Poultry manure is a useful nutrient source but recently it has raised environmental concern due to possible P movement from P saturated soils to waterbodies. This study was conducted to determine the effects of using phytase and high available phosphorous corn diets on the solubility and plant uptake of P, Cu, and Zn in poultry manure and soils amended with manure. Five diet treatments were used in the study: 1) normal phytic acid corn and 0.135% inorganic P (NPA), 2) normal phytic acid corn, 600 units phytase, and 0.135% inorganic P (NPA+Phytase), 3) normal phytic acid corn and 0.345% inorganic P (NPA+P), 4) high available phosphorous corn and 0.135% inorganic P (HAP), 5) high available phosphorous corn, 600 units phytase, and 0.135% inorganic P (HAP+Phytase). The NPA+P diet and NPA+Phytase diets are most similar to the conventional and alternative phytase supplemented diets currently used commercially. Three Virginia soils (Groseclose, Cecil, Mahan) were amended with manure from the diet treatments at rates of 25 and 50 g/kg and P and Cu were extracted with 0.01 M CaCl2 and Mehlich III extractant after incubation periods of 6 and 12 months. Corn was grown in a greenhouse experiment using these same Virginia soils and sand amended with 8.96 Mg/ha poultry manure from each of the five diet treatments. Poultry manure was nonsequentially extracted for determination of P, Cu, and Zn fractions. Comparing the alternative NPA+Phytase, HAP, and HAP+Phytase treatments to the conventional NPA+P treatment on an N- (nitrogen) basis all reduced both CaCl2 and Mehlich III-soluble P concentrations (P<0.05). Comparing these same treatments on a P-basis increased P extracted with CaCl2 24, 26, and 37%, respectively, and P extracted with Mehlich III P 5, 4, and 9%, respectively (P<0.05). The alternative NPA+Phytase and HAP+Phytase treatments increased water-soluble Cu compared to the conventional NPA+P on both a N- and P-basis, while no differences were observed in Mehlich III solubility between these treatments (P<0.05). The alternative NPA+Phytase treatment did not differ in Pand Cu in corn tissue or plant uptake when compared to the NPA or NPA+P (N- or P-basis) treatments. No difference in Zn in corn tissue was observed between these treatments on a N-basis, while NPA+Phytase was higher on a P-basis. Plant uptake of Zn was higher in the NPA+Phytase treatment compared to the NPA+P treatment on both a N- and P-basis. Addition of phytase reduced P solubility from all reagents except for CaCl2 (P<0.05). Replacing the conventional NPA+P treatment for the alternative NPA+Phytase treatment resulted in higher Cu concentrations for all reagents except for K-pyrophosphate and nitric acid. This same replacement increased Zn extracted by water, CaCl2, and CaNO3, while it reduced Zn extracted by HCl, acetic acid, PbNO3, K-pyrophosphate, and NH4-oxalate in the light. The use of phytase decreased P solubility from manure amended soils when treatments are compared on an equal N-basis, and increased P solubility when compared on an equal P-basis. No effect on plant uptake of P or Cu occurredfrom the NPA+Phytase treatment. / Master of Science

The objective of this dissertation was to investigate areas that needed further research with regards to phosphorus, phytate, and phytase in monogastric nutrition. To fulfill this objective, a total of 6 studies were carried out.<br>Study 1 was designed to evaluate the additivity of apparent ileal digestibility (AID) and standardized ileal digestibility (SID) of phosphorus (P) in mixed dies containing corn and soybean meal (SBM) and fed to broiler chickens. There were 7 dietary treatments in a randomized complete block design with body weight (BW) serving as the blocking factor. Treatments consisted of 4 semi-purified diets prepared to contain either corn or SBM as the sole source of P with or without phytase supplementation at 1,000 FYT/kg; 2 mixed diets containing corn and SBM with or without phytase addition; and a P-free diet (PFD) formulated to determine the basal endogenous loss (BEL) of P. A total of 512 day-old broiler chicks were fed a commercial starter diet for 21 days while experimental diets were fed for 3 days (day 22-24 post hatching). The BEL of P in broiler chickens as determined by the PFD was 166 mg/kg dry matter (DM). The SID of P in corn and SBM was 52.2 and 65.4 %, respectively. Phytase addition improved (P < 0.05) both the AID and SID of P in the ingredient and mixed diets. There was no difference between the predicted and determined AID and SID of P in the mixed diets and were thus additive. It was concluded that the AID and SID of P in corn and SBM were additive with or without phytase addition. However, we could not be certain if the assumption of additivity will hold true in mixed diets containing ingredients with a higher phytate content and if age of birds affected the BEL of P.