• Asian-Australasian Journal of Animal Sciences
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• v.16 no.1
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• pp.124-135
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• 2003

In order to prevent pollution from animal waste, the excretion of nutrients should be reduced through proper nutritional management. Among the many nutrients of concern, such as N, P, Cu, Zn and K, P is one of the most concerned nutrients to be managed. Seven feeding trials, three with layers and four with broilers, were conducted to determine if microbial phytase supplementation can reduce non-phytate phosphorus (NPP) level in diets and results in concomitant reductions of P excretion. The results showed that microbial phytase can be successfully used to achieve these purposes. Activity of natural phytase in certain plant feedstuffs is high enough to be considered in feed formulation. Three experiments have been conducted to study the characteristics of plant phytase and its application to feeding of broilers. Selected brands of wheat bran could be successfully used as a source of phytase in broiler feeding.

• Journal of Animal Science and Technology
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• v.44 no.4
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• pp.407-418
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• 2002

This study was conducted to evaluate the efficacy of wheat and wheat bran  as the source of phytase in a 5 week broiler feeding trial. One thousand day-old broiler chickens(Ross$^{(R)}$) were divided into 20 pens of 50 broilers(25 male and 25 female) each. Four pens were randomly arranged to one of the five dietary treatments: T1, control diet containing normal nonphytate P(NPP) ;  T2, T1 － 0.1% NPP; T3, T2 ＋ 600IU microbial phytase(NOVO$^{(R)}$) per kg diet; T4, T2 ＋ 600IU plant phytase from wheat and wheat bran; T5, T2 ＋ 600IU plant phytase from wheat and hydrothermally treated wheat bran. Reduction of NPP level by 0.1%(T2) reduced weight gain and feed intake but plant phytase treatments(T4 and T5) recovered the lost performance. Plant phytase treatments showed better (p<0.05) weight gain and intake than the microbial phytase treatment(T3). There was no difference between regular wheat bran treatment(T4) and hydrothermally treated wheat bran treatment(T5). Mortality was the highest by low NPP diet(T2). Availability of ether extract and crude ash of grower diet was the highest(p<0.05) in normal wheat bran diet(T4). Availability of Ca and P of grower diet was the highest(p<0.05) in T4 followed by T3 and T5. Availability of Mg, Fe and Zn was drastically improved by phytase treatments(T3, T4 and T5). Excretion of Ca, P, Mg, Fe and Zn was the lowest(p<0.05) with microbial phytase treatment(T3). Serum level of Ca and Mg was the highest(p<0.05) with the low NPP treatment(T2). Tibial ash content of T2 and T3 was lower(p<0.05) than that of T1, T4 and T5. However, tibial Ca content was higher(p<0.05) in T1 and T2 than other treatments. Tibial P and Mg contents were the highest(p<0.05) in T1. It was concluded that plant phytase from wheat bran can be effectively used to improve P utilization. Hydrothermal treatment of wheat bran prior to inclusion in the diet had no beneficial effects.

• Asian-Australasian Journal of Animal Sciences
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• v.10 no.6
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• pp.551-566
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• 1997

Phosphorus has been known as an essential component of animal body. However, the requirement has not been determined precisely because of the variable bioavailabilities of feedstuffs from plant origin. The bioavailability of P in various feedstuffs of plant origin varies from 10 to 60%. Digestibility and availability of the P differed considerably depending on the feed. The lowest values were found for maize (under 20%), the highest for wheat and triticale (over 50%). This is due to the proportion of phytate and the presence of intrinsic phytase. And the digestive tract of monogastric animals does not contain sufficient amounts of phytase, an enzyme that hydrolyses the unavailable phytate complexes to available, inorganic orthophosphates. Microbial phytase supplementation improves the P availability, and both intrinsic plant and microbial phytase improves the availability of P in feedstuffs of plant origin. In a mixture of feeds with low and high activity of intrinsic phytase and/or supplemented by commercial phytase, the P availability is additive. However, in the light of current results it seems that exceeding the P availability equal to 60-70% is unrealizable even at large microbial phytase doses.

