Native Phytase Research Articles

Can sprouting reduce phytate and improve the nutritional composition and nutrient bioaccessibility in cereals and legumes?

Sprouting is a traditional processing method which has been used for centuries to improve the nutritional value of cereals and legumes. There has been growing interest in sprouted products in recent years due to a high demand for more natural and healthy foods. Phytate is the primary storage form of phosphorus in plants. It is long recognised to affect human health as it forms insoluble complexes with minerals such as iron and zinc in cereals and legumes, thereby preventing their absorption in the body. Sprouting activates the enzyme phytase, which degrades phytate, thereby improving mineral bioaccessibility and bioavailability. The extent of phytate reduction varies depending on the sprouting conditions, cereal/legume species, cultivar and native phytase activity. Sprouting has been associated with increased iron, zinc and calcium bioaccessibility in many studies, but this appears to differ in cereals and legumes, which possibly is due to the presence of other 'antinutrients'. Protein digestibility also appears to be positively correlated with phytate reduction albeit less than for minerals. It is not possible to accurately predict the influence of sprouting on nutrient bioavailability because so few studies have been conducted. Further research is required to determine whether the commercial production of sprouted cereals and legumes can increase the nutritional value and health benefits of commercial end products.

Conditioning of Feed Material Prior to Feeding: Approaches for a Sustainable Phosphorus Utilization

A circular phosphorus (P) bioeconomy is not only worthwhile for conserving limited mineral P reservoirs, but also for minimizing negative environmental impacts caused by human-made alterations. Although P is an essential nutrient, most of the P in concentrates based on cereals, legumes and oilseed byproducts is organically bound to phytate. The latter cannot be efficiently utilized by monogastric animals and is therefore diluted into the environment through the manure pathway. This review examines various strategies for improved P utilization in animals and reflects the respective limitations. The strategies considered include feeding of debranned feedstuffs, pre-germinated feed, co-feeding of phytase and feeding material with high native phytase activity. All these approaches contribute to an improved P bioavailability. However, about half of the organic P content continues to be excreted and therefore remains unused by the animals. Nevertheless, technologies for an efficient utilization of P from cereal-based feed already exist; however, these are not industrially established. Conditioning feed material prior to feeding fosters P-reduced feed; meanwhile, P bound to phytate can be recovered. Based on known techniques for P separation and solubilisation from cereal products and phytate conversion, potential designs for feed material conditioning processes are proposed and evaluated.

Dephytinising efficacy of wheat phytase in enhancing the bioaccessibility of minerals in high phytate foods

Iron deficiency anemia and zinc deficiency are major public health problems in developing world which are attributable to the poor bioavailability due to the presence of phytic acid in plant foods. Dephytinisation by enzymatic application is identified to be ideal approach, however the commercial availability of phytases from food sources is scarce. This prompted us to undertake the investigation on the dephytinisation of the high phytate food matrices by utilising the indigenous phytase of wheat. Attempts were made to enhance the phytase activity by various approaches. Phytase activity of wheat was increased by fivefold on sprouting (96 h). The native phytase of wheat activated by incubation at optimum conditions of pH and temperature has shown an increase in the activity as a function of time. Isolation and partial purification of phytase from sprouted wheat further increased the activity by 1.5 fold. Thermostability of phytase was enhanced by additives such as sodium citrate, calcium chloride and magnesium sulphate. Addition of phytase activated wheat flour to soya flour enhanced the bioaccessible iron by 1.7 fold. Partially purified phytase enhanced the bioaccessible iron by 1.9 fold and zinc by 2.4 fold in defatted soya flour reflecting dephytinisation. Investigation on complete purification of phytase are warranted.

Heterologous Expression of Histidine Acid Phytase from Pantoea sp. 3.5.1 in Yarrowia lipolytica

The dimorphic yeasts Yarrowia lipolytica are used as effective expression system and are characterized by a high level of production of heterologous protein. In this work, we aimed to clone and express Pantoea sp. 3.5.1 agpP phytase gene in Yarrowia lipolytica. Genetic constructs containing the native phytase gene (agpP) under the control of the strong hybrid promoter hp4d and the signal peptide of the alkaline extracellular protease XPR2 gene, as well as the optimized gene (agpP-opt) of phytase under the control of two signal peptides—bacterial and yeast were obtained. Recombinant Y. lipolytica strains with integrated bacterial phytase genes were obtained and expression of phytase was analyzed.

