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A novel multifunctional GH9 enzyme from Paenibacillus curdlanolyticus B-6 exhibiting endo/exo functions of cellulase, mannanase and xylanase activities

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PcMulGH9, a novel glycoside hydrolase family 9 (GH9) from Paenibacillus curdlanolyticus B-6, was successfully expressed in Escherichia coli. It is composed of a catalytic domain of GH9, two domains of carbohydrate-binding module family 3 (CBM3) and two domains of fibronectin type 3 (Fn3). The PcMulGH9 enzyme showed broad activity towards the β-1,4 glycosidic linkages of cellulose, mannan and xylan, including cellulose and xylan contained in lignocellulosic biomass, which is rarely found in GH9. The enzyme hydrolysed substrates with bifunctional endo-/exotypes cellulase, mannanase and xylanase activities, but predominantly exhibited exo-activities. This enzyme released cellobiose as a major product from cellohexaose, while mannotriose and xylotriose were major hydrolysis products from mannohexaose and xylohexaose, respectively. Moreover, PcMulGH9 could hydrolyse untreated corn hull and rice straw into xylo- and cello-oligosaccharides. Enzyme kinetics, site-directed mutagenesis and molecular docking revealed that Met394, located at the binding subsite + 2, was involved in broad substrate specificity of PcMulGH9 enzyme. This study offers new knowledge of the multifunctional cellulase/mannanase/xylanase in GH9. The PcMulGH9 enzyme showed a novel function of GH9, which increases its potential for saccharification of lignocellulosic biomass into value-added products, especially oligosaccharides.

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This study was supported by the program Strategic Scholarships for Frontier Research Network for the Ph.D. Program Thai Doctoral degree from the Office of the Higher Education Commission (214/2551), Thailand. The authors also acknowledge the financial support provided by King Mongkut’s University of Technology Thonburi through the “KMUTT 55th Anniversary Commemorative Fund” and the Japan International Research Center for Agricultural Sciences.


The present work was funded by the program Strategic Scholarships for Frontier Research Network for the Ph.D. Program Thai Doctoral degree from the Office of the Higher Education Commission (214/2551), KMUTT 55th Anniversary Commemorative Fund, and the Japan International Research Center for Agricultural Sciences.

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Correspondence to Chakrit Tachaapaikoon.

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Phakeenuya, V., Ratanakhanokchai, K., Kosugi, A. et al. A novel multifunctional GH9 enzyme from Paenibacillus curdlanolyticus B-6 exhibiting endo/exo functions of cellulase, mannanase and xylanase activities. Appl Microbiol Biotechnol 104, 2079–2096 (2020).

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  • Carbohydrate-binding module family 3
  • Endo-/exotype enzyme
  • Glycoside hydrolase family 9
  • Multifunctional enzyme
  • Oligosaccharide
  • Paenibacillus curdlanolyticus