A genetically engineered Escherichia coli phytase improves nutrient utilization, growth performance, and bone strength of young swine fed diets deficient in available phosphorus. (73/247)

A 28-d experiment was conducted using 126 crossbred barrows to evaluate the addition of a genetically engineered Escherichia coli phytase to diets that were 0.15% deficient in available P. Growth performance, bone strength, ash weight, and the apparent absorption of P, Ca, Mg, N, energy, DM, Zn, Fe, and Cu were the response criteria. The pigs (2 pigs/pen) averaged 7.61 kg of BW and 30 d of age initially. The low-P basal diet was supplemented with 0, 100, 500, 2,500, or 12,500 units (U) of E. coli phytase/kg of diet, or 500 U of Peniophora lycii phytase/kg of diet. The positive control (PC) diet was adequate in available P. Pigs were fed the diets in meal form. Fecal samples were collected from each pig from d 22 to 27 of the experiment. There were linear and quadratic increases (P < 0.001) in 28-d growth performance (ADFI, ADG, and G:F), bone breaking strength and ash weight, and the apparent absorption (g/d and %) of P, Ca, and Mg (P < or = 0.01 for quadratic) with increasing concentrations of E. coli phytase. Pigs fed the low-P diets containing 2,500 or 12,500 U/kg of E. coli phytase had greater (P < or = 0.01 or P < 0.001, respectively) values for growth performance, bone breaking strength and ash weight, and the apparent absorption (g/d and %) of P, Ca, and Mg than pigs fed the PC diet. The addition of E. coli phytase did not increase the apparent percentage absorption of N, GE, DM, Zn, Fe, or Cu. There were no differences in the efficacy of the E. coli or P. lycii phytase enzymes at 500 U/kg of low-P diet for any criterion measured. In conclusion, there were linear increases in growth performance, bone breaking strength and ash weight, and the apparent absorption of P, Ca, and Mg with increasing addition of E. coli phytase up to 12,500 U/kg of diet. Also, all of these criteria were greater for pigs fed the low-P diets containing 2,500 or 12,500 U of E. coli phytase/kg than for pigs fed the PC diet. The addition of 500, 2,500, or 12,500 U of E. coli phytase/kg of low-P diet reduced P excretion (g/d) in manure by 35, 42, and 61%, respectively, compared with pigs fed the PC diet.  (+info)

Effect of graded doses and a high dose of microbial phytase on the digestibility of various minerals in weaner pigs. (74/247)

An experiment with 224 weaner pigs (initial BW of 7.8 kg) was conducted to determine the effect of dose of dietary phytase supplementation on apparent fecal digestibility of minerals (P, Ca, Mg, Na, K, and Cu) and on performance. Four blocks, each with 8 pens of 7 pigs, were formed. Eight dietary treatments were applied to each block in the 43-d experiment: supplementation of 0 (basal diet), 100, 250, 500, 750, 1,500, or 15,000 phytase units (FTU) or of 1.5 g of digestible P (dP; monocalcium phosphate; positive control) per kilogram of feed. The basal diet, with corn, barley, soybean meal, and sunflower seed meal as the main components, contained 1.2 g of dP per kilogram of feed. Fresh fecal grab samples were collected in wk 4 and 5 of the experiment. Average daily feed intake, ADG, G:F, and digestibility of all of the minerals increased (P < 0.001) with increasing phytase dose. Digestibility of P increased from 34% in the basal diet to a maximum of 84% in the diet supplemented with 15,000 FTU, generating 1.76 g of dP per kilogram of feed. At this level, 85% of the phytate phosphorus was digested, compared with 15% in the basal diet. Compared with the basal diet, digestibility of the monovalent minerals increased maximally at 15,000 FTU, from 81 to 92% (Na) and from 76 to 86% (K). In conclusion, phytase supplementation up to a level of 15,000 FTU/kg of a dP-deficient diet improved performance of weaner pigs and digestibility of minerals, including monovalent minerals. Up to 85% of the phytate-P was digested. Thus, dietary phytase supplementation beyond present day standards (500 FTU/kg) could further improve mineral use and consequently reduce mineral output to the environment.  (+info)

The intracellular fate of a recombinant protein is tissue dependent. (75/247)

