Lycii Phytase Research Articles

Performance of Microbial Phytases for Gastric Inositol Phosphate Degradation

Microbial phytases catalyze dephosphorylation of phytic acid, thereby potentially releasing chelated iron and improving human iron absorption from cereal-based diets. For this catalysis to take place in vivo, the phytase must be robust to low pH and proteolysis in the gastric ventricle. This study compares the robustness of five different microbial phytases, evaluating thermal stability, activity retention, and extent of dephosphorylation of phytic acid in a simulated low-pH/pepsin gastric environment and examines secondary protein structural changes at low pH via circular dichroism. The Peniophora lycii phytase was found to be the most thermostable, but the least robust enzyme in gastric conditions, whereas the Aspergillus niger and Escherichia coli phytases proved to be most resistant to gastric conditions. The phytase from Citrobacter braakii showed intermediate robustness. The extent of loss of secondary structure at low pH correlated positively with the extent of activity loss at low pH.

Organic Anion–Driven Solubilization of Precipitated and Sorbed Phytate Improves Hydrolysis by Phytases and Bioavailability to Nicotiana tabacum

Improved plant access to native soil phosphorus (P) species such as phytate (metal ion derivatives of myo-inositol hexakisphosphate (IHP)) could minimize agricultural dependence on nonrenewable mineral phosphates and reduce surface water pollution. Nicotiana tabacum plant lines with unique organic anion (OA) and phytase production patterns were used to investigate the effect of OA-driven solubilization on the bioavailability of precipitated and sorbed IHP. Organic anions released IHP sorbed to goethite (Gt) by chelation or reductive dissolution mechanisms in the order: ascorbate > citrate > oxalate > pyruvate > acetate. Transgenic tobacco overexpressing Peniophora lycii phytase (PHY) and a MATE-type citrate transporter (CIT) exuded 2.2- to 2.6-fold higher OA compared with that of wild-type plants. The PHY plants had 33-fold higher exudate phytase activity (6.0 × 10−2 nkat plant−1 day−1) compared with those of wild-type and CIT plants, produced the largest zone of iron-IHP hydrolysis in agar media, and incorporated the most shoot P (2.3 μg) when grown on Gt-IHP. Plants grown on IHP at 2× Gt saturation (0.26 mmol/L) were 10-fold higher in shoot P compared with the 1× Gt condition (0.13 mmol/L IHP) with PHY plants approaching excess P status (1 % shoot P). The addition of Gt diminished shoot P in plants grown without P (−70 to −80 %) and with phosphate (−50 %) or IHP (−100 %); the exception was the high citrate–exuding plant line (21 nmol citrate plant−1 day−1), for which phosphate uptake was only 20 % reduced. Plant OA production mitigates P inhibition by Gt when weakly sorbing phosphate species are supplied or when high phytase production by plants can maximize the hydrolysis of IHP.

In vitro assessment of the effects of phytate and phytase on nitrogen and phosphorus bioaccessibility within fish digestive tract

The aim of the present work was to assess different possible interactions between phytate (IP6), dietary protein and digestive proteases, as well as the preventive effect of phytase in the formation of protein-IP6 complexes, through the use of a gastrointestinal model simulating conditions existing in the digestive tract of rainbow trout ( Oncorhynchus mykiss). Two commercial phytases, expressed in P. lycii and E. coli, were evaluated. Fungal phytase showed an optimum at pH 5.5 and bacterial phytase showed two peaks at pH 2.5 and 4.5. These phytases evidenced different resistance against gastric proteolytic degradation; E. coli phytase retained 90% of its initial activity after 4 h incubation in the presence of either porcine pepsin or fish stomach proteases, while the dephosphorylation activity of P. lycii phytase was reduced by 90% (P<0.001). The presence of IP6 determined a reduction of 80% in protein solubility (P<0.001) when casein was used as substrate of fish acid proteases. The formation of IP6-protein complexes resulted in lower amino acids liberation from casein (P<0.05). Addition of phytase to soybean meal prevented the formation of such IP6-protein complexes and improved amino acid release by 60% (P<0.01). In contrast, IP6 had not effect on protein solubilisation or amino acid liberation during the intestinal digestion. Under conditions simulating gastric digestion a great amount of IP6 was combined to either porcine pepsin or soluble protein in fish stomach crude extract, precipitating 68 and 100% of such proteins, respectively. A similar, but lower reduction in soluble protein from either pancreatin or fish intestinal extract was observed when simulating intestinal digestion (47 and 33%, respectively). The formation of IP6-protein complexes reduced the activity of all gastric and intestinal proteases assayed (P<0.01) with the exception of chymotrypsin. A positive effect (P<0.05) of low pH on IP6-dephosphorylation was measured when the effect of bacterial phytase (2500 FTU/kg) on the release of soluble P from native IP6 present in soybean meal was tested under different digestion temperatures (6 and 16 °C) and pH (2.0, 3.0 and 4.0). A positive effect of temperature was also observed when the reaction was carried out at pH 2.0 and 3.0 (P<0.001), but not at 4.0, suggesting the existence of a limit in the solubility of IP6 and therefore in its availability for phytase action at this last pH value.