<br>In the 2nd study, the additivity of AID and SID of P in mixed diets containing corn and canola meal (CCM) was investigated in broiler chickens at 2 ages. A total of 588 broiler chickens was used in this study. Dietary treatments were arranged as a 2 × 3 × 2 factorial with 2 ages (day 13 and 21), 3 diets (corn, canola meal, and CCM), and 2 levels of phytase (0 and 1,000 FYT/kg) in a randomized complete block design. A PFD was fed to birds at both ages to determine the effects of age on the BEL of P. A commercial starter diet was fed from day 1 to 10 or 18 post hatching and then experimental diets were fed for 3 days until day 13 or 21, respectively. The AID and SID of P was higher (P < 0.05) in birds at day 13 as compared with birds at day 21 regardless of phytase addition. Similarly, the BEL of P in younger birds was higher (P < 0.01) than in older birds at d 21. Regardless of age or phytase supplementation, the predicted and determined AID and SID of P in the CCM diets were similar and thus additive. In conclusion, age influenced the BEL of P and the utilization of minerals in broiler chickens while the assumption of additivity held true when mixed diets containing CCM was fed to birds at both ages with or without phytase addition.<br>In order to evaluate the efficacy of a new consensus phytase variant, the 3rd and 4th studies investigated the responses of broiler chickens to varying concentrations of phytate phosphorus (PP) and the new consensus bacterial 6-phytase variant (PhyG) in the starter and grower phases, respectively. Responses evaluated included growth performance, tibia ash, AID and total tract retention (TTR) of nutrients. A total of 1,152 and 768 day-old male broiler chickens were used in the starter and grower phases, respectively. Dietary treatments were arranged as a 3 × 5 + 1 factorial with a nutrient-adequate positive control (PC) (2.8 g PP/kg) and 15 nutrient-reduced negative control (NC) diets with 3 levels of phytate (NC1, NC2, and NC3 with 2.3, 2.8, and 3.3 g PP/kg respectively), and 5 levels of PhyG (0, 500, 1,000, 2,000, and 4,000 FYT/kg). Rice bran served as the main source of PP in the experimental diets. All treatments had 6 replicates with 12 birds/cage in the starter phase (day 1 to 11 post hatching) and 8 birds/cage in the grower phase (day 12 to 23 post hatching). Birds fed the PC had greater responses (P < 0.05) as compared with birds fed the NC2 diets without phytase at the starter and grower phases. There was no interaction between PP and PhyG on responses of birds in the starter phase but there was an interaction effect (P < 0.05) on the AID of some amino acids (AA) in birds at the grower phase. Increasing levels of PP reduced (P < 0.05) the growth performance, Ca and P utilization of birds but had no effect on the tibia ash in the starter phase and grower phases. Phytase supplementation improved (P < 0.05) the growth performance, AID and TTR of nutrients, and tibia ash of birds in the starter and grower phases. In conclusion, increasing PP levels reduced some responses of birds in the starter and grower phase while PhyG inclusion improved all responses of broiler chickens at both phases.<br>The time effects of phytase on the P utilization of growing and finishing pigs was investigated in the 5th and 6th studies, respectively using growth performance, apparent total tract digestibility (ATTD) of nutrients, P excretion, and plasma mineral concentrations as evaluation parameters. In both studies (Exp 1 and 2), treatments were arranged as a 3 × 4 factorial in a randomized complete block design with 3 corn-SBM based diets including a P-adequate PC, a low-P NC, and NC + 1,000 FYT/kg; and 4 sampling time points at days 7, 14, 21, and 28 in Exp 1 and days 14, 26, 42, and 55 in Exp 2. Ninety-six growing pigs were used in both trials with an average BW of 19.8 ± 1.16 kg in Exp 1 and 49.8 ± 3.21 kg in Exp 2. Each