Recombinant proteins directed to the secretory pathway in plants require a signal peptide for entry into the endoplasmic reticulum. In the absence of further targeting information, such proteins are generally secreted via the default pathway to the apoplast. This has been well documented in protoplasts and leaf tissue, but the trafficking of recombinant proteins in seeds and other storage tissues has rarely been investigated. We used Aspergillus niger phytase as a model glycoprotein to compare the intracellular fate of a recombinant protein in the leaves and seeds of rice (Oryza sativa). Using fluorescence and electron microscopy we showed that the recombinant protein was efficiently secreted from leaf cells as expected. In contrast, within endosperm cells it was retained in endoplasmic reticulum-derived prolamin bodies and protein storage vacuoles. Consistent with our immunolocalization data, the phytase produced in endosperm cells possessed oligomannose and vacuolar-type N-glycans [Man(3)(Xyl)(Fuc)GlcNAc(2)], whereas the phytase produced in leaves contained predominantly secretion-type N-glycans [GlcNAc(2)Man(3)(Xyl)(Fuc)GlcNAc(2)]. The latter could not be detected in preparations of the endosperm-derived phytase. Our results show that the intracellular deposition and modification of a recombinant protein is tissue dependent.  (+info)

Shifting the pH profile of Aspergillus niger PhyA phytase to match the stomach pH enhances its effectiveness as an animal feed additive. (76/247)

Environmental pollution by phosphorus from animal waste is a major problem in agriculture because simple-stomached animals, such as swine, poultry, and fish, cannot digest phosphorus (as phytate) present in plant feeds. To alleviate this problem, a phytase from Aspergillus niger PhyA is widely used as a feed additive to hydrolyze phytate-phosphorus. However, it has the lowest relative activity at the pH of the stomach (3.5), where the hydrolysis occurs. Our objective was to shift the pH optima of PhyA to match the stomach condition by substituting amino acids in the substrate-binding site with different charges and polarities. Based on the crystal structure of PhyA, we prepared 21 single or multiple mutants at Q50, K91, K94, E228, D262, K300, and K301 and expressed them in Pichia pastoris yeast. The wild-type (WT) PhyA showed the unique bihump, two-pH-optima profile, whereas 17 mutants lost one pH optimum or shifted the pH optimum from pH 5.5 to the more acidic side. The mutant E228K exhibited the best overall changes, with a shift of pH optimum to 3.8 and 266% greater (P < 0.05) hydrolysis of soy phytate at pH 3.5 than the WT enzyme. The improved efficacy of the enzyme was confirmed in an animal feed trial and was characterized by biochemical analysis of the purified mutant enzymes. In conclusion, it is feasible to improve the function of PhyA phytase under stomach pH conditions by rational protein engineering.  (+info)

Avian multiple inositol polyphosphate phosphatase is an active phytase that can be engineered to help ameliorate the planet's "phosphate crisis". (77/247)

Contemporary phytase research is primarily concerned with ameliorating the problem of inadequate digestion of inositol hexakisphosphate (phytate; InsP6) in monogastric farm animal feed, so as to reduce the pollution that results from the high phosphate content of the manure. In the current study we pursue a new, safe and cost-effective solution. We demonstrate that the rate of hydrolysis of InsP6 by recombinant avian MINPP (0.7 micromol/mg protein/min) defines it as by far the most active phytase found to date in any animal cell (the corresponding activity of recombinant mammalian MINPP is only 0.006 micromol/mg protein/min). Although avian MINPP has less than 20% sequence identity with microbial phytases, we create a homology model of MINPP in which it is predicted that the structure of the phytase active site is well-conserved. This model is validated by site-directed mutagenesis and by use of a substrate analogue, scyllo-InsP6, which we demonstrate is only a weak MINPP substrate. In a model chicken cell line, we overexpressed a mutant form of MINPP that is secretion-competent. This version of the enzyme was actively secreted without affecting either cell viability or the cellular levels of any inositol phosphates. Our studies offer a genetic strategy for greatly improving dietary InsP6 digestion in poultry.  (+info)

Effect of a novel phytase on growth performance, bone ash, and mineral digestibility in nursery and grower-finisher pigs. (78/247)