Effects of phytase and 25-hydroxyvitamin D3 inclusions on the performance, mineral balance and bone parameters of grower–finisher pigs fed low-phosphorus diets

Two experiments, a performance experiment and a mineral balance study, were conducted on grower–finisher pigs (42 to 101 kg live weight) to investigate the effects of Peniophora lycii phytase enzyme and 25-hydroxyvitamin D3 (25-OHD3) on growth performance, carcass characteristics, nutrient retention and excretion, and bone and blood parameters. The two experiments were designed as a 2 × 2 factorial (two levels of phytase and two levels of 25-OHD3). The four diets were T1, low-phosphorous diet; T2, T1 + phytase; T3, T1 + 25-OHD3 and T4, T1 + phytase + 25-OHD3 diet. In all, 25 μg of 25-OHD3 was used to replace 1000 IU of vitamin D3 in diets T3 and T4. Diets were pelleted (70°C) and formulated to contain similar concentrations of energy (13.8 MJ DE/kg), lysine (9.5 g/kg) and digestible phosphorus (P; 1.8 g/kg). Neither the inclusion of phytase nor 25-OHD3 in the diet had any effect on pig performance. There was an interaction between phytase and 25-OHD3 on calcium (Ca) and P retention (P < 0.01) and on the apparent digestibility of ash (P < 0.01), P (P < 0.001) and Ca (P < 0.001). Pigs offered phytase diets only, had a higher retention of Ca and P and digestibility of ash (P < 0.01), P (P < 0.001) and Ca (P < 0.01) compared with pigs offered unsupplemented diets. However, when the combination of phytase and 25-OHD3 were offered, no effects were detected compared with 25-OHD3 diets only. Pigs fed phytase diets had higher bone ash (P < 0.01), bone P (P < 0.01) and bone Ca (P < 0.05) concentrations compared with pigs offered non-phytase diets. In conclusion, pigs offered phytase diets had a significantly increased bone ash, Ca and P than pigs offered unsupplemented phytase diets. However, there was no advantage to offering a combination of phytase and 25-OHD3 on either bone strength or mineral status compared to offering these feed additives separately.

Interrelationship of Steric Stabilization and Self-Crowding of a Glycosylated Protein

In the eukaryotic cell, protein glycosylation takes place in the crowded environment of the endoplasmatic reticulum. With the purpose of elucidating the impact of high concentration on the interactions of glycoproteins, we have conducted a series of small-angle x-ray scattering experiments on the heavily glycosylated enzyme Peniophora lycii phytase (Phy) and its deglycosylated counterpart (dgPhy). The small-angle x-ray scattering data were analyzed using an individual numerical form factor for each of the two glycoforms combined with two structure factors, a hard sphere and a screened coulomb potential structure factor, respectively, as determined by ab initio analysis. Based on this data analysis, three main conclusions could be drawn. First, at comparable protein concentrations (mg/ml), the relative excluded volume of Phy was ∼75% higher than that of dgPhy, showing that the glycans significantly increase excluded-volume interactions. Second, the relative excluded volume of dgPhy increased with concentration, as expected; however, the opposite effect was observed for Phy, where the relative excluded volume decreased in response to increasing protein concentration. Third, a clear difference in the effect of salinity on the excluded-volume interactions was observed between the two glycol forms. Although the relative excluded volume of dgPhy decreased with increasing ionic strength, the relative excluded volume of Phy was basically insensitive to increased salinity. We suggest that protrusion forces from the glycans contribute to steric stabilization of the protein, and that glycosylation helps to sustain repulsive electrostatic interactions under crowded conditions. In combination, this aids in stabilizing high concentrations of glycosylated proteins.

