Shifting the pH profile of Aspergillus niger PhyA phytase to match the stomach pH enhances its effectiveness as an animal feed additive

Abstract

Environmental pollution by phosphorus from animal waste is a major problem in agriculture because simple-stomached animals, such as swine, poultry, and fish, cannot digest phosphorus (as phytate) present in plant feeds. To alleviate this problem, a phytase from Aspergillus niger PhyA is widely used as a feed additive to hydrolyze phytate-phosphorus. However, it has the lowest relative activity at the pH of the stomach (3.5), where the hydrolysis occurs. Our objective was to shift the pH optima of PhyA to match the stomach condition by substituting amino acids in the substrate-binding site with different charges and polarities. Based on the crystal structure of PhyA, we prepared 21 single or multiple mutants at Q50, K91, K94, E228, D262, K300, and K301 and expressed them in Pichia pastoris yeast. The wild-type (WT) PhyA showed the unique bihump, two-pH-optima profile, whereas 17 mutants lost one pH optimum or shifted the pH optimum from pH 5.5 to the more acidic side. The mutant E228K exhibited the best overall changes, with a shift of pH optimum to 3.8 and 266% greater (P < 0.05) hydrolysis of soy phytate at pH 3.5 than the WT enzyme. The improved efficacy of the enzyme was confirmed in an animal feed trial and was characterized by biochemical analysis of the purified mutant enzymes. In conclusion, it is feasible to improve the function of PhyA phytase under stomach pH conditions by rational protein engineering.

FIG. 1.

(A) The region of the mutated amino acids (dotted circle) in the three-dimensional structure of A. niger PhyA. (B) The seven mutated amino acids (Q50, K91, K94, E228, D262, K300, and K301) and related amino acids in the circled region of panel A. The structure is based on Kostrewa et al. (10). The α-helices are shown in red and the β-sheets in blue in panel A; dotted lines represent hydrogen bonds in panel B.

FIG. 2.

Comparisons of pH profiles of the WT, the single mutant E228K, and the triple mutant E228K-K300R-K301E. In both panels, values are expressed as the mean specific activity ± standard error (n = 3) (for panel B, standard errors were too small to be seen) of the purified enzymes at each pH, and phytase activity was determined using sodium phytate dissolved in the designated assay buffer. (A) pH 2.0 to 3.5, 0.2 M glycine-HCl; pH 4.0 to 6.5, 0.2 M citrate; and pH 7.0 to 8.0, 0.2 M Tris-HCl. (B) pH 2.0 to 4.0, 0.2 M glycine-HCl; pH 3.5 to 6.5, 0.2 M citrate. Overlapping pH points between the two buffer systems and smaller intervals were used to compare the buffer effect and the exact optimal pH of the testing phytases.

FIG. 3.

Efficacies of phytate-phosphorus hydrolysis in soybean meal by the single mutant E228K and the triple mutant E228K-K300R-K301E at pH 2.5 (0.2 M glycine-HCl), 3.5 (0.2 M glycine-HCl), and 5.5 (0.2 M citrate) at 37°C compared with that of the WT. The hydrolysis rates were calculated as the percentage of the WT phytase at pH 5.5, and the values are means ± standard errors (n = 3). An asterisk indicates a difference (P < 0.05) between pH 5.5 and other pH values within each enzyme. Different letters indicate differences (P < 0.05) between the WT and the mutants within each pH point.

FIG. 4.

Effects of supplemental E228K and WT PhyA phytases at 250 U kg⁻¹ of a low-phosphorus corn-soybean meal diet for weanling pigs on plasma inorganic-phosphorus concentrations (A), plasma alkaline phosphatase activity (B), and body weight gain (C). The values are means ± standard errors (n = 8 for each group). An asterisk indicates a difference (P < 0.05) between treatment groups.

FIG. 5.

Time courses of phytate hydrolysis by the WT and mutant E228K PhyAs and the subsequent appearance and disappearance of the intermediate or end products from the hydrolysis. The values are expressed as the concentration relative to the possible complete (maximal) hydrolysis. The enzymes (2 U ml⁻¹) were incubated with 1% sodium phytate in 0.2 M glycine-HCl buffer, pH 3.5, for the designated times, and the hydrolytic metabolites were analyzed by HPLC. (A) Inositol hexaphosphate (IP6); (B) inositol pentaphosphate (IP5); (C) inositol tetraphosphate (IP4); (D) inositol triphosphate (IP3); (E) inositol diphosphate (IP2); (F) inositol monophosphate (IP1); (G) inorganic phosphate (Pi).