To compare the effectiveness of 2 phytase enzymes (Phyzyme and Natuphos), growth performance, fibula ash, and Ca and P digestibilities were evaluated in 4 studies. The first 3 studies used 832 pigs (i.e., 288 in the nursery phase, initial BW 8.1 kg; 288 in the grower phase, initial BW 24.2 kg; and 256 in the finisher phase, initial BW 57.8 kg) and were carried out over periods of 28, 42, and 60 d, respectively. Dietary treatments in each study consisted of a positive control [available P (aP) at requirement level]; negative control (Ca remained as in the positive control, and aP at 66, 56, and 40% of the requirement for the nursery, grower, and finisher studies, respectively); negative control plus graded levels of Phyzyme [250, 500, 750, or 1,000; measured as phytase units (FTU)/kg] or Natuphos (250 and 500 FTU/kg for the nursery and grower studies, or 500 and 1,000 FTU/kg for the finisher study) plus a very high dose of Phyzyme (tolerance level, at 10,000 FTU/kg) in the nursery and grower experiments. Across the 3 studies, there was no effect of any dietary treatment on ADFI, but the negative control reduced ADG (10%), G:F (7%), and bone ash (8%) compared with the positive control. In the nursery study, phytase addition increased G:F and bone ash linearly (P < 0.01). In the grower study, phytase increased ADG, G:F, and bone ash linearly (P < 0.01). In the finisher study, phytase addition increased ADG and bone ash linearly (P < 0.01) and increased G:F quadratically (P < 0.05); G:F was, on average, 5% greater (P < 0.05) with Phyzyme than with Natuphos. The fourth study was conducted to investigate the P-releasing efficacy of the 2 phytases. The apparent fecal digestibility of P, measured with chromic oxide as an external marker in 35 pigs (55.9 kg of BW), showed that aP increased (P < 0.001) by 0.17 and 0.06 g (+/- 0.023) per 100 FTU consumed for Phyzyme and Natuphos, respectively. Also, Phyzyme at 10,000 FTU/kg was not detrimental to animal health or growth performance. At doses intended for commercial conditions, Phyzyme proved to be effective in releasing phytate bound P from diets, with an efficacy superior to a commercially available enzyme.  (+info)

Recombinant, rice-produced yeast phytase shows the ability to hydrolyze phytate derived from seed-based feed, and extreme stability during ensilage treatment. (79/247)

When fresh rice leaves producing yeast Schwanniomyces occidentalis phytase were grounded and mixed with the whole extract of seed-based feed for pigs, the release of orthophosphate increased significantly. More specifically, phytate, a major source of phosphorus in the seeds, was hydrolyzed by heterologous phytase. Moreover, when transgenic rice plants were ensiled for up to 12 weeks, no decrease in the phytase activity of the heterologous enzyme was observed. This result strongly suggests that transgenic rice plants producing yeast phytase can be stored as silage without any loss of enzyme activity until usage as a feed additive.  (+info)

Effect of micronized pea and enzyme supplementation on nutrient utilization and manure output in growing pigs. (80/247)

An experiment was done to determine manure output, N and P excretion, and apparent digestibilities of AA, CP, P, and DM in growing pigs fed barley-based diets containing micronized or raw peas with or without supplementation with enzyme containing primarily beta-glucanase and phytase (Biogal S+). Eight barrows (21.5 +/- 1.2 kg of initial BW) fitted with T-cannulas at the distal ileum were used in a 40-d trial and housed in metabolism cages. Pigs were assigned in a replicated 4 x 4 Latin square design to 4 experimental diets: 1) barley-raw peas control (BRP), 2) barley-micronized peas (BMP), 3) BRP plus enzyme, and 4) BMP plus enzyme (BMP+E). Pigs received 2.6 times maintenance energy requirements based on BW at the beginning of each experimental period. During each experimental period, pigs were acclimatized to their respective diets for 5 d followed by a 3-d period of total fecal and urine collection and another 2-d period of ileal digesta collection. Samples were analyzed for DM, AA (diets and digesta only), N, and P. Wet fecal output of BRP plus enzyme-fed pigs tended to be lower (P = 0.07) than the amount produced by BMP-fed pigs. The amounts of dry feces and urine produced were not different among treatments (P > 0.10). Supplementing the BRP and BMP diet with enzyme increased (P = 0.002) the daily P retained per pig. Pigs fed the enzyme-supplemented diets tended to have lower (P = 0.06) fecal P excretion and greater urinary P excretion (P = 0.001) compared with pigs fed the nonsupplemented diets, but total P excretion was not influenced by diet (P > 0.10). Pigs fed the BMP+E diet retained more (P = 0.006) N per day than pigs fed the BMP diet. However, N excretion was not influenced by dietary treatment (P > 0.10), although BMP+E-fed pigs excreted 13.2% less N in the feces compared with those fed the nonenzyme supplemented controls. Inclusion of micronized peas with or without enzyme supplementation did not affect urinary or fecal N excretion (P > 0.10) compared with the BRP. Dietary treatment had no effect (P > 0.10) on ileal or fecal DM or CP digestibilities. Apparent ileal digestibilities of AA were usually lower (P < 0.05) in the BRP diet compared with the other diets. Enzyme supplementation improved P digestibility at the ileal and fecal level. The current results indicate that utilizing micronized peas in barley-based pig grower diets enhances P retention.  (+info)