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

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

Diversity,abundance and characterization of ruminal cysteine phytases suggest their important role in phytate degradation

A novel class of cysteine phytase showing ability to degrade phytate has recently been isolated from rumen bacteria. To expand our knowledge of this enzyme class, a total of 101 distinct cysteine phytase gene fragments were identified from the ruminal genomic DNA of Bore goats and Holstein cows, and most of them shared low identities (< 50%) with known sequences. By phylogenetic analysis, these sequences were separated into three clusters that showed substantial diversity. The two most abundant cysteine phytase genes of goat rumens were cloned and their protein products were characterized. Four findings were revealed based on our results. (i) Compared with soil and water environment, where β‐propeller phytase is the most important phytate‐degrading enzyme, cysteine phytase is the major phytate‐degrading enzyme in the anaerobic ruminal environment. (ii) Cysteine phytase fragments in the rumen contents of goat and cow have the same diversity profile, although most of the sequences and their abundance differ in the two species. (iii) Each species has their respective high‐abundance genes, which may play major roles for phytate degradation. (iv) Compared with previously reported cysteine phytases that have pH optimum at 4.5, the pH optima of the two most abundant secreted goat cysteine phytases are 6.5 and 6.0, which are within the pH range found in the rumens. This study provides valuable information about the diversity, abundance and enzymatic properties of the ruminal cysteine phytases and emphasizes the important role(s) of these cysteine phytases probably in the terrestrial cycle of phosphorus.     

Biochemical properties and substrate specificities of alkaline and histidine acid phytases

Phytases are a special class of phosphatase that catalyze the sequential hydrolysis of phytate to less-phosphorylated myo-inositol derivatives and inorganic phosphate. Phytases are added to animal feedstuff to reduce phosphate pollution in the environment, since monogastric animals such as pigs, poultry, and fish are unable to metabolize phytate. Based on biochemical properties and amino acid sequence alignment, phytases can be categorized into two major classes, the histidine acid phytases and the alkaline phytases. The histidine acid phosphatase class shows broad substrate specificity and hydrolyzes metal-free phytate at the acidic pH range and produces myo-inositol monophosphate as the final product. In contrast, the alkaline phytase class exhibits strict substrate specificity for the calcium–phytate complex and produces myo-inositol trisphosphate as the final product. This review describes recent findings that present novel viewpoints concerning the molecular basis of phytase classification.     

Phytase enzymology, applications, and biotechnology

Phytases are phosphohydrolases that initiate the step-wise removal of phosphate from phytate. These enzymes have been widely used in animal feeding to improve phosphorus nutrition and to reduce phosphorus pollution of animal waste. The potential of phytases in improving human nutrition of essential trace minerals in plant-derived foods is being explored. This review covers the basic biochemistry and application of phytases, and emphasizes the emerging biotechnology used for developing new effective phytases with improved properties.     

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

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

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

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

Novel phytases from Bifidobacterium pseudocatenulatum ATCC 27919 and Bifidobacterium longum subsp. infantis ATCC 15697

Two novel phytases have been characterized from Bifidobacterium pseudocatenulatum and Bifidobacterium longum subsp. infantis. The enzymes belong to a new subclass within the histidine acid phytases, are highly specific for the hydrolysis of phytate, and render myo-inositol triphosphate as the final hydrolysis product. They represent the first phytases characterized from this group of probiotic microorganisms, opening the possibilities for their use in the processing of high-phytate-content foods.     

Structural characteristics and catalytic mechanism of <Emphasis Type="Italic">Bacillus</Emphasis> β-propeller phytases

ß-Propeller phytases of Bacillus are unique highly conservative and highly specific enzymes capable of cleaving insoluble phytate compounds. In this review, we analyzed data on the properties of these enzymes, their differences from other phytases, and their unique spatial structures and substrate specificities. We considered influences of different factors on the catalytic activity and thermostability of these enzymes. There are few data on the hydrolysis mechanism of these enzymes, which makes it difficult to analyze their mechanism of action and their final products. We analyzed the available data on hydrolysis by ß-propeller phytases of calcium complexes with myo-inositol hexakisphosphate.     

