Alkaline Phytase Research Articles

The Enzyme Activity of Dual-Domain β-Propeller Alkaline Phytase as a Potential Factor in Improving Soil Phosphorus Fertility and Triticum aestivum Growth

The widespread use of non-renewable phosphate fertilizers in agriculture poses a significant pollution threat to soil, necessitating the exploration of sustainable alternatives for phosphate fertility. Releasing phytate phosphorus through microbial phytases presents an eco-friendly solution for sustainable phosphate fertility in agriculture. This study directly inoculated dual-domain β-propeller alkaline phytase (phyHT) derived from Bacillus sp. HJB17 into the soil. The study analyzed the impact of inoculated phyHT on the physicochemical properties of the soil, assessed the variations in enzyme activity of phyHT within the soil, and examined the effects of the treated soil on wheat growth. Additionally, the study explored the enhancement of the available phosphorus in the soil through the inoculation of phyHT in both crop residues and organic fertilizer. PhyHT exhibited the highest catalytic activity at 37 °C and pH 8.0. After soil adsorption, phyHT maintained stable enzymatic activity. PhyHT markedly boosted the available phosphorus in the soil while reducing the soil phytate content by about 20%, increasing the phosphorus levels and enhancing soil fertility. PhyHT effectively degraded phytates in an organic fertilizer and crop residues, increasing the available phosphorus. PhyHT supplementation enhanced growth, biomass, and phosphorus content in both the shoot and root weights of Triticum aestivum. This study establishes phyHT as a viable and eco-friendly method to enhance phosphorus fertility in soil. The direct application of microbial phytases can serve as a sustainable source of phosphate fertility in soil.

Cloning of Bacillus subtilis phytase gene construct in Escherichia coli.

Background and Objectives:Phytase has a hydrolysis function of phytic acid, which yields inorganic phosphate. Bacillus species can produce thermostable alkaline phytase. The aim of this study was to isolate and clone a Phytase gene (Phy) from Bacillus subtilis in Escherichia coli.Materials and Methods:In this study, the extracellular PhyC gene was isolated from Bacillus subtilis Phytase C. After purification of the bands, DNA fragment of Phy gene was cloned by T/A cloning technique, and the clone was transformed into Escherichia coli. Afterward, the pGEM-Phy was transferred into E. coli Top-10 strain and the recombinants were plated on LB agar containing 100 μg/ml ampicillin. The colonization of 1171 bp of gene Phytase C was confirmed by PCR. The presence of gene-targeting in vector was confirmed with enzymatic digestion by XhoI and XbaI restriction enzymes.Results:The Phytase gene was successfully cloned in E. coli. The result of cloning of 1171 bp Phytase gene was confirmed by PCR assay.Conclusion:Our impression of this article is that several methods, such as using along with microbial, plant phytase reproduction, or low-phytic acid corn may be the better way from a single phytase.

A dansyl-derivatized phytic acid analogue as a fluorescent substrate for phytases: experimental and computational approach

A new myo-inositol pentakisphosphate was synthesized, which featured a dansyl group at position C-5. The fluorescent tag was removed from the inositol by a 6-atom spacer to prevent detrimental steric interactions in the catalytic site of phytases. The PEG linker was used in order to enhance hydrophilicity and biocompatibility of the new artificial substrate. Computational studies showed a favorable positioning in the catalytic site of phytases. Enzymatic assays demonstrated that the tethered myo-inositol was processed by two recombinant phytases Phy-A and Phy-C, classified respectively as acid and alkaline phytases, with similar rates of phosphate release compared to their natural substrate.

Purification and characterization of an alkalistable phytase produced by Rhizopus microsporus var. microsporus in submerged fermentation

Abstract The filamentous fungus Rhizopus microsporus var. microsporus produces an alkaline phytase in submerged fermentation. This alkaline phytase was purified 83-fold with 1.9% protein yield. The molecular mass of the glycoprotein was estimated in 12% SDS-PAGE as 55 kDa and 63 kDa as revealed by gel filtration, indicating a monomeric structure for the enzyme. The alkaline phytase was optimally active at 65 °C and pH 9.5, with a half-life (T1/2) of 280 min at 50 °C. At 80 °C, the enzyme maintained 87% of its initial activity for 280 min. The phytase activity was characterized as alkalistable and this activity was stimulated when maintained at pH 8.0 and 9.0. It was inhibited mainly by KH2PO4, β-mercaptoethanol, EDTA, HgCl2, and urea. The alkaline phytase activity was also inhibited by detergents (Triton X-100, Tween 20, and SDS) and solvents (acetone, acetonitrile, ethanol, isopropanol, methanol, and n-butanol). The alkaline phytase showed a preference for sodium phytate as substrate (100%), followed by ATP (27.9%) and p-NPP (11.5%). The Km value for sodium phytate was 0.413 mmol L−1. This study represents the first description of the purification and characterization of a fungal alkaline phytase.