Zinc bioavailability in rats fed a plant-based diet: a study of fermentation and zinc supplementation

BackgroundZinc deficiency is a significant problem, in developing countries and in vegetarians, which can be caused by plant-based diets. Thus, dietary strategies, such as fermentation, to improve zinc bioavailability of diets should be investigated.ObjectiveTo improve zinc bioavailability in a plant-based diet by the inclusion of fermented food.DesignCassava tubers were fermented and made to replace the unfermented cassava in a basal plant-based diet, and compared with plant-based diets with and without zinc supplement. The zinc bioavailability of the diets was evaluated in Wistar rats that were fed these diets for 28 days. The evaluation was for zinc apparent absorption (ZnAA), serum zinc levels, and zinc deposits in liver and femur; in addition, the feed efficiency ratio (FER) of the diets and femur weight (FW) of the rats were evaluated.ResultsDuring the cassava fermentation, lactic acid increased and pH decreased (from 6.8 to 3.9), which is favorable for native phytase activity, resulting in a 90.2% reduction of phytate content in cassava. The diet containing fermented cassava showed significantly higher levels of ZnAA, FER, and FW (p<0.001). Moreover, the zinc levels in serum and femur were significantly higher (p<0.001) compared with the results of the diet with unfermented cassava. The results clearly show a higher zinc bioavailability in the diet containing fermented cassava and are comparable with the results obtained with the plant-based diet with zinc supplement.ConclusionsIn conclusion, the fermentation of cassava reduces the phytate content. The diet containing the fermented cassava represents a better nutritional alternative than the diet with unfermented cassava and is comparable with the zinc-supplemented diets.

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.

Ethanol Tolerance in &amp;lt;i&amp;gt;Aspergillus niger&amp;lt;/i&amp;gt; and &amp;lt;i&amp;gt;Escherichia coli&amp;lt;/i&amp;gt; Phytase

Despite yeast having its own native phytase, the high levels of phytate found in DDGS, a byproduct of ethanol (ETOH) fermentation, suggest that its activity is diminished in the presence of ETOH. Ethanol, a product of grain fermentation, is known to inactivate several hydrolytic enzymes but its effect on phytases is relatively unknown. In this study, two phytases, Aspergillus niger (PhyA) and Escherichia coli (AppA2), were tested for ETOH tolerance. The E. coli phytase displayed greater ethanol tolerance over fungal phytase in the 5% to 10% range. However, ETOH inactivation was found to be reversible for both the enzymes. These differences in ETOH tolerance do suggest that there is a potential to achieve higher ETOH tolerance in phytases by 'structure-function' studies to lower phytic acid levels in DDGS and for other applications.

Effects of supplemental phytase on nutrient digestibility and performance of sows fed diets with high or low native phytase activity

Two experiments were conducted to examine the influence of addition of microbial phytase to diets differing in native phytase content, on the performance of sows and digestibility of nutrients. Barley and wheat grains relatively poor in native phytase used in Experiment 1 mixtures were replaced by triticale and rye (rich in this enzyme) in Experiment 2 diets. There were 3 groups in each experiment. Group I (control) received basal diets enriched with dicalcium phosphate (10&amp;nbsp;g/kg), group II (negative control) was fed basal diets without any additive, and group III (negative control) was supplemented with microbial phytase (500&amp;nbsp;PU/kg). Body weights of sows were monitored at mating, before parturition, immediately after parturition and at weaning. Apparent ileal and total tract digestibility of basal nutrients, detergent fibre fractions, Ca and P of the investigated diets from particular treatments was determined. Microbial phytase (Natuphos&lt;sup&gt;&amp;reg;&lt;/sup&gt;, BASF AG, Ludwigshaven, Germany) supplemented at 500 PU/kg in pregnancy and lactation diets based on barley and wheat resulted in smaller body weight losses during lactation and higher weight gain over the whole cycle. Synergistic effects of microbial phytase and native phytase on body weight changes of sows, feed conversion ratio during pregnancy, and apparent (ileal and total tract) digestibility of both organic matter and minerals (total and phytic phosphorus and calcium) were found out.

