Production, purification and characterization of thermostable phytase from thermophilic fungus Thermomyces lanuginosus TL-7

Ten different strains of Thermomyces lanuginosus, isolated from composting soils were found to produce phytase when grown on PSM medium. The wild type strain CM was found to produce maximum amount ofphytase (4.33 units/g DW substrate). Culturing T. lanuginosus strain CM on medium containing wheat bran and optimizing other culture conditions (carbon source, media type, nitrogen source, level of nitrogen, temperature, pH, inoculum age, inoculum level and moisture), increased the phytase yield to 13.26 units/g substrate. This culture was further subjected to UV mutagenesis for developing phytase hyperproducing mutants. The mutant (TL-7) showed 2.29-fold increase in phytase activity as compared to the parental strain. Employing Box-Behnken factor factorial design of response surface methodology resulted in optimized phytase production (32.19 units/g of substrate) by mutant TL-7. A simple two-step purification (40.75-folds) ofphytase from mutant TL-7 was achieved by anion exchange and gel filtration chromatography. The purified phytase (approximately 54 kDa) was characterized to be optimally active at pH 5.0 and temperature 70 degrees C, though the enzyme showed approximately 70% activity over a wide pH and temperature range (2.0-10.0 and 30-90 degrees C, respectively). The phytase showed broad substrate specificity with activity against sodium phytate, ADP and riboflavin phosphate. The phytase from T. lanuginosus was thermoacidstable as it showed up to 70% residual activity after exposure to 70 degrees C at pH 3.0 for 120 min. The enzyme showed Km 4.55 microM and Vmax 0.833 microM/min/mg against sodium phytate as substrate.     

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

Abstract

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 K_m and V_max 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.     

Utilisation of phytate phosphorus by rumen bacteria in a semi-continuous culture system (Rusitec) in lactating goats fed on different forage to concentrate ratios

Experimental data on phytate phosphorus utilisation by ruminants are scarce. The aim of this study was to estimate the phytase activity of rumen micro-organisms when phytate phosphorus supply is high. A semi-continuous culture system fermentor (RUSITEC) was used. The inoculum was obtained from eight goats fed on either high or low forage level diets. Experimental buffers only differed by the nature of phosphorus monosodium phosphate vs. corn sodium phytate. The nylon bags containing 15 g DM of substrate were removed after a 48-hour incubation period. The system was maintained for 15 days: 5 days for adaptation, in order to obtain a steady state, and 10 days for sampling and recording. No significant differences were observed for DM digestibility, gas production, pH, N-NH3, and SCFA for the different treatments. Bacterial efficiency of phytate phosphorus utilisation was significantly higher (p < 0.001) with organic P, but remained lower than the data usually reported in the literature. These results may be explained by the relative saturation of bacterial phytase activity when the buffer contains a high level of phytate phosphorus.     

Biocatalytic potential of protease-resistant phytase of Aspergillus oryzae SBS50 in ameliorating food nutrition

Production of protease-resistant phytase by Aspergillus oryzae SBS50 was optimized in solid state fermentation using wheat bran as substrate. An integrated statistical optimization approach involving the Placket–Burman design followed by response surface methodology was employed. Among all the variables tested, incubation period, triton X-100, moisture ratio, and magnesium sulphate were identified as significant and further optimized using response surface methodology that resulted in 3.35-fold improvement in phytase production from 55.43 to 185.75 U/g dry mouldy bran (DMB). Optimal conditions for maximum phytase production (185.75 U/g DMB) included wheat bran 10 g per 250 ml flask moistened with 35 ml distilled water supplemented with 3.0% triton X-100, 0.04% magnesium sulphate, 1.0% sucrose and 0.5% yeast extract incubated at 30?°C for an incubation time of 48 h. Phytase titers were sustainable (179.55 to 185.75 U/g DMB), when the mould was grown in shake flasks of varied volumes and enamel-coated metallic trays under optimized conditions. Fermentation time was reduced to half from 96 h to 48 h after optimization resulting in a 6.7-fold enhancement in the phytase productivity from 577.39 to 3868.75 U/Kg/h and thus, reducing the cost of enzyme production. Phytase released inorganic phosphate, reducing sugars and soluble proteins from different food samples in a time dependent manner as a result of phytate hydrolysis.     

