Heterologous Expression and Characterization of an Acidic GH11 Family Xylanase from Hypocrea orientalis
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Heterologous Expression and Characterization of an Acidic GH11 Family Xylanase from Hypocrea orientalis Hailong Li 1,3,4 & Hongli Wu 2 & Fengjiao Jiang 2 & Jinlian Wu 2 & Yong Xue 2 & Lihui Gan 2 & Jian Liu 2 & Minnan Long 2
Received: 3 May 2017 / Accepted: 5 June 2017 # Springer Science+Business Media, LLC 2017
Abstract 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 °C, and retained more than 90% of the original activity after incubation at 50 °C for 60 min. The determined apparent Km and Vmax values using beechwood xylan were 10.43 mg/ml and 3246.75 IU/mg, respectively. The modes of action of recombinant HoXyn11B on xylooligosaccharides (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. Keywords Heterologous expression . Pichia pastoris . Xylan hydrolysis . Xylanases . Hypocrea orientalis . Xylo-oligosaccharides
* Jian Liu [email protected] * Minnan Long [email protected]
1
Guangzhou Institute of Energy Conversion, Chinese Academy of Sciences, Guangzhou 510640, People’s Republic of China
2
College of Energy, Xiamen University, Xiamen 361102, People’s Republic of China
3
CAS Key Laboratory of Renewable Energy, Guangzhou 510640, People’s Republic of China
4
Guangdong Provincial Key Laboratory of New and Renewable Energy Research and Development, Guangzhou 510640, People’s Republic of China
Appl Biochem Biotechnol
Introduction Hemicellulose is the second most abundant polysaccharides in plant cell wall. The efficient enzymatic hydrolysis of hemicellulose into fermentable monosaccharides or value-added xylo-oligosaccharides (XOSs) is of considerable interest [1, 2]. Hemicellulose is mainly composed of heteroxylans, which have a backbone of β-1,4-linked xylose residues and side chains of different substituent units such as L-arabinofuranose or 4-Omethyl-D-glucuronic acid [3]. Xylanases play a crucial role in the breakdown of heteroxylans, which randomly cleave xylan main chain and generate variously unsubstituted or branched XOS [4]. Xylanases in the various glycoside hydrolase (GH) family differ in their physicochemical properties, three-dimensional structure, and substrate specificity [5]. According to the sequence-based GH classification, most xylanases belong to GH10 and GH11 family, while minority xylanases are classified in GH5 and GH8 family. The substrate specificities of
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