Expression, gene cloning, and characterization of five novel phytases from four basidiomycete fungi: Peniophora lycii, Agrocybe pediades, a Ceriporia sp., and Trametes pubescens

Phytases catalyze the hydrolysis of phosphomonoester bonds of phytate (myo-inositol hexakisphosphate), thereby creating lower forms of myo-inositol phosphates and inorganic phosphate. In this study, cDNA expression libraries were constructed from four basidiomycete fungi (Peniophora lycii, Agrocybe pediades, a Ceriporia sp., and Trametes pubescens) and screened for phytase activity in yeast. One full-length phytase-encoding cDNA was isolated from each library, except for the Ceriporia sp. library where two different phytase-encoding cDNAs were found. All five phytases were expressed in Aspergillus oryzae, purified, and characterized. The phytases revealed temperature optima between 40 and 60 degrees C and pH optima at 5.0 to 6.0, except for the P. lycii phytase, which has a pH optimum at 4.0 to 5.0. They exhibited specific activities in the range of 400 to 1,200 U. mg, of protein(-1) and were capable of hydrolyzing phytate down to myo-inositol monophosphate. Surprisingly, (1)H nuclear magnetic resonance analysis of the hydrolysis of phytate by all five basidiomycete phytases showed a preference for initial attack at the 6-phosphate group of phytic acid, a characteristic that was believed so far not to be seen with fungal phytases. Accordingly, the basidiomycete phytases described here should be grouped as 6-phytases (EC 3.1.3.26).     

Prediction of substrate-binding site and elucidation of catalytic residue of a phytase from Bacillus sp

The present study primarily deals with the identification of substrate-binding site and elucidation of catalytic residue of the phytase from Bacillus sp. (Genbank Accession No. EF536824) employing molecular modeling and site-directed mutagenesis. Homology-based modeling of the Bacillus phytase revealed β-propeller structure with twelve active-site aminoacid residues, viz., D75, R77, Y78, H138, Q140, D189, D190, E191, Y238, Y239, N346 and R348. Docking of substrate Ins(1,2,3,4,5,6)hexakisphosphate with the phytase model disclosed interaction of Y78 residue with the sixth position phosphate, while D75 and R77 residues revealed hydrogen bonding with the fifth position phosphate of the phytate. Analysis of hydrolysis products of phytate indicated the sequential removal of alternate phosphates, resulting in the formation of final product Ins triphosphate. Mutant phytases Y78A/F, derived from site-directed mutagenesis, exhibited complete loss of enzyme activity despite substrate binding, thereby suggesting the intrinsic role of Y78 residue in the catalytic activity. The Bacillus mutant phytases can be used to generate enzyme crystals complexed with phytate and lower Ins phosphates for indepth analysis of substrate binding and catalytic activity of the enzyme.     

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

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

Glucose-1-phosphatase (AgpE) from Enterobacter cloacae displays enhanced phytase activity

Using a screening procedure developed for detection of phytate hydrolysing enzymes, the gene agpE encoding glucose-1-phosphatase was cloned from an Enterobacter cloacae VKPM B2254 plasmid library. Sequence analysis revealed 78% identity on nucleotide and 79% identity on peptide level to Escherichia coli glucose-1-phosphatase characterising the respective gene product as a representative of acid histidine phosphatases harbouring the RH(G/N)RXRP motif. The purified recombinant protein displayed maximum specific activity of 196 U mg⁻¹ protein against glucose-1-phosphate but was also active against other sugar phosphates and p-nitrophenyl phosphate. High-performance ion chromatography of hydrolysis products revealed that AgpE can act as a 3-phytase but is only able to cleave off the third phosphate group from the myo-inositol sugar ring. Based on sequence comparison and catalytic behaviour against phytate, we propose to classify bacterial acid histidine phosphatases/phytases in the three following subclasses: (1) AppA-related phytases, (2) PhyK-related phytases and (3) Agp-related phytases. A distinguished activity of 32 U mg⁻¹ of protein towards myo-inositol-hexa-phosphate, which is two times higher than that of E. coli Agp, suggests that possibly functional differences in terms of phytase activity between Agp- and AppA-like acid histidine phosphatases are fluent. Electronic supplementary material Supplementary material is available for this article at and accessible for authorised users.     

