Xylan degradation characteristics of strain XB
In order to determine the xylan degradation ability of strain XB, four kinds of xylan from different sources were used as substrates in the assay system. The result showed that much more reducing sugars were produced from the crude enzyme when using 1% OSX and CCX as substrate, with the highest value (3.28 mg/mL and 3.02 mg/mL, respectively) on the fifth day. For AX and BWX, there was less reducing sugar detected, and the highest peak appeared on the third (1.59 mg/mL) and fourth day (1.58 mg/mL), respectively (Fig. 1a). Thus, strain XB had the ability to degrade all tested xylans, but its degradation ability was different on different xylan types.
Genomic features and gene prediction of xylanolytic enzyme of strain XB
Genome assembly of strain XB resulted in 4 contigs, which were then combined into one scaffold. The genome was 4.83 Mb in size and the G+C content was 51.87 mol%. The NCBI Prokaryotic Genome Annotation Pipeline identified 4371 genes and 4198 proteins. Annotation with CAZy revealed strain XB had a relatively high proportion of CAZomes for 210 proteins (Additional file 2: Table S1). The glycoside hydrolase (GH) family accounted for more than 50% (107) of them, followed by the glycosyl transferase family (GT, 36), the carbohydrate esterase family (CE, 19) and the carbohydrate binding module family (CBM, 47). Only one axillary activity (AA) family and none of the polysaccharide lyase (PL) families were detected in the CAZomes of strain XB. (Additional file 1: Fig. S1).
When combining CAZy and COG/KEGG/GO/NR results, approximately 38 genes were considered putative xylan degradation-related genes (Table 1). Of these, two genes’ (Pph_0648 and Pph_1610) encoded proteins had endo-1,4-β-xylanase activity and belonged to the GH11 and GH10 families, respectively. One gene was annotated as xylanase and assigned to GH8 family (Pph_0002). In addition, there were 35 predicted proteins, belonging to GH43 (6), GH51 (3), GH31 (4), CBM 35 (1), GH36 (6), GH54 (1), GH67 (1) and CE4 (13) that encoded β- xylosidase, α-N-arabinofuranosidase, α-glucuronidase, α-galactosidase, α-xylosidase or peptidoglycan/xylan/chitin deacetylase, respectively.
Putative xylan degradation gene expression induced by CCX and xylose
The relative expression levels of genes encoding GH8, GH11, GH10, six of GH43 and three GH51 families were measured via RT-qPCR using recA as the reference gene (Additional file 2: Table S2; Fig. 1b). The majority of genes exhibited up-regulation in R2A broth with 1% CCX, and several were over-expressed more than 5-fold (p < 0.01), such as Pph_1610 (GH10), Pph_0648 (GH11), Pph_0602, Pph_2344, Pph_2078 and Pph_3723 of GH43, Pph_4538 (GH51). Only two genes (Pph_0602 and Pph_2344) displayed extremely high expression levels when 1% xylose was added to the medium, up-regulating more than 5-fold (p-value < 0.01). Thus, Pph_0602 and Pph_2344 not only could be induced to overexpress at high levels in a substrate of CCX but also could be prompted by xylose. They were further analyzed based on protein expression, purification, and enzymatic characteristics.
Gene cloning and purification of recombinant enzymes
The coding region of two GH43 family encoding genes Pph_0602 and Pph_2344 were linked with expression vector pET28a (+) based on the two restriction enzymes Bam HI and Xho I for construction of recombinant proteins Ppxyl43A (pET28a-Pph_0602), and EcoR I and Xho I for recombinant proteins Ppxyl43B (pET28a- Pph_2344) (Additional file 2:Table S2). Ppxyl43A and Ppxyl43B contained open reading frames encoding a protein of 329 (~36.9kDa) and 543 (~62.3kDa) amino acids, respectively (Additional file 2:Table S3). The SDS-PAGE showed single bands consistent with the expected sizes of Ppxyl43A and Ppxyl43B after purification and washing with buffer containing 50 and 100 mM imidazole (Fig. 2a and 2b).
In order to understand the reaction patterns of Ppxyl43A and Ppxyl43B, the reducing sugars and xylose amounts produced from 1% CCX assay showed that BL21 with either recombinant Ppxyl43A or Ppxyl43B could hydrolyze CCX and produce the significantly more reducing sugars (Fig.2c) and xylose (Fig. 2d) intracellularly than control (p<0.01). Whether for xylose or reducing sugars, recombinant Ppxyl43A exhibited an increasing trend intracellularly with induction time from 2 h to 18 h, and recombinant Ppxyl43B expressed higher activity levels from 2 h-induction and maintained a relatively stable level up to 18-h induction time. Moreover, Ppxyl43A and Ppxyl43B produced nearly similar amounts of reducing sugars (Fig.2c), while recombinant Ppxyl43B produced much more xylose than Ppxyl43A (Fig.2d). In addition, with a longer induction time (6 h and 18 h), some reducing sugars could be detected from both two recombinants and little xylose could be assayed mainly from recombinant Ppxyl43B extracellularly.
Substrate-specific and hydrolytic products analyses
The substrate specificity of purified Ppxyl43A and Ppxyl43B was determined by measuring the amount of reducing sugars toward several polysaccharides and xylose toward pNP derivatives, including pNPX and pNPAf, as substrates. Ppxyl43A and Ppxyl43B displayed similar substrate degradation ability, and both could hydrolyze pNPX, pNPAf, and various types of polysaccharides, containing CCX, XOS, and OSX. Ppxyl43A was more active toward pNPX, and other polysaccharides, such as CCX, XOS, X2, and X6 (p<0.01) than Ppxyl43B after 10 min’s digestion. However, neither Ppxyl43A nor Ppxyl43B could hydrolyze BWX and AX to reducing sugars (Table 2).
In order to determine the action model of Ppxyl43A and Ppxyl43B, hydrolytic products were further investigated by TLC and HPAEC method. Comparing the TLC profiles of different substrates with their hydrolytic products, CCX and OSX used in this survey mainly contained xylobiose, Xylotriose, Xylotetraose, et cetera—thus, in fact, should belong to xylo-oligosaccharides. When digested with Ppxyl43A and Ppxyl43B, xylose could be produced from CCX and OSX (Figure 3a), and their profiles were similar with those from XOS, but BWX could not be hydrolyzed by either Ppxyl43A or Ppxyl43B to produce xylo-oligosaccharides or xylose (Figure 3b) and only very little xylose was also produced from AX (Additional file1: Fig. S2). The TLC hydrolysis profile of X6 and X2 exhibited that both Ppxyl43A and Ppxyl43B could hydrolyze X6 and X2, even completely, to xylose (Figure 3c). Thus, All TLC profiles showed that Ppxyl43A and Ppxyl43B mainly hydrolyze CCX and OSX via β-xylosidase activity. In addition, although very weak TLC signals of arabinose could be observed from the substrate of pNPAf (Additional file1: Fig. S2), it also reflected Ppxyl43A and Ppxyl43B could hydrolyze pNPAf to arabinose and supposed that both had α-L-arabinofuranosidase activity. Thus, both Ppxyl43A and Ppxyl43B possess at least bifunctional enzyme activity for β-xylosidase and α-L-arabinofuranosidase. The hydrolytic products separated by HPAEC profiles also confirmed xylose could be produced from X6 (Fig. 4a, 4b and 4c) and X2 (Additional file1: Fig. S3) and arabinose was produced from pNPAf (Fig. 4d, 4e and 4f) after digestion by Ppxyl43A and Ppxyl43B.
In addition, According to the TLC hydrolytic profile at different reaction times, Ppxyl43A displayed more rapid CCX hydrolytic activity than Ppxyl43B. Xylose could be detected at 3 min post-incubation with Ppxyl43A, and the amount increased significantly within 1 h (Fig. 5a). The xylose signal could not be observed until 60 min post-incubation by Ppxyl43B (Fig. 5b).
