Improved thermal stability of phytase from Yersinia intermedia by physical adsorption immobilization on amino-multiwalled carbon nanotubes.

Phytase is used in poultry diets to hydrolyze and release of phytate-bound phosphorus. Immobilization on nanomaterials optimizes enzyme's thermal stability and reusability. This study aimed to immobilize the recombinant phytase from Yersinia intermedia on the surface of amino-multi-walled carbon nanotubes (amino-MWCNTs) by physical adsorption. For this, zeta potential measurement, FTIR spectroscopic analysis, scanning electron microscope (SEM), kinetic as well as thermodynamic parameters were used to characterize immobilized phytase on amino-MWCNTs. According to results, the optimum temperature of the immobilized phytase increased from 50 to 70 °C and also thermal and pH stability improved considerably. Moreover, immobilization led to an increase in the value of Km and kcat from 0.13 to 0.33 mM and 2220 to 2776 s-1, respectively. In addition, the changes in activation energy of thermal inactivation (ΔE#a (D)), the free energy of thermal inactivation (ΔG#D) and the enthalpy of thermal inactivation (ΔH#D) for immobilized phytase increased by +11.05, +24.7 and +11.4 kj/mole, respectively, while the value of the change in the entropy of thermal inactivation (ΔS#D) decreased by - 0.04 kj/mole.K. Overall, our results showed that adsorption immobilization of phytase on amino-MWCNTs increases thermal, pH and storage stability as well as some of kinetic parameters.

Purification of the phytase enzyme from Lactobacillus plantarum: The effect on pansy growth and macro-micro element content.

In the present study, the phytase enzyme was purified from Lactobacillus plantarum with a 3.08% recovery, 9.57-purification fold, and with a specific activity of 278.82 EU/mg protein. Then, the effects of the 5 EU and 10 EU purified phytase was determined on the plant growth, quality, the macro-micro nutrient content of pansy (Viola × wittrockiana), which is of great importance in ornamental plants industry. The research was established under greenhouse conditions with natural light in 2017. The pansy seeds were coated with phytase enzyme solution, sown in a peat environment, and transferred to pots at the seedling period. In general, the 5 EU and 10 EU applications increase plant height, the number of leaves per plant, the number of side branches per plant, and flower height parameters compared to control. Also, micro- and macronutrient values in soil and plant samples were examined. According to the results, the phytase application on pansy cultivation positively affected the properties and yielded high quality of plants.

Fabrication of PVA-chitosan-based nanofibers for phytase immobilization to enhance enzymatic activity.

In the present paper, polyvinyl alcohol (PVA)-chitosan (CS) nanofibers were fabricated by using the electrospinning method under a constant voltage (23 kV) and the distance (12 cm). Phytase was purified from cowpea seeds and it was immobilized into the PVA-CS nanofibers. The effects of the electrospinning parameters such as enzyme concentration and flow rate on the catalytic activity of the free and immobilized phytase were also studied. The fabricated nanofibers were characterized via FT-IR, SEM, and XRD techniques. PVA-CS nanofiber and phytase containing nanofiber (phytase-PVA-CS) were exhibited a homogenous morphology with an average diameter of about 43.6 ± 13.7 and 65.3 ± 26.0 nm and fiber diameter of nanofiber containing phytase was increased after enzyme immobilization into PVA-CS nanofiber. The optimum pH and temperature of the free and immobilized phytase were also investigated. The obtained results showed that the optimum pH and temperature values of the immobilized enzyme were shifted to higher pH and temperature values after the immobilization of phytase into PVA-CS nanofiber. According to these results of the immobilized phytase, it could be used in some industrial applications such as animal feed, agriculture, pharmaceutical area, and food industry.

Probiotic properties of a phytase producing Pediococcus acidilactici strain SMVDUDB2 isolated from traditional fermented cheese product, Kalarei.

