A novel phytase from Yersinia rohdei with high phytate hydrolysis activity under low pH and strong pepsin conditions

Two novel phytase genes belonging to the histidine acid phosphatase family were cloned from Yersinia rohdei and Y. pestis and expressed in Pichia pastoris. Both the recombinant phytases had high activity at pH 1.5-6.0 (optimum pH 4.5) with an optimum temperature of 55 degrees C. Compared with the major commercial phytases from Aspergillus niger, Escherichia coli, and a potential commercial phytase from Y. intermedia, the Y. rohdei phytase was more resistant to pepsin, retained more activity under gastric conditions, and released more inorganic phosphorus (two to ten times) from soybean meal under simulated gastric conditions. These superior properties suggest that the Y. rohdei phytase is an attractive additive to animal feed. Our study indicated that, in order to better hydrolyze the phytate and release more inorganic phosphorus in the gastric passage, phytase should have high activity and stability, simultaneously, at low pH and high protease concentration.     

PhyA gene product of Aspergillus ficuum and Peniophora lycii produces dissimilar phytases

PhyA gene products of Aspergillus ficuum (AF) and Peniophora lycii (PL) as expressed in industrial strains of Aspergillus niger and Aspergillus oryzae, respectively, were purified to homogeneity and then characterized for both physical and biochemical properties. The PL phytase is 26 amino acid residues shorter than the AF phytase. Dynamic light scattering studies indicate that the active AF phytase is a monomer while the PL phytase is a dimer. While both of the phytases retained four identical glycosylatable Asn residues, unique glycosylation sites, six for PL and seven for AF phytase, were observed. Global alignment of both the phytases has shown 38% sequence homology between the two proteins. At 58 degrees C and pH 5.0, the PL phytase gave a specific activity of 22,000 nKat/mg as opposed to about 3000 nKat/mg for AF phytase. However, the AF phytase is more thermostable than its counterpart PL phytase at 65 degrees C. Also, AF phytase is more stable at pH 7.5 than the PL phytase. The two phytases differed in K(m) for phytate, K(i) for myo-inositol hexasulfate (MIHS), and pH optima profile. Despite similarities in the active site sequences, the two phytases show remarkable differences in turnover number, pH optima profile, stability at higher temperature, and alkaline pH. These biochemical differences indicate that phytases from ascomycete and basidiomycete fungi may have evolved to degrade phytate in different environments.     

The type III secretion system is involved in the invasion and intracellular survival of Escherichia coli K1 in human brain microvascular endothelial cells

Two thermostable phytases were identified from Thai isolates of Aspergillus japonicus BCC18313 (TR86) and Aspergillus niger BCC18081 (TR170). Both genes of 1404 bp length, coding for putative phytases of 468 amino acid residues, were cloned and transferred into Pichia pastoris . The recombinant phytases, r-PhyA86 and r-PhyA170, were expressed as active extracellular, glycosylated proteins with activities of 140 and 100 U mL⁻¹, respectively. Both recombinant phytases exhibited high affinity for phytate but not for p -nitrophenyl phosphate. Optimal phytase activity was observed at 50 °C and pH 5.5. High thermostability, which is partly dependent on glycosylation, was demonstrated for both enzymes, as >50% activity was retained after heating at 100 °C for 10 min. The recombinant phytases also exhibited broad pH stability from 2.0 to 8.0 and are resistant to pepsin. In vitro digestibility tests suggested that r-PhyA86 and r-PhyA170 are at least as efficient as commercial phytase for hydrolyzing phytate in corn-based animal feed and are therefore suitable sources of phytase supplement.     

From DNA sequence to improved functionality: using protein sequence comparisons to rapidly design a thermostable consensus phytase

Naturally-occurring phytases having the required level of thermostability for application in animal feeding have not been found in nature thus far. We decided to de novo construct consensus phytases using primary protein sequence comparisons. A consensus enzyme based on 13 fungal phytase sequences had normal catalytic properties, but showed an unexpected 15-22 degrees C increase in unfolding temperature compared with each of its parents. As a first step towards understanding the molecular basis of increased heat resistance, the crystal structure of consensus phytase was determined and compared with that of Aspergillus niger phytase. Aspergillus niger phytase unfolds at much lower temperatures. In most cases, consensus residues were indeed expected, based on comparisons of both three-dimensional structures, to contribute more to phytase stabilization than non-consensus amino acids. For some consensus amino acids, predicted by structural comparisons to destabilize the protein, mutational analysis was performed. Interestingly, these consensus residues in fact increased the unfolding temperature of the consensus phytase. In summary, for fungal phytases apparently an unexpected direct link between protein sequence conservation and protein stability exists.     