treatment had 8 replicates evenly divided between barrows and gilts, and 4 pigs per pen. There was an interaction (P < 0.01) between time and diet on some growth performance parameters in pigs in Exp 1 but none in Exp 2. Pigs fed the PC had greater (P < 0.05) growth performance and ATTD of nutrients as compared to pigs fed the NC in both trials. Phytase supplementation improved (P < 0.05) the ATTD of P and Ca in pigs as compared with pigs fed the NC. There was an interaction (P < 0.05) between time and diet on the total and water-soluble P lost from pigs in Exp 1. Supplementing the NC with phytase reduced the water-soluble P by 45, 32, and 35 % over the growing, finishing and the entire grow-finish period, respectively. In Exp 2, plasma concentrations of P were increased (P < 0.05) over time. In conclusion, phytase improved the responses of growing pigs however, some of these responses were influenced over time.<br>In summary, the AID and SID of P in corn, SBM, and canola meal are additive in complete diets with or without the inclusion of phytase. The BEL of P could be affected by the age of birds. A new consensus phytase variant seems to be efficient in improving the productivity of broiler chickens regardless of the presence of varying concentrations of PP and the growth phase of the birds. Phytase supplementation improves the P utilization and reduces the P loss of growing and finishing pigs however, this may be influenced by time within each growth phase.<br><br>

Five experiments were conducted, utilizing 512 crossbred weanling pigs to determine the P (Exp. 1) and Ca (Exp. 2 and 3) equivalency values of microbial phytase based on performance, rib mineralization and P and Ca digestibility estimates, and to investigate the possible interactions of phytase and citric acid (Exp. 4 and 5). In Exp. 1, adding phytase to low P diets linearly increased ADG (P < .001), rib shear force (P < .01), shear energy (P < .02), ash weight (P < .001) and ash percent (P < .001), Ca (P < .001) and P (P < .001) digestibility and digestible Ca (P < .001) and P (P <.001). Added P linearly increased ADG (P < .003), rib shear force (P < .003) shear energy (P < .001), ash weight (P < .001) and ash percent (P < .01), Ca (P < .02) and P (P < .001) digestibility and digestible Ca (P < .02) and P (P <.001). Based on phytase and P linear or nonlinear response equations for ADG, rib shear force, shear energy, and ash weight, P digestibility, and digestible P, the average equivalency of 500 U/kg of phytase was .78 g of P per kg of diet. In Exp. 2, dietary addition of phytase linearly increased rib ash % (P < .03), Ca (P < .001) and P (P < .001) digestibilities, and digested Ca (P < .001) and P (P < .001), but had no effect (P > .10) on ADG and rib shear force and ash weight. Added Ca linearly increased ADG (wk 3-4, P < .04), and rib shear force (P < .001), ash percentage (P < .001) and ash weight (P < .01), and digested Ca (P < .001), but P digestibility (P = .07) and digested P (P = .08) were numerically decreased. In Exp. 3, added phytase linearly increased ADG (wk 3-4, P < .002), feed efficiency (wk 3-4, P < .02), rib ash weight (P < .001), Ca total tract digestibility (P < .001), and Ca (P < .001) and P (P < .001) ileal digestibilities. Added Ca linearly increased ADG (wk 3-4, P < .02), feed efficiency (wk 3-4, P < .01), rib ash percentage (P < .001) and ash weight ( P < .001), shear force (P < .03) and energy (P < .008), and total tract (P < .001) and ileal (P < .001) digestible Ca. Based on phytase and Ca linear or nonlinear response equations for ADG in wk 3-4, measurements of rib mineralization, and digestible Ca, 500 U of microbial phytase was estimated to be equivalent to 1.08 g and .78 g of Ca in Exp. 2 and 3, respectively. In Exp. 4 and 5, dietary phytase addition linearly increased rib shear force (P < .004 and P < .02), shear energy (P < .001), dry bone weight (P < .001), ash weight (P < .001) and ash percent (P < .001). Calcium (P < .001) and P (P < .001) digestibilities were also improved in both experiments when phytase was added. Addition of citric acid in both experiments, reduced dietary pH and stomach digesta pH (P < .05). The addition of citric acid improved ADG (P < .05), feed efficiency (P < .04) and Ca digestibility (P < .05) in Exp. 4, but decreased Ca digestibility in Exp. 5 and had no effect on performance. In Exp. 5, the addition of 2.0% citric acid to the diet supplemented with 500 U/kg of phytase caused a decrease (P < .04) in the phytase activity recovered in the stomach digesta resulting in a phytase by citric acid interaction (P < .02). In summary, the addition of 500 U/kg microbial phytase to weanling pig diets, causes the release of approximately .78 g of P and .93 g of Ca, thus decreasing the need for supplemental P and Ca. The addition of citric acid to phytase supplemented diets does not appear to enhance the efficacy of microbial phytase based on the results of these studies. / Master of Science

Measurement of soil phytase activity (PA) and delineation of the impact of this important phosphomonoesterase on the P-cycling process in soil and sediments suffer from the lack of a reliable assay. A method for measuring PA in soil that promises to be accurate and reliable has been recently published. The method involves the use of a novel chromophoric analog of phytic acid, referred to as T(tethered)InsP5 (5-O-[6-(benzoylamino)hexyl]-D-myo-inositol-1,2,3,4,6-pentakisphosphate). This study was conducted to measure PA in a Clymer forest soil, which contained over twice the amount of soil organic C as previously tested soils, using the TInsP5 PA assay. This investigation specifically addresses: (1) the development of a soil dilution technique for determining maximal PA, (2) identification of previously unsubstantiated soil-produced dephosphorylated intermediate probe species, (3) the impact of increasing assay buffer pH on soil PA and (4) testing stability of the probe's amide bond in a highly (bio)active forest soil. PA assays were conducted by measuring dephosphorylation of TInsP5 in citrate-acetate buffered (pH 4.2) active and autoclaved (Control) soil suspensions. Phosphorylated probe intermediates (i.e., TInsP4, TInsP3, TInsP2 and TInsP1) and T-myo-inositol were extracted from samples of soil suspension following incubation. Probe species were quantified using reversed phase high-performance liquid chromatography (RPHPLC) with UV detection. PA was calculated based on a mass balance approach. A soil dilution technique was developed to address the challenge of determining maximal PA in soils containing higher organic matter content. In the initial report on use of the TInsP5 method for measuring PA in soil, two "soil-generated" UV-adsorbing compounds (designated Y and Z) were observed, but never confirmed as probe species. The experimental evidence presented in this report supports inclusion of compound Y as a phosphorylated probe intermediate species (i.e. TInsPy), based primarily on its UV adsorption spectra (diode-array detection analysis). Compound Z could not be substantiated as a probe species based on the evidence presented in this study. PA of Claymer forest soil decreased with an increase in assay buffer pH. Further, the probe's amide bond linkage was stable in a forest soil exhibiting high PA. / Master of Science

Viele Bacillus Stämme sekretieren Phytasen, diese katalysieren die Dephosphorylierung von myo-Inositolhexakisphosphat (phytate). Das monocystronische phyC Gen aus dem im Boden lebenden Bacillus amyloliquefaciens FZB45 konnte als Teil des, durch Phosphatmangel induzierbaren, PhoPR-Regulons identifiziert werden. Der Transkriptionsstart des SigmaA-abhängigen Promotors wurde 27 bp upstream von Translationsstart bestimmt. Der Promotor weist jedoch eine ungewöhnliche Struktur auf, da die –35 und die –10 Region durch ein 21 bp Fenster voneinander getrennt sind. Die in vitro Transkriptionsanalyse zeigte, dass PhoP-P für die Initiation der phyC-Transkription notwendig ist. Die PhoP-Bindungsstellen der meisten B. subtilis Promotoren, die durch PhoP aktiviert werden, besitzen mindestens vier TTAACA-ähnliche Sequenzmotive, welche durch 5-6 bp- Intervalle getrennt sind. Die upstream Region von phyC aus B. amyloliquefaciens FZB45 weicht von dieser Struktur ab. Hier konnten nur zwei solcher Motive, die für die Bindung eines PhoP-Dimeres zuständig sind und die Positionen -47 und -35 überlagern, identifiziert werden. Ein weiteres Motiv befindet sich zwischen -13 und –8 und überlappt um eine Base mit der –10 Region. Die Funktionen der drei Bindungsstellen konnten durch DNaseI-Footprinting ermittelt werden. Ein PhoP-Dimer besetzt die –35 Region und dirigiert dabei die RNA-Polymerase wahrscheinlich in Richtung –10 Region. Durch die Bindung von PhoP an die PhoP-Erkennungsstelle bei –10 wird die Transkription gehemmt. Diese PhoP-vermittelte duale Kontrolle des phyC-Promotors scheint bis lang einzigartig für Pho-Regulongene zu sein. Der globale Regulator AbrB konnte als weiterer Repressor des phyC identifiziert werden. Die Bindungsstelle befinden sich upstream von –147 und downsteam von +29, müssen aber in weiteren Experimenten genauer spezifiziert werden. Diese Arbeit stellt als erste die Modelle der phyC-Regulation durch PhoP und AbrB vor. / Several Bacillus strains secrete phytase, an enzyme catalysing dephosphorylation of myo-inositol hexakisphosphate (phytate). The monocistronic phyC (phytase) gene from environmental Bacillus amyloliquefaciens FZB45 was identified as a member of the phosphate-starvation inducible PhoPR regulon. The transcriptional start was determined downstream of a sigmaA-like promoter region located 27 bp upstream of the translation start codon. Inspection of the phyC promoter sequence revealed an unusual structure, since the -35 and -10 region are separated by a window of 21 bp. In vitro transcription analysis established that PhoP-P is necessary to initiate the transcription from phyC promoter. PhoP binding boxes occurring in most B. subtilis promoters activated by PhoP consist of at least four TTAACA-like sequences repeated at intervals of 5-6 bps. The upstream region of the B. amyloliquefaciens FZB45 phyC gene deviates from this general architecture in that there is only one appropriate binding site for the dimeric PhoP protein, which consists of two motives centered at -47 and -35 and separated by five base pairs. A single PhoP binding site is located at -13 to -8, nearly matching the -10 consensus. Functionality of the three PhoP binding boxes was demonstrated by DNaseI footprinting suggesting that a pair of dimeric PhoP molecules cover the -35 region directing the RNA-polymerase to the –10 region. Binding of PhoP at a single PhoP binding site covering the -10 consensus repress the transcription. It seems to be a unique feature of the phyC promoter structure and has not been reported for any other member of the PhoPR regulon, previously. Furthermore an inhibitory effect via AbrB, a global regulator, could be verified. The binding regions could be determine upstream of –147 and downstream of +29, but have to be specify in further experiments. This work presents for the first time the models for the regulation of phyC in Bacillus via PhoP and AbrB.

L’optimisation de l’utilisation de phosphore reste un enjeu majeur pour assurer la durabilité de nos filières avicoles et porcines. Aujourd’hui, l’utilisation de coproduits, relativement riches en fibres alimentaires, est grandissante. Or, les fibres alimentaires sont reconnues pour moduler les processus de digestion et la digestibilité des nutriments. Ce travail de thèse a permis de progresser sur les mécanismes digestifs engendrés par l’apport de coproduits riches en fibres alimentaires et leurs effets sur la digestion des minéraux, en lien avec la phytase microbienne, spécifiquement chez le porc et le poulet. Leurs conséquences métaboliques en terme de balance minérale et d’équilibre acidobasique ont également été mises en évidence Les connaissances acquises vont contribuer à l’amélioration de l’apport en minéraux, notamment de phosphore et de calcium, dans des formules diversifiées chez le porc et le poulet. A terme, ceci permettra de mieux adapter nos systèmes d’alimentations au regard des enjeux de durabilité. / The optimisation of the use of phosphorus remains a major challenge to ensure the sustainability of poultry and pig industry. Today, the use of co-product, relatively rich in dietary fibre, is increasing. However, dietary fibre is known to modulate digestion processes and digestibility of nutrients. This work contributed to progress on the digestive mechanisms generated by the intake of dietary fibre from coproducts and their effects on the digestion of minerals, in relation with microbial phytase, specifically in pigs and chickens. Their metabolic consequences in terms of mineral and acid-base balance have also been demonstrated. The knowledge gained will contribute to the improvement of the intake of minerals, in particular phosphorus and calcium, in diversified formulas in pigs and the chicken. Finally, this will allow us to better adapt our feeding systems to the challenges of sustainability.

Die Verfügbarkeit des Makroelementes Phosphor ist für Lebewesen eingeschränkt. Besonders bei der Pflanzenproduktion und der Tierernährung spielt die Phosphorverfügbarkeit eine wichtige Rolle bei der ökonomischen Nutzung der Ressourcen. In den Fokus der Wissenschaft zur Lösung des Phosphorproblems gerieten die Phytasen, da monogastrische Tiere nicht in der Lage sind das in den Pflanzensamen gespeicherte Phytat zu nutzen. Die gentechnische Veränderung von Pflanzen stellt eine effiziente Möglichkeit zur Produktion von phosphatfreisetzenden Enzymen, zur Erhöhung der Biomasseproduktion und zur Veränderung der Inhaltstoffe dar. In dieser Arbeit wurden erfolgreich transgene Pflanzen der Arten Nicotiana tabacum L. cv. Samsun und Hordeum vulgare L. cv. Golden Promise erzeugt, die in der Lage waren die Phytase aus Klebsiella sp. ASR1 bzw. aus Bacillus amyloliquefaciens FZB45 zu produzieren. Es wurde für jedes Protein eine Strategie zur Reinigung des aktiven Enzyms aus den verschiedenen Wirtsorganismen entwickelt und seine biochemischen Eigenschaften charakterisiert. Es wurde gezeigt, dass die β-Propeller-Phytase aus Bacillus im Gegensatz zur sauren Phytase aus Klebsiella durch die posttranslationale Modifikation teilweise ihre Eigenschaften ändert. Die Auswirkungen der heterologen Expression der Phytasen auf die Veränderung der Anteile von Phytinsäure und anorganischem Phosphor, in Relation zum gesamten Phosphor, in den Gerstensamen wurden untersucht. Es wurde eine Reduktion des Phytinsäuregehaltes um 19 % und eine Erhöhung des Gehalts anorganischem Phosphor zwischen 27 % und 78 nachgewiesen. Mit Hilfe von spezifischen Signalsequenzen gelang es die Phytaseproteine aus der Wurzel in das umgebende Medium zu sekretieren. Die Sekretion der Bacillus Phytase führte zu einer Steigerung der Biomasseproduktion von Nicotiana tabacum L. unter unsterilen Wachstumsbedingungen mit Phytat als einziger P-Quelle um 34 %. / Due to the bad availability of phosphorus in natural habitats the improvement of phosphorus accessibility to organisms became an important topic of research, particularly for agriculture and animal nutrition. In plant seeds phosphorus is bound to D-myo-inositol to form phytic acid that is indigestible for mono gastric animals. Therefore the use of phytases to hydrolyze the phytic acid and to mobilize the anorganic phosphorus came in focus to science. Genetic engineering gave the opportunity to improve the phosphorus availability. Genetic manipulation of plants is a suitable tool to produce phosphorus releasing enzymes, thereby increasing the biomass and decreasing the content of phytic acid in plants. In this work I generated transgenic plants of Nicotiana tabacum and Hordeum vulgare vulgare which successfully express the phytase gene phyK from Klebsiella pneumonia ASR1 and phyC from Bacillus amyloliquefaciens FZB42. Chromatographically purification strategies were developed and biochemical properties were characterized for all phytase proteins. All enzymes were active and PhyC was posttranslational modified. The effect of the recombinant phytase activity to total phosphorus, phytic acid and anorganic phosphorus content of barley seeds were elucidated. The phyK-expression in barley seeds yield a reduction of phytic acid content of about 19 % and an increase of anorganic phosphorus of about 78 %. Transgenic barley seeds with phyC gene expression show an increase of anorganic phosphorus content from 27 % to 48 % but no decrease in phytic acid content. In growth experiments no special phenotypes of plant containing the recombinant protein were visible. The insertion of apoplastic signal sequences in front of the phytase genes resulted in secretion of the proteins into the rhizosphere. The secretion of PhyC led to an improved growth of Nicotiana tabacum under unsterile conditions with sodium phytate as single phosphorus source increasing the biomass up to 34 %.

