Aspergillus Niger Phytase Research Articles

Cloning of the phytases from Emericella nidulans and the thermophilic fungus Talaromyces thermophilus

Phytases (EC 3.1.3.8) belong to the family of histidine acid phosphatases. We have cloned the phytases of the fungi Emericella nidulans and Talaromyces thermophilus. The putative enzyme encoded by the E. nidulans sequence consists of 463 amino acids and has a M r of 51785. The protein deduced from the T. thermophilus sequence consists of 466 amino acids corresponding to a M r of 51450. Both predicted amino acid sequences exhibited high identity (48% to 67%) to known phytases. This high level of identity allowed the modelling of all available fungal phytases based on the three-dimensional structure coordinates of the Aspergillus niger phytase. By this approach we identified 21 amino acids which are conserved in fungal phyA phytases and are part of the residues forming the substrate pocket. Furthermore, potential glycosylation sites were identified and compared between the aforementioned phytases and the A. niger phytase.

Effects of phosphorus level and microbial phytase supplementation on the performance and bone mineralization in pigs

An experiment was conducted with 192 growing and finishing pigs in order to evaluate the phosphorus (P) requirement of the pigs and the efficacy of Aspergillus niger phytase on a barley based diet, and to study phase feeding of P and phytase. The performance of the pigs and certain chemical and physical parameters of their tibia and fibula bones were used as criteria for the effectiveness of the treatments. The variables in diet formulation were microbial phytase addition of 500 FTU kg−1 (‐/+ ) and three P levels—high (HP), medium (MP) and low (7.7, 6.1 and 4.5 g of total P kg−1 dry matter, respectively). Two additional diets were combinations of the former: MP—LP—and MP + LP —. The medium P level was sufficient for the pigs from 30 kg up to slaughter at 100 kg with the growth level of 900 g day and feed conversion ratio (FCR) of 2.60, but the lowest P level led to an impaired average daily gain (ADG) and FCR. Microbial phytase improved the ADG (P < 0.05) and FCR of pigs (P < 0.05) on LP diet. No signs of leg weakness were observed in the pigs on any of the diets. The tibia densities decreased with decreasing dietary P. The ash content of fibula bones increased and their P content decreased with declining dietary P. Lowering the P content of the diet, using an appropriate phase feeding of P, and adding phytase into the diet could support the normal growth rate of the pigs and reduce the amount of P excretion in faeces.

Expression of an Aspergillus niger Phytase (phyA) in Escherichia coli

The gene (phyA) for the Aspergillus niger phytase with optima at pH 5.5 and 2.2 was expressed in Escherichia coli under the control of the T7lac promoter. A 56 kDa fusion protein comprised of phytase linked to an S-Tag leader peptide accumulated in inclusion bodies at 30 °C. The yield of unglycosylated recombinant phytase purified from 50 mL cultures by anion exchange chromatography of solubilized inclusion body protein was 10 mg. The refolded enzyme had an activity of 1.5 μmol mg-1 min-1 at 37 °C, but most of the protein was in the form of inactive aggregates. Recombinant phytase displayed a single pH optimum at pH 5.1, was irreversibly denatured at pH 2.0 and was not active above 55 °C. As with A. niger phytase, the initial breakdown product observed was inositol 1,2,4,5,6-pentakis(phosphate). Km values for the hydrolysis of inositol hexakis(phosphate) and p-nitrophenylphosphate were 96 μM and 2.0 mM, respectively, at pH 4.5. Keywords: Phytase; phosphatase; phytic acid; inositol phosphates; Aspergillus ...

The effects of sow parity on digestibility of proximate components and minerals during lactation as influenced by diet and microbial phytase supplementation.