The influence of microbial phytase supplementation to diets with high or low native phytase activity on sow reproductive traits and composition of colostrum and milk

The influence of intrinsic phytase and a microbial phytase additive on reproductive traits of sows, the composition of colostrum and milk, and on piglet rearing indices was studied. The two trials differed in the kind of grains used in the diets to give them lower (420-440 PU . kg -1 , Trial 1) or higher (1000-1150 PU . kg -1 , Trial 2) levels of intrinsic phytase. There were 3 feeding groups (34-35 animals in Trial 1 and 25 in Trial 2) in each experiment. In both trials, group 1 received the basic diet enriched with dicalcium phosphate, group 2, the basic diet without this additive (NC), and group 3, the basic diet supplemented with 500 PU . kg -1 microbial phytase. A higher number of liveborn piglets in group 3 in comparison with group 2 in Trial 2 was noted. Also, the body weight of piglets was significantly higher in groups 1 and 3 in comparison with the NC group. The microbial phytase supplement increased the concentration of zn and Cu in colostrum and of zn in sow milk, regardless of native phytase activity.

Expression of a Bacillus Phytase C Gene in Pichia pastoris and Properties of the Recombinant Enzyme

The cloning and expression of a native gene encoding a Bacillus subtilis phytase using Pichia pastoris as the host is described. In addition, the influence of N-glycosylation on the biochemical properties of the B. subtilis phytase, the influence of pH on the thermostability of the recombinant and native B. subtilis phytases, and the resistance of both phytases to shrimp digestive enzymes and porcine trypsin are also described. After 48 h of methanol induction in shake flasks, a selected recombinant strain produced and secreted 0.82 U/ml (71 mg/liter) recombinant phytase. This phytase was N-glycosylated, had a molecular mass of 39 kDa after N-deglycosylation, exhibited activity within a pH range of 2.5 to 9 and at temperatures of 25 to 70 degrees C, had high residual activity (85% +/- 2%) after 10 min of heat treatment at 80 degrees C and pH 5.5 in the presence of 5 mM CaCl(2), and was resistant to shrimp digestive enzymes and porcine trypsin. Although the recombinant Bacillus phytase had pH and temperature activity profiles that were similar to those of the corresponding nonglycosylated native phytase, the thermal stabilities of the recombinant and native phytases were different, although both were calcium concentration and pH dependent.

Screening, cloning and overexpression of Aspergillus niger phytase (phyA) in Pichia pastoris with favourable characteristics

Using gene cloning and overexpression to obtain a potential industrial phytase as a feed additive to upgrade the nutritional quality of phytate-rich seed-based animal feed. A phyA gene from a high extracellular phytase-producing Aspergillus niger sp. was cloned and overexpressed in Pichia pastoris GS115 using the secretive expression vector pPICZalphaA. After cultivation for 4 days in buffered methanol complex medium (BMMY) containing methanol for induction, catalytically active phytase was secreted as a predominantly extracellular protein. The activity of the expressed phytase in fermented broth was 30 000-fold higher than that of native phytase with a specific activity of 503 U mg(-1). The Lineweaver-Burk plot indicated K(m) values of 0.196 mmol l(-1) for sodium phytate and 18.16 mmol l(-1) for p-nitrophenylphosphate (pNPP). Thermostability studies showed that recombinant phytase retained 70% activity after exposure to 90 degrees C for 5 min and 65% activity after 30 min, much higher than for commercial phytase. The higher activity and high thermostability of recombinant phytase enable it to withstand the temperatures of the feed pelleting process. The characteristics of this recombinant phytase, especially the good thermostability, are likely to render it of potential industrial importance.

Distribution of phytase activity, total phosphorus and phytate phosphorus in legume seeds, cereals and cereal by-products as influenced by harvest year and cultivar

