title: Functional and structural analysis of Pichia pastoris-expressed Aspergillus niger 1,4-β-endoglucanase.
abstract: Eukaryotic 1,4-β-endoglucanases (EC 3.2.1.4) have shown great potentials in many commercial applications 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-β-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 (β/α)8-TIM barrel architecture, resembling other GH5 members. In the substrate binding cavity, CTT is found to bind to -4 - -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.
authors.affiliation: School of Biotechnology, Jiangnan University, Wuxi 214122, China.
authors.forename: Junjie
authors.initials: J
authors.lastname: Yan
authors.affiliation: Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, China.
authors.forename: Weidong
authors.initials: W
authors.lastname: Liu
authors.affiliation: Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, China.
authors.forename: Yujie
authors.initials: Y
authors.lastname: Li
authors.affiliation: Genozyme Biotechnology Inc., Taipei 106, Taiwan; AsiaPac Biotechnology Co., Ltd., Dongguan, 523808, China.
authors.forename: Hui-Lin
authors.initials: HL
authors.lastname: Lai
authors.affiliation: Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, China.
authors.forename: Yingying
authors.initials: Y
authors.lastname: Zheng
authors.affiliation: Genozyme Biotechnology Inc., Taipei 106, Taiwan; AsiaPac Biotechnology Co., Ltd., Dongguan, 523808, China.
authors.forename: Jian-Wen
authors.initials: JW
authors.lastname: Huang
authors.affiliation: Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, China.
authors.forename: Chun-Chi
authors.initials: CC
authors.lastname: Chen
authors.affiliation: School of Pharmaceutical Sciences, Jiangnan University, Wuxi 214122, China.
authors.forename: Yun
authors.initials: Y
authors.lastname: Chen
authors.affiliation: School of Pharmaceutical Sciences, Jiangnan University, Wuxi 214122, China.
authors.forename: Jian
authors.initials: J
authors.lastname: Jin
authors.affiliation: School of Biotechnology, Jiangnan University, Wuxi 214122, China. Electronic address: hzhli@jiangnan.edu.cn.
authors.forename: Huazhong
authors.initials: H
authors.lastname: Li
authors.affiliation: Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, China. Electronic address: guo_rt@tib.cas.cn.
authors.forename: Rey-Ting
authors.initials: RT
authors.lastname: Guo
mesh_terms: D001234:Aspergillus niger / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D002480:Cellulase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D018360:Crystallography, X-Ray; D004795:Enzyme Stability; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D008958:Models, Molecular; D010843:Pichia / Q000235:genetics*; D011487:Protein Conformation
chemical_list: C407024:endoglucanase Cel5A; D002480:Cellulase
keywords: 1,4-β-Endoglucanase; Aspergillus niger; Crystal structure; Pichia pastoris