Journal
APPLIED BIOCHEMISTRY AND BIOTECHNOLOGY
Volume 184, Issue 1, Pages 228-238Publisher
HUMANA PRESS INC
DOI: 10.1007/s12010-017-2532-2
Keywords
Heterologous expression; Pichia pastoris; Xylan hydrolysis; Xylanases; Hypocrea orientalis; Xylo-oligosaccharides
Funding
- Natural Science Foundation of Guangdong Province, China [2016A030310124]
- National Natural Science Foundation of China [31600475]
- project of Guangzhou Science and Technology [201707010241]
- research fund from the Xiamen Southern Oceanographic Center [14GZP59HJ29]
- Fujian Provincial Department of Ocean and Fisheries [2015-27]
- President Fund of Xiamen University [20720150090]
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A gene encoding glycoside hydrolase family 11 xylanase (HoXyn11B) from Hypocrea orientalis EU7-22 was expressed in Pichia pastoris with a high activity (413 IU/ml). HoXyn11B was partly N-glycosylated and appeared two protein bands (19-29 kDa) on SDS-PAGE. The recombinant enzyme exhibited optimal activity at pH 4.5 and 55 A degrees C, and retained more than 90% of the original activity after incubation at 50 A degrees C for 60 min. The determined apparent K (m) and V (max) values using beechwood xylan were 10.43 mg/ml and 3246.75 IU/mg, respectively. The modes of action of recombinant HoXyn11B on xylo-oligosaccharides (XOSs) and beechwood xylan were investigated by thin-layer chromatography (TLC), high-performance liquid chromatography (HPLC), and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS), which indicated that the modes of action of HoXyn11B are different from HoXyn11A since it is able to release a significant amount of xylose from various substrates. This study provides an opportunity to better understand the hydrolysis mechanisms of xylan by xylanases from Trichoderma.
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