Samples of legume seeds, cereals and cereal by-products ( n = 113) grown in south-western Germany and originating from different cultivars and harvest years were analyzed for phytase activity, total phosphorus (P) and phytate P. Phytase activities determined by means of a direct incubation method were lowest in legume seeds and oats (262–496 U/kg dry matter), intermediate in cereals (except oats) (2323–6016 U/kg DM) and highest in cereal by-products (9241–9945 U/kg DM). However, the application of an extraction procedure for the determination of phytase activities in legume seeds resulted in values below the detection limit of 50 U/kg. On average, about 0.67 of total P in legume seeds, cereals and their by-products is bound to phytate. There was a significant influence (P<0.001) of harvest year (1998–2000) on phytate P contents in wheat. Furthermore, total P and phytate P concentrations differed (P<0.05) between different cultivars of wheat. Moreover, phytase activities differed (P=0.023) between different cultivars of barley. Total P and phytate P concentrations were highly correlated in legume seeds ( r = 0.95) and cereal by-products ( r = 0.96) and, to a smaller extent, in cereals ( r = 0.66). Milling of cereal grains to bran and flour revealed that phytase activity, total P and phytate P are highly concentrated (P<0.001) in the outer grain layers of cereals. The influence of preservation of intact legume seeds with propionic acid over a period of 4, 8 or 12 weeks resulted only in a marginal decrease in phytase activity. Due to high native phytase activities in cereals (except oats) and their by-products these feedstuffs may contribute substantially to the gastrointestinal hydrolysis of phytate in non-ruminant animals, whereas the contribution of native phytases originating from legume seeds in terms of improving the availability of plant P seems to be rather limited.

Nutrient utilization of Nile tilapia Oreochromis niloticus fed plant based low phosphorus diets supplemented with graded levels of different sources of microbial phytase

Experimental results regarding nutrient and phosphorus (P) utilization due to different sources of microbial phytase in tilapia nutrition are very scarce. An experiment was conducted with plant based diets (24.5% soybean meal; 11.5% wheat gluten; 22.5% corn; 32.5% wheat) containing moderate native phytase activity (< 300 FTU/kg). For comparison, a negative control diet (0.41% total P; 0.15% non-phytate P; no phytase added) and a positive control diet (0.88% total P; 0.62% non-phytate P; no phytase added) were used. Experimental diets with graded levels (500, 750, 1000, 1250 FTU/kg) of two different sources of microbial phytase (experimental phytase SP 1002; Ronozyme®P) were assigned in four repetition tanks and fed to juvenile all male Nile tilapia (average body weight 13.50 g) for 60 days. The efficiency of phytase supplementation was evaluated based on parameters of growth response, body composition, nutrient deposition and nutrient utilization following submission to ANOVA ( P < 0.01) and Tukey-test. Significant improvements due to phytase addition ( P < 0.01) were found for growth, feed conversion ratio, protein efficiency ratio, specific growth rate and nutrient deposition. Phytase supplementation increased energy-, protein- and phosphorus utilization significantly. In addition, the mineral composition of scale and vertebra was significantly affected. Ash content of scale and vertebra as well as P content in the ash of scale and vertebra were significantly increased due to improved P availability in the diet. Based on ingredients and composition of the diet under study, the experimental phytase SP1002 expressed superior performance data compared to Ronozyme®P. Supplementation of 750 FTU/kg from phytase SP1002 was similar to an inorganic P supplementation.

Biochemical characterisation of extracellular phytase (myo-inositol hexakisphosphate phosphohydrolase) from a hyper-producing strain of Aspergillus niger van Teighem

Aspergillus niger van Teighem, isolated in our laboratory from samples of rotten wood logs, produced extracellular phytase having a high specific activity of 22,592 units (mg protein)-1 . The enzyme was purified to near homogeneity using ion-exchange and gel-filtration chromatography. The molecular properties of the purified enzyme suggested the native phytase to be oligomeric, with a molecular weight of 353 kDa, the monomer being 66 kDa. The purified enzyme exhibited maximum activity at pH 2.5 and 52-55 degrees C. The enzyme retained 97% activity after a 24-h incubation at 55 degrees C in the presence of 10 mM glycine, while 87% activity was retained when no thermoprotectant was added. Phytase activity was not affected by most metal ions, inhibitors and organic solvents. Non-ionic and cationic detergents (0.1-5%) stabilise the enzyme, while the anionic detergent (SDS), even at a 0.1% level, severely inhibited enzyme activity. The chaotropic agents guanidinium hydrochloride, urea, and potassium iodide (0.5-8 M), significantly affected phytase activity. The maximum hydrolysis rate (Vmax) and apparent Michaelis-Menten constant (Km) were 1,074 IU/mL and 606 microM, respectively, with a catalytic turnover number of 3x10(5) s-1 and catalytic efficiency of 3.69x10(8) M-1 s-1.

Biochemical and haematological blood parameters of sows during pregnancy and lactation fed the diet with different source and activity of phytase

The aim of the study was to estimate the influence of microbial phytase supplementation to mixtures with low (425 ± 25 PU kg −1) or high (1075 ± 75 PU kg −1) native phytase on haematological and biochemical indices as well as contents of chosen mineral components in blood of sows during pregnancy and lactation. In two experiments conducted on 75 sows in each one, all the animals were randomized to three experimental groups. Diets with low content of native phytase used in experiment 1 were based on barley, wheat, oat and rapeseed meal. Diets with high native phytase level used in experiment 2 contained triticale, rye, oat and rapeseed meal. Additionally, lactating diets were supplemented with soybean meal. The experimental design was analogical in both experiments. The diet of group I (positive control) was supplemented with the standard mixture of nutrients, containing Ca and P according to NRC recommendations (1998). Sows from group II (negative control) were fed a mixture without inorganic phosphorus supplementation. In group III, the animals were fed the same mixture as in group II, but with microbial phytase addition (500 PU kg −1 of mixture). Blood samples were taken on 8th and 14th week of pregnancy and 21st day of lactation. The following indices in whole blood were determined: hematocrit (Ht), haemoglobin (Hb) level, erythrocyte (RBC) and leukocyte (WBC) number. In the blood the activity of AP, ALT, AST, LDH, Na +K +ATP-ase has estimated and concentration of P, Ca, Na, K, Mg, Fe, Cu and Zn. Supplementing diet with the microbial phytase increased the availability of Ca, Fe, Zn in both experiments and P, Cu in experiment with high native phytase activity (experiment 2). The microbial phytase addition to the high native phytase diets diminished WBC number and increased Hb level in blood. The levels of the rest haematological indices were not changed by the exogenous microbial phytase. The increase of the phosphorous availability in the diet affected by the addition of microbial phytase is in line with the increased AP activity. The higher level of native phytase increased Mg and P, Ca, Cu and Zn availability and activity of the following enzymes: AP, ALT and LDH. Irrespective of phytase activity in diet, a higher content of sodium and the elevated RBC and Ht levels, higher AP and LDH activity but lower activity of ALT and ATP-aseNa +K + in the blood of sows during pregnancy in comparison with lactation was noted.

Expression and characterization of extracellular fungal phytase in transformed sesame hairy root cultures

A recombinant fungal phytase was produced by cultures of sesame hairy roots transformed with Agrobacterium rhizogenes, purified and its molecular properties were characterized. Its transcription level and the phytase production were rapidly increased after 4 weeks of the cultures, suggesting that its transcription and protein synthesis might concur. Western blot analysis provided evidence that the recombinant fungal phytase was secreted into the liquid culture medium of the hairy roots. The phytase enzyme secreted was purified by three steps of ultrafiltration, DEAE–Sepharose ion exchange chromatography, and Sephadex G-100 size-exclusion chromatography. As a result, one single band signal was observed with SDS–PAGE, indicating that the purification step was reasonable. The positive signs of both the zymogram and the PAS staining on SDS–PAGE suggested that the activity of the final product phytase was active and glycosylated. The optimal reaction temperature of the phytase was between 50 and 60 °C and at over 60 °C its activity was reduced by 30–90%, depending on the temperatures applied. Pre-incubation at temperatures of 20–50 °C showed stable catalytic activity, while at over 50 °C the phytase activity was gradually decreased by 90%. The optimal pH was between 4 and 5 pH values for the recombinant fungal phytase, while for native phytase it was at pH 5.0. Addition of iron ion inhibited the phytase activity but treatments of some cations, EDTA, and PMSF showed no effect on the activity or slightly stimulated it positively.

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

Fungal phyA gene from Aspergillus ficuum ( niger) was cloned and expressed in potato leaves. The recombinant enzyme was stable and catalytically active. The expressed protein in the leaves of the dicotyledonous plant retained most physical and catalytic properties of the benchmark A. ficuum phytase. The expressed enzyme was, however, 15% less glycosylated than the native phytase. The usual bi-hump pH optima profile, which is characteristic of the fungal phytase, was altered; however, the pH optimum at 5.0 was unchanged for phytate and at 4.0 for synthetic substrate p-nitrophenyl phosphate. The temperature was, however, unchanged. The expressed phytase was found to be as sensitive as the native enzyme to the inhibitory action of pseudo substrate, myo-inositol hexasulfate, while losing about 90% of the activity at 20 μM inhibitor concentration. Similar to the benchmark phytase, the expressed phytase in leaves was completely inactivated by Arg modifier phenylglyoxal at 60 nM. In addition, the expressed phytase in the leaves was inhibited by antibody raised against a 20-mer internal peptide, which is present on the surface of the molecule as shown by the X-ray deduced 3D structure of fungal phytase. Taken together, the biochemical evidences indicate that fungal phytase when cloned and expressed in potato leaves produces a stable and active biocatalyst. ‘Biofarming,’ therefore, is an alternative way to produce functional hydrolytic enzymes as exemplified by the expression of A. ficuum ( niger) phyA gene in potato leaf.