The Effect of Glycosylation on Interparticle Interactions and Dimensions of Native and Denatured Phytase

Glycosylation affects the physical properties of proteins in a number of ways including solubility and aggregation behavior. To elucidate the mechanism underlying these effects, we have measured second virial coefficients ( A 2 ) of the heavily glycosylated pheniophora lycii phytase (Phy) and its enzymatically deglycosylated counterpart (dgPhy) in native and in denatured form by means of small angle x-ray scattering. The measured A 2-values show that the native forms of Phy and dgPhy are equally repulsive at the studied pH 8 where A 2 equals 10.9 ± 0.1 × 10 4 mL mol g −2. However, when thermally denatured, the A 2 of dgPhy decreases to 9.0 ± 0.2 × 10 4 mL mol g −2 whereas it remained unchanged for Phy. In accord with earlier investigations, the p( r) -function measured here suggested that the glycans did not affect the peptide structure of the native protein. Conversely, glycosylation markedly changed the structure of thermally denatured protein. This was evident from the radius of gyration, which increased by 32% for Phy and only 11% for dgPhy on denaturation. We suggest that this expanding effect of the glycans on the denatured protein conformation relies on steric hindrance that limits the range of torsion angles available to the polypeptide.

Peniophora lycii phytase is stabile and degrades phytate and solubilises minerals in vitro during simulation of gastrointestinal digestion in the pig

Microbial phytases (EC 3.1.3) are widely used in diets for monogastric animals to hydrolyse phytate present in the feed and thereby increase phosphorus and mineral availability. Previous work has shown that phytate solubility is strongly affected by calcium in the feed and by pH in the gastrointestinal (GI) tract, which may have an effect on phytase efficacy. An in vitro model simulating the GI tract of pigs was used to study the survival of Peniophora lycii phytase and the effect of the phytase on phytate degradation, inositol phosphate formation and mineral solubilisation during in vitro digestion of a 30:70 soybean meal/maize meal blend with different calcium levels. The phytase retained 76 and 80% of its initial activity throughout the gastric in vitro digestion. Total phytate hydrolysis by P. lycii phytase was in the same range at total calcium levels of 1.2 and 6.2 mg g(-1) dry matter (DM), despite very large differences in phytate solubility at these calcium levels. However, at 11.2 and 21.2 mg Ca g(-1) DM, phytate hydrolysis was significantly lower. The amount of soluble mineral was generally increased by P. lycii phytase. Stability of P. lycii phytase during gastric digestion was not found to be critical for phytate hydrolysis. Furthermore, original phytate solubility was not an absolute requirement for phytate degradation; phytate solubility seemed to be in a steady state, allowing insoluble phytate to solubilise as soluble phytate was degraded. This is new and interesting knowledge that adds to the current understanding of phytate-phytase interaction. Copyright © 2007 Society of Chemical Industry.

Effects of Various Phytase Concentrations in Diets with Low-phytate Corn on Broiler Chick Performance and Nutrient Use

Research indicates a reduction in phosphorus content of broiler excreta with the supplementation of phytase to corn-soybean meal based diets or with the use of Low Phytate Corn (LPC). This study examined how broiler chicks are impacted by phytase supplementation to a LPC [0.136% Phytate Phosphorus (PP) by analysis] based diet. Different dietary inclusion levels from a 2,500 or a 5,000 FYT/g of Peniophora lycii phytase product were used as experimental treatments. Following a six day pretest, 576 broiler chicks were randomly assigned to one of 12 dietary treatments, using eight diet replicates and six birds per cage. Live performance, tibia ash, mineral digestibility and apparent metabolizable energy were determined. The percent of phosphorus that may have been spared due to each phytase treatment was calculated using response curves created with increasing levels of monocalcium phosphate. Results demonstrated that phytase supplementation to a LPC based diet had a positive impact on broiler chick growth. However, this impact is expressed to a lesser extent than would be expected for normal corn. In addition, phytase efficacy did not vary based on the concentration of phytase product, likely due to adequate mix uniformity.

