Modular glucuronoxylan-specific xylanase with a family CBM35 carbohydrate-binding module

Abstract

Xyn30D from the xylanolytic strain Paenibacillus barcinonensis has been identified and characterized. The enzyme shows a modular structure comprising a catalytic module family 30 (GH30) and a carbohydrate-binding module family 35 (CBM35). Like GH30 xylanases, recombinant Xyn30D efficiently hydrolyzed glucuronoxylans and methyl-glucuronic acid branched xylooligosaccharides but showed no catalytic activity on arabinose-substituted xylans. Kinetic parameters of Xyn30D were determined on beechwood xylan, showing a K(m) of 14.72 mg/ml and a k(cat) value of 1,510 min(-1). The multidomain structure of Xyn30D clearly distinguishes it from the GH30 xylanases characterized to date, which are single-domain enzymes. The modules of the enzyme were individually expressed in a recombinant host and characterized. The isolated GH30 catalytic module showed specific activity, mode of action on xylan, and kinetic parameters that were similar to those of the full-length enzyme. Computer modeling of the three-dimensional structure of Xyn30D showed that the catalytic module is comprised of a common (β/α)(8) barrel linked to a side-associated β-structure. Several derivatives of the catalytic module with decreasing deletions of this associated structure were constructed. None of them showed catalytic activity, indicating the importance of the side β-structure in the catalysis of Xyn30D. Binding properties of the isolated carbohydrate-binding module were analyzed by affinity gel electrophoresis, which showed that the CBM35 of the enzyme binds to soluble glucuronoxylans and arabinoxylans. Analysis by isothermal titration calorimetry showed that CBM35 binds to glucuronic acid and requires calcium ions for binding. Occurrence of a CBM35 in a glucuronoxylan-specific xylanase is a differential trait of the enzyme characterized.

Fig 1

Secretome analysis of Paenibacillus barcinonensis by two-dimensional electrophoresis. (A) Zymogram of activity on birchwood xylan. (B) Protein-stained two-dimensional SDS polyacrylamide gel. The arrow points to Xyn30D. The positions of molecular mass standards and pI markers are indicated.

Fig 2

SDS-PAGE analysis of Xyn30D, Xyn-CM, and Xyn-CBM35. (A) Protein staining; (B) zymogram of xylanase activity. Lanes: 1, purified Xyn30D; 2, purified Xyn-CM; 3, purified Xyn-CBM35; M, positions of molecular mass standards.

Fig 3

Effect of pH and temperature on activity and stability of Xyn30D and Xyn-CM. (A) Effect of temperature on activity of Xyn30D (○) and Xyn-CM (●); (B) effect of pH on activity of Xyn30D (○) and Xyn-CM (●); (C) effect of temperature on stability of Xyn30D (open symbols) and Xyn-CM (closed symbols). The samples were incubated in 50 mM phosphate buffer (pH 6.5) at 50°C (○, ●), 55°C (♢, ♦), or 60°C (□, ■), and residual activity after different time intervals was determined. (D) Effect of pH on stability of Xyn30D (○) and Xyn-CM (●). The samples were incubated at 50°C in buffers at different pH for 3 h, and residual activity was determined.

Fig 4

Thin-layer chromatography analysis of the mode of action of Xyn30D. (A) Hydrolysis products from beechwood xylan. Lanes: 1, control, no digested samples; 2, products of xylan hydrolysis by Xyn30D; M, size markers of xylose (X), xylobiose (X₂), xylotriose (X₃), xylotetraose (X₄), and xylopentaose (X₅). (B) Hydrolysis products from glucuronic acid-substituted xylooligomers. Lanes: 3, control, no digested aldotetraouronic acid (MeGlcA³Xyl₃); 4, products of aldotetraouronic acid hydrolysis by Xyn30D; 5, control, no digested aldopentaouronic acid (MeGlcA³Xyl₄); 6, products of aldopentaouronic acid hydrolysis by Xyn30D; C, size markers of a mixture containing aldotetraouronic, aldotriouronic, and aldobiouronic acids and xylose.

Fig 5

SDS-PAGE analysis of binding to insoluble polysaccharides. (A) Binding of Xyn30D. (B) Binding of Xyn-CM. (C) Binding of Xyn-CBM35. Proteins were mixed with Avicel or with the insoluble fraction of oat spelt xylan for 1 h; bound and unbound fractions were separated by centrifugation and analyzed by SDS-PAGE. Lanes: 1, unbound fraction; 2, wash; 3, fraction adsorbed to the polymer; C, control protein. The positions of molecular mass standard proteins are indicated.

Fig 6

Affinity gel electrophoresis analysis of Xyn-CBM35 binding to soluble polysaccharides. Xyn-CBM35 was analyzed on nondenaturing polyacrylamide gels containing no ligand (control) or soluble xylan from oat spelt, beechwood, or wheat arabinoxylan. Lanes: 1, BSA; 2, Xyn-CBM35.

Fig 7

ITC analysis of Xyn-CBM35 binding to glucuronic acid. The upper part of each panel shows the raw binding heats, and the lower part shows the integrated binding heats minus the dilution control heats fitted to a single-site binding model with MicroCal Origin. Calcium was at 5 mM when included in titrations with glucuronic acid and was at 3 mM (in the syringe) when titrated into apo-Xyn-CBM35.