Structure Prediction and Enzymatic Properties of Phytase phyS

Phytase is a kind of enzyme that hydrolyzes phytic acid and its salts to produce inositol and phosphoric acid. As a new feed additive, phytase has great potential in animal nutrition and environmental protection. Because of its good stability, large-scale production and high activity, microbial phytase has become a hot spot in industrial application. Here, we reported the predicted structure and enzymatic properties of a phytase from Bacillus subtilis, which was named as phyS. It was clear that the optimal temperature is 35°C, and the optimal pH is 8. Meanwhile, the enzyme activity was kept at above 90% in the range of pH 8 - 9, this result demonstrated that phyS is an alkaline phytase. This study lays a foundation for the extensive application of phyS.

New Bacterial Phytase through Metagenomic Prospection.

Alkaline phytases from uncultured microorganisms, which hydrolyze phytate to less phosphorylated myo-inositols and inorganic phosphate, have great potential as additives in agricultural industry. The development of metagenomics has stemmed from the ineluctable evidence that as-yet-uncultured microorganisms represent the vast majority of organisms in most environments on earth. In this study, a gene encoding a phytase was cloned from red rice crop residues and castor bean cake using a metagenomics strategy. The amino acid identity between this gene and its closest published counterparts is lower than 60%. The phytase was named PhyRC001 and was biochemically characterized. This recombinant protein showed activity on sodium phytate, indicating that PhyRC001 is a hydrolase enzyme. The enzymatic activity was optimal at a pH of 7.0 and at a temperature of 35 °C. β-propeller phytases possess great potential as feed additives because they are the only type of phytase with high activity at neutral pH. Therefore, to explore and exploit the underlying mechanism for β-propeller phytase functions could be of great benefit to biotechnology.

Comparative Study on Different Expression Hosts for Alkaline Phytase Engineered in Escherichia coli.

The application of alkaline phytase as a feed additive is restricted by the poor specific activity. Escherichia coli is a frequently used host for directed evolution of proteins including alkaline phytase towards improved activity. However, it is not suitable for production of food-grade products due to potential pathogenicity. To combine the advantages of different expression systems, mutants of the alkaline phytase originated from Bacillus subtilis 168 (phy168) were first generated via directed evolution in E. coli and then transformed to food-grade hosts B. subtilis and Pichia pastoris for secretory expression. In order to investigate the suitability of different expression systems, the phy168 mutants expressed in different hosts were characterized and compared in terms of specific activity, pH profile, pH stability, temperature profile, and thermostability. The specific activity of B. subtilis-expressed D24G/K70R/K111E/N121S mutant at pH 7.0 and 60°C was 30.4U/mg, obviously higher than those in P. pastoris (22.7U/mg) and E. coli (19.7U/mg). Moreover, after 10min incubation at 80°C, the B. subtilis-expressed D24G/K70R/K111E/N121S retained about 70% of the activity at pH 7.0 and 37°C, whereas the values were only about 25 and 50% when expressed in P. pastoris and E. coli, respectively. These results suggested B. subtilis as an appropriate host for expression of phy168 mutants and that the strategy of creating mutants in one host and expressing them in another might be a new solution to industrial production of proteins with desired properties.

Extracellular expression of alkaline phytase in Pichia pastoris: Influence of signal peptides, promoters and growth medium

Alkaline phytase isolated from pollen grains of Lilium longiflorum (LlALP) possesses unique catalytic and thermal stability properties that suggest it has the potential to be used as a feed supplement. However, substantial amounts of active enzymes are needed for animal feed studies and endogenous levels of LlALP in lily pollen are too low to provide the required amounts. Active rLlALP2 (coded by LlAlp2, one of two isoforms of alkaline phytase cDNA identified in lily pollen) has been successfully expressed in intracellular compartments of Pichia pastoris, however enzyme yields have been modest (25–30mg/L) and purification of the enzyme has been challenging. Expression of foreign proteins to the extracellular medium of P. pastoris greatly simplifies protein purification because low levels of endogenous proteins are secreted by the yeast. In this paper, we first describe the generation of P. pastoris strains that will secrete rLlALP2 to the extracellular medium. Data presented here indicates that deletion of native signal peptides at the N- and C-termini of rLlALP2 enhanced α-mating factor (α-MF)-driven secretion by four-fold; chicken egg white lysozyme signal peptide was ineffective in the extracellular secretion of rLlALP2. Second, we describe our efforts to increase expression levels by employing a constitutive promoter from the glyceraldehyde-3-phosphate dehydrogenase gene (PGAP) in place of the strong, tightly controlled promoter of alcohol oxidase 1 gene (PAOX1). PGAP enhanced the extracellular expression levels of rLlALP2 compared to PAOX1. Finally, we report on the optimization of the culture medium to enhance yields of rLlALP2. The strength of PGAP varies depending on the carbon source available for cell growth; secreted expression of rLlALP2 was highest when glycerol was the carbon source. The addition of histidine and Triton X-100 also enhanced extracellular expression. Taken together, the employment of PGAP under optimized culture conditions resulted in approximately eight-fold (75–80mg/L) increase in extracellular activity compared to PAOXI (8–10mg/L). The P. pastoris expression system can be employed as a source of active alkaline phytase for animal feed studies.

Enhanced activity of an alkaline phytase from Bacillus subtilis 168 in acidic and neutral environments by directed evolution

As a feed additive, the desirable features of phytase include thermostability, strict substrate specificity, and high stability in a wide pH range. Alkaline phytases from Bacillus subtilis are promising candidates because of their high inherent thermostability and substrate specificity, but the application is restricted due to their poor specific activity in acidic and neutral conditions. Directed evolution was utilized in this paper to improve the activity of phytase from B. subtilis 168 in neutral and acidic environments. After two rounds of evolution, a number of positive mutants were picked out from the mutant library. Compared with the wild type, variants D24G, S51A, D24G/K265N, D24G/K70R/K111E/N121S showed 29.7%, 13.5%, 42.7%, 42.8% improvement in specific activity under the optimal reaction conditions (pH 7.0, 60°C) and 76.6%, 79.5%, 84.2%, 121.1% improvement at pH 4.5 and 37°C, respectively. The catalytic efficiency (kcat/Km) of D24G, D24G/K265N, D24G/K70R/K111E/N121S at pH 7.0 was increased by 132%, 110%, 131% as compared with that of the wild type, and the improvement at pH 4.5 was 98.0%, 114.5%, 163.3%, respectively. Molecular docking results revealed that D24, S51 and K70 had a relatively large influence on the catalytic activity probably because they are adjacent to the active site cleft.

Improving mineral availability in soymilk by dephosphorylation of phytic acid using an alkaline phytase from Bacillus amyloliquefaciens DS11

To enhance mineral availability in soymilk, the alkaline phytase from Bacillus amyloliquefaciens DS11 was applied to degrade the phytic acid in soymilk, and resulting effects on mineral profiles were assessed. Nearly 60% of phytic acid was degraded within 25 min by treatment with 0.090 unit/mg of phytate, whereas most phytic acid was removed in 125 min using 0.18 unit/mg of phytate. Compared to a control, the free calcium, magnesium, phosphorus, and iron contents significantly (p<0.05) increased by 1.9, 1.8, 4.0, and 4.0×, respectively, after 125 min of reaction using 0.18 unit/mg of phytate. Free minerals were released from the phytate complex by enzymatic dephosphorylation, resulting in enhancement of mineral availability in soymilk. Alkaline phytase DS11 has a great potential for enhancing the mineral availability in neutral pH phytate-rich foods, like soymilk.

Cloning and Expression of a Novel Phytase Gene (phyMS) from &amp;lt;em&amp;gt;Mycobacterium smegmatis&amp;lt;/em&amp;gt;

Phytase, also known as phytate-degrading enzyme, catalyzes the hydrolysis of phytate (inositol hexakisphosphate) with sequential release of phosphate and lower inositol phosphate. We report here a new plasmid construct designated as pMSuia from pBAD-TOPO that harbors a 1.1 kb phytase gene (phyMS) from Mycobacterium smegmatis, and expression as well as characterization of the purified recombinant M. smegmatis phytase. DNA sequencing analysis and multiple alignment exercise indicated that the M. smegmatis phytase is different from both known acid and alkaline phytases. The active ~45 kDa recombinant enzyme was expressed and confirmed by enzyme assay and Western blot analyses. Ni-NTA affinity purified recombinant M. smegmatis phytase exhibited specific activity of 233.51 U/mg, optimal pH of 3 and 7 and optimal temperature of 60°C. The purified enzyme retains almost 30% of the initial activity after incubation at 90°C for 60 min. The enzyme showed broad substrate specificity with Km and Vmax of the recombinant enzyme for sodium phytate substrate of 0.105 ± 0.016 mM and 26.93 ± 1.21 mM min-1, respectively.

Effect of metals ions on thermostable alkaline phytase from Bacillus subtilis YCJS isolated from soybean rhizosphere soil

A Bacillus sp.YCJS strain showing phytase activity was isolated, and the phytase-encoding gene was cloned and expressed in Escherichia coli. The 1,149-bp full-length gene encoded a 26-residue putative signal peptide and a 356-residue mature protein. The molecular weight was estimated to be 47.5 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified recombinant enzyme phy(ycE) from E. coli exhibited a specific activity of 14 U mg-1 protein. The optimum pH and temperature were 6.0 and 50 °C, respectively. The thermal stability of phy(ycE) was drastically improved in the presence of calcium ions (Ca2+). Fluorescence analysis results indicated that compared with phy(ycE) without added Ca2+, phy(ycE) in the presence of Ca2+ was more stable and the melting temperature improved from 47.8 to 62.4 °C. Circular dichroism spectrometric analysis revealed that the loss of enzymatic activity was most likely due to a conformational change, as the circular dichroism spectra of the holoenzyme and metal-depleted enzyme were significantly different. Compared with the Ca2+-reactivated enzyme, the La3+-reactivated enzyme did not undergo a significant recovery with respect to its conformation. The aromatic-sensitized terbium (Tb3+) fluorescence results indicated that five Tb3+ could bind to each molecule of phy(ycE) and that there were two high-affinity and three low-affinity binding sites.

Enhancement of alkaline phytase production in Pichia pastoris: Influence of gene dosage, sequence optimization and expression temperature

Supplementation of animal feed with phytases has proven to be an effective strategy to alleviate phosphorous contamination of soil and water bodies. The inability of non-ruminant animals to digest phytates in corn and soybeans contributes to environmental contamination. Alkaline phytase from lily pollen (LlALP) exhibits unique catalytic and thermal stability properties that could be useful as a feed supplement. rLlALP2 was successfully expressed in Pichia pastoris; however, enzyme yields were modest (8–10mg/L). In this paper, we describe our efforts to enhance rLlALP2 yield by investigating the influence of the following potential limiting factors: transgene copy number, codon bias, sequence optimization, and temperature during expression. Data presented indicate that increasing rLlAlp2 copy number was detrimental to heterologous expression, clones with one copy of wt-rLlAlp2 produced the highest activity, clones with two, four and seven or more copies produced 70%, 25% and 10% respectively, of enzyme activity implying that gene dosage is not limiting rLlALP2 yield. Use of a sequence-optimized rLlAlp2 increased the yield of the active enzyme by 25–50% in one/two copy clones, suggesting that translational efficiency is not a major bottleneck for rLlALP2 expression. Reducing the temperature during heterologous expression led to increases of 1.2–20-fold suggesting that protein folding and post-translational processes may be the dominant factors limiting rLlALP2 expression. Early knowledge of the transgene copy number allowed us to develop a more rational strategy for yield enhancement. Cumulatively, sequence optimization and temperature reduction led to the doubling of rLlALP2 enzyme activity in P. pastoris.

Supplementation of Alkaline Phytase (Ds11) in Whole‐Wheat Bread Reduces Phytate Content and Improves Mineral Solubility

In this study, alkaline phytase was added to whole-wheat bread and the phytate content and mineral profiles were compared to commercially available acidic phytase. At neutral pH, some phytate (approximately 20%) was degraded by endogenous phytase in wheat flour, while 40% of phytate was hydrolyzed by alkaline phytase DS11 and a 35% reduction was observed with acidic phytase. Most of the enzymatic activity occurred during the proofing stage, and the rate of reaction depended on pH. DS11 phytase effectively degraded the phytate level within a 30 min treatment at pH 7; however, at least 60 min was needed with acidic phytase to achieve the same hydrolysis level. Mineral profiles were also dramatically affected by the phytate reduction. The biggest increase was observed in Fe²⁺ by the phytase treatment. The Fe²⁺ content increased 10-fold at pH 7 and 8-fold at pH 5 with alkaline phytase DS11. Alkaline phytase DS11 was shown to be effective at phytate reduction in whole-wheat bread preparation. Additionally, phytate degradation enhanced the mineral availability of bread.