Ninety-six (Finnish Landrace x Dutch Landrace) reproductive sows were used at parities 1, 3, 5, and 7 + 8 from d 107 of gestation to d 21 of lactation to investigate the effects of diet and parity on apparent total tract digestibility (ATTD). Animals were randomly assigned to one of four dietary treatments. Dietary treatments were 1) a P-deficient (1.1 g digestible P [dP]/kg) Dutch semipractical diet; 2) Diet 1 supplemented with 400 FTU Aspergillus niger phytase per kilogram of diet (1.7 g dP/kg); 3) a corn-soybean meal-based diet (1.3 g dP/kg); and 4) Diet 3 supplemented with extra monocalcium phosphate (MCP; 2.4 g dP/kg of diet). Animals were fed twice daily at 2.8 times maintenance (418 kJ ME/ BW75) from d 8 to the end of lactation. Feces and urine were collected during d 11 to 13 and d 18 to 20 of lactation. The ATTD of DM, OM, ash, CP, Ca, Mg, and total P (P < .01) were higher for the corn-soybean meal-based diets than for the Dutch semipractical diet not supplemented with phytase. Addition of MCP enhanced total P ATTD by an average of 6.7 percentage units. Addition of microbial phytase improved Ca, Mg, and total P ATTD, but the effects were dependent on the stage of lactation. Lower ATTD of OM and CP were seen for first parity animals compared with higher parity sows. The ATTD of Mg increased with increasing parity. Parity had little effect on the ATTD of minerals during lactation, and dietary effects were prominent and followed a similar trend to those seen in growing-finishing pigs.

The efficacy of Aspergillus niger phytase in rendering phytate phosphorus available for absorption in pigs is influenced by pig physiological status.

We performed an experiment with 112 piglets, 32 growing-finishing pigs, and 12 sows during pregnancy and lactation to test the hypotheses that apparent total tract digestibilities of P and Ca as well as the efficacy of Aspergillus niger phytase in rendering phytate P available in pigs depend on pig physiological status. All pigs were fed diets with identical feedstuff composition either without or with added microbial phytase (Natuphos, 500 FTU/kg diet). The diets contained 6.2 g Ca, 4.8 g total P, and 3.7 g phytate P per kilogram, and intrinsic phytase activity of 120 FTU/kg. The digestibility of P increased by 8.3 percentage units when BW of pigs increased from 30 to 60 kg and then remained stable until 100 kg. Pregnant sows had a lower efficiency of P absorption than piglets and growing-finishing pigs. During lactation, the efficiency of P absorption was 3.4 percentage units higher than during pregnancy but was still 6.6 percentage units lower than for growing-finishing pigs. The efficacy of the phytase in generating digestible P decreased in the order or lactating sows, growing-finishing pigs, sows at the end of pregnancy, piglets, and sows at midpregnancy; the average amounts of generated digestible P were 1.03, .83, .74, .66, and .32 g/kg diet, respectively. The addition of phytase to the diet raised apparent Ca digestibility in the piglets and growing-finishing pigs (by 4.6 and 4.0 percentage units, respectively) but not in the sows. We conclude that in the formulation of swine diets the amount of phytase to be added should be tailored to the target category.

Dietary Aspergillus niger Phytase Increases Iron Absorption in Humans

Phytate is an inhibitor of iron absorption that can be removed before the intestinal site of absorption by microbial phytase, thereby increasing iron absorption from a meal. The effects of two kinds of dietary phytase, cereal phytase and microbial phytase from Aspergillus niger, on iron absorption were investigated. Iron absorption was measured from single meals containing white wheat rolls supplemented with wheat bran with or without phytase activity (expt. 1) and phytase-deactivated wheat bran with or without addition of microbial phytase from A. niger (expt. 2). Each experiment had 10 subjects and two different radio iron tracers: 55Fe and 59Fe were used for comparison of the absorption from the test meals in each experiment. No differences in iron absorption were found between meals containing wheat bran with or without phytase activity. Addition of microbial phytase to the meal containing phytase-deactivated wheat bran increased iron absorption from 14.3 ± 2.6% to 26.1 ± 3.8% (P < 0.0001). Two pH optima, one at pH 2.0 and one at pH 6.0, were found for A. niger phytase at 37°C, but activity occurred at all pH values between 1.0 and 7.5. The results suggest that effective and complete degradation of phytate occurred in the stomach when A. niger phytase was given with the meal. This may be explained by high activity of microbial phytase at physiological pH conditions of the stomach, whereas wheat phytase has a different pH optimum.

Stable accumulation of Aspergillus niger phytase in transgenic tobacco leaves.

Phytase from Aspergillus niger increases the availability of phosphorus from feed for monogastric animals by releasing phosphate from the substrate phytic acid. A phytase cDNA was constitutively expressed in transgenic tobacco (Nicotiana tabacum) plants. Secretion of the protein to the extracellular fluid was established by use of the signal sequence from the tobacco pathogen-related protein S. The specific phytase activity in isolated extracellular fluid was found to be approximately 90-fold higher than in total leaf extract, showing that the enzyme was secreted. This was confirmed by use of immunolocalization. Despite differences in glycosylation, specific activities of tobacco and Aspergillus phytase were identical. Phytase was found to be biologically active and to accumulate in leaves up to 14.4% of total soluble protein during plant maturation. Comparison of phytase accumulation and relative mRNA levels showed that phytase stably accumulated in transgenic leaves during plant growth.

Availability for growing pigs of minerals and protein of a high phytate barley-rapeseed meal diet treated with Aspergillus niger phytase or soaked with whey

The improvement of phytin-phosphorus utilization in barley-rapeseed meal (800 and 200 g kg −1) diet due to Aspergillus niger phytase (EC 3.1.3.8) supplement was assayed in a 8 × 6 cyclic change over designed digestibility and balance trial with eight growing pigs of 28–70 kg live weight. The 2 × 2 × 2 factorially arranged diets were either fortified with dicalcium phosphate to supply total phosphorus (P) 6.7 and available P (aP) 2.8 g kg −1 or without inorganic P supplement, P 5.3, aP 1.4 g kg −1; were fed either as soaked with whey at 40 °C for 3 h or without soaking; and half the diets were supplemented with phytase 1000 PU g −1 (Finase ® FP500). Each diet contained calcium (Ca) 8.0 g kg −1. Other nutrients were at similar levels in each diet. Soaking of the meal with whey significantly improved ash and organic matter digestibilities ( P < 0.001). The digestibilities of ash ( P < 0.001) and ether extract ( P < 0.05) were enhanced (2 and 3% units) by phytase treatment. No effect on nitrogen (N) utilization was found due to phytase addition, but soaking significantly improved N retention in relation to N intake. Both the soaking of the diet and addition of microbial phytase significantly improved ( P < 0.01, P < 0.001) the apparent absorption of P (3 and 9% units). The retained P in diets with supplementary phytase was significantly higher ( P < 0.01) than that without, 3.8 vs. 2.8 g day −1. The soaking had an enhancing effect on P retention. P retention in relation to intake was greater ( P < 0.001) in diets with added phytase than without (36 vs. 31%). From the diet with inorganic P supplementation a significantly lower retention value, 29% ( P < 0.001), was achieved compared with the P-unsupplemented diets. Phytase supplementation did not affect Ca absorption but significantly increased Ca retention due to lower urinary excretion. The soaking had no effect on Ca absorption or retention. Utilization of magnesium and zinc was not affected by the treatments. The results indicate that microbial phytase and soaking with whey improved the utilization of phytate P in barley-rapeseed meal diet and reduced the amount of P excretion in faces.

Effects of supplemental microbial phytase on phosphorus digestibility and utilization in rainbow trout (oncorhynchus mykiss)

Potential effects of supplemental microbial phytase on digestibility and utilization of phosphorus (P) originating from plant materials were studied in trout. Diets were mainly based on soybean products and contained P only from plant materials. Concentration of total P was suboptimal and about 60% of total P were bound as phytate P. Diets were prepared either without enzyme supplementation or after supplementation with an Aspergillus niger phytase (1000 units/kg diet). Apparent digestibility of P was determined at a water temperature of 15°C. Utilization of P was measured in two growth trials at water temperatures of either 15 or 10°C. P retention of trout was measured using the comparative body analysis. At 15°C, both digestibility and utilization of dietary P were considerably improved when microbial phytase was supplemented. P digestibility and P utilization increased from 25 to 57% and from 17 to 49%, respectively. At 10°C, P utilization increased from 6 to 25%, but intake of the diet as well as growth rate of trout were very low, irrespective of whether phytase was supplemented or not. Practical relevance of the findings seems to apply only to diets in which protein is supplied almost entirely by plant products.