Effect of phytase supplementation on phosphorus digestibility in low-phytate barley fed to finishing pigs

Forty crossbred barrows (Camborough 15 Line female x Canabred sire) weighing an average of 79.6 +/- 8.0 kg were used in a factorial design experiment (5 barleys x 2 enzyme levels) conducted to determine the effects of phytase supplementation on nutrient digestibility in low-phytate barleys fed to finishing pigs. The pigs were assigned to one of 10 dietary treatments comprised of a normal 2-rowed, hulled variety of barley (CDC Fleet, 0.26% phytate) or 2 low-phytate hulled genotypes designated as LP422 (0.14% phytate) and LP635 (0.09% phytate). A normal, hulless barley (CDC Dawn, 0.26% phytate) and a hulless genotype designated as LP422H (0.14% phytate) were also included. All barleys were fed with and without phytase (Natuphos 5000 FTU/kg). The diets fed contained 98% barley, 0.5% vitamin premix, 0.5% trace mineral premix, 0.5% NaCl and 0.5% chromic oxide but no supplemental phosphorus. The marked feed was provided for a 7-day acclimatization period, followed by a 3-day faecal collection. In the absence of phytase, phosphorus digestibility increased substantially (P < 0.05) as the level of phytate in the barley declined. For the hulled varieties, phosphorus digestibility increased from 12.9% for the normal barley (0.26% phytate) to 35.3 and 39.8% for the two low-phytate genotypes (0.14 and 0.09% phytate respectively). For the hulless varieties, phosphorus digestibility increased from 9.2% for the normal barley (0.26% phytate) to 34.7% for the hulless variety with 54% of the normal level of phytate (0.14% phytate). In contrast, when phytase was added to the diet, there was little difference in phosphorus digestibility between pigs fed normal barley and those fed the low-phytate genotypes (significant barley x enzyme interaction, P = 0.01). For the hulled varieties, phosphorus digestibility was 50.1% for the barley with the normal level of phytate (0.26% phytate) compared with 51.1 and 52.4% for the varieties with 54 and 35% of the normal level of phytate (0.14 and 0.09% phytate respectively). For the hulless varieties, phosphorus digestibility increased from 47.1% for the normal barley (0.26% phytate) to 54.4% for the hulless variety with 54% of the normal level of phytate (0.14% phytate). In conclusion, both supplementation with phytase and selection for low-phytate genotypes of barley were successful in increasing the digestibility of phosphorus for pigs. Unfortunately, the effects did not appear to be additive. Whether or not swine producers will choose low-phytate barley or supplementation with phytase as a means to improve phosphorus utilization, will likely depend on the yield potential of low-phytate barley and the additional costs associated with supplementation with phytase.     

Production and characterization of thermostable alkaline phytase from <Emphasis Type="Italic">Bacillus laevolacticus</Emphasis> 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 °C and between pH 7.0 and pH 8.0. The enzyme exhibited thermostability with ∼80% activity at 70 °C and pH 8.0 for up to 3 h in the presence/absence of 5 mM CaCl₂. 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 μmole/min/mg of activity against sodium phytate.     

