title: N-Glycosylation Improves the Pepsin Resistance of Histidine Acid Phosphatase Phytases by Enhancing Their Stability at Acidic pHs and Reducing Pepsin's Accessibility to Its Cleavage Sites.
abstract: N-Glycosylation can modulate enzyme structure and function. In this study, we identified two pepsin-resistant histidine acid phosphatase (HAP) phytases from Yersinia kristensenii (YkAPPA) and Yersinia rohdei (YrAPPA), each having an N-glycosylation motif, and one pepsin-sensitive HAP phytase from Yersinia enterocolitica (YeAPPA) that lacked an N-glycosylation site. Site-directed mutagenesis was employed to construct mutants by altering the N-glycosylation status of each enzyme, and the mutant and wild-type enzymes were expressed in Pichia pastoris for biochemical characterization. Compared with those of the N-glycosylation site deletion mutants and N-deglycosylated enzymes, all N-glycosylated counterparts exhibited enhanced pepsin resistance. Introduction of the N-glycosylation site into YeAPPA as YkAPPA and YrAPPA conferred pepsin resistance, shifted the pH optimum (0.5 and 1.5 pH units downward, respectively) and improved stability at acidic pH (83.2 and 98.8% residual activities at pH 2.0 for 1 h). Replacing the pepsin cleavage sites L197 and L396 in the immediate vicinity of the N-glycosylation motifs of YkAPPA and YrAPPA with V promoted their resistance to pepsin digestion when produced in Escherichia coli but had no effect on the pepsin resistance of N-glycosylated enzymes produced in P. pastoris. Thus, N-glycosylation may improve pepsin resistance by enhancing the stability at acidic pH and reducing pepsin's accessibility to peptic cleavage sites. This study provides a strategy, namely, the manipulation of N-glycosylation, for improvement of phytase properties for use in animal feed.
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: Canfang
authors.initials: C
authors.lastname: Niu
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: 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: Huoqing
authors.initials: H
authors.lastname: Huang
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: Yaru
authors.initials: Y
authors.lastname: Wang
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: Peilong
authors.initials: P
authors.lastname: Yang
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 binyao@caas.cn.
authors.forename: Bin
authors.initials: B
authors.lastname: Yao
mesh_terms: D010832:6-Phytase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D000135:Acid Phosphatase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004795:Enzyme Stability; D015870:Gene Expression; D006031:Glycosylation; D006863:Hydrogen-Ion Concentration; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D010434:Pepsin A / Q000378:metabolism*; D010843:Pichia / Q000235:genetics / Q000378:metabolism; D059748:Proteolysis; D015007:Yersinia / Q000201:enzymology*
chemical_list: D050505:Mutant Proteins; D000135:Acid Phosphatase; D010832:6-Phytase; D010434:Pepsin A