The nutritional challenge faced by the monogastric animals due to the chelation effects of phytic acid, fuel the research on bioprospecting of probiotics for phytase production. Pediococcus acidilactici SMVDUDB2 isolated from Kalarei, exhibited extracellular phytase activity of 5.583 U/mL after statistical optimization of fermentation conditions viz. peptone (1.27%); temperature (37 °C); pH (6.26) and maltose (1.43%). The phytase enzyme possessed optimum pH and temperature of 5.5 and 37 °C, respectively and was thermostable at 60 °C. The enzyme was purified 6.42 fold with a specific activity of 245.12 U/mg with hydrophobic interaction chromatography. The purified enzyme had Km and Vmax values of 0.385 mM and 4.965 µmol/min respectively, with sodium phytate as substrate. The strain depicted more than 80% survival rate at low pH (pH 2.0, 3.0), high bile salt concentration (0.3 and 0.5%), after gastrointestinal transit, highest hydrophobicity affinity with ethyl acetate (33.33 ± 0%), autoaggregation (77.68 ± 0.68%) as well as coaggregation (73.57 ± 0.47%) with Staphylococcus aureus (MTCC 3160). The strain exhibited antimicrobial activity against Bacillus subtilis (MTCC 121), Mycobacterium smegmatis (MTCC 994), Staphylococcus aureus (MTCC 3160), Proteus vulgaris (MTCC 426), Escherichia coli (MTCC 1652) and Lactobacillus rhamnosus (MTCC 1408). The amount of exopolysaccharide produced by the strain was 2 g/L. This strain having the capability of phytate degradation and possessing probiotic traits could find application in food and feed sectors.

Partial characterization and purification of phytase from Lactobacillus plantarum CRL1964 isolated from pseudocereals.

Cereals and pseudocereals are a rich source of nutrients and trace elements, but their dietary bioavailability is low due to the presence of phytate (IP6), an antinutritional compound with the ability to chelate cations and proteins. Phytase is an enzyme that catalyzes the hydrolysis of IP6 and it is used as an additive improving the nutritional quality of grain-based foods. The aim of this study was to select lactic acid bacteria (LAB) isolated from pseudocereals with phytase activity, characterize their production and activity, and purify the enzyme. LAB strains isolated from grains and spontaneous sourdough of quinoa and amaranth were grown in the Man Rogosa and Sharpe medium where the inorganic phosphate (Pi) was replaced by 1% of IP6. Phytase activity was determined by measuring the Pi released from IP6. Phytase of Lactobacillus (L.) plantarum CRL1964 (PhyLP) showed the highest specific activity from 73 LAB evaluated. IP6 induces PhyLP production, which is at its maximum at the end of the exponential phase. PhyLP was thermostable and maintained its activity under acidic conditions. The enzymatic activity is stimulated by ethylenediaminetetraacetic acid, Co2+, and ascorbic acid. PhyLP was partially purified and showed a molecular mass of 55 kDa. L. plantarum CRL1964 and/or PhyLP have the potential to be included in the processing of cereal/pseudocereals based products for animal feed and/or the food industry improving its nutritional value.

Improving catalytic efficiency and maximum activity at low pH of Aspergillus neoniger phytase using rational design.

A preferable phytase for use in animal feeds for industrial applications should have high, optimal activity at low pH in the monogastric gut environment and high thermostability. To obtain enzymes with enhanced catalytic efficiency (pH 5.5) and excellent activity in acidic pH range, we performed structure-based rational design of a thermostable phytase (PhyAn). For this, six mutants based on different rational design strategies were constructed and heterologously expressed in Pichia pastoris. Particularly, the extracellular enzymatic activity was assessed to ensure that the produced enzymes met requirements of further analyses. Several positive mutants with enhanced catalytic efficiency or pH-profile shifts were carefully examined. Biochemical and kinetic investigations of purified mutants revealed that E79K, E80K, E79K + E80K and D68K had higher catalytic efficiency than the parent enzyme by approximately 49%, 67%, 86% and 15%, respectively. Moreover, the optimum pH of mutant Y65H was shifted from 5.0 to 3.0, and the peak of D68K shifted to pH 5.5. Analysis of the structural-functional relationships revealed that changes in amino acid charges, structural flexibility and space hindrance could significantly influence certain enzyme characteristics. Our results illustrate the feasibility and present a structural foundation for enhancing the phytase-catalytic efficiency and acid resistance by assembling mutations derived using rational design.

Purification and Biochemical Characterization of Phytase Enzyme from Lactobacillus coryniformis (MH121153).