Effects of pH and temperature on activity and stability
The effects of pH on β--xylosidase and α-L-arabinofuranosidase activity of Ppxyl43A and Ppxyl43B further examined using pNPX and pNPAf displayed that the optimum pH value was 6.0 for β--xylosidase but 5.0 for α-L-arabinofuranosidase for Ppxyl43A (Fig. 5a). Ppxyl43A retained more than 60% β--xylosidase activity in pH range 3.0 to 8.0 but only retained ~60% of α-L-arabinofuranosidase activity in pH range 4.0-6.0. The pH stability assay showed that highest stability occurred in pH range 5.0-8.0 for β--xylosidase and 6.0-8.0 for α-L-arabinofuranosidase activity (Fig. 5b). However, for Ppxyl43B, the optimal pH for β--xylosidase was 5.0 and 3.0 for α-L-arabinofuranosidase activity (Fig. 5c). β--xylosidase could act in the widest pH range, retaining 80% or more activity over the entire detected pH range of 2.0 to 9.0; whereas, the relative activity of α-L-arabinofuranosidase was only 60% of total within pH range 3.0-6.0. The pH stability assay showed that both enzymes could retain 60% relative activity from pH 3.0-9.0 (Fig. 5d). Thus, there were notable differences in suitable pH ranges for the two enzymes—Ppxyl43B had a wider pH range and better pH stability than Ppxyl43A.
The optimal temperature of purified Ppxyl43A for α-L-arabinofuranosidase activity was 40°C, while that for β--xylosidase was 50°C (Fig. 6a). Thermostability assays showed that Ppxyl43A had a relatively high activity only when the temperature was lower than the optimum and could retain more than 50% activity across the temperature range 10-40°C for α-L-arabinofuranosidase and 20-50°C for β--xylosidase (Fig. 6b). While the optimal reaction temperature of Ppxyl43B was 40°C for both enzymes, β--xylosidase activity level were similar across the entire range of 20-70°C (Fig. 6c), and they displayed high thermostability in temperatures ranging from 10 to 70°C (Fig. 6d). Thus, Ppxyl43B exhibited a wider temperature range and higher thermostability than Ppxyl43A, especially when using pNPX as substrate for β--xylosidase activity.
Effects of metal ion on enzyme activity
Among the 10 kinds of metal ions detected, the β--xylosidase activity of Ppxyl43A and Ppxyl43B could be enhanced by 10 mM Ca2+ and Mn2+ by approximately 10% over the control group, slightly inhibited by Fe3+and Ni2+ (10-30% loss of activity) and strongly inhibited by Zn2+and Cu2+ by loss of 70-80% activity. In addition, Mg2+ and K+ enhanced β--xylosidase activity of Ppxyl43B but decreased its activity in Ppxyl43A. However, the α-L-arabinofuranosidase activity of Ppxyl43A could be stimulated by Mg2+, Co2+, Ca2+, K+, Ba2+ and Mn2+, especially by Ca2+ (increasing by approximately 50% activity), but strongly inhibited by Fe3+ (43% loss of activity), Cu2+, and Zn2+ (82-90% decrease of activity); whereas the α-L-arabinofuranosidase activity of Ppxyl43B could only be significantly enhanced by Fe3+, Ni2+, and K+, strongly inhibited by Zn2+, and no remarkable differences were observed in activity levels in the presence or absence of other metal ions (Table 3).
Kintetic properties
Kinetic parameters such as Vmax and Km were determined from regression lines of Lineweaver-Burk plots using pNPX and pNPAf, respectively (Table 4). The Vmax and Km values obtained for β--xylosidase of Ppxyl43A based on the substrate of pNPX were 8.64±1.87 U/mg and 2.37±0.54 mM, while those for Ppxyl43B were 13.49±0.27 U/mg and 2.12±0.05 mM, respectively; when using CCX as the substrate, the detected Vmax and Km values were 481.48±159.30 U/mg and 88.44±48.16 mg/ml for Ppxyl43A, and 316.80±217.95 U/mg and 75.16±34.35 mg/ml for Ppxyl43B, and there were no significant differences between Km values of two proteins (p>0.05). In addition, the kinetic parameters for pNPAf showed that the average Vmax and Km values were 5.73±5.35 U/mg and 3.03±1.64 mM for Ppxyl43A, were slightly higher than those of Ppxyl43B (0.86±0.60 U/mg and 0.82±0.28 mM) but not significantly. Thus, these two proteins showed similar affinity to both chemical and natural substrates.