Title
Characterization of an alkali-stable xyloglucanase/mixed-linkage β-glucanase Pgl5A from Paenibacillus sp. S09
Author
Rui Cheng, Long Cheng, Lei Wang, Renjie Fu, Xiaqing Sun, Jing Li, Shiming Wang, Jianfa Zhang
Year
2019
Journal
International Journal of Biological Macromolecules
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.
Instrument
J-1500
Keywords
Circular dichroism, Secondary structure, Chemical stability, Biochemistry