Salt effect on the pH profile and kinetic parameters of microbial phytases

The pH profiles of two microbial phytases were determined using four different general purpose buffers at different pH values. The roles of calcium chloride, sodium chloride, and sodium fluoride on activity were compared in these buffers. For Aspergillus niger phytase, calcium extended the pH range to 8.0. A high concentration of sodium chloride affected the activity of fungal phytase in the pH 3-4 range and shifted the pH optimum to 2.0 from 5.5 in Escherichia coli phytase. As expected, both of the microbial phytases were inhibited by sodium fluoride at acidic pH values. Because the Km for phytate increased nearly 2-fold for fungal phytase while Vmax increased about 75% in a high concentration of sodium chloride, it is possible that salt enhanced the product to dissociate from the active site due to an altered electrostatic environment. Modeling studies indicate that while the active site octapeptide's orientation is very similar, there are some differences in the arrangements of alpha-helices, beta-sheets, and coils that could account for the observed catalytic and salt effect differences.     

Kinetic characterization of O-phospho-L-tyrosine phosphohydrolase activity of two fungal phytases

Fungal phytases belonging to "histidine acid phosphatase" or HAP class of phosphohydrolases that catalyze the hydrolysis of phytic acid could also hydrolyze O-phospho-L-tyrosine, which is also called phosphotyrosine. Two phytases from Aspergillus niger and Aspergillus awamori with pH optima 2.5 were tested for phosphotyrosine hydrolase activity; both enzymes cleaved the phosphomonoester bond of phosphotyrosine efficiently at acidic pH. The Km for phosphotyrosine ranged from 465 to 590 microM as opposed to 135 to 160 microM for phytate. The Vmax, however, is 2-4 times higher for phosphotyrosine than it is for phytate. The catalytic efficiency of phytase for phosphotyrosine is on the same order as it is for phytate (3.5 x 10(6) to 1.6 x 10(7) M(-1) s(-1)); the pH versus activity profile for phosphotyrosine is, however, different from what it is for phytate. The temperature optima shifted 5 degrees C higher to 70 degrees C when phosphotyrosine was used as the substrate. Taken together, the kinetic data show that fungal HAPs that are known as PhyB are capable of cleaving the phosphomonoester bond in phosphotyrosine. This is the first time that phosphotyrosine phosphatase (PTPase) activity has been reported for the subgroup of HAP known as phytase.     

Unfolding and refolding of Aspergillus niger PhyB phytase: role of disulfide bridges

The role of disulfide bridges in the folding of Aspergillus niger phytase pH 2.5-optimum (PhyB) was investigated using dynamic light scattering (DLS). Guanidinium chloride (GuCl) at 1.0 M unfolded phytase; however, its removal by dialysis refolded the protein. The thiol reagent tris(2-carboxyethyl)phosphine (TCEP) reduces the refolding activity by 68%. The hydrodynamic radius (R(H)) of PhyB phytase decreased from 5.5 to 4.14 nm when the protein was subjected to 1.0 M GuCl concentration. The active homodimer, 183 kDa, was reduced to a 92 kDa monomer. The DLS data taken together with activity measurements could indicate whether refolding took place or not in PhyB phytase. The correlation between molecular mass and the state of unfolding and refolding is a very strong one in fungal phytase belonging to histidine acid phosphatase (HAP). Unlike PhyA phytase, for which sodium chloride treatment boosted the activity at 0.5 M salt concentration, PhyB phytase activity was severely inhibited under identical condition. Thus, PhyA and PhyB phytases are structurally very different, and their chemical environment in the active site and substrate-binding domain may be different to elicit such an opposite reaction to monovalent cations.     