Phytase (myo-inositol hexaphosphate phosphohydrolase) belongs to phosphatases. It catalyzes the hydrolysis of phytate to less-phosphorylated inorganic phosphates and phytate. Phytase is used primarily for the feeding of simple hermit animals in order to increase the usability of amino acids, minerals, phosphorus and energy. In the present study, phytase isolation from the Lactobacillus coryniformis strain, isolated from Lor cheese sources, phytase purification and characterization were studied. The phytase was purified in simple three steps. The enzyme was obtained with 2.60% recovery and a specific activity of 202.25 (EU/mg protein). The molecular mass of the enzyme was determined to be 43.25 kDa with the sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) method. The optimum temperature and pH for the enzyme were found as 60 °C and 5.0 and respectively. To defined the substrate specificity of the phytase, the hydrolysis of several phosphorylated compounds by the purified enzyme was studied and sodium phytate showed high specificity. Furthermore, the effects of Ca2+, Ag+, Mg2+, Cu2+, Co2+, Pb2+, Zn2+ and Ni2+ metal ions on the enzyme were studied.

Purification and kinetics of a protease-resistant, neutral, and thermostable phytase from Bacillus subtilis subsp. subtilis JJBS250 ameliorating food nutrition.

A novel protease-resistant and thermostable phytase from Bacillus subtilis subsp. subtilis JJBS250 was purified 36-fold to homogeneity with a combination of ammonium sulfate precipitation followed by Q-Sepharose and Sephadex G-50 chromatographic techniques. The estimated molecular mass of the purified phytase was 46 kDa by electrophoresis with optimal activity at pH 7.0 and 70 °C. About 19% of original activity was maintained at 80 °C for 10 min. Phytase activity was stimulated in presence of surfactants like Tween-20, Tween-80, and Triton X-100 and metal ions like Ca+2, K+, and Co+2 and it was inhibited by SDS and Mg+2, Al+2, and Fe+2. Purified enzyme showed specificity to different salts of phytic acid and values of Km and Vmax were 0.293 mM and 11.49 nmoles s-1, respectively for sodium phytate. The purified enzyme was resistant to proteases (trypsin and pepsin) that resulted in amelioration of food nutrition with simultaneous release of inorganic phosphate, reducing sugars, and soluble protein.

Production of extracellular heterologous proteins in Streptomyces rimosus, producer of the antibiotic oxytetracycline.

Among the Streptomyces species, Streptomyces lividans has often been used for the production of heterologous proteins as it can secrete target proteins directly into the culture medium. Streptomyces rimosus, on the other hand, has for long been used at an industrial scale for oxytetracycline production, and it holds 'Generally Recognised As Safe' status. There are a number of properties of S. rimosus that make this industrial strain an attractive candidate as a host for heterologous protein production, including (1) rapid growth rate; (2) growth as short fragments, as for Escherichia coli; (3) high efficiency of transformation by electroporation; and (4) secretion of proteins into the culture medium. In this study, we specifically focused our efforts on an exploration of the use of the Sec secretory pathway to export heterologous proteins in a S. rimosus host. We aimed to develop a genetic tool kit for S. rimosus and to evaluate the extracellular production of target heterologous proteins of this industrial host. This study demonstrates that S. rimosus can produce the industrially important enzyme phytase AppA extracellularly, and analogous to E. coli as a host, application of His-Tag/Ni-affinity chromatography provides a simple and rapid approach to purify active phytase AppA in S. rimosus. We thus demonstrate that S. rimosus can be used as a potential alternative protein expression system.

A novel purple acid phytase from an earthworm cast bacterium.

BACKGROUND: Phytases are a diverse group of enzymes initiating the dephosphorylation of phytate. Phytate is considered as an anti-nutritional compound because of its capability to chelate nutrients such as Fe2+ , Zn2+ , Mg2+ , and Ca2+ . In this study, several bacterial isolates obtained from earthworm casts were evaluated for their phytate degrading capability. Enzymatic properties and the sequence of the corresponding phytase-encoding gene of the selected isolate were determined. RESULTS: The phytase exhibited its highest activity at pH 4.0 and was stable from pH 3 up to pH 9. The temperature optimum was determined to be 65 °C. The strongest inhibitors of enzymatic activity were identified as vanadate, Cu2+ , and Zn2+ . High-performance ion chromatography analysis of enzymatic phytate dephosphorylation revealed that the first dephosphorylation product was d/l-myo-inositol(1,2,3,4,5)pentakisphosphate. CONCLUSION: Owing to its enzymatic properties, such as tolerance to tartrate and the presence of the consensus motifs PDTVY, GNHE, DLG, VLFH, and GHDH, this phytase could be classified as a purple acid phytase. To the best of our knowledge, this is the first report describing a bacterial purple acid phytase. © 2017 Society of Chemical Industry.