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.

High level expression of a synthetic gene encoding Peniophora lycii phytase in methylotrophic yeast Pichia pastoris

Phytase is widespread in nature. It has been used as a cereal feed additive that can enhance the phosphorus and mineral absorption in monogastric animals to reduce the level of phosphorus output in manure. Phytase of Peniophora lycii is a 6'-phytase, which owns high specific activity. To achieve a high expression level of 6'-phytase in Pichia pastoris, the 1,230-bp phytase gene of P. lycii was synthesized and optimized for codon usage, G+C content, as well as mRNA secondary structures. The gene constructs containing wild type or modified phytase gene coding sequences under the control of the highly-inducible alcohol oxidase gene (AOX1) promoter, the synthetic signal peptide (designated MF4I), which is a codon-modified Saccharomyces cerevisiae mating factor alpha-prepro-leader sequence, were used to transform P. pastoris. The P. pastoris strain that expressed the modified phytase gene (phy-pl-sh) with MF4I sequence produced 12.2 g phytase per liter of fluid culture, with the phytase activity of 10,540 U ml(-1). The yield of the modified phytase gene, with bias codon usage and MF4I signal, is 4.4 times higher than that of the wild type gene with MF4I signal and 13.6 times higher than that of the wild type gene with wild type S. cerevisiae signal. The recombinant phytase had one optimum pH (pH 4.5) and an optimum temperature of 50 degrees C. The P. pastoris strain expressed the modified 6-phytase gene, with the MF4I signal peptide showing great potential as a commercial phytase production system.

Interrelationships of Glycosylation and Aggregation Kinetics for Peniophora lycii Phytase

The kinetics of thermally induced aggregation of the glycoprotein Peniophora lycii phytase (Phy) and a deglycosylated form (dgPhy) was studied by dynamic (DLS) and static (SLS) light scattering. This provided a detailed insight into the time course of the formation of small aggregates ( approximately 10-100 molecules) of the enzyme. The thermodynamic stability of the two forms was also investigated using scanning calorimetry (DSC). It was found that the glycans strongly promoted kinetic stability (i.e., reduced the rate of irreversible denaturation) while leaving the equilibrium denaturation temperature, T(d), defined by DSC, largely unaltered. At pH 4.5-5.0, for example, dgPhy aggregated approximately 200 times faster than Phy, even though the difference in T(d) was only 1-3 degrees C. To elucidate the mechanism by which the glycans promote kinetic stability, we measured the effect of ionic strength and temperature on the aggregation rate. Also, the second virial coefficients (B(22)) for the two forms were measured by SLS. These results showed that the aggregation rate of Phy scaled with the concentration of thermally denatured protein. This suggested first-order kinetics with respect to the concentration of the thermally denatured state. A similar but less pronounced correlation was found for dgPhy, and it was suggested that while the aggregation process for the deglycosylated form is dominated by denatured protein, it also involves a smaller contribution from associating molecules in the native state. The measurements of B(22) revealed that dgPhy had slightly higher values than Phy. This suggests that dgPhy interacts more favorably with the buffer than Phy and hence rules out strong hydration of the glycans as the origin of their effect on the kinetic stability. On the basis of this and the effects of pH and ionic strength, we suggest that the inhibition of aggregation is more likely to depend on steric hindrance of the glycans in the aggregated form of the protein.

Phytase activity along the digestive tract of the broiler chick: A comparative study of an Escherichia coli-derived and Peniophora lycii phytase

Residual activity of an Escherichia coli-derived phytase and a commercially available Peniophora lycii phytase along the digestive tract of broiler chicks was compared in order to evaluate their relative resistance to hydrolysis in the digestive tract. Seventy-two 7-d-old male broiler chicks were grouped by weight into six blocks of three cages with four birds per cage. Three corn-soybean meal-based diets were randomly assigned to cages within each block. The three diets were a low P diet containing 3.9 g P kg-1 diet; and low P diet plus either E. coli-derived phytase or the P. lycii phytase at 1000 units kg-1 of feed. The chicks were fed experimental diets from 8 to 22 d of age. At the end of the study, chicks were killed and the contents of the crop, proventriculus and gizzard, jejunum and ileum were collected, freeze-dried, ground and analyzed for phytase activity. Escherichia coli-derived phytase had more residual activity at the proventriculus and gizzard, jejunum and ileum when compared with the P. lycii phytase (P &lt; 0.0001). The E. coli-derived phytase may be more resistant to hydrolysis in the digestive tract when compared with the P. lycii phytase. Key words: Broiler chick, Escherichia coli phytase, residual phytase activity