Purification and physico-chemical characterisation of genetically modified phytases expressed in Aspergillus awamori

Two heterologous phytases from Aspergillus awamori and Aspergillus fumigatus obtained from submerged cultures of genetically modified fungal strains in addition to two commercially available phytase preparations (Allzyme and Natuphos phytases) were purified to homogeneity using a combination of ultrafiltration, gel filtration and ion exchange. The purified preparations were used in subsequent characterisation studies, in which Western Immunoblot analysis, pH and temperature optima, thermal stability and substrate specificity were assessed. A. fumigatus phyA phytase expressed in A. awamori exhibited activity over a broad pH range together with an increased temperature optimum, and slightly enhanced thermal stability compared to the other phytases tested, and is thus a promising candidate for animal feed applications. This particular phytase retains activity over a wide range of pH values characteristic of the digestive tract and could conceivably be more suited to the increasingly higher feed processing temperatures being utilised today, than the corresponding phytases from Aspergillus niger.     

Biochemical characteristics of phytases from fungi and the transformed microorganism

Five sources of phytases were used to study their biochemical characteristics. Phytase E was from an original Escherichia coli (E. coli), phytase PI and PG from the transformed Pichia pastoris (P. pastoris) with phytase gene of E. coli, phytase B and R from Aspergillus niger (A. niger). The results showed that the relative phytase activities had no significant changes when temperature was below 60 °C (P>0.05), and then decreased significantly with temperature increasing (P<0.01). The fungal phytase with the phytase gene from A. niger had the higher thermostability than the bacterial phytase with the phytase gene from E. coli; i.e. at 70 °C, 27–58% of phytase activity (compared with 30 °C) was retained for the bacterial phytase, and 73–96% for the fungal phytase; at 90 °C, 20–47% was retained for the bacterial phytase, and 41–52% for the fungal phytase, especially for the most thermostable phytase R (P<0.01). The optimum pH ranges were 3.0–4.5 for the bacterial phytases and 5.0–5.5 for the fungal phytases (P<0.01). When pH levels were 1, 7 and 8, only 3–7% of phytase activity (compared with the maximum phytase activity at a pH point) was retained for both bacterial and fungal phytases. The amount of inorganic P released from soybean meal was significantly increased when the levels of phytase activity in the soybean meal increased from 0 to 1.0 U/g soybean meal (P<0.01), except for phytase PI. The maximum P released was obtained at 1 U/g soybean meal for all five kinds of phytases (P<0.01). The most economical phytase concentration for P released was 0.25 U/g for phytase PI and B, and 0.50–1.0 U/g for phytase PG, E and R. In addition, the linear and non-linear regression models were established to estimate phytase activity and its characteristics very easily and economically.     

Comparative studies on the in vitro properties of phytases from various microbial origins

The physical and chemical properties of six crude phytase preparations were compared. Four of these enzymes (Aspergillus A, Aspergillus R, Peniophora and Aspergillus T) were produced at commercial scale for the use as feed additives while the other two (E. coli and Bacillus) were produced at laboratory scale. The encoding genes of the enzymes were from different microbial origins (4 of fungal origin and 2 of bacterial origin, i.e., E. coli and Bacillus phytases). One of the fungal phytases (Aspergillus R) was expressed in transgenic rape. The enzymes were studied for their pH behaviour, temperature optimum and stability and resistance to protease inactivation. The phytases were found to exhibit different properties depending on source of the phytase gene and the production organism. The pH profiles of the enzymes showed that the fungal phytases had their pH optima ranging from 4.5 to 5.5. The bacterial E. coli phytase had also its pH optimum in the acidic range at pH 4.5 while the pH optimum for the Bacillus enzyme was identified at pH 7.0. Temperature optima were at 50 and 60°C for the fungal and bacterial phytases, respectively. The Bacillus phytase was more thermostable in aqueous solutions than all other enzymes. In pelleting experiments performed at 60, 70 and 80°C in the conditioner, Aspergillus A, Peniophora (measurement at pH 5.5) and E. coli phytases were more heat stable compared to other enzymes (Bacillus enzyme was not included). At a temperature of 70°C in the conditioner, these enzymes maintained a residual activity of approximately 70% after pelleting compared to approximately 30% determined for the other enzymes. Incubation of enzyme preparations with porcine proteases revealed that only E. coli phytase was insensitive against pepsin and pancreatin. Incubation of the enzymes in digesta supernatants from various segments of the digestive tract of hens revealed that digesta from stomach inactivated the enzymes most efficiently except E. coli phytase which had a residual activity of 93% after 60 min incubation at 40°C. It can be concluded that phytases of various microbial origins behave differently with respect to their in vitro properties which could be of importance for future developments of phytase preparations. Especially bacterial phytases contain properties like high temperature stability (Bacillus phytase) and high proteolytic stability (E. coli phytase) which make them favourable for future applications as feed additives.     