Biochemical Characterization of a Psychrophilic Phytase from an Artificially Cultivable Morel Morchella importuna.

Psychrophilic phytases suitable for aquaculture are rare. In this study, a phytase of the histidine acid phosphatase (HAP) family was identified in Morchella importuna, a psychrophilic mushroom. The phytase showed 38% identity with Aspergillus niger PhyB, which was the closest hit. The M. importuna phytase was overexpressed in Pichia pastoris, purified, and characterized. The phytase had an optimum temperature at 25°C, which is the lowest among all the known phytases to our best knowledge. The optimum pH (6.5) is higher than most of the known HAP phytases, which is fit for the weak acidic condition in fish gut. At the optimum pH and temperature, MiPhyA showed the maximum activity level (2,384.6 ± 90.4 µmol·min⁻¹·mg⁻¹, suggesting that the enzyme possesses a higher activity level over many known phytases at low temperatures. The phytate-degrading efficacy was tested on three common feed materials (soybean meal/rapeseed meal/corn meal) and was compared with the well-known phytases of Escherichia coli and A. niger. When using the same amount of activity units, MiPhyA could yield at least 3× more inorganic phosphate than the two reference phytases. When using the same weight of protein, MiPhyA could yield at least 5× more inorganic phosphate than the other two. Since it could degrade phytate in feed materials efficiently under low temperature and weak acidic conditions, which are common for aquacultural application, MiPhyA might be a promising candidate as a feed additive enzyme.

Purification and characterization of a protease-resistant phytase of Aspergillus oryzae SBS50 whose properties make it exceptionally useful as a feed supplement.

An extracellular phytase of Aspergillus oryzae SBS50 was purified to homogeneity using ammonium sulphate precipitation, ion-exchange and gel filtration chromatography. Purified phytase has a monomeric molecular mass of ∼80kDa exhibiting its optimal activity at pH 5.0 and 50°C with a T 1/2 of 300min at 50°C. Phytase of A. oryzae displayed broad substrate specificity with Vmax and Km values of 58.82µmol/ml/min and 1.14mM, respectively, for calcium phytate. Purity and homogeneity of the phytase was confirmed by high performance liquid chromatography and MALDI-TOF analysis revealed the identification of a peptide showing homology with acid phosphatase of Aspergillus oryzae RIB40. Among the inhibitors, 2,3-butanedione and sodium molybdate significantly inhibited the enzyme activity. Phytase of A. oryzae showed protease-resistance and was more stable during storage at 4°C and -20°C as compared to room temperature. Among all the feed samples, mustard oil cake was dephytinized more efficiently than other feed samples. These unique properties suggested that the phytase has the potential to be useful as an animal feed supplement.

Molecular advancements in the development of thermostable phytases.

Since the discovery of phytic acid in 1903 and phytase in 1907, extensive research has been carried out in the field of phytases, the phytic acid degradatory enzymes. Apart from forming backbone enzyme in the multimillion dollar-based feed industry, phytases extend a multifaceted role in animal nutrition, industries, human physiology, and agriculture. The utilization of phytases in industries is not effectively achieved most often due to the loss of its activity at high temperatures. The growing demand of thermostable phytases with high residual activity could be addressed by the combinatorial use of efficient phytase sources, protein engineering techniques, heterologous expression hosts, or thermoprotective coatings. The progress in phytase research can contribute to its economized production with a simultaneous reduction of various environmental problems such as eutrophication, greenhouse gas emission, and global warming. In the current review, we address the recent advances in the field of various natural as well as recombinant thermotolerant phytases, their significance, and the factors contributing to their thermotolerance.

Strain improvement of Pichia kudriavzevii TY13 for raised phytase production and reduced phosphate repression.

