Aspergillus Niger Phytase Research Articles

Role of metal complexation on the solubility and enzymatic hydrolysis of phytate.

Phytate is a dominant form of organic phosphorus (P) in the environment. Complexation and precipitation with polyvalent metal ions can stabilize phytate, thereby significantly hinder the hydrolysis by enzymes. Here, we studied the stability and hydrolyzability of environmentally relevant metal phytate complexes (Na, Ca, Mg, Cu, Zn, Al, Fe, Al/Fe, Mn, and Cd) under different pHs, presence of metal chelators, and thermal conditions. Our results show that the order of solubility of metal phytate complexes is as follows: i) for metal species: Na, Ca, Mg > Cu, Zn, Mn, Cd > Al, Fe, ii) under different pHs: pH 5.0 > pH 7.5), and iii) in the presence of chelators: EDTA> citric acid. Phytate-metal complexes are mostly resistant towards acid hydrolysis (except Al-phytate), and dry complexes are generally stable at high pressure and temperature under autoclave conditions (except Ca phytate). Inhibition of metal complex towards enzymatic hydrolysis by Aspergillus niger phytase was variable but found to be highest in Fe phytate complex. Strong chelating agents such as EDTA are insufficient for releasing metals from the complexes unless the reduction of metals (such as Fe) occurs first. The insights gained from this research are expected to contribute to the current understanding of the fate of phytate in the presence of various metals that are commonly present in agricultural soils.

Performance of Aspergillus niger (NCIM 563) phytase based feed supplement for broiler growth and phosphorus excretion

Despite availability of commercial enzymes, the phytase produced from relatively inexpensive systems with high yields are gaining global attention in the feed industries in post-antibiotic era. We studied A. niger NCIM 563 Phytase produced in solid state fermentation (SSF) derived Koji powder and evaluated its utility in the poultry feed for broiler growth performance and phosphorous (P) excretion. The ability of phytase in the dried powder was estimated to dephytinize the poultry feed under simulated gastric conditions. Poultry feed was formulated using A. niger NCIM 563 phytase followed by a 42 days feed trial on broilers. After supplementation of phytase to the diet, there was a reduction of dietary P, maintained growth performance, skeletal development of broilers and reduced levels of phytic acid and available P in the litter. Extracellular phytase was able to replace up to 0.1% P in poultry feed. Minimal downstream processing a low-cost feed supplement with significant phytase activity could provide added advantage for anti-nutrition free poultry feed. • Fungal phytase was produced in lab by solid state fermentation. • Dried koji was directly used as phytase source in poultry feed. • Phytase at 500U/kg of feed can replace 0.1% NPP without affecting growth performance.

Recovery and purification of Aspergillus niger phytase from crude extract using AOT / isooctane reversed micelles

This work describes the successful extraction of Aspergillus niger phytase from a crude extract (CE) obtained from solid-state fermentation by reversed micelle system using anionic surfactant sodium bis (2-ethylhexyl) sulfosuccinate (AOT) in isooctane achieved in two simple steps: forward and backward extractions. The effects of potassium chloride (KCl) concentration, pH of the aqueous solution, and AOT concentration that affect the system were examined. The best result for the forward extraction was obtained with the CE solution at pH 4.0, 50 mM KCl, and 100 mM AOT, while for the backward extraction the best result was achieved with a stripping aqueous solution at pH 5.5 containing 200 mM KCl, achieving a purification factor of 4.03, 1.15 times higher than that reported for the conventional purification process. Phytase purity was demonstrated by SDS-PAGE (89 kDa) and its activity by zymogram, confirming the efficiency of the process with low time consumption (∼40 min).

Microbial and Fungal Phytases Can Affect Growth Performance, Nutrient Digestibility and Blood Profile of Broilers Fed Different Levels of Non-Phytic Phosphorous.

