title: Functional and structural analyses of a 1,4-β-endoglucanase from Ganoderma lucidum.
abstract: Ganoderma lucidum is a saprotrophic white-rot fungus which contains a rich set of cellulolytic enzymes. Here, we screened an array of potential 1,4-β-endoglucanases from G. lucidum based on the gene annotation library and found that one candidate gene, GlCel5A, exhibits CMC-hydrolyzing activity. The recombinant GlCel5A protein expressed in Pichia pastoris is able to hydrolyze CMC and β-glucan but not xylan and mannan. The enzyme exhibits optimal activity at 60°C and pH 3-4, and retained 50% activity at 80 and 90°C for at least 15 and 10min. The crystal structure of GlCel5A and its complex with cellobiose, solved at 2.7 and 2.86Å resolution, shows a classical (β/α)8 TIM-barrel fold as seen in other members of glycoside hydrolase family 5. The complex structure contains a cellobiose molecule in the +1 and +2 subsites, and reveals the interactions with the positive sites of the enzyme. Collectively, the present work provides the first comprehensive characterization of an endoglucanase from G. lucidum that possesses properties for industrial applications, and strongly encourages further studying in the cellulolytic enzyme system of G. lucidum.
authors.affiliation: School of Biotechnology, Jiangnan University, Wuxi 214122, China.
authors.forename: Guizhi
authors.initials: G
authors.lastname: Liu
authors.affiliation: Industrial Enzymes National Engineering Laboratory, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, China.
authors.forename: Qian
authors.initials: Q
authors.lastname: Li
authors.affiliation: Industrial Enzymes National Engineering Laboratory, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, China.
authors.forename: Na
authors.initials: N
authors.lastname: Shang
authors.affiliation: Genozyme Biotechnology Inc., Taiwan 196, Taiwan; AsiaPac Biotechnology Co., Ltd., Dongguan 523808, China.
authors.forename: Jian-Wen
authors.initials: JW
authors.lastname: Huang
authors.affiliation: Institute of Biological Chemistry, Academia Sinica, Taipei 11529, Taiwan.
authors.forename: Tzu-Ping
authors.initials: TP
authors.lastname: Ko
authors.affiliation: Industrial Enzymes National Engineering Laboratory, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, China.
authors.forename: Weidong
authors.initials: W
authors.lastname: Liu
authors.affiliation: Industrial Enzymes National Engineering Laboratory, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, China.
authors.forename: Yingying
authors.initials: Y
authors.lastname: Zheng
authors.affiliation: Industrial Enzymes National Engineering Laboratory, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, China.
authors.forename: Xu
authors.initials: X
authors.lastname: Han
authors.affiliation: School of Pharmaceutical Sciences, Jiangnan University, Wuxi, Jiangsu 214122, China.
authors.forename: Yun
authors.initials: Y
authors.lastname: Chen
authors.affiliation: Industrial Enzymes National Engineering Laboratory, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, China. Electronic address: ccckate0722@gmail.com.
authors.forename: Chun-Chi
authors.initials: CC
authors.lastname: Chen
authors.affiliation: School of Pharmaceutical Sciences, Jiangnan University, Wuxi, Jiangsu 214122, China. Electronic address: jinjian31@126.com.
authors.forename: Jian
authors.initials: J
authors.lastname: Jin
authors.affiliation: Industrial Enzymes National Engineering Laboratory, 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: D020134:Catalytic Domain; D002480:Cellulase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D018360:Crystallography, X-Ray; D005656:Fungal Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D005800:Genes, Fungal; D006868:Hydrolysis; D008958:Models, Molecular; D010843:Pichia / Q000201:enzymology / Q000235:genetics; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020940:Reishi / Q000201:enzymology* / Q000235:genetics; D055672:Static Electricity; D013379:Substrate Specificity
chemical_list: D005656:Fungal Proteins; D011994:Recombinant Proteins; D002480:Cellulase
keywords: Crystal structure; Endoglucanase; Ganoderma lucidum