Effect of phytase inclusion and calcium/phosphorus ratio on the performance and nutrient retention of grower–finisher pigs fed barley/wheat/soya bean meal‐based diets

AbstractTwo experiments were conducted to evaluate the effects of phosphorus (P) level and calcium (Ca)/total P (tP) ratio on the efficacy of microbial phytase. Experiment 1 examined the effects of P concentration and microbial phytase inclusion on mineral excretion and pig performance, while experiment 2 examined the effects of Ca/tP ratio and microbial phytase inclusion on mineral excretion and pig performance. In experiment 1, nutrient and mineral digestibility (n = 4) and growth performance (n = 12) were determined in pigs individually fed diets containing (T1) 5.5 g kg−1 tP, 2.3 g kg−1 available P (aP) and 8.0 g kg−1 Ca, (T2) 5.5 g kg−1 tP, 2.3 g kg−1 aP, 8.0 g kg−1 Ca and 750 FYT kg−1 Peniophora lycii phytase, (T3) 4.3 g kg−1 tP, 1.4 g kg−1 aP and 8.0 g kg−1 Ca and (T4) 4.3 g kg−1 tP, 1.4 g kg−1 aP, 8.0 g kg−1 Ca and 750 FYT kg−1 P lycii phytase. In experiment 2, nutrient and mineral digestibility (n = 4) and growth performance (n = 12) were determined in pigs individually fed diets containing (TT1) 4.3 g kg−1 tP and 8.0 g kg−1 Ca, (TT2) 4.3 g kg−1 tP, 8.0 g kg−1 Ca and 750 FYT kg−1 P lycii phytase, (TT3) 4.3 g kg−1 tP and 5.0 g kg−1 Ca and (TT4) 4.3 g kg−1 tP, 5.0 g kg−1 Ca and 750 FYT kg−1 P lycii phytase. All diets were formulated, using standard feeding values for the ingredients, to have similar concentrations of digestible energy (DE) and lysine. In experiment 1, pigs offered the low‐P diets had significantly lower P intake (P &lt; 0.001), faecal P excretion (P &lt; 0.05), Ca intake (P &lt; 0.05) and faecal Ca excretion (P &lt; 0.05) compared with pigs given the adequate‐P diets. These pigs also had significantly lower daily gain (P &lt; 0.01), feed intake (P &lt; 0.05) and feed conversion ratio (FCR) (P &lt; 0.05). The inclusion of phytase in both the adequate‐ and low‐P diets increased the digestibility of energy (P &lt; 0.05) and Ca (P &lt; 0.01) but had no effect on pig performance. In experiment 2, lowering the Ca/tP ratio from 1.85:1 to 1.15:1 increased the DE content of the diet (P &lt; 0.05). The inclusion of phytase increased (P &lt; 0.05) the digestibility of protein (0.874 versus 0.840, SEM 0.009) and Ca (0.427 versus 0.380, SEM 0.019) as well as the DE content of the diet (14.47 versus 14.26 MJ kg−1, SEM 0.073). There was a significant ratio × phytase interaction (P &lt; 0.5) for P digestibility. Microbial phytase significantly increased P digestibility when added to the 1.15:1 ratio diet but had no effect when added to the 1.85:1 ratio diet. The inclusion of microbial phytase increased feed intake (2.16 versus 2.00 kg day−1, SEM 0.05; P &lt; 0.05) and weight gain (0.893 versus 0.818 kg day−1, SEM 0.022; P &lt; 0.05). Lowering the Ca/tP ratio resulted in a significant improvement in FCR (2.32 versus 2.40 kg kg−1, SEM 0.03; P &lt; 0.05). In conclusion, the beneficial effects of microbial phytase supplementation of pig diets are adversely affected by a wide Ca/tP ratio.© 2002 Society of Chemical Industry