Effect of phytase supplementation on phosphorus digestibility in low-phytate barley fed to finishing pigs

Forty crossbred barrows (Camborough 15 Line female×Canabred sire) weighing an average of 79.6±8.0?kg were used in a factorial design experiment (5 barleys×2 enzyme levels) conducted to determine the effects of phytase supplementation on nutrient digestibility in low-phytate barleys fed to finishing pigs. The pigs were assigned to one of 10 dietary treatments comprised of a normal 2-rowed, hulled variety of barley (CDC Fleet, 0.26% phytate) or 2 low-phytate hulled genotypes designated as LP422 (0.14% phytate) and LP635 (0.09% phytate). A normal, hulless barley (CDC Dawn, 0.26% phytate) and a hulless genotype designated as LP422H (0.14% phytate) were also included. All barleys were fed with and without phytase (Natuphos 5000 FTU/kg). The diets fed contained 98% barley, 0.5% vitamin premix, 0.5% trace mineral premix, 0.5% NaCl and 0.5% chromic oxide but no supplemental phosphorus. The marked feed was provided for a 7-day acclimatization period, followed by a 3-day faecal collection. In the absence of phytase, phosphorus digestibility increased substantially (P<0.05) as the level of phytate in the barley declined. For the hulled varieties, phosphorus digestibility increased from 12.9% for the normal barley (0.26% phytate) to 35.3 and 39.8% for the two low-phytate genotypes (0.14 and 0.09% phytate respectively). For the hulless varieties, phosphorus digestibility increased from 9.2% for the normal barley (0.26% phytate) to 34.7% for the hulless variety with 54% of the normal level of phytate (0.14% phytate). In contrast, when phytase was added to the diet, there was little difference in phosphorus digestibility between pigs fed normal barley and those fed the low-phytate genotypes (significant barley×enzyme interaction, P=0.01). For the hulled varieties, phosphorus digestibility was 50.1% for the barley with the normal level of phytate (0.26% phytate) compared with 51.1 and 52.4% for the varieties with 54 and 35% of the normal level of phytate (0.14 and 0.09% phytate respectively). For the hulless varieties, phosphorus digestibility increased from 47.1% for the normal barley (0.26% phytate) to 54.4% for the hulless variety with 54% of the normal level of phytate (0.14% phytate). In conclusion, both supplementation with phytase and selection for low-phytate genotypes of barley were successful in increasing the digestibility of phosphorus for pigs. Unfortunately, the effects did not appear to be additive. Whether or not swine producers will choose low-phytate barley or supplementation with phytase as a means to improve phosphorus utilization, will likely depend on the yield potential of low-phytate barley and the additional costs associated with supplementation with phytase.     

Optimization of cold-active protease production by the psychrophilic bacterium Colwellia sp. NJ341 with response surface methodology

Culture conditions were optimized for an extracellular cold-active protease production by the psychrophilic bacterium Colwellia sp. NJ341. Response surface methodology was applied for the most significant fermentation parameters (casein, citrate sodium, temperature and Tween-80) identified earlier by one-factor-at-a-time approach. A 2(4) full factorial central composite design was employed to determine the maximum protease production. Using this methodology, the quadratic regression model of producing cold-active protease was built and the optimal combinations of media constituents for maximum protease production (183.21 U/mL) were determined as casein 5.18 g/L, citrate sodium 3.84 g/L, temperature 7.96 degrees C, Tween-80 0.23 g/L. Protease production obtained experimentally coincident with the predicted value and the model was proven to be adequate.     

Dietary intrinsic phytate protects colon from lipid peroxidation in pigs with a moderately high dietary iron intake.

High iron consumption has been proposed to relate to an increase in the risk of colon cancer, whereas high levels of supplemental sodium phytate effectively reduce iron-induced oxidative injury and reverse iron-dependent augmentation of colorectal tumorigenesis. However, the protective role of intrinsic dietary phytate has not been determined. In this study, we examined the impact of removing phytate present in a corn-soy diet by supplemental microbial phytase on susceptibility of pigs to the oxidative stress caused by a moderately high dietary iron intake. Thirty-two weanling pigs were fed the corn-soy diets containing two levels of iron (as ferrous sulfate, 80 or 750 mg/kg diet) and microbial phytase (as Natuphos, BASF, Mt. Olive, NJ, 0 or 1200 units/kg). Pigs fed the phytase-supplemented diets did not receive any inorganic phosphorus to ensure adequate degradation of phytate. After 4 months of feeding, liver, colon, and colon mucosal scrapings were collected from four pigs in each of the four dietary groups. Colonic lipid peroxidation, measured as thiobarbituric acid reacting substances (TBARS), was increased by both the high iron (P< 0.0008) and phytase (P< 0.04) supplementation. Both TBARS and F2-isoprostanes, an in vivo marker of lipid peroxidation, in colonic mucosa were affected by dietary levels of iron (P< 0.03). Mean hepatic TBARS in pigs fed the phytase-supplemented, high iron diet was 43%-65% higher than that of other groups although the differences were nonsignificant. Moderately high dietary iron induced hepatic glutathione peroxidase activity (P= 0.06) and protein expression, but decreased catalase (P< 0.05) in the colonic mucosa. In conclusion, intrinsic phytate in corn and soy was protective against lipid peroxidation in the colon associated with a moderately high level of dietary iron.     