Effect of a supplemental Aspergillus niger phytase on the utilization of plant phosphorus by rainbow trout (Oncorhynchus mykiss)

Effects of a supplemental Aspergillus niger-phytase on digestibility and utilization of dietary phosphorus (P) were studied in three experiments with rainbow trout. P concentration in the diets was 4.8 and 5.8 g/kg DM, respectively. The P contained in the diet originated solely from plants, mainly soy-products. Digestibility of P was studied using the stripping method and hydrochloride insoluble ash as marker. Utilization was studied in growth trials by use of the comparative body analysis. At a water temperature of 15 degrees C, both digestibility and utilization of P were increased from 25 to 57% and from 17 to 49%, respectively when 1000 U/kg phytase were supplemented. Feed consumption and gain of trout were significantly increased. At a water temperature of 10 degrees C, utilization of P was also increased from 6 to 25%. However, feed consumption and gain of trout were very low at this water temperature and not influenced by the supplemental phytase.

The effect of microbial phytase in diets for pigs on apparent ileal and faecal digestibility, pH and flow of digesta measurements in growing pigs fed a high-fibre diet

The effect of Aspergillus niger phytase on digestibility of some nutrients, pH and flow of digesta in post valvular T-caecum (PVTC) cannulated growing pigs fed an oat (80.6%) diet was studied. Four barrows weighing on average 40 kg were assigned to four diets in a 4 × 4 Latin square design. Diet 1 contained 1.11% monocalcium phosphate (MCP); diet 2 had neither MCP nor phytase; diet 3 included phytase at a concentration of 487 phytase units kg−1 feed; and diet 4 had 0.55% MCP and 320 phytase units kg−1 feed. Ileal digestibility of phosphorus (P) increased by 19.4 and 26.8% in diets containing microbial phytase compared with the control diets 1 and 2, respectively. Corresponding increases in faecal digestibility values were 18.3 and 23.6%. Ileal digestibility of calcium (Ca) was significantly higher in diet 3 but there was no effect of diet on the faecal digestibility of Ca. There was no effect of microbial phytase on the digestibility of magnesium (Mg), dry matter (DM) or crude protein (CP). Ileal digestibility of crude fibre (CF) was not affected by diet, but faecal CF digestibility was lower (P &lt; 0.05) in diets containing microbial phytase. There was no effect of phytase on excretion of Ca, Mg and N and Mg and N retention. There was a time-related response of pH of ileal digesta, with hourly pH measurements ranging between 7.1 and 5.9, irrespective of diet. Flow of digesta peaked 3–6 h after the morning feed and 1–2 hours after the evening feed. Key words: Pigs, cannulation, phytase, digestibility, pH

Effects of supplemental microbial phytase on phosphorus digestibility and utilization in rainbow trout (Oncorhynchus mykiss)

Potential effects of supplemental microbial phytase on digestibility and utilization of phosphorus (P) originating from plant materials were studied in trout. Diets were mainly based on soybean products and contained P only from plant materials. Concentration of total P was suboptimal and about 60% of total P were bound as phytate P. Diets were prepared either without enzyme supplementation or after supplementation with an Aspergillus niger phytase (1000 units/kg diet). Apparent digestibility of P was determined at a water temperature of 15°C. Utilization of P was measured in two growth trials at water temperatures of either 15 or 10°C. P retention of trout was measured using the comparative body analysis. At 15°C, both digestibility and utilization of dietary P were considerably improved when microbial phytase was supplemented P digestibility and P utilization increased from 25 to 57% and from 17 to 49%, respectively. At 10°C, P utilization increased from 6 to 25%, but intake of the diet as well as growth rate of trout were very tow, irrespective of whether phytase was supplemented or not. Practical relevance of the findings seems to apply only to diets in which protein is supplied almost entirety by plant products.

