Iterative key-residues interrogation of a phytase with thermostability increasing substitutions identified in directed evolution

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• 作者：Amol V. Shivange ; Danilo Roccatano…
• 关键词：Directed evolution ; KeySIDE ; Molecular dynamics simulations ; Phytase ; Thermostability ; Protein engineering
• 刊名：Applied Microbiology and Biotechnology
• 出版年：2016
• 出版时间：January 2016
• 年：2016
• 卷：100
• 期：1
• 页码：227-242
• 全文大小：2,575 KB
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• 作者单位：Amol V. Shivange (1) (2) (3)
  Danilo Roccatano (2)
  Ulrich Schwaneberg (1) (2)
  
  1. Lehrstuhl für Biotechnologie, RWTH Aachen University, Worringerweg 1, 52074, Aachen, Germany
  2. School of Engineering and Science, Jacobs University Bremen gGmbH, Campus Ring 1, 28759, Bremen, Germany
  3. Present Address: Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA, 91125, USA
  
• 刊物类别：Chemistry and Materials Science
• 刊物主题：Chemistry
  Biotechnology
  Microbiology
  Microbial Genetics and Genomics
  
• 出版者：Springer Berlin / Heidelberg
• ISSN：1432-0614

文摘

Bacterial phytases have attracted industrial interest as animal feed supplement due to their high activity and sufficient thermostability (required for feed pelleting). We devised an approach named KeySIDE,  an iterative Key-residues interrogation of the wild type with Substitutions Identified in Directed Evolution for improving Yersinia mollaretii phytase (Ymphytase) thermostability by combining key beneficial substitutions and elucidating their individual roles. Directed evolution yielded in a discovery of nine positions in Ymphytase and combined iteratively to identify key positions. The “best” combination (M6: T77K, Q154H, G187S, and K289Q) resulted in significantly improved thermal resistance; the residual activity improved from 35 % (wild type) to 89 % (M6) at 58 °C and 20-min incubation. Melting temperature increased by 3 °C in M6 without a loss of specific activity. Molecular dynamics simulation studies revealed reduced flexibility in the loops located next to helices (B, F, and K) which possess substitutions (Helix-B: T77K, Helix-F: G187S, and Helix-K: K289E/Q). Reduced flexibility in the loops might be caused by strengthened hydrogen bonding network (e.g., G187S and K289E/K289Q) and a salt bridge (T77K). Our results demonstrate a promising approach to design phytases in food research, and we hope that the KeySIDE might become an attractive approach for understanding of structure–function relationships of enzymes.