• Journal of Microbiology and Biotechnology
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• v.20 no.11
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• pp.1491-1499
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• 2010

Rhizosphere microorganisms possessing phytase activity are considered important for rendering phytate-phosphorus (P) available to plants. In the present study, the Citrobacter braakii phytase gene (appA) was overexpressed in rhizobacteria possessing plant growth promoting (PGP) traits, for increasing their potential as bioinoculants. AppA was cloned under the lac promoter in the broadhost-range expression vector pBBR1MCS-2. Transformation of the recombinant construct pCBappA resulted in high constitutive phytase activity in all of the eight rhizobacterial strains belonging to genera Pantoea, Citrobacter, Enterobacter, Pseudomonas (two strains), Rhizobium (two strains), and Ensifer that were studied. Transgenic rhizobacterial strains were found to display varying levels of phytase activity, ranging from 10-folds to 538-folds higher than the corresponding control strains. The transgenic derivative of Pseudomonas fluorescens CHA0, a well-characterized plant growth promoting rhizobacterium, showed the highest expression of phytase (~8 U/mg) activity in crude extracts. Although all transformants showed high phytase activity, rhizobacteria having the ability to secrete organic acid showed significantly higher release of P from Ca-phytate in buffered minimal media. AppA overexpressing rhizobacteria showed increased P content, and dry weight (shoot) or shoot/ root ratio of mung bean (Vigna radiata) plants, to different extents, when grown in semisolid agar (SSA) medium containing Na-phytate or Ca-phytate as the P sources. This is the first report of the overexpression of phytase in rhizobacterial strains and its exploitation for plant growth enhancement.

• Journal of Animal Science and Technology
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• v.44 no.1
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• pp.55-60
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• 2002

Two experiments were conducted to investigate the effect of dietary supplementation of plant phytase (Phytazyme$^{(R)}$) in corn-soybean meal based diets on utilization of phytase-bound phosphorus in laying hens and evaluate nitrogen(N) digestibility and phosphorus(P) availability in breeders. In the experiment one, three levels of the Phytazyme$^{(R)}$(0.05, 0.1, and 0.2% of diet) were added to diets containing a half of control TCP level(0.96%) for 4 wks. Feed consumption, egg production rate, egg weight and eggshell quality were recorded weekly. At the end of experiment, 8 birds per treatment were sacrificed, liver weight were weighed and right tibiae were removed for determination of P content. The second experiment was conducted to evaluate the P availability and nitrogen digestibility in breeders fed same diets for 2 wks. Feed and excreta were collected to determine the P and N contents for the last three days of experiment two. Addition of Phytazyme$^{(R)}$ resulted in no effects on feed intake, egg product rate, egg weight and egg shell quality. P excretion decreased and its availability enhanced as phytase supplementation increased in diets. Dietary supplementation of Phytazyme$^{(R)}$ above 0.1% level in corn-soybean meal based diets did not have an adverse effect on production and decreased level of phosphorus in excreta.

• Asian-Australasian Journal of Animal Sciences
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• v.27 no.1
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• pp.1-9
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• 2014

Calcium (Ca) and phosphorus (P) are minerals that have important physiological functions in the body. For formulation of diets for pigs, it is necessary to consider an appropriate Ca:P ratio for an adequate absorption and utilization of both minerals. Although both minerals are important, much more research has been conducted on P digestibility than on Ca digestibility. Therefore, this review focuses on aspects that are important for the digestibility of Ca. Only values for apparent total tract digestibility (ATTD) of Ca have been reported in pigs, whereas values for both ATTD and standardized total tract digestibility (STTD) of P in feed ingredients have been reported. To be able to determine STTD values for Ca it is necessary to determine basal endogenous losses of Ca. Although most Ca is absorbed in the small intestine, there are indications that Ca may also be absorbed in the colon under some circumstances, but more research to verify the extent of Ca absorption in different parts of the intestinal tract is needed. Most P in plant ingredients is usually bound to phytate. Therefore, plant ingredients have low digestibility of P due to a lack of phytase secretion by pigs. During the last 2 decades, inclusion of microbial phytase in swine diets has improved P digestibility. However, it has been reported that a high inclusion of Ca reduces the efficacy of microbial phytase. It is possible that formation of insoluble calcium-phytate complexes, or Ca-P complexes, not only may affect the efficacy of phytase, but also the digestibility of P and Ca. Therefore, Ca, P, phytate, and phytase interactions are aspects that need to be considered in Ca digestibility studies.