In this work, we present the development and characterization of a strain of Pichia kudriavzevii (TY1322), with highly improved phytate-degrading capacity. The mutant strain TY1322 shows a biomass-specific phytate degradation of 1.26 mmol g-1  h-1 after 8 h of cultivation in a high-phosphate medium, which is about 8 times higher compared with the wild-type strain. Strain TY1322 was able to grow at low pH (pH 2), at high temperature (46°C) and in the presence of ox bile (2% w/v), indicating this strain's ability to survive passage through the gastrointestinal tract. The purified phytase showed two pH optima, at pH 3.5 and 5.5, and one temperature optimum at 55°C. The lower pH optimum of 3.5 matches the reported pH of the pig stomach, meaning that TY1322 and/or its phytase is highly suitable for use in feed production. Furthermore, P. kudriavzevii TY1322 tolerates ethanol up to 6% (v/v) and shows high osmotic stress tolerance. Owing to the phenotypic characteristics and non-genetically modified organisms nature of TY1322, this strain show great potential for future uses in (i) cereal fermentations for increased mineral bioavailability, and (ii) feed production to increase the phosphate bioavailability for monogastric animals to reduce the need for artificial phosphate fortification.

A Novel Phytase Derived from an Acidic Peat-Soil Microbiome Showing High Stability under Acidic Plus Pepsin Conditions.

Four novel phytases of the histidine acid phosphatase family were identified in two publicly available metagenomic datasets of an acidic peat-soil microbiome in northeastern Bavaria, Germany. These enzymes have low similarity to all the reported phytases. They were overexpressed in Escherichia coli and purified. Catalytic efficacy in simulated gastric fluid was measured and compared among the four candidates. The phytase named rPhyPt4 was selected for its high activity. It is the first phytase identified from unculturable Acidobacteria. The phytase showed a longer half-life than all the gastric-stable phytases that have been reported to date, suggesting a strong resistance to low pH and pepsin. A wide pH profile was observed between pH 1.5 and 5.0. At the optimum pH (2.5) the activity was 2,790 µmol/min/mg at the physiological temperature of 37°C and 3,989 µmol/min/mg at the optimum temperature of 60°C. Due to the competent activity level as well as the high gastric stability, the phytase could be a potential candidate for practical use in livestock and poultry feeding.

Characterization of a Thermotolerant Phytase Produced by Rhizopus microsporus var. microsporus Biofilm on an Inert Support Using Sugarcane Bagasse as Carbon Source.

The Rhizopus microsporus var. microsporus biofilm was able to produce increased levels of an extracellular thermotolerant phytase using polyethylene and viscose as an inert support in both modified NBRIP medium and modified Khanna medium containing sugarcane bagasse as the carbon source. The enzyme production was strictly regulated by the phosphorus content with optimal production at 0.5 mM of sodium phytate and KH2PO4. The extracellular phytase, RMPhy1, was purified 4.18-fold with 4.78 % recovery using DEAE-cellulose and CM-cellulose. A single protein band with a molecular mass of 35.4 kDa was obtained when the samples were subjected to 10 % SDS-PAGE. The optimum temperature for activity was 55 °C and the optimum pH was 4.5. R. microsporus var. microsporus phytase exhibited high stability at 30 and 40 °C with a half-life of 115 min at 60 °C. The enzyme activity increased in the presence of Ca (2+) and was inhibited by Zn(2+), arsenate, and sodium phosphate. Phytase demonstrated high substrate specificity for sodium phytate with K m = 0.72 mM and V max = 94.55 U/mg of protein and for p-NPP with K m = 0.04 mM and V max = 106.38 U/mg of protein. The enzyme also hydrolyzed ATP, AMPc, glucose 6-phosphate, glucose 1-phosphate, and UDPG. This is the first report on phytase characterization delivered with biofilm technology. The properties of the enzyme account for its high potential for use in biotechnology and the possibility of application in different industrial sectors as feed in the future.

Cloning and Expression of Phytase appA Gene from Shigella sp. CD2 in Pichia pastoris and Comparison of Properties with Recombinant Enzyme Expressed in E. coli.

The phytase gene appAS was isolated from Shigella sp. CD2 genomic library. The 3.8 kb DNA fragment contained 1299 bp open reading frame encoding 432 amino acid protein (AppAS) with 22 amino acid signal peptide at N-terminal and three sites of N-glycosylation. AppAS contained the active site RHGXRXP and HDTN sequence motifs, which are conserved among histidine acid phosphatases. It showed maximum identity with phytase AppA of Escherichia coli and Citrobacter braakii. The appAS was expressed in Pichia pastoris and E. coli to produce recombinant phytase rAppAP and rAppAE, respectively. Purified glycosylated rAppAP and nonglycosylated rAppAE had specific activity of 967 and 2982 U mg(-1), respectively. Both had pH optima of 5.5 and temperature optima of 60°C. Compared with rAppAE, rAppAP was 13 and 17% less active at pH 3.5 and 7.5 and 11 and 18% less active at temperature 37 and 50°C, respectively; however, it was more active at higher incubation temperatures. Thermotolerance of rAppAP was 33% greater at 60°C and 24% greater at 70°C, when compared with rAppAE. Both the recombinant enzymes showed high specificity to phytate and resistance to trypsin. To our knowledge, this is the first report on cloning and expression of phytase from Shigella sp.