Les phytases hydrolysent les phytates représentant la forme majeure de stockage du P dans les céréales. Ces phytates sont aussi des facteurs anti-nutritionnels qui chélatent les cations réduisant leur absorption. Dans le premier volet de cette thèse, une nouvelle souche bactérienne produisant une phytase extracellulaire a été isolée et identifiée comme Bacillus amyloliquefaciens US573. L’enzyme «PHY US573» a été purifiée et caractérisée en comparaison avec deux phytases commerciales Ronozyme PL et Natuphos. PHY US573 se distingue par sa forte thermostabilité en présence de calcium. En outre, PHY US573 se caractérise aussi par une tolérance remarquable aux sels comme le NaCl et LiCl. L’ensemble de ces propriétés montre que PHY US573 pourrait être une candidate intéressante pour des applications en alimentation animale ou en agriculture pour améliorer la biodisponibilité du P-phytique pour les plantes. Dans le deuxième volet, la souche Streptomyces sp. US42 produisant une activité phytase extracellulaire a été sélectionnée. L’enzyme «PHY US42» a été purifiée et caractérisée. PHY US42 est calcium dépendante également une grande stabilité en présence de sels biliaires et des protéases digestives. La modélisation moléculaire de PHY US42 indique qu'elle appartient au groupe des β-propeller phytases qui sont généralement calcium-dépendantes. Vu ses propriétés biochimiques intéressantes, PHY US42 constitue une bonne candidate comme additif dans les aliments pour animaux monogastriques en combinaison avec une histidine acide phytase. Enfin dans un troisième volet, nous nous sommes intéressés à l’étude de la régulation de l'expression du gène phytase de S. coelicolor M145 (sco7697) chez S. coelicolor M145, S. lividans TK24 ainsi que chez ses deux mutants ppk et phoP. Ainsi, en plus des boites pho localisées en amont de la région promotrice -35 siège de la régulation positive PhoP-dépendante, nous avons révélé pour la première fois que la RD localisée en aval de la région promotrice -10 est le siège d’une forte régulation négative par un répresseur inconnu. Ce dernier empêcherait l’activation PhoP-dépendante de l’expression du gène phytase. / Phytases hydrolyse phytate representing the major storage form of P in cereal. phytates are also anti-nutritional factors that chelate cations such as Ca²⁺, Mg²⁺, Fe²⁺, Z²⁺ reducing their absorption. The low bioavailability of phytic phosphorus in monogastric animals require their food supplementation with Pi to meet the needs of the animal in P. This creates an extra cost and increases the environmental pollution by the manure excretion highly charged phosphate. In the first part of this thesis, from soil samples taken near hot hydrothermal waters of the region Elhamma in southern Tunisian, a new bacterial strain producing extracellular phytase was isolated and identified as Bacillus amyloliquefaciens US573. The enzyme referred "PHY US573" was purified and characterized in comparison with two commercial acid histidine phytases Ronozyme PL and Natuphos. PHY US573 is calcium dependent and has an optimum activity at pH 7.5 (5 for Ronozyme and 5.5 for Natuphos) and 70°C (55°C for Ronozyme and Natuphos). PHY US573 is distinguished by its high thermostability, in fact, it keeps 93% of its activity after incubation for 10 min at 75°C in the presence of calcium while Ronozyme and Natuphos keep only 45% and 53% of their activity, respectively. This enzyme is specific for phytic acid and also has a very good stability at pH 3 to 9 and a perfect stability in presence of bile salts. In addition, PHY US573 is also characterized by a remarkable salt tolerance because it retains 80 to 95% of its activity in the presence of 20 g/l of NaCl and LiCl, respectively. All these properties shows that PHY US573 could be an interesting candidate for applications in feed industry alone or in combination with an histidine acid phytase. In a second part of this thesis, from the Streptomyces collection of LMB-CBS, a strain producing extracellular phytase activity was selected and identified as Streptomyces sp. US42. The enzyme "PHY US42" was purified and characterized. PHY US42 has a calcium-dependent activity (such as Bacillus phytases), optimally active at pH 7 and 65°C. PHY US42 is perfectly stable at pHs ranging from 5 to 10 and its thermal stability is greatly increased in the presence of calcium. Indeed, PHY US42 maintains 80% of its activity after 10 min of incubation at 75 °C in the presence of calcium. PHY US42 has also a high stability in the presence of bile salts and digestive proteases. Molecular modeling of PHY US42 indicates that it belongs to the β-propeller phytase group which are usually calcium-dependent. Given its interesting biochemical properties, PHY US42 which would operate mainly in the intestine, is a good candidate for use as an additive in agastriques fish food or in combination with an histidine acid phytase in feed industry. Finally in a third part, we are interested in studying the regulation of the expression of the phytase gene of S. coelicolor M145 (sco7697) in S.coelicolor M145, S.lividans TK24 and among its two mutants ppk and phoP. To do this, we merged the wild promoter regions (phyWT) or mutated (phym1, phym2, phym1+2) of sco7697 gene with the GUS reporter gene encoding ß-glucuronidase activity. Thus as expected, we demonstrated that the deletion of the PHO box located upstream of the -35 reduces the level of induction of sco7697 in conditions of Pi limitation. Moreover, we have revealed for the first time that the alteration of RD located downstream of -10 correlates with a dramatic increase of GUS expression when PhoP is present. Our results demonstrate that this RD is the seat of a strong negative regulation by an unknown repressor. This would prevent the PhoP-dependent activation of expression of the phytase gene.