弗氏柠檬酸杆菌植酸酶的分离纯化及其酶学性质研究

从弗氏柠檬酸杆菌(Citrobacter freundii)中分离纯化了一种植酸酶并进行了酶学性质研究,其反应最适pH为4.0~4.5,最适温度为40℃,在37℃下以植酸钠为底物的Km值为0.85nmol/L,Vmax为0.53IU/(mg.min),具有较好的抗胰蛋白酶的能力。酶蛋白的分子量大小约为45kDa,成熟酶蛋白N端序列为QCAPEGYQLQQVLMM。     

Crystal structures of Escherichia coli phytase and its complex with phytate

Phytases catalyze the hydrolysis of phytate and are able to improve the nutritional quality of phytate-rich diets. Escherichia coli phytase, a member of the histidine acid phosphatase family has the highest specific activity of all phytases characterized. The crystal structure of E. coli phytase has been determined by a two-wavelength anomalous diffraction method using the exceptionally strong anomalous scattering of tungsten. Despite a lack of sequence similarity, the structure closely resembles the overall fold of other histidine acid phosphatases. The structure of E. coli phytase in complex with phytate, the preferred substrate, reveals the binding mode and substrate recognition. The binding is also accompanied by conformational changes which suggest that substrate binding enhances catalysis by increasing the acidity of the general acid.     

Engineering of phytase for improved activity at low pH

For industrial applications in animal feed, a phytase of interest must be optimally active in the pH range prevalent in the digestive tract. Therefore, the present investigation describes approaches to rationally engineer the pH activity profiles of Aspergillus fumigatus and consensus phytases. Decreasing the negative surface charge of the A. fumigatus Q27L phytase mutant by glycinamidylation of the surface carboxy groups (of Asp and Glu residues) lowered the pH optimum by ca. 0.5 unit but also resulted in 70 to 75% inactivation of the enzyme. Alternatively, detailed inspection of amino acid sequence alignments and of experimentally determined or homology modeled three-dimensional structures led to the identification of active-site amino acids that were considered to correlate with the activity maxima at low pH of A. niger NRRL 3135 phytase, A. niger pH 2.5 acid phosphatase, and Peniophora lycii phytase. Site-directed mutagenesis confirmed that, in A. fumigatus wild-type phytase, replacement of Gly-277 and Tyr-282 with the corresponding residues of A. niger phytase (Lys and His, respectively) gives rise to a second pH optimum at 2.8 to 3.4. In addition, the K68A single mutation (in both A. fumigatus and consensus phytase backbones), as well as the S140Y D141G double mutation (in A. fumigatus phytase backbones), decreased the pH optima with phytic acid as substrate by 0.5 to 1.0 unit, with either no change or even a slight increase in maximum specific activity. These findings significantly extend our tools for rationally designing an optimal phytase for a given purpose.     

Optimization of the catalytic properties of Aspergillus fumigatus phytase based on the three-dimensional structure