Phytate degradation by micro-organisms in synthetic media and pea flour

AIMS: To screen micro-organisms for the ability to produce phytase enzyme(s) and to use promising strains for the fermentation of pea flour. METHODS AND RESULTS: Two methods using the indirect estimation of phytate degradation were evaluated and both shown to be inadequate. A third method, measuring the inositol phosphate (IP3-IP6) content directly during fermentation, was used instead of the indirect estimations of phytate degradation. In synthetic media, some strains required customized conditions, with no accessible phosphorus sources other than phytate, to express phytase activity. The repression of phytase synthesis by inorganic phosphorus was not detected during fermentation with pea flour as substrate and seemed to be less significant with a higher composition complexity of the substrate. None of the tested lactic acid bacteria strains showed phytase activity. CONCLUSIONS: The methodology for the phytase screening procedure was shown to be critical. Some of the screening methods and media used in previous publications were found to be inadequate. SIGNIFICANCE AND IMPACT OF THE STUDY: This paper highlights the pitfalls and difficulties in the evaluation of phytase production by micro-organisms. The study is of great importance for future studies in this area.     

Extracellular secretion of Aspergillus phytase from Arabidopsis roots enables plants to obtain phosphorus from phytate

Phosphorus (P) deficiency in soil is a major constraint for agricultural production worldwide. Despite this, most soils contain significant amounts of total soil P that occurs in inorganic and organic fractions and accumulates with phosphorus fertilization. A major component of soil organic phosphorus occurs as phytate. We show that when grown in agar under sterile conditions, Arabidopsis thaliana plants are able to obtain phosphorus from a range of organic phosphorus substrates that would be expected to occur in soil, but have only limited ability to obtain phosphorus directly from phytate. In wild-type plants, phytase constituted less than 0.8% of the total acid phosphomonoesterase activity of root extracts and was not detectable as an extracellular enzyme. By comparison, the growth and phosphorus nutrition of Arabidopsis plants supplied with phytate was improved significantly when the phytase gene (phyA) from Aspergillus niger was introduced. The Aspergillus phytase was only effective when secreted as an extracellular enzyme by inclusion of the signal peptide sequence from the carrot extensin (ex) gene. A 20-fold increase in total root phytase activity in transgenic lines expressing ex::phyA resulted in improved phosphorus nutrition, such that the growth and phosphorus content of the plants was equivalent to control plants supplied with inorganic phosphate. These results show that extracellular phytase activity of plant roots is a significant factor in the utilization of phosphorus from phytate and indicate that opportunity exists for using gene technology to improve the ability of plants to utilize accumulated forms of soil organic phosphorus.     