Vanadate inhibition of fungal PhyA and bacterial AppA2 histidine acid phosphatases

The fungal PhyA protein, which was first identified as an acid optimum phosphomonoesterase (EC 3.1.3.8), could also serve as a vanadate haloperoxidase (EC 1.11.1.10) provided the acid phosphatase activity is shut down by vanadate. To understand how vanadate inhibits both phytate and pNPP degrading activities of fungal PhyA phytase and bacterial AppA2 phytase, kinetic experiments were performed in the presence and absence of orthovanadate and metavanadate under various acidic pHs. Orthovanadate was found to be a potent inhibitor at pH 2.5 to 3.0. A 50% activity of fungal phytase was inhibited at 0.56 μM by orthovanadate. However, metavanadate preferentially inhibited the bacterial AppA2 phytase (50% inhibition at 8 μM) over the fungal phytase (50% inhibition at 40 μM). While in bacterial phytase the K(m) was not affected by ortho- or metavanadate, the V(max) was reduced. In fungal phytase, both the K(m) and V(max) was lowered. The vanadate exists as an anion at pH 3.0 and possibly binds to the active center of phytases that has a cluster of positively charged Arg, Lys, and His residues below the enzymes' isoelectric point (pI). The active site fold of haloperoxidase was shown to be very similar to fungal phytase. The vanadate anions binding to cationic residues in the active site at acidic pH thus serve as a molecular switch to turn off phytase activity while turning on the haloperoxidase activity. The fungal PhyA phytase's active site housing two distinct reactive centers, one for phosphomonoesterase and the other for haloperoxidase, is a unique example of how one protein could catalyze two dissimilar reactions controlled by vanadate.     

The importance of lactic acid bacteria for phytate degradation during cereal dough fermentation

Lactic acid fermentation of cereal flours resulted in a 100 (rye), 95-100 (wheat), and 39-47% (oat) reduction in phytate content within 24 h. The extent of phytate degradation was shown to be independent from the lactic acid bacteria strain used for fermentation. However, phytate degradation during cereal dough fermentation was positively correlated with endogenous plant phytase activity (rye, 6750 mU g(-1); wheat, 2930 mU g(-1); and oat, 23 mU g(-1)), and heat inactivation of the endogenous cereal phytases prior to lactic acid fermentation resulted in a complete loss of phytate degradation. Phytate degradation was restored after addition of a purified phytase to the liquid dough. Incubation of the cereal flours in buffered solutions resulted in a pH-dependent phytate degradation. The optimum of phytate degradation was shown to be around pH 5.5. Studies on phytase production of 50 lactic acid bacteria strains, previously isolated from sourdoughs, did not result in a significant production of intra- as well as extracellular phytase activity. Therefore, lactic acid bacteria do not participate directly in phytate degradation but provide favorable conditions for the endogenous cereal phytase activity by lowering the pH value.     

Degradation of phytate by high-phytase Saccharomyces cerevisiae strains during simulated gastrointestinal digestion

Hydrolysis of extracellular phytate (InsP(6)) by high-phytase yeast strains and survival of yeast cells were studied at simulated digestive conditions using yeast peptone dextrose growth medium and wheat gruel as model meals. An in vitro digestion method was modified to better correlate with the gastric pH gradient following food intake in vivo. High-phytase yeast gave a strong reduction of InsP(6) (up to 60%) in the early gastric phase, as compared to no degradation by wild-type strains. The degree of InsP(6) degradation during digestion was influenced by the type of yeast strain, cell density, and InsP(6) concentration. Despite high InsP(6) solubility, high resistance against proteolysis by pepsin, and high cell survival, degradation in the late gastric and early intestinal phases was insignificant. Dependency on pH for phytase expression and/or activity seemed thus to be an important limiting factor. Although further studies are needed, our results show the potential of using yeast as a phytase carrier in the gastrointestinal tract.     

Reducing, radical scavenging, and chelation properties of in vitro digests of alcalase-treated zein hydrolysate

The objective of the study was to assess the antioxidant potential of alcalase-treated zein hydrolysate (ZH) during a two-stage (1 h of pepsin --> 0.5-2 h of pancreatin, 37 degrees C) in vitro digestion. Sephadex gel filtration and high-performance size exclusion chromatography were used to separate ZH into fractions. The amino acid composition, 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS(+*)) and 1,1-diphenyl-2-picrylhydrazyl (DPPH*) free radical scavenging activity, reducing power, and Cu (2+) chelation ability were tested to determine the antioxidant efficacy of ZH. Results showed that in vitro digests of ZH contained up to 16.5% free amino acids, with short peptides (<500 Da) making up the rest of the mass. The ABTS(+*) scavenging activity of ZH was decreased by 27% (P<0.05) after pepsin treatment but was fully recovered upon subsequent pancreatin digestion, while the DPPH* scavenging activity of ZH was substantially less than ABTS(+*) scavenging activity and showed a 7-fold reduction following pancreatin treatment. The reducing power of ZH increased 2-fold (P<0.05) following pancreatin digestion when compared with nondigested ZH. The ability of ZH to sequester Cu (2+) was reduced by pepsin digestion but was reestablished following pancreatin treatment. The antioxidant activity demonstrated by in vitro digests of ZH (1-8 mg/mL) was comparable to or exceeded (P<0.05) that of 0.1 mg/mL of ascorbic acid or BHA. The results suggested that dietary zein alcalase hydrolysate may have the benefit to promote the health of the human digestive tract.     