Site-directed mutagenesis of an alkaline phytase: Influencing specificity, activity and stability in acidic milieu

Site-directed mutagenesis of a thermostable alkaline phytase from Bacillus sp. MD2 was performed with an aim to increase its specific activity and activity and stability in an acidic environment. The mutation sites are distributed on the catalytic surface of the enzyme (P257R, E180N, E229V and S283R) and in the active site (K77R, K179R and E227S). Selection of the residues was based on the idea that acid active phytases are more positively charged around their catalytic surfaces. Thus, a decrease in the content of negatively charged residues or an increase in the positive charges in the catalytic region of an alkaline phytase was assumed to influence the enzyme activity and stability at low pH. Moreover, widening of the substrate-binding pocket is expected to improve the hydrolysis of substrates that are not efficiently hydrolysed by wild type alkaline phytase. Analysis of the phytase variants revealed that E229V and S283R mutants increased the specific activity by about 19% and 13%, respectively. Mutation of the active site residues K77R and K179R led to severe reduction in the specific activity of the enzyme. Analysis of the phytase mutant–phytate complexes revealed increase in hydrogen bonding between the enzyme and the substrate, which might retard the release of the product, resulting in decreased activity. On the other hand, the double mutant (K77R–K179R) phytase showed higher stability at low pH (pH 2.6–3.0). The E227S variant was optimally active at pH 5.5 (in contrast to the wild type enzyme that had an optimum pH of 6) and it exhibited higher stability in acidic condition. This mutant phytase, displayed over 80% of its initial activity after 3 h incubation at pH 2.6 while the wild type phytase retained only about 40% of its original activity. Moreover, the relative activity of this mutant phytase on calcium phytate, sodium pyrophosphate and p-nitro phenyl phosphate was higher than that of the wild type phytase.

Thermostable alkaline phytase from Bacillus sp. MD2: Effect of divalent metals on activity and stability

Phytate, the major source of phosphorus in seeds, exists as a complex with different metal ions. Alkaline phytases are known to dephosphorylate phytate complexed with calcium ions in contrast to acid phytases that act only on phytic acid. A recombinant alkaline phytase from Bacillus sp. MD2 has been purified and characterized with respect to the effect of divalent metal ions on the enzyme activity and stability. The presence of Ca 2+ on both the enzyme and the substrate is required for optimal activity and stability. Replacing Ca 2+ with Ba 2+, Mn 2+, Mg 2+ and Sr 2+ in the phytase resulted in the expression of > 90% of the maximal activity with calcium-phytate as the substrate, while Fe 2+ and Zn 2+ rendered the enzyme inactive. On the other hand, the calcium loaded phytase showed significant activity (60%) with sodium phytate and lower activity (17–20%) with phytate complexed with only Mg 2+, Sn 2+ and Sr 2+, respectively. On replacing Ca 2+ on both the enzyme and the substrate with other metal ions, about 20% of the maximal phytase activity was obtained only with Mg 2+ and Sr 2+, respectively. Only Ca 2+ resulted in a marked increase in the melting temperature (T m) of the enzyme by 12–21 °C, while Ba 2+, Mn 2+, Sr 2+ or Cu 2+ resulted in a modest (2–3.5 °C) increase in T m. In the presence of 1–5 mM Ca 2+, the optimum temperature of the phytase activity was increased from 40 °C to 70 °C, while optimum pH of the enzyme shifted by 0.4–1 pH unit towards the acidic region.