Previously, we determined the DNA and amino acid sequences as well as biochemical and biophysical properties of a series of fungal phytases. The amino acid sequences displayed 49-68% identity between species, and the catalytic properties differed widely in terms of specific activity, substrate specificity, and pH optima. With the ultimate goal to combine the most favorable properties of all phytases in a single protein, we attempted, in the present investigation, to increase the specific activity of Aspergillus fumigatus phytase. The crystal structure of Aspergillus niger NRRL 3135 phytase known at 2.5 A resolution served to specify all active site residues. A multiple amino acid sequence alignment was then used to identify nonconserved active site residues that might correlate with a given favorable property of interest. Using this approach, Gln27 of A. fumigatus phytase (amino acid numbering according to A. niger phytase) was identified as likely to be involved in substrate binding and/or release and, possibly, to be responsible for the considerably lower specific activity (26.5 vs. 196 U x [mg protein](-1) at pH 5.0) of A. fumigatus phytase when compared to Aspergillus terreus phytase, which has a Leu at the equivalent position. Site-directed mutagenesis of Gln27 of A. fumigatus phytase to Leu in fact increased the specific activity to 92.1 U x (mg protein)(-1), and this and other mutations at position 27 yielded an interesting array of pH activity profiles and substrate specificities. Analysis of computer models of enzyme-substrate complexes suggested that Gln27 of wild-type A. fumigatus phytase forms a hydrogen bond with the 6-phosphate group of myo-inositol hexakisphosphate, which is weakened or lost with the amino acid substitutions tested. If this hydrogen bond were indeed responsible for the differences in specific activity, this would suggest product release as the rate-limiting step of the A. fumigatus wild-type phytase reaction.     

Biophysical Characterization of Fungal Phytases (myo-Inositol Hexakisphosphate Phosphohydrolases): Molecular Size, Glycosylation Pattern, and Engineering of Proteolytic Resistance

Phytases (myo-inositol hexakisphosphate phosphohydrolases) are found naturally in plants and microorganisms, particularly fungi. Interest in these enzymes has been stimulated by the fact that phytase supplements increase the availability of phosphorus in pig and poultry feed and thereby reduce environmental pollution due to excess phosphate excretion in areas where there is intensive livestock production. The wild-type phytases from six different fungi, Aspergillus niger, Aspergillus terreus, Aspergillus fumigatus, Emericella nidulans, Myceliophthora thermophila, and Talaromyces thermophilus, were overexpressed in either filamentous fungi or yeasts and purified, and their biophysical properties were compared with those of a phytase from Escherichia coli. All of the phytases examined are monomeric proteins. While E. coli phytase is a nonglycosylated enzyme, the glycosylation patterns of the fungal phytases proved to be highly variable, differing for individual phytases, for a given phytase produced in different expression systems, and for individual batches of a given phytase produced in a particular expression system. Whereas the extents of glycosylation were moderate when the fungal phytases were expressed in filamentous fungi, they were excessive when the phytases were expressed in yeasts. However, the different extents of glycosylation had no effect on the specific activity, the thermostability, or the refolding properties of individual phytases. When expressed in A. niger, several fungal phytases were susceptible to limited proteolysis by proteases present in the culture supernatant. N-terminal sequencing of the fragments revealed that cleavage invariably occurred at exposed loops on the surface of the molecule. Site-directed mutagenesis of A. fumigatus and E. nidulans phytases at the cleavage sites yielded mutants that were considerably more resistant to proteolytic attack. Therefore, engineering of exposed surface loops may be a strategy for improving phytase stability during feed processing and in the digestive tract.     

Degradation of phytate in the gut of pigs ‐ pathway of gastrointestinal inositol phosphate hydrolysis and enzymes involved

The present study gives an overview on the whole mechanism of phytate degradation in the gut and the enzymes involved. Based on the similarity of the human and pigs gut, the study was carried out in pigs as model for humans. To differentiate between intrinsic feed phytases and endogenous phytases hydrolysing phytate in the gut, two diets, one high (control diet) and the other one very low in intrinsic feed phytases (phytase inactivated diet) were applied. In the chyme of stomach, small intestine and colon inositol phosphate isomers and activities of phytases and alkaline phosphatases were determined. In parallel total tract phytate degradation and apparent phosphorus digestibility were assessed. In the stomach chyme of pigs fed the control diet, comparable high phytase activity and strong phytate degradation were observed. The predominant phytate hydrolysis products were inositol phosphates, typically formed by plant phytases. For the phytase inactivated diet, comparable very low phytase activity and almost no phytate degradation in the stomach were determined. In the small intestine and colon, high activity of alkaline phosphatases and low activity of phytases were observed, irrespective of the diet fed. In the colon, stronger phytate degradation for the phytase inactivated diet than for the control diet was detected. Phytate degradation throughout the whole gut was nearly complete and very similar for both diets while the apparent availability of total phosphorus was significantly higher for the pigs fed the control diet than the phytase inactivated diet. The pathway of inositol phosphate hydrolysis in the gut has been elucidated.     