Simple SummaryTo reduce the environmental pollution is a must to preserve the health of the world. The environmental impact of poultry farming is receiving an increasing attention due to several emissions among these is phosphorus. This element is in general present in the commercial diets of broilers or laying hens in an amount exceeding the real needing of the animals, and, therefore, a great amount of phosphorus ends in the excreta. Thus, optimizing the amount of phosphorous in the diets of poultry could partially alleviate the environmental impact of these farms.A total of 420 day old chicks were divided into seven groups (5 replicates of 12 chicks/group) fed isoproteic and isoenergetic diets. The control group was fed diets containing 0.50%, 0.45% and 0.40% of non-phytic phosphorous (nPP) in starter (1–35), grower (37–56) and finisher (57–64 d) periods, respectively. The three intermediate nPP (IntnPP) groups were fed diets with 0.40%, 0.35% and 0.30% nPP according to the growth period and were submitted to three dietary treatments: unsupplemented; supplemented with 500 FTU/kg diet of an Aspergillus niger phytase (IntnPP_fp) and supplemented with 500 FTU/kg diet of an Escherichia coli phytase (IntnPP_bp). The three low nPP groups fed diets contained 0.30%, 0.25% and 0.20% nPP and were submitted to the same dietary treatments than IntnPP to obtain LnPP, LnPP_fp and LnPP_bp groups. IntnPP and LnPP groups had lower body weight gain and feed, crude protein (CP) and metabolizable energy (ME) intake (p < 0.05) than the control. Feed conversion ratio of IntnPP was more favorable (p < 0.01) than the LnPP group. CP and ME conversion ratios worsened (p < 0.01) in IntnPP and LnPP groups in comparison to the control. The nPP conversion ratio improved (p < 0.01) from the control to the LnPP group. Fungal phytase reduced (p < 0.05) feed, CP, ME and nPP intake than the bacterial one. IntnPP and LnPP diets had a lower digestibility of CP (p < 0.01) and CF (p = 0.01) than the control. IntnPP and LnPP groups showed a higher (p < 0.05) economic efficiency than the control. Blood total protein was the lowest (p < 0.05) in the LnPP group, the control group showed the lowest (p < 0.05) level of albumin and IntnPP group had the lowest (p < 0.01) globulin level. The use of bacterial phytase increased (p < 0.01) total protein and globulin and decreased (p < 0.05) the plasma cholesterol in comparison to fungal phytase. Decreasing nPP levels in colored slow-growing broilers diet negatively affects growth performance and the use of phytase can partly alleviate these negative effects, but the efficiency of different enzyme sources (bacterial or fungal) was tied to the dietary nPP levels.

The effect of microbial phytase supplementation of sorghum-canola meal diets with no added inorganic phosphorus on growth performance, apparent total-tract phosphorus, calcium, nitrogen and energy utilization, bone measurements, and serum variables of growing and finishing swine

Two experiments were conducted with growing and finishing swine to determine the effect of phytase supplementation of sorghum-canola meal diets without added inorganic phosphorus (iP) on growth performance, apparent total-tract P, calcium (Ca), nitrogen (N), and energy utilization, bone strength, bone ash, serum Ca and P concentrations, and serum phosphatase activity. For Exp. 1, 42 crossbred barrows in individual metabolism cages were fed a P-deficient diet for 7 d before starting a 28-d experiment at 24.8 kg body weight (BW). Experiment 1 had 7 treatments that were a negative control (NC) diet supplemented with 0, 400, 800, 1200, or 1600 U of Aspergillus niger (AN) phytase/kg, a positive control (PC) diet, and the PC diet supplemented with 1200 U of AN phytase/kg. Fecal and urine collections were made from d 21–26. Blood samples were collected on d 0, 14, and 28. For Exp. 2, 126 growing-finishing crossbred swine with an initial BW of 21.6 kg were group-fed (3 pigs/pen) in 3 phases (grower, early-finisher, and late-finisher). Experiment 2 had 6 treatments that were a NC diet for each phase supplemented with 0, 200, 400, 600, or 800 U of AN phytase/kg and a PC diet. Fecal collections were made from d 49–54. Blood samples were collected on d 35, 63, and 91. For both experiments, 400 U of AN phytase/kg of diet was adequate (P ≤ 0.05) for growth performance compared with the PC treatment groups. For Exp. 1, apparent total-tract P absorption and retention plateaued at 1200 U of AN phytase/kg of diet (P < 0.01), with fecal Ca and P excretion reduced 22.4% and 28.6%, respectively, compared with the PC treatment group. However, pigs fed the PC + 1200 U of AN phytase/kg absorbed and retained more P (g/d, P < 0.01) than any other treatment group. For Exp. 2, apparent total-tract P absorption, bone strength, and bone ash weight plateaued at 600 U of AN phytase/kg of diet (P < 0.01), with fecal P excretion reduced 29.4% compared with the PC treatment group. For both experiments, the AN phytase treatments did not increase Ca, N, or energy absorption (amount/d or % of intake), and had no effect on serum Ca and P concentrations, or the serum activity of alkaline phosphatase, total acid phosphatase, or tartrate-resistant acid phosphatase.