• Biotechnology and Bioprocess Engineering:BBE
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• v.10 no.6
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• pp.576-581
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• 2005

Phytase improves the bioavailability of phytate phosphorus in plant foods to humans and animals, and reduces the phosphorus pollution of animal waste. We have engineered the cell surface of the yeast. Saccharomyces cerevisiae, by anchoring active fungal phytase on its cell wall, in order to apply it as a dietary supplement containing bioconversional functions in animal foods and a whole cell bio-catalyst for the treatment of waste. The phytase gene (phyA) of Aspergillus niger with a signal peptide of rice amylase 1A (Ramy1A) was fused with the gene encoding the C-terminal half (320 amino acid residues from the C-terminus) of yeast ${\alpha}-agglutinin$, a protein which is involved in mating and is covalently anchored to the cell wall. The resulting fusion construct was introduced into S. cerevisiae and expressed under the control of the constitutive glyceraldehydes-3-phosphate dehydrogenase (GPD) promoter. Phytase plate assay revealed that the surface-engineered cell exhibited a catalytically active opaque zone which was restricted to the margin of the colony. Additionally, the phytase activity was detected in the cell fraction, but was not detected in the culture medium when it was grown in liquid. These results indicate that the phytase was successfully anchored to the cell surface of yeast and was displayed as its active form. The amount of recombinant phytase on the surface of yeast cells was estimated to be 16,000 molecules per cell.

• Journal of Microbiology and Biotechnology
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• v.11 no.6
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• pp.915-921
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• 2001

Phytase improves the bioavailability of phytate phosphorus in plant foods to humans and animals, and reduces the phosphorus pollution of animal waste. In order to express a high level of fungal phytase in Saccharomyces cerevisiae, various expression vectors were constructed with different combinations of promoters, translation enhancers, signal peptides, and terminator. Three different promoters fused to the phytase gene (phyA) from Aspergillus niger were tested: a galactokinase (GAL1) promoter, glyceraldehyde-3-phosphate dehydrogenase (GPD) promoter, and yeast hybrid ADH2-GPD promoter consisting of alcohol dehydrogenase II (ADH2) and a GPD promoter. The signal peptides of phytase, glucose oxidase (GO), and rice amylase 1A(RAmy1A) were included. Plus, the translation enhancers of the ${\Omega}$ sequence and UTR70 from the tobacco mosaic virus (TMV) and spinach, respectively, were also tested. Among the recombinant vectors, pGphyA06 containing the GPD promoter, the ${\Omega}$ sequence, RAmy1A, and GAL7 terminator expressed the highest phytase activity in a culture filtrate, which was estimated at 20 IU/ml. An intracellular localization of the expressed phytase activity in a culture filtrate, which was estimated at 20 IU/ml. An intracellular localization of the expressed phytase was also performed by inserting an endoplasmic reticulum (ER) retention signal, KDEL sequence, into the C-terminus of the phytase within the vector pHphyA-6. It appeared that the KDEL sequence directed most of the early expression of phytase into the intracellular compartment yet more than $60\%$ of the total phytase activity was still retained within the cell even after the prolonged (>3 days) incubation of the transformant. However, the intracellular enzyme activity of the transformant without a KDEL sequence was as high as that of the extracellular one, thereby strongly suggesting that the secretion of phytase in S. cerevisiae appeared to be the rate-limiting step for the expression of a large amount of extracellular recombinant phytase, when compared with other yeasts.

• The Plant Pathology Journal
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• v.17 no.6
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• pp.378.1-378
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• 2001

• Asian-Australasian Journal of Animal Sciences
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• v.27 no.1
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• pp.77-82
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• 2014

The study aimed to evaluate the potential effects of feeding with phytase transgenic corn (PTC) on organ weight, serum biochemical parameters and nutrient digestibility, and to determine the fate of the transgenic DNA in laying hens. A total of 144 50-week-old laying hens were grouped randomly into 2 treatments, with 8 replicates per treatment and 9 hens per replicate. Each treatment group of hens was fed with diets containing 62.4% non-transgenic conventional corn (CC) or PTC for 16 weeks. The phytase activity for CC was 37 FTU/kg of DM, whereas the phytase activity for PTC was 8,980 FTU/kg of DM. We observed that feeding PTC to laying hens had no adverse effect on organ weight or serum biochemical parameters (p>0.05). A fragment of a poultry-specific ovalbumin gene (ov) was amplified from all tissues of hens showing that the DNA preparations were amenable to PCR amplification. Neither the corn-specific invertase gene (ivr) nor the transgenic phyA2 gene was detected in the breast muscle, leg muscle, ovary, oviduct and eggs. The digestibility data revealed no significant differences between the hens that received the CC- and PTC-based diets in the digestibility of DM, energy, nitrogen and calcium (p>0.05). Phosphorus digestibility of hens fed the PTC-based diet was greater than that of hens fed the CC-based diet (58.03% vs 47.42%, p<0.01). Based on these results, it was concluded that the PTC had no deleterious effects on the organ weight or serum biochemical parameters of the laying hens. No recombinant phyA2 gene was detected in muscle tissues and reproductive organs of laying hens. The novel plant phytase was efficacious in improving the phosphorus digestibility of laying hens.