Expression of an Aspergillus niger phytase gene (phyA) in Saccharomyces cerevisiae. (1/247)

Phytase improves the bioavailability of phytate phosphorus in plant foods to humans and animals and reduces phosphorus pollution of animal waste. Our objectives were to express an Aspergillus niger phytase gene (phyA) in Saccharomyces cerevisiae and to determine the effects of glycosylation on the phytase's activity and thermostability. A 1.4-kb DNA fragment containing the coding region of the phyA gene was inserted into the expression vector pYES2 and was expressed in S. cerevisiae as an active, extracellular phytase. The yield of total extracellular phytase activity was affected by the signal peptide and the medium composition. The expressed phytase had two pH optima (2 to 2.5 and 5 to 5.5) and a temperature optimum between 55 and 60 degrees C, and it cross-reacted with a rabbit polyclonal antibody against the wild-type enzyme. Due to the heavy glycosylation, the expressed phytase had a molecular size of approximately 120 kDa and appeared to be more thermostable than the commercial enzyme. Deglycosylation of the phytase resulted in losses of 9% of its activity and 40% of its thermostability. The recombinant phytase was effective in hydrolyzing phytate phosphorus from corn or soybean meal in vitro. In conclusion, the phyA gene was expressed as an active, extracellular phytase in S. cerevisiae, and its thermostability was affected by glycosylation.  (+info)

Phytase improves iron bioavailability for hemoglobin synthesis in young pigs. (2/247)

Dietary phytase supplementation improves bioavailabilities of phytate-bound minerals such as P, Ca, and Zn to pigs, but its effect on Fe utilization is not clear. The efficacy of phytase in releasing phytate-bound Fe and P from soybean meal in vitro and in improving dietary Fe bioavailability for hemoglobin repletion in young, anemic pigs was examined. In Exp. 1, soybean meal was incubated at 37 degrees C for 4 h with either 0, 400, 800, or 1,200 units (U) of phytase/kg, and the released Fe and P concentrations were determined. In Exp. 2, 12 anemic, 21-d-old pigs were fed either a strict vegetarian, high-phytate (1.34%) basal diet alone, or the diet supplemented with 50 mg Fe/kg diet (ferrous sulfate) or phytase at 1,200 U/kg diet (Natuphos, BASF, Mt. Olive, NJ) for 4 wk. In Exp. 3, 20 anemic, 28-d-old pigs were fed either a basal diet with a moderately high phytate concentration (1.18%) and some animal protein or the diet supplemented with 70 mg Fe/kg diet, or with one of two types of phytase (Natuphos or a new phytase developed in our laboratory, 1,200 U/kg diet) for 5 wk. In Exp. 2 and 3, diets supplemented with phytase contained no inorganic P. In Exp. 1, free P concentrations in the supernatant increased in a phytase dose-dependent fashion (P<.05), whereas free Fe concentrations only increased at the dose of 1,200 U/kg (P<.10). In Exp. 2 and 3, dietary phytase increased hemoglobin concentrations and packed cell volumes over the unsupplemented group; these two measures, including growth performance, were not significantly different than those obtained with dietary supplemental Fe. In conclusion, both sources of phytase effectively degraded phytate in corn-soy diets and subsequently released phytate-bound Fe from the diets for hemoglobin repletion in young, anemic pigs.  (+info)

Occupational IgE sensitisation to phytase, a phosphatase derived from Aspergillus niger. (3/247)

