Insight into glycoside hydrolases for debranched xylan degradation from extremely thermophilic bacterium Caldicellulosiruptor lactoaceticus

Abstract

Caldicellulosiruptor lactoaceticus 6A, an anaerobic and extremely thermophilic bacterium, uses natural xylan as carbon source. The encoded genes of C. lactoaceticus 6A for glycoside hydrolase (GH) provide a platform for xylan degradation. The GH family 10 xylanase (Xyn10A) and GH67 α-glucuronidase (Agu67A) from C. lactoaceticus 6A were heterologously expressed, purified and characterized. Both Xyn10A and Agu67A are predicted as intracellular enzymes as no signal peptides identified. Xyn10A and Agu67A had molecular weight of 47.0 kDa and 80.0 kDa respectively as determined by SDS-PAGE, while both appeared as homodimer when analyzed by gel filtration. Xyn10A displayed the highest activity at 80 °C and pH 6.5, as 75 °C and pH 6.5 for Agu67A. Xyn10A had good stability at 75 °C, 80 °C, and pH 4.5-8.5, respectively, and was sensitive to various metal ions and reagents. Xyn10A possessed hydrolytic activity towards xylo-oligosaccharides (XOs) and beechwood xylan. At optimum conditions, the specific activity of Xyn10A was 44.6 IU/mg with beechwood xylan as substrate, and liberated branched XOs, xylobiose, and xylose. Agu67A was active on branched XOs with methyl-glucuronic acids (MeGlcA) sub-chains, and primarily generated XOs equivalents and MeGlcA. The specific activity of Agu67A was 1.3 IU/mg with aldobiouronic acid as substrate. The synergistic action of Xyn10A and Agu67A was observed with MeGlcA branched XOs and xylan as substrates, both backbone and branched chain of substrates were degraded, and liberated xylose, xylobiose, and MeGlcA. The synergism of Xyn10A and Agu67A provided not only a thermophilic method for natural xylan degradation, but also insight into the mechanisms for xylan utilization of C. lactoaceticus.

Figure 1. Modular and genomic organization of Xyn10A and Agu67A from C. lactoaceticus.

A. Modular organization for Xyn10A and Agu67A. NCBI conserved domains database and SignalP 4.1 Server were used for analysis. B. Genomic organization for Xyn10A. Calla_1331 was annotated as a putative endo-β-1,4-xylanase, and upstream of the xylanase is the gene predicted to encode a polysaccharide deacetylase. C. Genomic organization for Agu67A. Calla_1259 was annotated as a putative α-glucuronidase. Gene annotations were performed using the rapid annotations using subsystems technology (RAST) Server.

Figure 2. Phylogenetic analyses of Xyn10A and Agu67A.

A. Phylogenetic tree of xylanases in different organisms. B. Phylogenetic tree of α-glucuronidases in different organisms. Trees were constructed using MEGA 5.05 by the Neighbor-Joining method with 1000 bootstrap replicates, and Genbank accession numbers of each protein sequence were given at the end of each species name.

Figure 3. Purification of Xyn10A and Agu67A.

A. SDS-PAGE analysis of purified Xyn10A. B. Quaternary structure analysis of Xyn10A by gel filtration chromatography. C. SDS-PAGE analysis of purified Agu67A. D. Quaternary structure analysis of Agu67A by gel filtration chromatography. Both Xyn10A and Agu67A were purified by Ni-affinity chromatography, followed by Superdex 200 gel filtration.

Figure 4. Hydrolytic activities of Xyn10A and Agu67A against different polysaccharide substrates.

A. Identifications of Xyn10A and Agu67A activity on agar plate. The capacity of enzymes was assessed by incubating each protein on agar plates infused with different substrates at 60°C for 12 h, followed by staining with Congo red. B. The activity of Xyn10A and Agu67A on different substrates with produced reducing sugar assay. Both Xyn10A and Agu67A (0.5 µM each, final concentration) were incubated with different substrates at 80°C and pH 6.5 for 40 min. 1, beechwood xylan; 2, xylo-oligosaccharides; 3, locust bean gum; 4, soluble starch; 5, Avicel; 6, carboxymethyl cellulose. All of the tested substrates were at a fixed concentration of 1.0% (w/v).

Figure 5. Effects of temperature and pH on the activity and stability of Xyn10A and Agu67A.

A. Temperature profile of Xyn10A. Xylanase activity determination was performed in a temperature range of 40–95°C at pH 6.0 for 3 min. B. pH profile of Xyn10A. Xylanase activity assay was carried out by a 3 min incubation using phosphate-citrate buffers (pH 4.0–8.5) at 80°C. C. Thermostability profile of Xyn10A. The purified Xyn10A was incubated in pH 8.5 buffer at 75, 80 and 85°C, respectively for 0.5, 1, 2, 3, 4 and 6 h, and residual activity was detected under optimal conditions. D. pH stability profile of Xyn10A. The purified Xyn10A was pre-incubated in pH 4.0–8.5 buffers at room temperature for 10 h, and then the residual activity was measured under optimal conditions. E. Temperature profile of Agu67A. The α-glucuronidase activity determination was performed in a temperature range of 40-95°C at pH 6.5 for 5 min. F. pH profile of Agu67A. The α-glucuronidase activity assay was carried out by a 5 min incubation using phosphate-citrate buffers (pH 4.0–8.5) at 75°C. The maximum activity was defined as 100% and values shown were the means of three replicates.

Figure 6. Hydrolysis of beechwood xylan at different concentration with constant loading of Xyn10A.

A. TLC analysis of each hydrolysis products. B. The produced reducing sugar assay in each hydrolysis products. 0.1–2.0% (w/v) beechwood xylan was incubated with Xyn10A (0.5 µM, final concentration) at 80°C and pH 6.5 for 4 hours.

Figure 7. Hydrolysis products released from XOs and beechwood xylan by Xyn10A and Agu67A.

A. TLC analysis of the XOs hydrolysis products. The detectable xylose produced by Agu67A was marked with a box. B. Produced reducing sugar assay of the XOs hydrolysis products. C. TLC analysis of the beechwood xylan hydrolysis products after different incubation times. D. Produced reducing sugar assay of the beechwood xylan hydrolysis products. The differences of hydrolysis products after 1 hour were marked with a box. E. HPLC analysis of the beechwood xylan hydrolysis products of 2 hours. F. Details of the HPLC analysis (retention time 13.5–14.5 min). XOs hydrolysis was performed by incubating single or mixed enzyme (2.0 µM each, final concentration) with 2.0% (w/v) XOs at 80°C and pH 6.5 for 4 hours. Beechwood xylan hydrolysis was conducted by incubating Xyn10A (1.75 µM, final concentration), or Agu67A (0.85 µM, final concentration), or Xyn10A (1.75 µM, final concentration) and Agu67A (0.85 µM, final concentration) mixture with 1.0% (w/v) beechwood xylan at 80°C and pH 6.5 for different times (0, 0.25, 1, 2, 6, 12 hours). Xylose (X₁), xylobiose (X₂), xylotriose (X₃), and xylotetraose (X₄) were used as standards and labeled.