Effect of germination temperature on characteristics of phytase production from barley

The effects of germination temperature on the growth of barley seedlings for phytase production were studied at 15, 20 and 25 degrees C for 6-10 days. The growth rate of the barley seedlings was increased as the germination temperature was increased. The initial rate of total protein production was closely coupled to that of the barley growth, and the rate of total protein production tended to increase as the germination temperature was increased. SDS-PAGE analysis of total protein from the barley seedlings showed time-dependent appearance and disappearance of protein bands. Although no significant phytase activity was detected at zero time of germination, a significant increase in phytase activity up to 7.9-fold occurred during the first several days of germination then decreased. Phosphate production (viz. phytate degradation) in the barley seedlings occurred rapidly at the beginning of germination. However, the rate of production continued to decrease with further germination. A time lag of about 1-2 days between the rate of total protein production and that of phytase production was observed. Unlike the extent of total protein production, that of phytase production was similar irrespective of germination temperature. Partial purification of a crude enzyme extract by hydrophobic interaction chromatography resulted in two phytase fractions (PI and PII). Zymogram analysis demonstrated that PI had two bands with molecular masses of about 66 and 123 kDa while PII had one band corresponding to a molecular mass of about 96 kDa. The optimal temperature for PI was found to be 55 degrees C, while it was 50 degrees C for PII. The enzyme fraction PI had a pH optimum at 6.0, whereas the optimum pH for PII was found to be 5.0. Addition of 0.1% (v/v) Tween 80 was found to increase enzyme activity significantly (i.e., 167% for PI and 137% for PII). Phytate in cereals including barley, rice, corn and soybean degraded effectively by the treatment of the barley phytases.     

Phytase from antarctic yeast strain Cryptococcus laurentii AL27

Cryptococcus laurentii strain AL(27) demonstrating significant potential for intracellular phytase production was selected by 2-step screening of Antarctic yeasts. The strain showed increased phytase activity in a culture medium with 40 g/L sucrose, KH(2)PO(4) providing 5 mg/L phosphorus, and cultivation temperature of 24 degrees C, which relates it to psychrotrophic microorganisms. The enzyme kinetic characteristics according to sodium phytate were K (m) = 0.98 mmol/L, v (lim) = 33.3 mumol g(-1) min(-1). The enzyme had maximum activity at 40 degrees C and acted within a wide pH range: from 2.0 to 5.5, which is of positive significance for its direct inclusion into the feed of monogastric animals.     

Purification and Biochemical Characterization of Phytase Enzyme from <Emphasis Type="Italic">Lactobacillus coryniformis</Emphasis> (<Emphasis Type="Italic">MH121153</Emphasis>)

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.     

Production of phytase by a marine yeast Kodamaea ohmeri BG3 in an oats medium: optimization by response surface methodology

Statistical experimental designs were applied for the optimization of phytase production by a marine yeast Kodamaea ohmeri BG3 in a cost-effective oats medium. Using Plackett-Burman design, oats, ammonium sulfate and initial pH were identified as significant factors and these factors were subsequently optimized using a central composite design (CCD). The optimum variables that supported maximum enzyme activity were oats 1.0%, ammonium sulfate 2.3%, glucose 2.0%, NaCl 2.0% and initial pH 6.3. The validity of the optimized variables was verified in shake-flasks level. An overall 9-fold enhancement in phytase activity (62.0-->575.5 U/ml) was attained due to the optimization.     

Effect of particle size and microbial phytase on phytate degradation in incubated maize and soybean meal

