title: Role of N-linked glycosylation in the enzymatic properties of a thermophilic GH 10 xylanase from Aspergillus fumigatus expressed in Pichia pastoris.
abstract: N-Glycosylation is a posttranslational modification commonly occurred in fungi and plays roles in a variety of enzyme functions. In this study, a xylanase (Af-XYNA) of glycoside hydrolase (GH) family 10 from Aspergillus fumigatus harboring three potential N-glycosylation sites (N87, N124 and N335) was heterologously produced in Pichia pastoris. The N-glycosylated Af-XYNA (WT) exhibited favorable temperature and pH optima (75°C and pH 5.0) and good thermostability (maintaining stable at 60°C). To reveal the role of N-glycosylation on Af-XYNA, the enzyme was deglycosylated by endo-β-N-acetylglucosaminidase H (DE) or modified by site-directed mutagenesis at N124 (N124T). The deglycosylated DE and mutant N124T showed narrower pH adaptation range, lower specific activity, and worse pH and thermal stability. Further thermodynamic analysis revealed that the enzyme with higher N-glycosylation degree was more thermostable. This study demonstrated that the effects of glycosylation at different degrees and sites were diverse, in which the glycan linked to N124 played a key role in pH and thermal stability of Af-XYNA.
authors.affiliation: College of Biological Sciences and Engineering, Jiangxi Agricultural University, Nanchang, People's Republic of China.|Key Laboratory for Feed Biotechnology of the Ministry of Agriculture, Feed Research Institute, Chinese Academy of Agricultural Sciences, Beijing, People's Republic of China.
authors.forename: Xiaoyu
authors.initials: X
authors.lastname: Chang
authors.affiliation: College of Biological Sciences and Engineering, Jiangxi Agricultural University, Nanchang, People's Republic of China.
authors.forename: Bo
authors.initials: B
authors.lastname: Xu
authors.affiliation: Key Laboratory for Feed Biotechnology of the Ministry of Agriculture, Feed Research Institute, Chinese Academy of Agricultural Sciences, Beijing, People's Republic of China.
authors.forename: Yingguo
authors.initials: Y
authors.lastname: Bai
authors.affiliation: Key Laboratory for Feed Biotechnology of the Ministry of Agriculture, Feed Research Institute, Chinese Academy of Agricultural Sciences, Beijing, People's Republic of China.
authors.forename: Huiying
authors.initials: H
authors.lastname: Luo
authors.affiliation: Key Laboratory for Feed Biotechnology of the Ministry of Agriculture, Feed Research Institute, Chinese Academy of Agricultural Sciences, Beijing, People's Republic of China.
authors.forename: Rui
authors.initials: R
authors.lastname: Ma
authors.affiliation: Key Laboratory for Feed Biotechnology of the Ministry of Agriculture, Feed Research Institute, Chinese Academy of Agricultural Sciences, Beijing, People's Republic of China.
authors.forename: Pengjun
authors.initials: P
authors.lastname: Shi
authors.affiliation: Key Laboratory for Feed Biotechnology of the Ministry of Agriculture, Feed Research Institute, Chinese Academy of Agricultural Sciences, Beijing, People's Republic of China.
authors.forename: Bin
authors.initials: B
authors.lastname: Yao
mesh_terms: D001232:Aspergillus fumigatus / Q000201:enzymology* / Q000235:genetics; D004795:Enzyme Stability; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006031:Glycosylation; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase / Q000378:metabolism; D020125:Mutation, Missense; D010843:Pichia / Q000201:enzymology* / Q000235:genetics; D011134:Polysaccharides / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D014995:Xylosidases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D005656:Fungal Proteins; D011134:Polysaccharides; D014995:Xylosidases; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase