Design of thermostable beta-propeller phytases with activity over a broad range of pHs and their overproduction by Pichia pastoris

Abstract

Thermostable phytases, which are active over broad pH ranges, may be useful as feed additives, since they can resist the temperatures used in the feed-pelleting process. We designed new beta-propeller phytases, using a structure-guided consensus approach, from a set of amino acid sequences from Bacillus phytases and engineered Pichia pastoris strains to overproduce the enzymes. The recombinant phytases were N-glycosylated, had the correct amino-terminal sequence, showed activity over a pH range of 2.5 to 9, showed a high residual activity after 10 min of heat treatment at 80°C and pH 5.5 or 7.5, and were more thermostable at pH 7.5 than a recombinant form of phytase C from Bacillus subtilis (GenBank accession no. AAC31775). A structural analysis suggested that the higher thermostability may be due to a larger number of hydrogen bonds and to the presence of P257 in a surface loop. In addition, D336 likely plays an important role in the thermostability of the phytases at pH 7.5. The recombinant phytases showed higher thermostability at pH 5.5 than at pH 7.5. This difference was likely due to a different protein total charge at pH 5.5 from that at pH 7.5. The recombinant beta-propeller phytases described here may have potential as feed additives and in the pretreatment of vegetable flours used as ingredients in animal diets.

FIG. 1.

SDS-polyacrylamide gels of FTE (A), FTEII (B), and FBA (C) phytases concentrated by ultrafiltration and treated with or without endo H_f. Lanes M, molecular size marker. (A) Lanes: 1, endo H_f; 2, FTE treated with endo H_f; 3, FTE without endo H_f treatment. (B) Lanes: 1, FTEII treated with endo H_f; 2, endo H_f; 3, FTEII without endo H_f treatment. (C) Lanes: 1, FBA treated with endo H_f; 2, FBA without endo H_f treatment; 3, endo H_f.

FIG. 2.

Effects of pH (A) and temperature (B) on specific phytase activity at 37°C and pH 7.5 for FTE (□), FTEII (▪), and FBA (•). Points represent the means for at least three independent enzyme assays (coefficient of variation, <5%).

FIG. 3.

Residual activities of FTE (□), FTEII (▪), FBA (•), and PhyC-R (○) after 10 min of heat treatment at pH 7.5 (A and B) or 5.5 (C and D) in the presence of 1.0 (A and C) or 5.0 (B and D) mM CaCl₂. Points represent the means for at least three independent determinations (coefficient of variation, <5%).

FIG. 4.

(A) Molecular model of FTEII constructed by homology modeling using SWISS-MODEL 8.05, with the 2POO structure as a template. (B) Hydrogen bond between T37 and K361 in the structure for FTE, FTEII, and FBA, binding the ninth residue from the N-terminal end with the alpha helix of the protein. (C) P257 in a surface loop in FTE, FTEII, and FBA. (D) Salt bridges between D336 and a Ca²⁺ ion or K351 in FTEII, FBA, and PhyC. Other salt bridges with the Ca²⁺ ion are also shown.