Evaluation of aspergillus niger phytase and phosphate with weaned piglets

A high phytic acid diet (barley, wheat, soya bean meal, 4 g P/kg diet, of that 2/3 phytate P) without added phytase, with phytase supplement (1000 U/kg diet) or with supplementary phosphate (2.2 g P/kg diet) was examined with 3 x 12 weaned piglets. The three diets contained 8 g Ca/kg. At the end of experiment 6 pigs/group were slaughtered. In animal body (as empty body) the content and gain of ash, P, Ca, protein and fat were detected. P supplementation and supplementary phytase had no effect on dry matter, protein and fat content of animal body. The enzyme but more the supplementary phosphate increased mineralization of skeleton and made the animal body higher in ash, P and Ca content. Piglets without supplementary phytase and P gained 1.1 g P daily. Phytase increased daily P gain by 0.5 g (P < 0.05), the phosphate by 1.4 g (P < 0.001). The daily Ca gain was 1.7; 2.8 and 5.1 g in the different groups. A piglet (body weight 20 kg) with sufficient P and Ca in the diet gains 5 g P and 10 g Ca per kg body weight gain (empty body).

Supplementing corn-soybean meal diets with microbial phytase linearly improves phytate phosphorus utilization by weanling pigs.

Two experiments were conducted with weanling pigs to determine the effectiveness of a dietary supplement of Aspergillus niger phytase in improving the availability of phytate-P in corn-soybean meal diets without supplemental inorganic P. Experiment 1 consisted of two P and Ca balance trials and two feeding trials. Twelve pigs (8.18 +/- .44 kg BW) were housed individually in stainless steel metabolism cages. Six pigs received 750 phytase units (PU)/g of basal diet and the other six pigs received the basal diet without supplemental phytase as control. In Exp. 2, 96 pigs (8.81 +/- .75 kg BW) were allotted to 16 partially slotted floor pens and their basal diets were supplemented with either 0, 250, 500, or 750 PU/g for 4 wk. Individual pig weights and pen feed consumption were measured weekly. Blood samples were taken from all pigs at the end of each trial in Exp. 1 and from three pigs per pen weekly in Exp. 2 to measure serum (plasma) inorganic P (P) and Ca concentrations and alkaline phosphatase (AP) activities. The results of Exp. 1 indicated that dietary phytase increased P retention by 50% (P < .0001) and decreased fecal P excretion by 42% (P < .0001). Pigs that received dietary phytase had serum P and Ca concentrations and serum AP activities that were nearly normal, whereas control pigs had values indicative of a moderate P deficiency. Favorable effects of phytase disappeared when the phytase was removed from the diet.(ABSTRACT TRUNCATED AT 250 WORDS)

Supplementing corn-soybean meal diets with microbial phytase maximizes phytate phosphorus utilization by weanling pigs.

Two experiments were conducted with crossbred weanling pigs to determine the optimal dietary supplement of Aspergillus niger phytase activity to a low-P, corn-soybean meal basal diet (BD). In Exp. 1, 50 pigs (7.61 +/- .56 kg BW) received the BD supplemented with 750, 1,050, 1,250, or 1,350 phytase units (PU)/g, or .21% P as mono-dibasic calcium phosphate (MDCaP) for 4 wk. In Exp. 2, 12 pigs (6.39 +/- .74 kg BW) were individually housed in metabolism cages and received BD, BD plus the optimal phytase activity (1,200 PU/g), or BD plus .21% P as MDCaP for 2 wk. In Exp. 1, additions of phytase > 1,050 PU/g of BD did not improve ADG, ADFI, gain/feed, or plasma AP activity. Quadratic relationships between dietary phytase activity and these measures were found and their stationary points were at approximately 1,200 PU/g of BD. Estimated maximum responses of these measures in pigs fed phytase were > or = 90% compared with MDCaP. Pigs fed 1,250 PU/g of BD maintained normal plasma P and Ca concentrations. In Exp. 2, pigs that received 1,200 PU/g of BD utilized dietary P more effectively (P < .05) than pigs fed the BD or the BD plus MDCaP. Although they consumed 44% less P per day, these pigs retained only 7% less P than pigs that received MDCaP. One thousand units of phytase activity supported retention of 1.1 mg of P from the BD, and this level of phytase supplementation was equivalent in effect to .91 mg of P from MDCaP.(ABSTRACT TRUNCATED AT 250 WORDS)