Exchanging the active site between phytases for altering the functional properties of the enzyme

By using a novel consensus approach, we have previously managed to generate a fully synthetic phytase, consensus phytase-1, that was 15-26 degrees C more thermostable than the parent fungal phytases used in its design (Lehmann et al., 2000). We now sought to use the backbone of consensus phytase-1 and to modify its catalytic properties. This was done by replacing a considerable part of the active site (i.e., all the divergent residues) with the corresponding residues of Aspergillus niger NRRL 3135 phytase, which displays pronounced differences in specific activity, substrate specificity, and pH-activity profile. For the new protein termed consensus phytase-7, a major - although not complete - shift in catalytic properties was observed, demonstrating that rational transfer of favorable catalytic properties from one phytase to another is possible by using this approach. Although the exchange of the active site was associated with a 7.6 degrees C decrease in unfolding temperature (Tm) as measured by differential scanning calorimetry, consensus phytase-7 still was >7 degrees C more thermostable than all wild-type ascomycete phytases known to date. Thus, combination of the consensus approach with the selection of a "preferred" active site allows the design of a thermostabilized variant of an enzyme family of interest that (most closely) matches the most favorable catalytic properties found among its family members.     

Phytase isozymes from Aspergillus niger NCIM 563 under solid state fermentation: Biochemical characterization and their correlation with submerged phytases

Aspergillus niger NCIM 563 produces dissimilar phytase isozymes under solid state and submerged fermentation conditions. Biochemical characterization and applications of phytase Phy III and Phy IV in SSF and their comparison with submerged fermentation Phy I and Phy III were studied. SSF phytases have a higher metabolic potential as compared to SmF. Phy I is tetramer and Phy II, III and IV are monomers. Phy I and IV have pH optima of 2.5 and Phy II and III have pH optima of 5.0 and 5.6, respectively. Phy I, III and IV exhibited very broad substrate specificity while Phy II was more specific for sodium phytate. SSF phytase is less thermostable as compared to SmF phytase. Phy I and II show homology with other known phytases while Phy III and IV show no homology with SmF phytases and any other known phytases from the literature suggesting their unique nature. This is the first report about differences among phytase produced under SSF and SmF by A. niger and this study provides basis for explanation of the stability and catalytic differences observed for these enzymes. Exclusive biochemical characteristics and multilevel application of SSF native phytases determine their efficacy and is exceptional.     

Acid phosphatase production by Aspergillus niger N402A in continuous flow culture

The production of acid phosphatases (E.C.3.1.3.2, ACPs) by Aspergillus niger N402A is regulated by specific growth rate, as well as phosphate availability and pH, as demonstrated by studies in continuous flow culture. Specific ACP activity was highest when A. niger was grown at pH 6.3 (64±8 U g⁻¹) or pH 2.8 (99±11 U g⁻¹), at a dilution rate of 0.07 h⁻¹ and phosphate concentrations below 0.46 mM. ACP production was growth correlated for specific growth rates between 0.07 and 0.13 h⁻¹. Four different ACPs, including two phytases, were produced by A. niger N402A. The ACP and the phytase with maximal activities at pH 5.5 were differentially expressed at different culture pH values, with greater production at low pH.     

Adsorption immobilization of Escherichia coli phytase on probiotic Bacillus polyfermenticus spores

The immobilization of enzymes on edible matrix supports is of great importance for developing stabilized feed enzymes. In this study, probiotic Bacillus spores were explored as a matrix for immobilizing Escherichia coli phytase, a feed enzyme releasing phosphate from phytate. Because Bacillus spore is inherently resistant to heat, solvents and drying, they were expected to be a unique matrix for enzyme immobilization. When mixed with food-grade Bacillus polyfermenticus spores, phytases were adsorbed to their surface and became immobilized. The amount of phytase attached was 28.2 ± 0.7 mg/g spores, corresponding to a calculated activity of 63,960 U/g spores; however, the measured activity was 41,120 ± 990.1 U/g spores, reflecting a loss of activity upon adsorption. Immobilization increased the half life (t_1/2) of the enzyme three- to ten-fold at different temperatures ranging from 60 to 90 °C. Phytase was bound to the spore surface to the extent that ultrasonication treatment was not able to detach phytases from spores. Desorption of spore-immobilized phytase was only achieved by treatment with 1 M NaCl, 10% formic acid in 45% acetonitrile, SDS, or urea, suggesting that adsorption of phytase to the spore might be via hydrophobic and electrostatic interactions. We propose here that Bacillus spore is a novel immobilization matrix for enzymes that displays high binding capacity and provides food-grade safety.     

Cloning, sequencing, and expression of an Escherichia coli acid phosphatase/phytase gene (appA2) isolated from pig colon

Bacterial strains were isolated from the pig colon to screen for phytase and acid phosphatase activities. Among 93 colonies, Colony 88 had the highest activities for both enzymes and was identified as an Escherichia coli strain. Using primers derived from the E. coli pH 2.5 acid phosphatase appA sequence (Dassa et al. (1990), J. Bacteriol. 172, 5497-5500), we cloned a 1482 bp DNA fragment from the isolate. In spite of 95% homology between the sequenced gene and the appA, 7 amino acids were different in their deduced polypeptides. To characterize the properties and functions of the encoded protein, we expressed the coding region of the isolated DNA fragment and appA in Pichia pastoris, respectively, as r-appA2 and r-appA. The recombinant protein r-appA2, like r-appA and the r-phyA phytase expressed in Aspergillus niger, was able to hydrolyze phosphorus from sodium phytate and p-nitrophenyl phosphate. However, there were distinct differences in their pH profiles, Km and Vmax for the substrates, specific activities of the purified enzymes, and abilities to release phytate phosphorus in soybean meal. In conclusion, the DNA fragment isolated from E. coli in pig colon seems to encode for a new acid phosphatase/phytase and is designated as E. coli appA2.     

Production of two Highly Active Bacterial Phytases with Broad pH Optima in Germinated Transgenic Rice Seeds

Phytate is the main storage form of phosphorus in many plant seeds, but phosphate bound in this form is not available to monogastric animals. Phytase, an enzyme that hydrolyzes phosphate from phytate, has the potential to enhance phosphorus availability in animal diets when engineered in rice seeds as a feed additive. Two genes, derived from a ruminal bacterium Selenomonas ruminantium (SrPf6) and Escherichia coli (appA), encoding highly active phytases were expressed in germinated transgenic rice seeds. Phytase expression was controlled by a germination inducible alpha-amylase gene (alphaAmy8) promoter, and extracellular phytase secretion directed by an betaAmy8 signal peptide sequence. The two phytases were expressed in germinated transgenic rice seeds transiently and in a temporally controlled and tissue-specific manner. No adverse effect on plant development or seed formation was observed. Up to 0.6 and 1.4 U of phytase activity per mg of total extracted cellular proteins were obtained in germinated transgenic rice seeds expressing appA and SrPf6 phytases, respectively, which represent 46-60 times of phytase activities compared to the non-transformant. The appA and SrPf6 phytases produced in germinated transgenic rice seeds had high activity over broad pH ranges of 3.0-5.5 and 2.0-6.0, respectively. Phytase levels and inheritance of transgenes in one highly expressing plant were stable over four generations. Germinated transgenic rice seeds, which produce a highly active recombinant phytase and are rich in hydrolytic enzymes, nutrients and minerals, could potentially be an ideal feed additive for improving the phytate-phosphorus digestibility in monogastric animals.     

Purification and characterisation of an acid phosphatase with phytase activity from Mucor hiemalis Wehmer

An acid phosphatase with phytase activity, produced by Mucor hiemalis Wehmer, was purified to homogeneity by a combination of anion exchange, gel filtration and hydrophobic interaction chromatography. The monomeric, glycosylated enzyme displayed maximum activity at 55 degrees C and pH 5.0-5.5. When compared to commercialised products, the enzyme is more thermostable (80 degrees C, 5min), displays a broader pH versus activity profile and greater stability under simulated digestive tract conditions. Unlike commercial phytases, the Mucor enzyme should retain some activity in the small intestine as well as in the stomach, facilitating a longer duration of action and hence more extensive substrate hydrolysis. Substrate specificity studies and protein database similarity searching using mass spectrometry-derived sequence data indicate that the enzyme is an acid phosphatase with activity on phytate. Cocktails containing acid phosphatases in combination with true phytases have been shown to promote more extensive phytate degradation than do true phytases alone. This, coupled to the enzyme's functionally relevant physicochemical characteristics, suggests its likely suitability for inclusion in second generation phytase cocktails for application in animal feed.