title: Enhancing thermostability of Escherichia coli phytase AppA2 by error-prone PCR.
abstract: Phytases are used to improve phosphorus nutrition of food animals and reduce their phosphorus excretion to the environment. Due to favorable properties, Escherichia coli AppA2 phytase is of particular interest for biotechnological applications. Directed evolution was applied in the present study to improve AppA2 phytase thermostability for lowering its heat inactivation during feed pelleting (60-80 degrees C). After a mutant library of AppA2 was generated by error-prone polymerase chain reaction, variants were expressed initially in Saccharomyces cerevisiae for screening and then in Pichia pastoris for characterizing thermostability. Compared with the wild-type enzyme, two variants (K46E and K65E/K97M/S209G) showed over 20% improvement in thermostability (80 degrees C for 10 min), and 6-7 degrees C increases in melting temperatures (T (m)). Structural predictions suggest that substitutions of K46E and K65E might introduce additional hydrogen bonds with adjacent residues, improving the enzyme thermostability by stabilizing local interactions. Overall catalytic efficiency (k (cat) / K (m)) of K46E and K65E/K97M/S209G was improved by 56% and 152% than that of wild type at pH 3.5, respectively. Thus, the catalytic efficiency of these enzymes was not inversely related to their thermostability.
authors.affiliation: Department of Animal Science and Graduate Field of Food Science, Cornell University, Ithaca, NY 14853, USA.
authors.forename: Moon-Soo
authors.initials: MS
authors.lastname: Kim
authors.forename: Xin Gen
authors.initials: XG
authors.lastname: Lei
mesh_terms: D010832:6-Phytase / Q000235:genetics / Q000378:metabolism*; D000135:Acid Phosphatase / Q000235:genetics / Q000378:metabolism*; D002152:Calorimetry, Differential Scanning; D002384:Catalysis; D019020:Directed Molecular Evolution; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D006860:Hydrogen Bonding; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008958:Models, Molecular; D009097:Multienzyme Complexes / Q000235:genetics / Q000378:metabolism*; D016296:Mutagenesis; D016133:Polymerase Chain Reaction; D015202:Protein Engineering; D017433:Protein Structure, Secondary; D044366:Transition Temperature
chemical_list: D029968:Escherichia coli Proteins; D009097:Multienzyme Complexes; D000135:Acid Phosphatase; D010832:6-Phytase; C118716:AppA2 protein, E coli