title: Iterative key-residues interrogation of a phytase with thermostability increasing substitutions identified in directed evolution.
abstract: 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.
authors.affiliation: Lehrstuhl für Biotechnologie, RWTH Aachen University, Worringerweg 1, 52074, Aachen, Germany.|School of Engineering and Science, Jacobs University Bremen gGmbH, Campus Ring 1, 28759, Bremen, Germany.|Present Address: Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA, 91125, USA.
authors.forename: Amol V
authors.initials: AV
authors.lastname: Shivange
authors.affiliation: School of Engineering and Science, Jacobs University Bremen gGmbH, Campus Ring 1, 28759, Bremen, Germany.
authors.forename: Danilo
authors.initials: D
authors.lastname: Roccatano
authors.affiliation: Lehrstuhl für Biotechnologie, RWTH Aachen University, Worringerweg 1, 52074, Aachen, Germany. u.schwaneberg@biotec.rwth-aachen.de.|School of Engineering and Science, Jacobs University Bremen gGmbH, Campus Ring 1, 28759, Bremen, Germany. u.schwaneberg@biotec.rwth-aachen.de.
authors.forename: Ulrich
authors.initials: U
authors.lastname: Schwaneberg
mesh_terms: D010832:6-Phytase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D019943:Amino Acid Substitution; D019020:Directed Molecular Evolution / Q000379:methods*; D004795:Enzyme Stability; D056004:Molecular Dynamics Simulation; D015202:Protein Engineering / Q000379:methods*; D013696:Temperature; D015007:Yersinia / Q000201:enzymology* / Q000235:genetics*
chemical_list: D010832:6-Phytase
keywords: Directed evolution; KeySIDE; Molecular dynamics simulations; Phytase; Protein engineering; Thermostability