Striped catfish (Pangasianodon hypophthalmus) is freshwater fish that is raising mainly in the Mekong Delta. The research was implemented at the Model Farm (College of Aquaculture and Fisheries), Can Tho University during 6 months from May to November of 2016 and aimed to evaluate the water quality between two treatments of feed in the pangasius production for proposing a better environmental management method. The treatment 1 (control treatment, CT) has two ponds used feed without adding phytase and treatment 2 (experiment treatment, ET) includes two ponds used feed with adding phytase. The results showed that the temperature, pH, DO, TSS, COD, BOD, N-NO2-, N-NO3-, TKN, TAN were not significantly different (p>0.05) between the CT and ET ponds. At the experiment pond (EP), the factors (TP: 2.28 mg/L; P-PO4 3-: 1.24 mg/L) which has P release to the environment are less than control pond (CP) (TP: 2.62 mg/L; P-PO4 3-: 2.13 mg/L). These results suggested that the addition of phytase in feed could contribute to reducing the nutrient to the water body of pangasius pond. / Cá tra (Pangasianodon hypophthalmus) là loài thủy sản nước ngọt được nuôi chủ yếu ở các tỉnh Đồng bằng sông Cửu Long. Nghiên cứu được thực hiện trong 6 tháng từ tháng 05 – 11/2016 tại Trang trại mẫu đặt tại Khoa Thủy sản, Trường Đại học Cần Thơ nhằm đánh giá chất lượng nước giữa 2 nghiệm thức thức ăn trong giai đoạn nuôi cá thịt để đề xuất biện pháp quản lý chất lượng nước ao nuôi tốt hơn. Nghiệm thức 1 (nghiệm thức đối chứng, CT) gồm 2 ao nuôi sử dụng thức ăn không bổ sung enzyme phytase, nghiệm thức 2 (nghiệm thức thí nghiệm, ET) gồm 2 ao nuôi sử dụng thức ăn có bổ sung enzyme phytase. Kết quả thí nghiệm cho thấy giá trị nhiệt độ, pH, DO, TSS, COD, BOD, H2S, N-NO2-, N-NO3-, TKN, TAN ở mức phù hợp cho ao đối chứng (CT). Tại ao thí nghiệm (ET) (TP:2,28 mg/L; P-PO4 3-: 1,24 mg/L) lượng P thải ra ít hơn so với ao CT (TP: 2,62 mg/L; P-PO4 3-: 2,13 mg/L) chứng tỏ việc bổ sung enzyme phytase trong thức ăn đã góp phần làm giảm lượng lân thải ra môi trường nước trong nuôi cá tra.

Effects of dietary supplementation with low levels of organic sources of trace minerals in place of normal levels of their inorganic salts and phytase on growth performance and mineral metabolism were evaluated in two studies using pullets of white and brown shell laying strains. The organic sources were proteinates of copper, iron, manganese and zinc and selenium yeast. A corn-soybean meal diet was fed alone, plus inorganic minerals or plus organic minerals, and with or without phytase in a 3 x 2 factorial arrangement. Twelve groups of 16 pullets, 2 weeks old, were used per treatment. Compared with inorganic minerals, feeding no mineral supplement or organic minerals significantly (P<0.05) decreased manure Cu, Fe and Zn for white pullets and Cu, Fe, Mn and Zn for brown pullets. Dietary phytase significantly reduced manure Fe, P and Ca for white pullets and Fe, Mn, Zn, P and Ca for brown pullets. Adding phytase to diets containing inorganic minerals reduced manure Zn concentration for white pullets and manure Fe, Mn, Zn, P and Ca concentrations for brown pullets. These studies indicate manure levels of trace minerals can be decreased by using low levels of organic mineral supplements and phytase in pullet diets.