OBJECTIVE: Phytase is a phosphatase derived from Aspergillus niger that enhances phosphate bioavailability in the gut, and therefore has been increasingly used as an animal feed additive since the early 1990s. The aim of this study was to assess whether work related respiratory symptoms among workers in a so called premix factory producing animal feed additives, could be due to type I (mediated by immunoglobulin E (IgE) allergic sensitisation to phytase. METHODS: Preparations of specific IgE against phytase as used in the factory were assessed by enzyme immunoassay (EIA) in serum samples of 11 exposed workers who regularly handled the enzyme, in 11 office and laboratory workers of the same plant (non-exposed internal controls), and in 19 laboratory animal workers as external controls. The factory workers also completed a questionnaire on common and work related respiratory symptoms. RESULTS: Depending on the cut off level in the EIA for IgE, and the preparation used as coated allergen, antiphytase sensitisation was found in one to four of the 19 external controls, in one to five of the 11 internal controls, and in four to 10 of the 11 exposed workers. Strongest IgE reactions were found in four exposed workers who reported work related respiratory symptoms, particularly wheezing, and in one internal control who possibly had become sensitised because the structure of the factory building did not preclude airborne exposure in the offices and corridors of the plant. Experiments with inhibition EIA for IgE showed that (a) phytase of another commercial source was only partially cross reactive with phytase as used in the premix factory, and (b) phytase used as an animal feed additive did not cross react with common mould extracts, except for extracts from the species of origin, Aspergillus niger. The amount of IgE binding phytase in Aspergillus niger was estimated to be between 0.1% and 1% of the extractable mould proteins. CONCLUSIONS: Phytase is a potentially important new occupational allergen causing specific IgE immune responses among exposed workers. Such IgE sensitisation could probably be the cause of work related asthmatic and other respiratory symptoms if no effective measures are taken to prevent airborne occupational exposure at sites where phytase is handled, particularly during addition of enzyme preparations to animal feed.  (+info)

Dephosphorylation of phytate by using the Aspergillus niger phytase with a high affinity for phytate. (4/247)

A phytase (EC 3.1.3.8) with a high affinity for phytic acid was found in Aspergillus niger SK-57 and purified to homogeneity in four steps by using ion-exchange chromatography (two types), gel filtration, and chromatofocusing. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified enzyme gave a single stained band at a molecular mass of approximately 60 kDa. The Michaelis constant of the enzyme for phytic acid (18.7 +/- 4.6 microM) was statistically analyzed. In regard to the orthophosphate released from phytic acid, a significant difference between a low K(m) phytase from A. niger SK-57 and a high K(m) phytase from Aspergillus ficuum was recognized.  (+info)

Comparative enzymatic hydrolysis of phytate in various animal feedstuff with two different phytases. (5/247)

Bacillus amyloliquefaciens DS11 phytase (DS11 phytase) and Aspergillus ficuum phytase (AF phytase) activities were investigated by measuring the release of phosphate from phytate in animal feedstuff such as wheat bran, corn meal, soybean meal and rice flour at pH 5 and 7. In all the tested feedstuff, the enzymatic activity of DS11 phytase was more active at pH 7, but that of AF phytase was more active at pH 5. From these results, the phytate in the gastrointestinal tract could be degraded in the small intestine or stomach by DS11 or AF phytase, respectively. In conclusion, the results presented in this paper indicated that different combination ratios of DS11 and AF phytase, depending on the kind of feedstuff, might effectively induce more enzymatic activity both in the stomach and small intestine in terms of the pH of the gastrointestinal tract.  (+info)

From DNA sequence to improved functionality: using protein sequence comparisons to rapidly design a thermostable consensus phytase. (6/247)

Naturally-occurring phytases having the required level of thermostability for application in animal feeding have not been found in nature thus far. We decided to de novo construct consensus phytases using primary protein sequence comparisons. A consensus enzyme based on 13 fungal phytase sequences had normal catalytic properties, but showed an unexpected 15-22 degrees C increase in unfolding temperature compared with each of its parents. As a first step towards understanding the molecular basis of increased heat resistance, the crystal structure of consensus phytase was determined and compared with that of Aspergillus niger phytase. Aspergillus niger phytase unfolds at much lower temperatures. In most cases, consensus residues were indeed expected, based on comparisons of both three-dimensional structures, to contribute more to phytase stabilization than non-consensus amino acids. For some consensus amino acids, predicted by structural comparisons to destabilize the protein, mutational analysis was performed. Interestingly, these consensus residues in fact increased the unfolding temperature of the consensus phytase. In summary, for fungal phytases apparently an unexpected direct link between protein sequence conservation and protein stability exists.  (+info)