The objective of the study was to evaluate the effect of screen size (1, 2 and 3 mm) and microbial phytase (0 and 1000 FTU/kg as-fed) on phytate degradation in maize (100% maize), soybean meal (100% SBM) and maize–SBM (75% maize and 25% SBM) incubated in water for 0, 2, 4, 8 and 24 h at 38°C. Samples were analysed for pH, dry matter and phytate phosphorus (P). Particle size distribution (PSD) and average particle size (APS) of samples were measured by the Laser Diffraction and Bygholm method. PSD differed between the two methods, whereas APS was similar. Decreasing screen size from 3 to 1 mm reduced APS by 48% in maize, 30% in SBM and 26% in maize–SBM. No interaction between screen size and microbial phytase on phytate degradation was observed, but the interaction between microbial phytase and incubation time was significant (P<0.001). This was because microbial phytase reduced phytate P by 88% in maize, 84% in maize–SBM and 75% in SBM after 2 h of incubation (P<0.05), whereas the reduction of phytate P was limited (<50%) in the feeds, even after 24 h when no microbial phytase was added. The exponential decay model was fitted to the feeds with microbial phytase to analyse the effect of screen size and feed on microbial phytase efficacy on phytate degradation. The interaction between screen size and feed affected the relative phytate degradation rate (R_d) of microbial phytase as well as the time to decrease 50% of the phytate P (t1/2) (P<0.001). Thus, changing from 3 to 1 mm screen size increased R_d by 22 and 10%/h and shortened t1/2 by 0.4 and 0.2 h in maize and maize–SBM, respectively (P<0.05), but not in SBM. Moreover, the screen size effect was more pronounced in maize and maize–SBM compared with SBM as a higher phytate degradation rate constant (K_d) and R_d, and a shorter t1/2 was observed in maize compared with SBM in all screen sizes (P<0.05). However, a higher amount of degraded phytate was achieved in SBM than in maize because of the higher initial phytate P content in SBM. In conclusion, reducing screen size from 3 to 1 mm increased K_d and R_d and decreased t1/2 in maize and maize–SBM with microbial phytase. The positive effect of grinding on improving microbial phytase efficacy, which was expressed as K_d, R_d and t1/2, was greater in maize than in SBM.     

The effect of surfactants on the phytase production and the reduction of the phytic acid content in canola meal byAspergillus carbonarius during a solid state fermentation process

Summary During the growth of A. carbonarius, the rates of biomass growth, phytase production and phytic acid content reduction in canola meal media during solid state fermentation were higher in the presence of Na-oleate or Tween-80 than in the control medium which was not supplemented with these surfactants. Addition of Triton X-100 had a negative effect on the studied processes.The optimum concentration of Na-oleate in solid state culture media was 1%.     

Novel screening method for extracellular phytase-producing microorganisms

A bioassay was developed to screen extracellular phytase-producing microorganisms. Washed cells of Corynebacterium glutamicum, which cannot use sodium phytate as source of phosphate, were mixed with phytate-minimal agar as indicator strain. By this method, we could easily obtain phytase-producing strains from soil samples and 71 % of the isolates had phytase activities above 0.01 U/ml when they were grown in modified phytase screening medium. © Rapid Science Ltd. 1998     

Phytase production by the thermophilic fungus Rhizomucor pusillus

A thermophilic fungus, Rhizomucor pusillus, isolated from composting soil, was studied for phytase production using solid-state fermentation. The optimization of phytase production was carried out by Box–Behnken design of experiments, using three independent variables (pH of medium, culture age and incubation period), resulting in a maximal level of phytase (9.18 units/g substrate). The partially purified phytase was optimally active at 70 °C and pH 5.4, though the enzyme showed 80% activity over a wide pH range, 3.0–8.0. The phytase was found to have broad substrate specificity.     

Phytate degradation by immobilized<Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> phytase in soybean-curd whey

Saccharomyces cerevisiae CY phytase-producing cells were immobilized in calcium alginate beads and used for the degradation of phylate. The maximum activity and immobilization yield of the immobilized phytase reached 280 mU/g-bead and 43%, respectively. The optimal pH of the immobilized cell phytase was not different from that of the free cells. However, the optimum temperature for the immobilized phytase was 50°C, which was 10°C higher than that of the free cells; pH and thermal stability were enhanced as a consequence of immobilization. Using the immobilized phytase, phytate was degraded in a stirred tank bioreactor. Phytate degradation, both in a buffer solution and in soybean-curd whey mixture, showed very similar trends. At an enzyme dosage of 93.9 mU/g-phytate, half of the phytate was degraded after 1 h of hydrolysis. The operational stability of the immobilized beads was examined with repeated batchwise operations. Based on 50% conversion of the phytate and five times of reuse of the immobilized beads, the specific degradation (g phytate/g dry cell weight) for the immobilized phytase increased 170% compared to that of the free phytase.