Use of Lactobacilli in Cereal-Legume Fermentation and as Potential Probiotics towards Phytate Hydrolysis

Phytate is a potent inhibitor of mineral absorption in humans occurring in plant-based food. Application of lactobacilli that produce phytate-degrading enzymes (phytases) to reduce phytate is an interesting yet a not much explored sector of research. Therefore, phytate dephosphorylation by Lactobacillus plantarum MTCC 1325 was evaluated. Cells at stationary phase showed phytase activity which was maximal at 24 h of growth. Glucose concentration and the type of phosphorous source in the media modulated the enzyme activity. Fermentation of cereal and/or legume flours with the strain resulted in phytate reduction with the highest in sorghum (73%) and the lowest in horse gram (34%). Further, the strain showed tolerance to acid, bile, and simulated gastrointestinal fluid. Significant phytase activity in the presence of simulated gastrointestinal fluids along with the ability to produce phytases post-exposure to simulated gastrointestinal fluids is of interest. To the best of our knowledge, this is the first report on the effect of simulated gastrointestinal fluid on cell-associated phytases of lactobacilli. The results of the investigation indicate that L. plantarum MTCC 1325 could be used as a starter in cereal-legume fermentation and as potential probiotics to achieve phytate hydrolysis in food matrices and also in gastrointestinal tract.     

Purification and properties of phytate-specific phosphatase from Bacillus subtilis.

An enzyme which liberates Pi from myo-inositol hexaphosphate (phytic acid) was shown to be present in culture filtrates of Bacillus subtilis. It was purified until it was homogeneous by ultracentrifugation, but it still showed two isozymes on polyacrylamide gel electrophoresis. The enzyme differed from other previously known phytases in its metal requirement and in its specificity for phytate. It had a specific requirement for Ca2+ for its activity. The enzyme hydrolyzed only phytate and had no action on other phosphate esters tested. This B. subtilis phytase is the only known phytate-specific phosphatase. The products of hydrolysis of phytate by this enzyme were Pi and myo-inositol monophosphate. The enzyme showed optimum activity at pH 7.5. It was inhibited by Ba2+, Sr2+, Hg2+, Cd2+, and borate. Its activity was unaffected by urea, diisopropylfluorophosphate, arsenate, fluoride, mercaptoethanol, trypsin, papain, and elastase.     

Structures of Selenomonas ruminantium phytase in complex with persulfated phytate: DSP phytase fold and mechanism for sequential substrate hydrolysis

Various inositide phosphatases participate in the regulation of inositol polyphosphate signaling molecules. Plant phytases are phosphatases that hydrolyze phytate to less-phosphorylated myo-inositol derivatives and phosphate. The phytase from Selenomonas ruminantium shares no sequence homology with other microbial phytases. Its crystal structure revealed a phytase fold of the dual-specificity phosphatase type. The active site is located near a conserved cysteine-containing (Cys241) P loop. We also solved two other crystal forms in which an inhibitor, myo-inositol hexasulfate, is cocrystallized with the enzyme. In the "standby" and the "inhibited" crystal forms, the inhibitor is bound, respectively, in a pocket slightly away from Cys241 and at the substrate binding site where the phosphate group to be hydrolyzed is held close to the -SH group of Cys241. Our structural and mutagenesis studies allow us to visualize the way in which the P loop-containing phytase attracts and hydrolyzes the substrate (phytate) sequentially.     