Characteristics of Recombinant Phytase (rSt-Phy) of the Thermophilic mold Sporotrichum thermophile and its applicability in dephytinizing foods.

Sporotrichum thermophile produces very low titres of phytase (St-Phy) extracellularly, which is acidstable, thermostable, and protease insensitive with broad substrate specificity, and therefore, the gene encoding phytase (St-Phy) has been cloned and expressed in E. coli. The purified recombinant phytase (rSt-Phy) has the molecular mass of 55 kDa with Km and Vmax (calcium phytate), kcat and kcat/Km of 0.143 mM, 185.05 nmoles mg(-1)  s(-1), 5.1 × 10(3) s(-1), and 3.5 × 10(7) M(-1) s(-1), respectively. Mg(2+) and Ba(2+) display slight stimulatory effect on the enzyme, while it is inhibited by other ions to a varied extent. The enzyme is also inhibited by chaotropic agents (guanidinium hydrochloride, potassium iodide, and urea), Woodward's reagent K, and 2,3-butanedione but resistant to both pepsin and trypsin. The rSt-Phy is useful in dephytinization of tandoori and naan (unleavened flat Indian breads), and bread, liberating soluble inorganic phosphate that mitigates anti-nutrient effects of phytic acid.

Purification, sequencing and evaluation of a divergent phytase from Penicillium oxalicum KCTC6440.

A fungal strain producing high levels of phytase was purified to homogeneity from Penicillium oxalicum KCTC6440 (PhyA). The molecular mass of the purified PhyA was 65 kDa and optimal activity occurred at 55°C. The enzyme was stable in a pH range of 4.5-6.5, with an optimum performance at pH 5.5. The Km value for the substrate sodium phytate was 0.48 mM with a Vmax of 672 U/mg. The enzyme was inhibited by Ca(2+), Cu(2+), and Zn(2+), and slightly enhanced by EDTA. The PhyA efficiently released phosphate from feedstuffs such as soybean, rich bran and corn meal. The PhyA gene was cloned in two steps of degenerate PCR and inverse PCR and found to comprise 1501 bp and encode 461 amino acid residues. The enzyme was found to have only 13 amino acids differing to the known PhyA from other Penicillium sp., but has distinct enzyme characteristics. Computational analysis showed that PhyA possessed more positively charged residues in the active sites compared to other PhyA molecules, which may explain the broader pH spectrum.

Isolation of a thermostable acid phytase from Aspergillus niger UFV-1 with strong proteolysis resistance.

An Aspergillus niger UFV-1 phytase was characterized and made available for industrial application. The enzyme was purified via ultrafiltration followed by acid precipitation, ion exchange and gel filtration chromatography. This protein exhibited a molecular mass of 161 kDa in gel filtration and 81 kDa in sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), indicating that it may be a dimer. It presented an optimum temperature of 60 °C and optimum pH of 2.0. The K M for sodium phytate hydrolysis was 30.9 mM, while the k cat and k cat / K M were 1.46 ×10 (5) s (-1) and 4.7 × 10 (6) s (-1) .M (-1) , respectively. The purified phytase exhibited broad specificity on a range of phosphorylated compounds, presenting activity on sodium phytate, p-NPP, 2- naphthylphosphate, 1- naphthylphosphate, ATP, phenyl-phosphate, glucose-6-phosphate, calcium phytate and other substrates. Enzymatic activity was slightly inhibited by Mg (2+) , Cd (2+) , K (+) and Ca (2+) , and it was drastically inhibited by F (-) . The enzyme displayed high thermostability, retaining more than 90% activity at 60 °C during 120 h and displayed a t 1/2 of 94.5 h and 6.2 h at 70 °C and 80 °C, respectively. The enzyme demonstrated strong resistance toward pepsin and trypsin, and it retained more than 90% residual activity for both enzymes after 1 h treatment. Additionally, the enzyme efficiently hydrolyzed phytate in livestock feed, liberating 15.3 µmol phosphate/mL after 2.5 h of treatment.