In vitro efficacies of phosphorolytic enzymes synthesized in mycelial cells of Aspergillus niger AbZ4 grown by a liquid surface fermentation

Activities of phytase, a pH 6.0 optimum nonspecific phosphomonoesterase and phosphodiesterase assayed toward bis(p-nitrophenyl)phosphate (phosphodiesterase I) and against p-nitrophenylphosphorylcholine (phosphodiesterase II), were partially purified from mycelial extracts of Aspergillus niger AbZ4 cultivated on a molasses medium by a liquid surface fermentation method. After elimination of phosphate from the medium, 7.3- and 3.5-fold enhancements in specific activities of phytase and phosphodiesterase II were observed. Efficacies of mycelial protein fractions in dephosphorylating a wheat-based broiler feed were determined in vitro according to a procedure that simulated digestion in the intestinal tract of poultry. The addition of 0.052 mg of protein from fractions, each of which was high in either pH 6.0 optimum phosphomonoesterase, phosphodiesterase I, phosphodiesterase II, or phytase per gram of a feed sample resulted in the enhancement of phosphorus release by 10, 11, 27, and 88%, respectively. In the presence of an excess of commercial phytase, the addition of the mycelial fraction high in phytase increased the dephosphorylation rate by 56%. The fraction high in phosphodiesterase II enhanced feed dephosphorylation by 8% in the presence of an excess of commercial phytase and commercial acid phosphatase.     

Differential interaction of Aspergillus niger and Peniophora lycii phytases with soil particles affects the hydrolysis of inositol phosphates

The effect of the soil environment on the mobility, stability and catalytic activity of phytase from two sources was compared, as these factors have important implications for the efficacy of enzyme function in soil. Phytase from an ascomycete fungus (Aspergillus niger) and a basidiomycete fungus (Peniophora lycii) was added to soil suspensions from three contrasting soils and activities in the solution and solid phase were monitored. The two enzymes were compared because the P. lycii phytase was known to have greater specific activity and a more acidic isoelectric point (pI) than A. niger and therefore predicted to have different adsorption characteristics. When added to soil suspensions buffered at pH 7.5, both phytases remained in solution in all of the soils. In contrast at near natural soil pH (pH 5.5), only the P. lycii phytase remained in solution, while the A. niger phytase was rapidly adsorbed to the soil solid phase. The extent of this adsorption was reduced, however, in a soil-dependent manner by prior addition of bovine serum albumin (BSA) to the soil suspensions. At the natural pH of the soil, the stability of the P. lycii phytase in soil solution was improved under sterile conditions, whereas degradation of the A. niger phytase was unaffected. Subsequently, P. lycii phytase was shown to be more effective at hydrolysing myo-inositol hexakisphosphate added to the soil. Moreover, the P. lycii phytase also hydrolysed more organic phosphate that was endogenous to a range of soils. This research indicates that the physicochemical properties of fungal phytases affect their mobility and temporal stability and their capacity to hydrolyse inositol phosphates in soil environments.     

