title: Engineering the residual side chains of HAP phytases to improve their pepsin resistance and catalytic efficiency.
abstract: Strong resistance to proteolytic attack is important for feed enzymes. Here, we selected three predicted pepsin cleavage sites, L99, L162, and E230 (numbering from the initiator M of premature proteins), in pepsin-sensitive HAP phytases YkAPPA from Yersinia kristensenii and YeAPPA from Y. enterocolitica, which corresponded to L99, V162, and D230 in pepsin-resistant YrAPPA from Y. rohdei. We constructed mutants with different side chain structures at these sites using site-directed mutagenesis and produced all enzymes in Escherichia coli for catalytic and biochemical characterization. The substitutions E230G/A/P/R/S/T/D, L162G/A/V, L99A, L99A/L162G, and L99A/L162G/E230G improved the pepsin resistance. Moreover, E230G/A and L162G/V conferred enhanced pepsin resistance on YkAPPA and YeAPPA, increased their catalytic efficiency 1.3-2.4-fold, improved their stability at 60 °C and pH 1.0-2.0 and alleviated inhibition by metal ions. In addition, E230G increased the ability of YkAPPA and YeAPPA to hydrolyze phytate from corn meal at a high pepsin concentration and low pH, which indicated that optimization of the pepsin cleavage site side chains may enhance the pepsin resistance, improve the stability at acidic pH, and increase the catalytic activity. This study proposes an efficient approach to improve enzyme performance in monogastric animals fed feed with a high phytate content.
authors.affiliation: National Engineering Research Center of Biological Feed, Key Laboratory for Feed Biotechnology of the Ministry of Agriculture, Feed Research Institute, Chinese Academy of Agricultural Sciences, Beijing 100081, People’s Republic of China.
authors.forename: Canfang
authors.initials: C
authors.lastname: Niu
authors.affiliation: National Engineering Research Center of Biological Feed, Key Laboratory for Feed Biotechnology of the Ministry of Agriculture, Feed Research Institute, Chinese Academy of Agricultural Sciences, Beijing 100081, People’s Republic of China.
authors.forename: Peilong
authors.initials: P
authors.lastname: Yang
authors.affiliation: National Engineering Research Center of Biological Feed, Key Laboratory for Feed Biotechnology of the Ministry of Agriculture, Feed Research Institute, Chinese Academy of Agricultural Sciences, Beijing 100081, People’s Republic of China.
authors.forename: Huiying
authors.initials: H
authors.lastname: Luo
authors.affiliation: National Engineering Research Center of Biological Feed, Key Laboratory for Feed Biotechnology of the Ministry of Agriculture, Feed Research Institute, Chinese Academy of Agricultural Sciences, Beijing 100081, People’s Republic of China.
authors.forename: Huoqing
authors.initials: H
authors.lastname: Huang
authors.affiliation: National Engineering Research Center of Biological Feed, Key Laboratory for Feed Biotechnology of the Ministry of Agriculture, Feed Research Institute, Chinese Academy of Agricultural Sciences, Beijing 100081, People’s Republic of China.
authors.forename: Yaru
authors.initials: Y
authors.lastname: Wang
authors.affiliation: National Engineering Research Center of Biological Feed, Key Laboratory for Feed Biotechnology of the Ministry of Agriculture, Feed Research Institute, Chinese Academy of Agricultural Sciences, Beijing 100081, People’s Republic of China.
authors.forename: Bin
authors.initials: B
authors.lastname: Yao
mesh_terms: D010832:6-Phytase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D019943:Amino Acid Substitution; D000821:Animal Feed / Q000032:analysis; D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D055162:Biocatalysis; D020134:Catalytic Domain; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005765:Gastrointestinal Agents / Q000737:chemistry* / Q000378:metabolism; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010434:Pepsin A / Q000737:chemistry; D010833:Phytic Acid / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding; D015202:Protein Engineering / Q000379:methods*; D054730:Protein Interaction Domains and Motifs; D017433:Protein Structure, Secondary; D059748:Proteolysis; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D015007:Yersinia / Q000737:chemistry* / Q000201:enzymology
chemical_list: D001426:Bacterial Proteins; D005765:Gastrointestinal Agents; D011994:Recombinant Proteins; D010833:Phytic Acid; D010832:6-Phytase; D010434:Pepsin A