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.

Influence of the Environment on the Morphological and Biochemical Characteristics of Different Aspergillus niger Wild Type Strains

“…Our results are in agreement with the results obtained by Tsang et al [9]. Differences in the activity and amount of other enzymes connected with low and very low pH were found by different authors [9,[23][24][25]. Long-term effect of heavy metal ions, low pH and changes in the gene expression were represented on the morphological characteristics of hyphae and heads carrying spores (Fig.…”

Expression, purification and characterization of a phyA m -phyCs fusion phytase

“…Recently phyA was site-mutated to phyA m and phyCs was synthesized without altering the protein sequence of phyC according to the Pichia pastoris codon usage bias because of the low productions of wild-type phyA and phyC phytases in P. pastoris, and we overexpressed them in yeast as an active and extracellular enzyme (Chen et al, 2005;Zou et al, 2006). The phyA (phyA m ) phytase exhibits an optimum pH at 2.5 to 5.5, having a pH-activity profile ideally suited for maximal activity at low pH point (Chen et al, 2003;Kim et al, 2006). The phyC (phyCs) phytase exhibits an optimum pH around 6 to 9, suitable for neutral tracts and is more thermostable (Janne et al, 1998;Kim et al, 1998).…”

Ethanol Tolerance in &lt;i&gt;Aspergillus niger&lt;/i&gt; and &lt;i&gt;Escherichia coli&lt;/i&gt; Phytase

“…Both of these enzymes not only share a common catalytic mechanism, but they also display considerable amino acids divergence in their molecular structure. Previous studies have shown that a change of just a single amino acid can alter physical properties of these enzymes [7,8]. In addition, researchers have shown that significant differences exist in these two enzymes in their response to sodium chloride [9].…”