A novel phosphorus biofertilization strategy using cattle manure treated with phytase–nanoclay complexes

The aim of this work was to evaluate the treatment of cattle manure with phytases stabilized in allophanic nanoclays as a potential novel phosphorus (P) biofertilization technology for crops grown in volcanic soils (Andisol). Furthermore, because the optimal pH for commercial phytase catalysis does not match the natural pH of manure, a complementary experiment was set up to evaluate the effect of manure inoculation with an alkaline phytase-producing bacterium. Finally, phytase-treated soil, manure, and soil–manure mixtures were evaluated for their P-supplying capacity to wheat plants grown under greenhouse conditions. Treating cattle manure with phytases stabilized in nanoclays resulted in a significant (P?≤?0.05) increase of inorganic P in soil extracts (NaOH-EDTA and Olsen). The use of phytase-treated cattle manure increased dry weights by 10 % and the P concentration by 39 % in wheat plants grown under greenhouse conditions, which is equivalent to a P fertilizer rate of about 150 kg of P per hectare. The inoculation of cattle manure with β-propeller phytase-producing bacteria led to an ～10 % increase in inorganic P in the manure extracts. However, applying inoculated manure to soil did not significantly increase wheat yield or P acquisition responses. Our results suggest that the novel approach of incubating cattle manure with phytases stabilized in nanoclay enhances the organic P cycling and P nutrition of plants grown in P-deficient soils.     

Purification and characterization of a phytate-degrading enzyme from germinated faba beans (Vicia faba Var. Alameda)

A phytate-degrading enzyme was purified approximately 2190-fold from germinated 4-day-old faba bean seedlings to apparent homogeneity with a recovery of 6% referred to the phytase activity in the crude extract. It behaves as a monomeric protein of a molecular mass of approximately 65 kDa. The phytate-degrading enzyme belongs to the acidic phytases. It exhibits a single pH optimum at 5.0. Optimal temperature for the degradation of sodium phytate is 50 degrees C. Kinetic parameters for the hydrolysis of sodium phytate are K(M) = 148 micromol L(-1) and k(cat) = 704 s(-1) at 35 degrees C and pH 5.0. The faba bean phytase exhibits a broad affinity for various phosphorylated compounds and hydrolyzes phytate in a stepwise manner. The first hydrolysis product was identified as D/L-myo-inositol(1,2,3,4,5)pentakisphosphate.     

Changes in Phytate Content in Whole Meal Wheat Dough and Bread Fermented with Phytase‐Active Yeasts

The degradation of inositol hexakisphosphate (IP6) was evaluated in whole meal wheat dough fermented with baker's yeast without phytase activity, different strains of Saccharomyces cerevisiae (L1.12 or L6.06), or Pichia kudriavzevii with extracellular phytase activity to see if the degradation of IP6 in whole meal dough and the corresponding bread could be increased by fermentation with phytase‐active yeasts. The IP6 degradation was measured after the dough was mixed for 19 min, after the completion of fermentation, and in bread after baking. Around 60–70% of the initial value of IP6 in the flour (10.02 mg/g) was reduced in the dough already after mixing, and additionally 10–20% was reduced after fermentation. The highest degradation of IP6 was seen in dough fermented with the phytase‐active yeast strains S. cerevisiae L1.12 and P. kudriavzevii L3.04. Activity of wheat phytase in whole meal wheat dough seems to be the primary source of phytate degradation, and the degradation is considerably higher in this study with a mixing time of 19 min compared with earlier studies. The additional degradation of IP6 by phytase‐active yeasts was not related to their extracellular phytase activities, suggesting that phytases from the yeasts are inhibited differently. Therefore, the highest degradation of IP6 and expected highest mineral bioavailability in whole meal wheat bread can be achieved by use of a phytase‐active yeast strain with less inhibition. The strain S. cerevisiae L1.12 is suitable for this because it was the most effective yeast strain in reducing the amount of IP6 in dough during a short fermentation time.     

Purification and characterization of three phytases from germinated lupine seeds (Lupinus albus var. amiga)

Three phytases were purified about 14200-fold (LP11), 16000-fold (LP12), and 13100-fold (LP2) from germinated 4-day-old lupine seedlings to apparent homogeneity with recoveries of 13% (LP11), 8% (LP12), and 9% (LP2) referred to the phytase activity in the crude extract. They behave as monomeric proteins of a molecular mass of about 57 kDa (LP11 and LP12) and 64 kDa (LP2), respectively. The purified proteins belong to the acid phytases. They exhibit a single pH optimum at 5.0. Optimal temperature for the degradation of sodium phytate is 50 degrees C. Kinetic parameters for the hydrolysis of sodium phytate are K(M) = 80 microM (LP11), 300 microM (LP12), and 130 microM (LP2) and k(cat) = 523 s(-1) (LP11), 589 s(-1) (LP12), and 533 s(-1) (LP2) at pH 5.0 and 35 degrees C. The phytases from lupine seeds exhibit a broad affinity for various phosphorylated compounds and hydrolyze phytate in a stepwise manner.     