Characterization of Recombinant Fungal Phytase (phyA) Expressed in Tobacco Leaves

The phyA gene from Aspergillus ficuum coding for a 441-amino-acid full-length phytase was expressed in Nicotiana tabacum (tobacco) leaves. The expressed phytase was purified to homogeneity using ion-exchange column chromatography. The purified phytase was characterized biochemically and its kinetic parameters were determined. When the recombinant phytase was compared with its counterpart from Aspergillus ficuum for physical and enzymatic properties, it was found that catalytically the recombinant protein was indistinguishable from the native phytase. Except for a decrease in molecular mass, the overexpressed recombinant phytase was virtually the same as the native fungal phytase. While the temperature optima of the recombinant protein remain unchanged, the pH optima shifted from pH 5 to 4. The results are encouraging enough to open the possibility of overexpressing phyA gene from Aspergillus ficuum in other crop plants as an alternative means of commercial production of this important enzyme.

Effect of Traditional Food Processing on Phytate Degradation in Wheat and Millets

For cereal-based vegetarian meals, processing such as soaking cereal flour prior to heating can activate native phytases. This activation will result in improving the zinc bioavailability since degraded products of phytate have a lower affinity for zinc. The effect of increasing the time for soaking wheat batter at 10 °C for 0−48 h and the effect of roti making with millet and sorghum flour batters was investigated. Phytate (IP6) degradation was studied using ion-exchange chromatography on a column of Dowex 1X8 resin (200−400 mesh). A soaking time of 12 h for wheat batter resulted in a 40% decrease in IP6. The decreasing trend for wheat with increased soaking time was significant (p < 0.01). Soaking also resulted in an increase of zinc solubility by 38.5%. Degradation of IP6 due to roti making without soaking of batter for all three cereals was 14−19% with a marginal decrease in zinc solubility. Keywords: Phytate degradation; traditional methods; zinc solubility

Cloning and characterization of a cDNA encoding a maize seedling phytase

During germination, maize seedlings express a phytase able to hydrolyse the large amount of phytin stored in the dry seed. Previous studies allowed purification and characterization of this enzyme as a homodimer of 38 kDa subunits [Laboure, Gagnon and Lescure, Biochem. J. (1993) 295, 413-419]. In the present work, an antibody against the purified maize phytase has been used to screen a maize seedling cDNA expression library. Several positive clones containing an insert of about 1400 bp were isolated. The nucleotide sequence of the insert of one of these clones has been established. This cDNA, called phy S11, was 1335 bp long and contained an open reading frame of 387 amino acids. The sequence of N-terminal residues (23 amino acids) of the purified phytase has been established. These residues are found at positions 19-41 of the amino acid sequence encoded by phy S11. This confirms that this cDNA codes for the maize phytase. The deduced amino acid sequence appears to be very different from those of published Aspergillus niger phytases; however, an homologous region of 33 amino acids was detected. This region of the fungal sequence contains the RHGxRxP consensus motif found in various high molecular mass acid phosphatases and believed to be the acceptor site for phosphate. Expression of the phy S11 cDNA in Escherichia coli allowed the production of the phytase subunit and its assembly to give a protein of the same size as the native phytase. The time course of phy S11 mRNA accumulation during germination showed that no transcript was present in dry seeds. The mRNA accumulated during the first day of germination, to reach a maximum after 2 days (radicle protrusion), and then decreased in young seedlings. Genomic Southern blot analyses suggest the existence of at least two genes and genetic mapping reveals two loci separated by 1 cM on chromosome 3 of maize. The cloning of this first cDNA coding for a plant phytase, will allow the isolation of the corresponding genes and the study of their regulation during germination.