol, stimulated the activity of XynNF (Table 2). The strong inhibition by SDS and EDTA (Table 2) indicates that hydrophobic interactions may be important in maintaining the structure of XynNF and certain metal ions were required for its activity.

Table 2.aThe activity of the enzymes assayed under the optimum condition in the absence of additives was defined as 100%. Data are presented as means ± SD (n = 3).

Substrate Specificity and Kinetic Studies

Specificity studies indicate that XynNF could hydrolyze all the tested xylans, but did not act on CMC and Avicel. The purified XynNF exhibited the highest xylanase activity when oat spelt xylan or birchwood xylan was used as the substrate (Table 3), with relatively lower activities being measured for rice hull xylan (82.23%) and bran xylan (79.86%). In addition, the purified XynNF showed Km values of 5.64 and 6.32 mg/ml and Vmax values of 3,364.57 and 3,216.44 μ mol/min/mg of protein for oat spelt xylan and birchwood xylan, respectively. The values of Km for these two substrates indicated that XynNF had greater affinity for oat spelt xylans. Strong specificity toward birchwood xylan and oat spelt xylan was also verified for xylanases from P. campinasensis BL11 [8], T. clypeatus [18], and P. sclerotiorum [7].

Table 3.aRelative activity (%): the maximum specific activity was taken as 100%. bND: not detectable. Data are presented as means ± SD (n = 3).

Analysis of XOS in Xylanase Hydrolysates by HPLC

When oat spelt xylan was incubated with the purified XynNF for 2 h at 60℃ and pH 6.0, the major hydrolysis products were xylobiose, identified by HPLC (Table 4 and Fig. 5). After a 10 min reaction time, about half of the larger polysaccharides (≥xylopentaose) was hydrolyzed to xylobiose, xylotriose, and xylotetraose, but no xylose was liberated (Table 4). Xylobiose and xylotriose contents increased steadily during further incubation for 1 h; however, xylotetraose and xylose were not increased (Table 4). The amount of xylotetraose remained almost constant from 10 min to 2 h reaction and there were no higher xylooligosaccharides (≥xylopentaose) in the hydrolysis products until the end of the hydrolysis at the reaction time of 2 h (Table 4, Fig. 5D). It indicates that XynNF was more efficient in conversion than S. thermophile xylanase [13], which took 8 h for complete hydrolysis of oat spelt xylan. By HPLC analysis of xylanase hydrolysates against oat spelt xylan, xylotetraose and higher xylooligosaccharides were found to be efficiently hydrolyzed mainly to xylobiose, a small part of xylotriose appeared to be further hydrolyzed to xylobiose and xylose until 2 h, while xylotetraose was mainly hydrolyzed to xylobiose, but no xylobiose were hydrolyzed to xylose (Table 4 and Fig. 5). Therefore, XynNF was confirmed to be an endoxylanase rather than a β-xylosidase that hydrolyzes xylobiose to xylose. When the reaction time was increased from 10 min to 2 h, the xylooligosacharides (DP2-4) content finally increased to 95.8% (Table 4) and xylobiose was the majority of the xylooligosacharides (DP2-4) products (Fig. 5). With the time course of hydrolysis, the amount of xylobiose increased further reaching to 61.5% in 2 h (Table 4). The high catalytic efficiency and enrichment effect of xylobiose makes XynNF a highly potential enzyme in xylooligosaccharides production.

Fig. 5.Chromatograms from HPLC analysis of the oat spelt xylan hydrolysates by the purified xylanase (XynNF). Enzyme-substrate mixtures containing 1.5 ml of 1% (w/v) oat spelt xylan and 30 IU of the diluted purified xylanase were incubated at 60℃ and pH 6.0 for 0 min (A), 10 min (B), 1 h (B), and 2 h (D), respectively. The oligomeric state (DP2-4) of each peak was determined by comparing it with the retention time of the xylooligomer standard marker.

Table 4.HPLC analysis of the products liberated by the action of the purified xylanase (XynNF) on oat spelt xylan.

In conclusion, the endoxylanase (XynNF) of Paenibacillus sp. NF1 was purified to homogeneity by multistep chromatographies. Its stability at high temperature (70℃-80℃) and low pH range (pH 4.0-7.0) makes it a suitable biocatalyst for various industrial applications. In particular, its application in oat spelt xylan hydrolysis, showing 95.8% xylooligosaccharides (DP 2-4) products, makes it a promising candidate for xylooligosaccharides production.