Interaction between protein, phytate, and microbial phytase. In vitro studies

The interaction between protein and phytate was investigated in vitro using proteins extracted from five common feedstuffs and from casein. The appearance of naturally present soluble protein-phytate complexes in the feedstuffs, the formation of complexes at different pHs, and the degradation of these complexes by pepsin and/or phytase were studied. Complexes of soluble proteins and phytate in the extracts appeared in small amounts only, with the possible exception of rice pollards. Most proteins dissolved almost completely at pH 2, but not after addition of phytate. Phytase prevented precipitation of protein with phytate. Pepsin could release protein from a precipitate, but the rate of release was increased by phytase. Protein was released faster from a protein-phytate complex when phytase was added, but phytase did not hydrolyze protein. Protein was released from the complex and degraded when both pepsin and phytase were added. It appears that protein-phytate complexes are mainly formed at low pH, as occurs in the stomach of animals. Phytase prevented the formation of the complexes and aided in dissolving them at a faster rate. This might positively affect protein digestibility in animals.     

Moderate decrease of pH by sourdough fermentation is sufficient to reduce phytate content of whole wheat flour through endogenous phytase activity

Whole wheat bread is an important source of minerals but also contains considerable amounts of phytic acid, which is known to impair their absorption. An in vitro trial was performed to assess the effect of a moderate drop of the dough pH (around 5.5) by way of sourdough fermentation or by exogenous organic acid addition on phytate hydrolysis. It was shown that a slight acidification of the dough (pH 5.5) with either sourdough or lactic acid addition allowed a significant phytate breakdown (70% of the initial flour content compared to 40% without any leavening agent or acidification). This result highlights the predominance of wheat phytase activity over sourdough microflora phytase activity during moderate sourdough fermentation and shows that a slight drop of the pH (pH value around 5.5) is sufficient to reduce significantly the phytate content of a wholemeal flour. Mg "bioaccessibility"of whole wheat dough was improved by direct solubilization of the cation and by phytate hydrolysis.     

Characterization of phytase activity from cultivated edible mushrooms and their production substrates

Phytase is used commercially to maximize phytic acid degradation and to decrease phosphorus levels in poultry and swine manure. To determine phytase content in edible mushrooms, basidiomata of Agaricus bisporus and three specialty mushrooms (Grifola frondosa, Lentinula edodes, and Pleurotus cornucopiae) and spent mushroom substrate (SMS) were surveyed. Enzyme activity ranged from 0.046 to 0.074 unit/g of tissue for four A. bisporus types (closed and open whites and closed and open browns) grown at The Pennsylvania State University's Mushroom Test Demonstration Facility (MTDF). The addition of various nutrient supplements to phase II mushroom production substrate did not alter phytase activity in A. bisporus. Portabella mushrooms (open brown) obtained from a commercial farm had significantly higher levels of phytase activity (0.211 unit/g of tissue) compared to A. bisporus grown at the MTDF. Of the specialty mushrooms surveyed, maitake (G. frondosa) had 20% higher phytase activity (0.287 unit/g of tissue) than commercial portabella mushrooms. The yellow oyster mushroom (P. cornucopiae) ranked second in level of phytase activity (0.213 unit/g of tissue). Shiitake (L. edodes) contained the least amount of phytase in basidiomata (0.107 unit/g of tissue). Post-crop steam treatment (60 degrees C, 24 h) of SMS reduced phytase activity from 0.074 to 0.018 unit/g. Phytase was partially purified from commercially grown portabella basidiomata 314-fold with an estimated molecular mass of 531 kDa by gel filtration chromatography. The optimum pH for activity was 5.5, but appreciable phytase activity was observed over the range of pH 5.0-8.0. Partially purified A. bisporus phytase was inactivated following a 10-min incubation at > or =60 degrees C.     