Efficacy of yeast phytase in improving phosphorus bioavailability in a corn-soybean meal-based diet for growing pigs. (7/247)

Crossbred barrows (n = 66; 6 wk old) were used in a 6-wk experiment to evaluate the efficacy of phytase from yeast or Aspergillus niger on performance, tibial characteristics, and serum inorganic P concentration. We also investigated the stability of these phytases in acidic solutions with pepsin, which simulated gastric conditions. Pigs were fed a P-adequate diet containing .34% nonphytate-P or a low-P diet containing .20% nonphytate-P. The low-P diet was supplemented with 0, 1,000, 2,000, or 4,000 phytase units (PU; the activity at optimal pH, i.e., pH 4.2 for yeast phytase and pH 5.5 for phytase from Aspergillus niger)/kg of yeast phytase, or 1,000 PU/kg phytase from Aspergillus niger. The graded level of yeast phytase linearly increased ADG (P = .047), tibial weight (P = .091), tibial density (P < .001), and P concentration in tibial cortex (P = .018). Aspergillus niger phytase also increased ADG (P = .022), serum inorganic P concentration (P < .001), tibial density (P = .007), and tibial P concentration (P = .025). The pigs given 1,000 PU/kg Aspergillus niger phytase showed greater ADG (P = .091), tibial density (P= .001), and tibial P concentration (P = .062) than those given 1,000 PU/kg yeast phytase. No measurements differed (P > .31) between the pigs given 1,000 PU/kg Aspergillus niger phytase and those given 4,000 PU/kg yeast phytase. These results suggested that yeast phytase improves bioavailability of P in the diet for growing pigs but the efficacy of yeast phytase is less than that of Aspergillus niger phytase. During incubation in acidic solutions with pepsin, yeast phytase (P < .001) lost more of its activity than Aspergillus niger phytase. This lesser stability of yeast phytase may be responsible for the poorer efficacy of yeast phytase than that of Aspergillus niger. In summary, supplementation of swine diets with yeast phytase is beneficial, but its efficacy is less than that of Aspergillus niger phytase.  (+info)

Effects of dietary calcium:phosphorus ratios on apparent absorption of calcium and phosphorus in the small intestine, cecum, and colon of pigs. (8/247)

Thirty-two crossbred barrows were used to investigate the effects of dietary Ca:total P (tP) ratios in phytase-supplemented diets on the apparent absorption of P and Ca in the small intestine, cecum, and colon. Three Ca:tP ratio treatments (1.5:1, 1.3:1, or 1.0:1) were created by adjusting the amount of ground limestone added to the basal low-P grower (.39% tP including .07% added inorganic P) and finisher (.32% tP without added inorganic P) diets. All low-P ratio diets were supplemented with Natuphos phytase at 500 units/kg. A positive control diet without phytase supplementation contained adequate P and Ca to meet dietary requirements. At 123 kg, the pigs were slaughtered and the contents of ileum, cecum, and colon were collected. Lowering the dietary Ca:tP ratio in the diets containing phytase linearly increased (P < .01) the apparent absorption (% and g/d) of P in the small intestine, but Ca absorption was not affected. Pigs fed the low-P diet with a Ca:tP ratio of 1.0:1 had an apparent absorption (g/d) of P or Ca similar to that of pigs fed the control diet, which was adequate in Ca and P. Averaged across all diets, the apparent absorption of P was highest when measured at the cecum, and the apparent absorption of Ca was highest when measured at the colon. In conclusion, lowering the dietary Ca:tP ratio to 1.0:1 in a low-P diet containing phytase increased the apparent absorption of P in the small intestine. Furthermore, a significant amount of P was absorbed in the cecum.  (+info)

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