Phytase, a new life for an "old" enzyme.

Phytases are phosphohydrolytic enzymes that initiate stepwise removal of phosphate from phytate. Simple-stomached species such as swine, poultry, and fish require extrinsic phytase to digest phytate, the major form of phosphorus in plant-based feeds. Consequently, this enzyme is supplemented in these species' diets to decrease their phosphorus excretion, and it has emerged as one of the most effective and lucrative feed additives.

Vanadate inhibition of fungal PhyA and bacterial AppA2 histidine acid phosphatases.

The fungal PhyA protein, which was first identified as an acid optimum phosphomonoesterase (EC 3.1.3.

Site-directed mutagenesis of disulfide bridges in Aspergillus niger NRRL 3135 phytase (PhyA), their expression in Pichia pastoris and catalytic characterization.

Earlier studies have established the importance of five disulfide bridges (DBs) in Aspergillus niger phytase. In this study, the relative importance of each of the individual disulfide bridge is determined by its removal by site-directed mutagenesis of specific cysteines in the cloned A. niger phyA gene.

Impact of assay conditions on activity estimate and kinetics comparison of Aspergillus niger PhyA and Escherichia coli AppA2 phytases.

Aspergillus niger PhyA and Escherichia coli AppA2 are increasingly used in animal feed for phosphorus nutrition and environmental protection. The objective of this study was to determine the impacts of assay conditions on activity estimates of these two phytases and to compare their biochemical characteristics at a pH similar to the stomach environment. The activities of the unpurified AppA2 were more variable than those of PhyA with three commonly used phytase activity assays.

Unfolding and refolding of Aspergillus niger PhyB phytase: role of disulfide bridges.

The role of disulfide bridges in the folding of Aspergillus niger phytase pH 2.5-optimum (PhyB) was investigated using dynamic light scattering (DLS). Guanidinium chloride (GuCl) at 1.

Kinetic characterization of O-phospho-L-tyrosine phosphohydrolase activity of two fungal phytases.

Fungal phytases belonging to "histidine acid phosphatase" or HAP class of phosphohydrolases that catalyze the hydrolysis of phytic acid could also hydrolyze O-phospho-L-tyrosine, which is also called phosphotyrosine. Two phytases from Aspergillus niger and Aspergillus awamori with pH optima 2.5 were tested for phosphotyrosine hydrolase activity; both enzymes cleaved the phosphomonoester bond of phosphotyrosine efficiently at acidic pH.

Salt effect on the pH profile and kinetic parameters of microbial phytases.

The pH profiles of two microbial phytases were determined using four different general purpose buffers at different pH values. The roles of calcium chloride, sodium chloride, and sodium fluoride on activity were compared in these buffers. For Aspergillus niger phytase, calcium extended the pH range to 8.

Altering the substrate specificity site of Aspergillus niger PhyB shifts the pH optimum to pH 3.2.

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.

Adopting selected hydrogen bonding and ionic interactions from Aspergillus fumigatus phytase structure improves the thermostability of Aspergillus niger PhyA phytase.

Although it has been widely used as a feed supplement to reduce manure phosphorus pollution of swine and poultry, Aspergillus niger PhyA phytase is unable to withstand heat inactivation during feed pelleting. Crystal structure comparisons with its close homolog, the thermostable Aspergillus fumigatus phytase (Afp), suggest associations of thermostability with several key residues (E35, S42, R168, and R248) that form a hydrogen bond network in the E35-to-S42 region and ionic interactions between R168 and D161 and between R248 and D244. In this study, loss-of-function mutations (E35A, R168A, and R248A) were introduced singularly or in combination into seven mutants of Afp.

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

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.

Conservation of cysteine residues in fungal histidine acid phytases.

Amino acid sequence analysis of fungal histidine acid phosphatases displaying phytase activity has revealed a conserved eight-cysteine motif. These conserved amino acids are not directly associated with catalytic function; rather they appear to be essential in the formation of disulfide bridges. Their role is seen as being similar to another eight-cysteine motif recently reported in the amino acid sequence of nearly 500 plant polypeptides.

Monitoring of unfolding and refolding in fungal phytase (phyA) by dynamic light scattering.

Role of disulfide bridges in phytase's unfolding-refolding was probed using dynamic light scattering. Phytase was unfolded by guanidinium chloride and then refolded by removing the denaturant by dialysis. Thiol reagents prevented refolding; thus, disulfide bridge formation is an integral step in phytase folding.

Fungal phyA gene expressed in potato leaves produces active and stable phytase.

Fungal phyA gene from Aspergillus ficuum (niger) was cloned and expressed in potato leaves. The recombinant enzyme was stable and catalytically active. The expressed protein in the leaves of the dicotyledonous plant retained most physical and catalytic properties of the benchmark A.

Site-directed mutagenesis of Aspergillus niger NRRL 3135 phytase at residue 300 to enhance catalysis at pH 4.0.

Increased phytase activity for Aspergillus niger NRRL 3135 phytaseA (phyA) at intermediate pH levels (3.0-5.0) was achieved by site-directed mutagenesis of its gene at amino acid residue 300.

Cloned and expressed fungal phyA gene in alfalfa produces a stable phytase.

The phyA gene from Aspergillus ficuum that codes for a 441-amino-acid full-length phosphomonoesterase (phytase) was cloned and expressed in Medicago sativa (alfalfa) leaves. The expressed enzyme from alfalfa leaves was purified to homogeneity and biochemically characterized, and its catalytic properties were elucidated. The expressed phytase in alfalfa leaves retained all the biochemical properties of the benchmark A.