~(60)Co γ射线诱变黑曲霉菌株产植酸酶的研究

研究了60 Coγ射线对黑曲霉 447 92菌种产植酸酶的诱变效应 ,筛选出产植酸酶较高的A .niger 496 1菌株 ,并初步分析了其植酸酶的基本性质。     

Use of Phytases in Ethanol Production from E‐Mill Corn Processing

Effects of phytase addition, germ, and pericarp fiber recovery were evaluated for the E‐Mill dry grind corn process. In the E‐Mill process, corn was soaked in water followed by incubation with starch hydrolyzing enzymes. For each phytase treatment, an additional phytase incubation step was performed before incubation with starch hydrolyzing enzymes. Germ and pericarp fiber were recovered after incubation with starch hydrolyzing enzymes. Preliminary studies on phytase addition resulted in germ with higher oil (40.9%), protein (20.0%), and lower residual starch (12.2%) contents compared to oil (39.1%), protein (19.2%), and starch (18.1%) in germ from the E‐Mill process without phytase addition. Phytase treatment resulted in lower residual starch contents in pericarp fiber (19.9%) compared to pericarp fiber without phytase addition (27.4%). Results obtained led to further investigation of effects of phytase on final ethanol concentrations, germ, pericarp fiber, and DDGS recovery. Final ethanol concentrations were higher in E‐Mill processing with phytase addition (17.4% v/v) than without addition of phytase (16.6% v/v). Incubation with phytases resulted in germ with 4.3% higher oil and 2.5% lower residual starch content compared to control process. Phytase treatment also resulted in lower residual starch and higher protein contents (6.58 and 36.5%, respectively) in DDGS compared to DDGS without phytase incubations (8.14 and 34.2%, respectively). Phytase incubation in E‐Mill processing may assist in increasing coproduct values as well as lead to increased ethanol concentrations.     

Degradation of HMW Glutenins During Wheat Sourdough Fermentations

Bakeries use sourdoughs to improve bread properties such as flavor and shelf life. The degradation of gluten proteins during fermentation may, however, crucially alter the gluten network formation. We observed changes that occurred in the HMW glutenins during wheat sourdough fermentations. As fermentation starters, we used either rye sourdough or pure cultures of lactobacilli and yeast. In addition, we incubated wheat flour (WF) in the presence of antibiotics under different pH conditions. The proteolytic activities of cereal and sourdough‐derived proteinases were studied with edestin and casein. During sourdough fermentations, most of the highly polymerized HMW glutenins degraded. A new area of alcohol‐soluble proteins (≈30.000 MW) appeared as a result of the proteolytic breakdown of gluten proteins. Very similar changes were observable as WF was incubated in the presence of antibiotics at pH 3.7. Cereal and sourdough‐derived proteinases hydrolyzed edestin at pH 3.5 but showed no activity at pH 5.5. An aspartic proteinase inhibitor (pepstatin A) arrested 88–100% of the activities of sourdough enzymes. According to these results, the most active proteinases in wheat sourdoughs were the cereal aspartic proteinases. Acidic conditions present in sourdoughs create an ideal environment for cereal aspartic proteinases to be active against gluten proteins.     

Iron availability from whey protein hydrogels: an in vitro study

The influence of whey protein hydrogel microstructure, filamentous versus particulate, on iron delivery was studied under different conditions, including simulated gastrointestinal conditions. Experiments were initially conducted to determine the impact of pH and enzymes on iron release. The results show that different iron release profiles can be obtained from filamentous and particulate gels. Particulate gels released more iron than filamentous gels at acidic pH, but the opposite was observed at alkaline pH. In the presence of pepsin at pH 1.2 or pancreatin at pH 7.5, both gel types showed increased protein hydrolysis, but only filamentous gels showed increased iron release, suggesting that matrix structure plays an important role in iron delivery. A dissolution test was carried out under gastrointestinal conditions to mimic the in vivo dissolution process. Filamentous gel released most of its iron during the intestinal phase of a simulated digestion, hence protecting iron during its transit in the gastric zone. Absorption of iron by the Caco-2 system, used to estimate intestinal absorption, revealed that filamentous gels favored intracellular iron absorption. These results suggest that filamentous gels show promise as matrices for transporting iron and promoting its absorption and therefore should be of major interest in the development of innovative functional foods.