Identification and Cloning of a Second Phytase Gene (phyB) from Aspergillus niger (ficuum)

An Aspergillus niger (ficuum) genomic DNA λEMBL3 library was probed with a 354-bp DNA fragment obtained by polymerase chain reaction of A. niger DNA with oligonucleotides based on partial amino acid sequence of a pH 2.5 optimum acid phosphatase. A clone containing a 1605 bp segment (phyB) encoding the 479 amino acid enzyme was isolated and found to contain four exons. Global alignment revealed 23.5% homology to Aspergillus niger phytase (PhyA); four regions of extensive homology were identified. Some of these regions may contain catalytic sites for phosphatase function.

Effect of supplementaryAspergillus nigerphytase in diets for piglets on their performance and apparent digestibility of phosphorus

AbstractA total of 384 piglets from a three-way cross Yorkshire ♂ × (Finnish Landrace ♂ × Dutch Landrace♀) ♀ in the live-weight range of 11 to 25 kg were used in a feeding trial. The effect of supplementary microbial phytase from Aspergillus niger var. Van Tieghem on performance (average daily gain (ADG), average daily food intake (ADFI) and food conversion ratio (FCR)) and apparent digestibility of total phosphorus (P), in comparison with a non-supplemented basal diet or a diet supplemented with one or two levels of monocalcium phosphate (MCP), were studied.The piglets were allotted to 12 blocks. Within each block, four experimental diets were offeredad libitumfor a period of 4 weeks i.e. (1) basal diet; (2) basal diet + 1·0 g P, from MCP per kg diet; (3) basal diet + 1·9 g P, from MCP per kg diet; and (4) basal diet + 1450 phytase units (PTU) per kg diet. The diets, with added chromium III oxide as a marker, were cold pelleted without steam at a temperature not above 60°C to avoid a possible inactivation of the phytase. In all diets the ratio calcium: digestible P was intended to be constant.Adding 1450 PTU of Aspergillus niger phytase enhanced the apparent digestibility of P by proportionately 0·216 when compared with diet 1 and by proportionately 0·100 compared with diet 3. In addition, significantly higher ADG and ADFI and better FCR were noted in pigs receiving supplementary microbial phytase than in pigs on any other treatment. The content of digestible P of the diet with added phytase, however, zvas between the two levels of the diets with added MCP.

The effect of supplementary Aspergillus niger phytase in diets for pigs on concentration and apparent digestibility of dry matter, total phosphorus, and phytic acid in different sections of the alimentary tract.

Six barrows of approximately 37 kg BW, fitted with two simple T-cannulas in the duodenum (25 cm posterior to the pylorus) and terminal ileum (12 to 15 cm anterior to the ileocecal junction), were fed two diets containing 2.1 g of P/kg in the form of phytic acid and a low intrinsic phytase activity (corn-soybean meal based diet [Diet A] or a typical Dutch diet [Diet B]) without or with supplementary microbial phytase from Aspergillus niger (var. ficuum) equal to 1,500 phytase units per kilogram of diet, in a crossover design. The apparent duodenal, ileal, and total tract (overall) digestibilities of DM, total P, and phytate P (phytic acid x .282) were calculated using both Cr-NDR (neutral detergent residue mordanted with Cr) and Co-EDTA as dual-phase markers. Concentration of total P in the ileal digesta (P less than .01) and feces (P less than .001) of pigs fed microbial phytase was lower than without this enzyme, irrespective of the diet. Ileal digestibility of total P was 18.5 and 29.8 percentage units higher (which was a 1.7- to 2.9-fold increase) due to added Aspergillus niger phytase (P less than .05). Also, total tract (overall) digestibility increased by 27.0 to 29.7 percentage units (P less than .01). Phytic acid concentration in the duodenal and ileal digesta of pigs receiving microbial phytase was lower (P less than .01 or .001), resulting in its higher ileal digestibility (dephosphorylation rate) by 50.1 percentage units for Diet A and by 75.4 percentage units for Diet B. Irrespective of the treatment, no phytase activity could be detected in the ileal digesta of pigs.