Molecular characterization,physicochemical properties,known and potential applications of phytases: An overview

Phytases (myo-inositol hexakisphosphate phosphohydrolases) hydrolyze the phosphate ester bonds of phytate-releasing phosphate and lower myo-inositol phosphates and/or myo-inositol. Phytases, in general, are known to enhance phosphate and mineral uptake in monogastric animals such as poultry, swine, and fish, which cannot metabolize phytate besides reducing environmental pollution significantly. In this study, the molecular, biophysical, and biochemical properties of phytases are reviewed in detail. Alterations in the molecular and catalytic properties of phytases, upon expression in heterologous hosts, are discussed. Diverse applications of phytases as feed additives, as soil amendment, in aquaculture, development of transgenic organisms, and as nutraceuticals in the human diet also are dealt with. Furthermore, phytases are envisaged to serve as potential enzymes that can produce versatile lower myo-inositol phosphates of pharmaceutical importance. Development of phytases with improved attributes is an important area being explored through genetic and protein engineering approaches, as no known phytase can fulfill all the properties of an ideal feed additive.     

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.     

Effect of exogenous factors on metabolism of phosphorous compounds in the chicken tissues

The activity of plant and microbic phytases depending on the medium pH was studied. The factors have been investigated as follows: decomposition efficiency of seed ingredients and the releasing of phytate phosphorus; the efficiency of the adsorption of phosphorus under the in situation of microbic phytase; the influence of microbic phytase feeding in the ratios with low content of accessible phosphorus for assimilation of phytate phosphorus and poultry production indices. It has been stated that the microbic phytase has a wider optimum of action depending on pH value. The microbic phytase positive action on the metabolism of phosphorus in the chicken organism has been determined through the experimental investigations.     

Two Types of Phytases (Histidine Acid Phytase and β-Propeller Phytase) in <Emphasis Type="Italic">Serratia</Emphasis> sp. TN49 from the Gut of <Emphasis Type="Italic">Batocera horsfieldi</Emphasis> (Coleoptera) Larvae

Microbial phytases play a major role in the mineralization of organic phosphorous, especially in symbiotic plants and animals. In this study, we identified two types of phytases in Serratia sp. TN49 that was harbored in the gut of Batocera horsfieldi (Coleoptera) larvae. The two phytases, an acidic histidine acid phosphatase (PhyH49) and an alkaline β-propeller phytase (PhyB49), shared low identities with known phytases (61% at most). PhyH49 and PhyB49 produced in Escherichia coli exhibited maximal activities at pH 5.0 (60°C) and pH 7.5–8.0 (45°C), respectively, and are complementary in phytate degradation over the pH range 2.0–9.0. Serratia sp. TN49 harboring both PhyH49 and PhyB49 might make it more adaptive to environment change, corresponding to the evolution trend of microorganism.     

Conformational dynamics of active site loop in Escherichia coli phytase

Phytases catalyze the release of phosphate by stepwise hydrolysis of phytate, a major source of phosphate in cereal grains, legumes, and oilseeds. Phytase improves, as a feed supplement, the nutritional quality of phytate rich diets and eventually reduce environmental pollution. Recently, phytases from enterobacteriaceae family have attracted industrial interest due to their high specific activity (2500–4000 U/mg). However, only limited information is available concerning structural dynamics of this class of enzymes. In this study, 50 nanosecond molecular dynamics simulation was performed on two Escherichia coli phytase structures (closed and open active site loop) to investigate conformational dynamics of the active site loop. Cluster analysis and principal component analysis (PCA) reveal significant difference in the conformational dynamics of active site compared to reported crystal structure. Molecular dynamic studies indicated that the movement in the active site of E. coli phytase is mainly confined by the active site loop resulted in wider opening of the loop in absence of phytate. The molecular dynamics studies highlight the possible role of loop residues as prerequisite for highly active phytases. © 2010 Wiley Periodicals, Inc. Biopolymers 93: 994–1002, 2010.