title: Altering the substrate specificity site of Aspergillus niger PhyB shifts the pH optimum to pH 3.2.
abstract: Phytases are of biotechnological importance as animal feed additives for their ability to catalyze the hydrolysis of phosphate from phytate for absorption by simple-stomached animals, and to reduce their fecal phosphorus excretion. Aspergillus niger PhyB has high catalytic activity at low pHs around 2.5, but has little activity at the commonly observed gastric pH of young animals (3.0-3.5). Our objective was to determine if the pH optima of PhyB could be broadened to a more characteristic pH range in the stomach of young animals through site-directed mutagenesis. We created two mutants, E272K and E272Q, each with a single amino acid substitution of the same residue in the substrate specificity site. Mutants were designed to replace an acidic amino acid, with either a neutral amino acid (E272Q) or basic amino acid (E272K), and were overexpressed in the yeast Pichia pastoris. While the wild-type (WT) pH optimum was 2.5, mutant E272K shifted to a new optimum of pH 3.2. E272K had a concomitant reduction in K (m) of 36-fold at pH 2.5 and 6-fold at pH 3.2 compared to the WT. Our results indicate that the pH optimum of PhyB can be altered to match the stomach pH, along with an improved substrate affinity.
authors: Department of Animal Science, Cornell University, Ithaca, NY 14853, USA.
authors: Jeremy D
authors: JD
authors: Weaver
authors: Edward J
authors: EJ
authors: Mullaney
authors: Xin Gen
authors: XG
authors: Lei
mesh_terms: D010832:6-Phytase / Q000235:genetics / Q000378:metabolism*; D019943:Amino Acid Substitution; D001234:Aspergillus niger / Q000201:enzymology* / Q000235:genetics; D001665:Binding Sites / Q000235:genetics; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D010833:Phytic Acid / Q000378:metabolism; D010843:Pichia / Q000378:metabolism; D017354:Point Mutation; D011994:Recombinant Proteins / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D005656:Fungal Proteins; D011994:Recombinant Proteins; D010833:Phytic Acid; D010832:6-Phytase