Crystal Structures of Bacillus Alkaline Phytase in Complex with Divalent Metal ions and Inositol Hexasulfate

Alkaline phytases from Bacillus species, which hydrolyze phytate to less phosphorylated myo-inositols and inorganic phosphate, have great potential as additives to animal feed. The thermostability and neutral optimum pH of Bacillus phytase are attributed largely to the presence of calcium ions. Nonetheless, no report has demonstrated directly how the metal ions coordinate phytase and its substrate to facilitate the catalytic reaction. In this study, the interactions between a phytate analog (myo-inositol hexasulfate) and divalent metal ions in Bacillus subtilis phytase were revealed by the crystal structure at 1.25 Å resolution. We found all, except the first, sulfates on the substrate analog have direct or indirect interactions with amino acid residues in the enzyme active site. The structures also unraveled two active site-associated metal ions that were not explored in earlier studies. Significantly, one metal ion could be crucial to substrate binding. In addition, binding of the fourth sulfate of the substrate analog to the active site appears to be stronger than that of the others. These results indicate that alkaline phytase starts by cleaving the fourth phosphate, instead of the third or the sixth that were proposed earlier. Our high-resolution, structural representation of Bacillus phytase in complex with a substrate analog and divalent metal ions provides new insight into the catalytic mechanism of alkaline phytases in general.

Characterization of a thermostable alkaline phytase from Bacillus licheniformis ZJ-6 in Pichia pastoris

A novel neutral phytase gene (phyC) from Bacillus licheniformis was cloned and expressed in Pichia pastoris under the control of AOX1 promoter. The gene is 1,146 bp in size and encodes a polypeptide of 381 amino acids. The recombinant PhyCm (rePhyCm), driven by the Saccharomyces cerevisiae α-mating factor, was secreted into culture medium. After 0.5% methanol induction for 96 h, the activity of rePhyCm in culture supernatant reached 0.23 U/ml. The optimum temperature and pH of purified rePhyCm were 60°C and 7.5, respectively. The rePhyCm was stable in a wide pH range of 5.0–9.0, especially for alkaline pH. The residual activities of rePhyCm retained over 80% after being incubated at pH 5.0–9.0, 37°C for 1 h in the presence of 1 mM CaCl2. Interestingly, supplemental Ca2+ upgraded both the thermostability and pH stability of rePhyCm. Substrate specificity of rePhyCm, effects of metal ions and chemicals on phytase activity were also investigated in current study.

Heterologous expression and functional characterization of a plant alkaline phytase in Pichia pastoris

Phytases catalyze the sequential hydrolysis of phytic acid ( myo-insositol hexakisphosphate), the most abundant inositol phosphate in cells. Phytic acid constitutes 3–5% of the dry weight of cereal grains and legumes such as corn and soybean. The high concentration of phytates in animal feed and the inability of non-ruminant animals such as swine and poultry to digest phytates leads to phosphate contamination of soil and water bodies. The supplementation of animal feed with phytases results in increased bioavailability to animals and decreased environmental contamination. Therefore, phytases are of great commercial importance. Phytases with a range of properties are needed to address the specific digestive needs of different animals. Alkaline phytase (LlALP1 and LlALP2) which possess unique catalytic properties that have the potential to be useful as feed and food supplement has been identified in lily pollen. Substantial quantities of alkaline phytase are needed for animal feed studies. In this paper, we report the heterologous expression of LlALP2 from lily pollen in Pichia pastoris. The expression of recombinant LlALP2 (rLlALP2) was optimized by varying the cDNA coding for LlALP2, host strain and growth conditions. The catalytic properties of recombinant LlALP2 were investigated extensively (substrate specificity, pH- and temperature dependence, and the effect of Ca 2+, EDTA and inhibitors) and found to be very similar to that of the native LlALP2 indicating that rLlALP2 from P. pastoris can serve as a potential source for structural and animal feed studies.

Production and characterization of thermostable alkaline phytase from Bacillus laevolacticus isolated from rhizosphere soil

A novel phytase producing thermophilic strain of Bacillus laevolacticus insensitive to inorganic phosphate was isolated from the rhizosphere soil of leguminous plant methi (Medicago falacata). The culture conditions for production of phytase by B. laevolacticus under shake flask culture were optimized to obtain high levels of phytase (2.957 +/- 0.002 U/ml). The partially purified phytase from B. laevolacticus strain was optimally active at 70 degrees C and between pH 7.0 and pH 8.0. The enzyme exhibited thermostability with approximately 80% activity at 70 degrees C and pH 8.0 for up to 3 h in the presence/absence of 5 mM CaCl(2). The phytase from B. laevolacticus showed high specificity for phytate salts of Ca(+) > Na(+). The enzyme showed an apparent K (m) 0.526 mM and V (max) 12.3 mumole/min/mg of activity against sodium phytate.