Functional and structural analysis of Pichia pastoris-expressed Aspergillus niger 1,4-b-endoglucanase Junjie Yan a, 1 , Weidong Liu b, 1 , Yujie Li b , Hui-Lin Lai c, d , Yingying Zheng b , Jian-Wen Huang c, d , Chun-Chi Chen b , Yun Chen e , Jian Jin e , Huazhong Li a, ** , Rey-Ting Guo b, * a School of Biotechnology, Jiangnan University, Wuxi 214122, China b Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, China c Genozyme Biotechnology Inc., Taipei 106, Taiwan d AsiaPac Biotechnology Co., Ltd., Dongguan, 523808, China e School of Pharmaceutical Sciences, Jiangnan University, Wuxi 214122, China article info Article history: Received 20 April 2016 Accepted 2 May 2016 Available online 3 May 2016 Keywords: 1,4-b-Endoglucanase Aspergillus niger Pichia pastoris Crystal structure abstract Eukaryotic 1,4-b-endoglucanases (EC 3.2.1.4) have shown great potentials in many commercial applica- tions because they effectively catalyze hydrolysis of cellulose, the main component of the plant cell wall. Here we expressed a glycoside hydrolase family (GH) 5 1,4-b-endoglucanase from Aspergillus niger (AnCel5A) in Pichia pastoris, which exhibits outstanding pH and heat stability. In order to further investigate the molecular mechanism of AnCel5A, apo-form and cellotetraose (CTT) complex enzyme crystal structures were solved to high resolution. AnCel5A folds into a typical (b/a) 8 -TIM barrel archi- tecture, resembling other GH5 members. In the substrate binding cavity, CTT is found to bind to 4 e 1 subsites, and several polyethylene glycol molecules are found in positive subsites. In addition, several unique N-glycosylation motifs that may contribute to protein higher stability were observed from crystal structures. These results are of great importance for understanding the molecular mechanism of AnCel5A, and also provide guidance for further applications of the enzyme. © 2016 Elsevier Inc. All rights reserved. 1. Introduction Cellulose is the major component of terrestrial plant cell walls, thus is the most abundant renewable resource on earth. Cellulose degradation has found manifold useful applications in a wide va- riety of industries, including textile industries, feed and food manufacture, and biofuel production [1]. The core structure of cellulose is long-chain polysaccharides composed of 1,4-b-glyco- sidic bond-linked glucoses. 1,4-b-Endoglucanase (endoglucanase, EC 3.2.1.4) is so far the most powerful and specific biocatalyst which is capable of randomly hydrolyzing the cellulose main chain into smaller fragments to facilitate subsequent processes. Searching an effective endoglucanase is among the most important tasks to develop a good enzyme product. Therefore, studying the molecular mechanism of these enzymes is of great interest in both academic and commercial areas. Endoglucanases are classified by the Carbohydrate-Active Enzyme database (CAZy) into 13 glycoside hydrolase (GH) fam- ilies (5e9, 12, 44, 45, 48, 51, 74, 124, and 131) based on protein sequence similarity and catalytic domain structure (http://www. cazy.org/) [2]. Among them, GH5 family contains the largest num- ber of characterized enzymes second to GH13. GH5 enzymes fold into (b/a) 8 TIM barrel architecture and utilize two catalytic gluta- mate residues to catalyze hydrolytic reaction via a retaining mechanism. Despite many archaeal and prokaryotic GH5 endo- glucanases have been extensively studied, only four eukaryotic endoglucanase structures from a ruminal fungus Piromyces rhizin- flata (PrEglA) [3], a thermophilic fungus Thermoascus aurantiacus (TaCel5A) [4,5] a filamentous fungus Hypocrea jecorina (TrCel5A) [6], and a saprotrophic fungus Ganoderma lucidum (GlCel5A) [7] are available. Among them, only PrEglA structure that contains a cel- lotriose molecule in minus subsites and GlCel5A structure that contains a cellobiose in positive subsites have been solved to * Corresponding author. ** Corresponding author. E-mail addresses: hzhli@jiangnan.edu.cn (H. Li), guo_rt@tib.cas.cn (R.-T. Guo). 1 JY and WL contribute equally. Contents lists available at ScienceDirect Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc http://dx.doi.org/10.1016/j.bbrc.2016.05.012 0006-291X/© 2016 Elsevier Inc. All rights reserved. Biochemical and Biophysical Research Communications 475 (2016) 8e12