Characterization of an alkali-stable xyloglucanase/mixed-linkage β-glucanase Pgl5A from Paenibacillus sp. S09

Abstract Xyloglucans and mixed-linkage β-glucans are the major components of hemicelluloses in lignocellulosic biomass. In this study, a novel β-1,4-glucanase Pgl5A belonging to the glycoside hydrolase family 5 subfamily 4 (GH5_4), was identified from Paenibacillus sp. S09. Pgl5A is a 70.9-kDa protein containing an N-terminal GH5_4 module, a carbohydrate-binding module (CBM)_X2 and a CBM3. Full-length Pgl5A and its CBM deletion mutants Pgl5A∆C and Pgl5A-CD were expressed in E. coli. All three enzymes showed maximal activity at 55 °C and pH 4.5–5.0, and possessed similar activity toward xyloglucan, barley β-glucan, and lichenan. Deletion of the CBM modules can improve thermostability and acid-tolerant properties of Pgl5A. Circular dichroism (CD) and intrinsic fluorescence spectroscopy analysis verified that C-terminus truncation improves the enzyme acid-tolerant properties. Homology modeling and CD spectra indicated that Pgl5A has an architectural (β/α)8 fold of GH5_4 enzymes. The catalytic efficiency (kcat/Km) of Pgl5A toward xyloglucan, but not mixed-linkage β-glucan, was reduced due to C-terminus truncation. TLC and LC-MS analysis showed that Pgl5A cleaves xyloglucan and mixed-linkage β-glucan into a series of xyloglucan oligosaccharides and gluco-oligosaccharides, respectively. The favorable enzymatic characteristics and high catalytic activities toward both xyloglucan and mixed-linkage β-glucan make Pgl5A a promising candidate for biotechnological industrial applications.