title: Improving specific activity and thermostability of Escherichia coli phytase by structure-based rational design.
abstract: Escherichia coli phytase (EcAppA) which hydrolyzes phytate has been widely applied in the feed industry, but the need to improve the enzyme activity and thermostability remains. Here, we conduct rational design with two strategies to enhance the EcAppA performance. First, residues near the substrate binding pocket of EcAppA were modified according to the consensus sequence of two highly active Citrobacter phytases. One out of the eleven mutants, V89T, exhibited 17.5% increase in catalytic activity, which might be a result of stabilized protein folding. Second, the EcAppA glycosylation pattern was modified in accordance with the Citrobacter phytases. An N-glycosylation motif near the substrate binding site was disrupted to remove spatial hindrance for phytate entry and product departure. The de-glycosylated mutants showed 9.6% increase in specific activity. On the other hand, the EcAppA mutants that adopt N-glycosylation motifs from CbAppA showed improved thermostability that three mutants carrying single N-glycosylation motif exhibited 5.6-9.5% residual activity after treatment at 80°C (1.8% for wild type). Furthermore, the mutant carrying all three glycosylation motifs exhibited 27% residual activity. In conclusion, a successful rational design was performed to obtain several useful EcAppA mutants with better properties for further applications.
authors: Institute of Biotechnology, National Taiwan University, Taipei 106, Taiwan.
authors: Tzu-Hui
authors: TH
authors: Wu
authors: Industrial Enzymes National Engineering Laboratory, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, China.
authors: Chun-Chi
authors: CC
authors: Chen
authors: Genozyme Biotechnology Inc., Taipei 106, Taiwan; AsiaPac Biotechnology Co. Ltd., Dongguan 523808, China.
authors: Ya-Shan
authors: YS
authors: Cheng
authors: Institute of Biological Chemistry, Academia Sinica, Taipei 115, Taiwan.
authors: Tzu-Ping
authors: TP
authors: Ko
authors: Genozyme Biotechnology Inc., Taipei 106, Taiwan; AsiaPac Biotechnology Co. Ltd., Dongguan 523808, China.
authors: Cheng-Yen
authors: CY
authors: Lin
authors: Genozyme Biotechnology Inc., Taipei 106, Taiwan; AsiaPac Biotechnology Co. Ltd., Dongguan 523808, China.
authors: Hui-Lin
authors: HL
authors: Lai
authors: Genozyme Biotechnology Inc., Taipei 106, Taiwan; AsiaPac Biotechnology Co. Ltd., Dongguan 523808, China.
authors: Ting-Yung
authors: TY
authors: Huang
authors: Institute of Biotechnology, National Taiwan University, Taipei 106, Taiwan; Department of Animal Science and Technology, National Taiwan University, Taipei 106, Taiwan; Agricultural Biotechnology Research Center, Academia Sinica, Taipei 115, Taiwan. Electronic address: jrliu@ntu.edu.tw.
authors: Je-Ruei
authors: JR
authors: Liu
authors: 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: Rey-Ting
authors: RT
authors: Guo
mesh_terms: D010832:6-Phytase / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D000135:Acid Phosphatase / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D000595:Amino Acid Sequence; D020134:Catalytic Domain; D016384:Consensus Sequence; D004795:Enzyme Stability; D004926:Escherichia coli / Q000737:chemistry / Q000201:enzymology*; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D006031:Glycosylation; D007700:Kinetics; D008958:Models, Molecular; D015202:Protein Engineering / Q000379:methods; D017433:Protein Structure, Secondary; D013696:Temperature
chemical_list: D029968:Escherichia coli Proteins; D000135:Acid Phosphatase; D010832:6-Phytase; C406788:appA protein, E coli
keywords: Animal feed; Phytate; Protein engineering; Site-specific mutagenesis