Purification and biochemical characterization of an Aspergillus niger phytase produced by solid-state fermentation using triticale residues as substrate

In this study, an extracellular phytase produced by Aspergillus niger 7A-1, was biochemically characterized for possible industrial application. The enzyme was purified from a crude extract obtained by solid-state fermentation (SSF) of triticale waste. The extract was obtained by microfiltration, ultrafiltration (300, 100 and 30 kDa) and DEAE-Sepharose column chromatography. The molecular weight of the purified enzyme was estimated to be 89 kDa by SDS-PAGE. The purified enzyme was most active at pH 5.3 and 56 °C, and retained 50% activity over a wide pH range of 4 to 7. The enzymatic thermostability assay showed that the enzyme retained more than 70% activity at 80 °C for 60 s, 40% activity for 120 s and 9% after 300 s. The phytase showed broad substrate specificity, a Km value of 220 μM and Vmax of 25 μM/min. The purified phytase retained 50% of its activity with phosphorylated compounds such as phenyl phosphate, 1-Naphthyl phosphate, 2-Naphthyl phosphate, p-Nitrophenyl phosphate and Glycerol-2-phosphate. The inhibition of phytase activity by metal ions was observed to be drastically inhibited (50%) by Ca++ and was slightly inhibited (10%) by Ni++, K+, and Na+, at 10 and 20 mM concentrations. A positive effect was obtained with Mg++, Mn++, Cu++, Cd++ and Ba++ at 25 and 35% with stimulatory effect on the phytase activity.

Improvement in biochemical characteristics of glycosylated phytase through immobilization on nanofibers

Abstract The objective of the current study was to enzymatically characterize immobilized phytase as a glycoprotein on an optimal blend of starch/PAAm nanofibers. Aspergillus niger phytase (phyA) was first expressed in Hansenula polymorpha using formate dehydrogenase promoter and α-mating factor signal peptide. Response surface methodology was then used to optimize the different levels of the enzyme, temperature and pH on the immobilized phytase. The electrospun nanofibers showed uniform morphology with an average diameter of approximately 269 nm. Afterward, the kinetic parameters of Km (56 μM) and Vmax (401 μmol/min mg) of the immobilized enzyme improved, but the optimum pH and temperature had not changed in comparison with the soluble phytase. The applicability of the nanofiber was confirmed by its outstanding immobilization efficiency (193%) and suitable retention of catalytic activity of the immobilized phytase (50.4% after the tenth repetition). Optimal orientation of the enzyme on the nanofiber using the in silico approach was predicted, which could improve the kinetic properties. The results demonstrate the good potential of H. polymorpha as an efficient expression system for the production of glycoproteins for immobilization.

Effect of citrate on Aspergillus niger phytase adsorption and catalytic activity in soil

Phytase enzymes from bacteria, fungus and plant root exudates are known to hydrolyse organic phosphorus (Po) to bioavailable inorganic orthophosphate in soil. Exploiting such biochemical functions in agricultural systems, offers the potential for alternative sustainable phosphorus sources. Phytase adsorption to soil particles and phytate metal complexation has been shown to inhibit phytate (InsP6) dephosphorylation. Organic acid anions such as citrate increase phytase catalytic efficiency towards complexed forms of InsP6, but the mechanisms are poorly understood. The aim of this work was to evaluate Aspergillus niger phytase inactivation and changes in its catalytic properties upon addition to soil, as well as the effect of citrate on phytase adsorption and activity towards free, precipitated and adsorbed InsP6.We performed a series of enzyme hydrolysis activity assays when the enzymes were free, in soil solution and adsorbed to soil under varying chemical conditions, with and without citrate. A. niger phytase showed a relatively low absorption affinity for the Cambisol test soil. Phytase activity reduced by 37.3% due to adsorption. Citrate had no effect on the rate or total amount of phytase adsorption or soil adsorbed phytase activity thereafter. Free phytases and phytases in soil solution showed optimum activity (≥80%) at pH4.5–5.5. Activity decreased slightly for soil adsorbed enzymes compared to enzymes which were free or in soil solution>pH5 and <4 in the pH dependency curve. A decrease in activity was seen only at ionic strengths (NaCl)>0.6M in adsorbed and free enzymes, while activity from enzymes in soil solution reduced in all tested ionic strengths. Citrate significantly increased phytase activity towards InsP6 adsorbed to soil when the phytases were free (p≤0.003) but not in other treatments (Na, Al and Ca-phytate). These results suggest that the effect of citrate on soil InsP6 dephosphorylation is associated with the availability of the substrate (InsP6) rather than its effect on the enzyme per se. The ionic strength and pH of soil solution has been shown to impact on phytase activity, suggesting that salinity, quality of irrigation water, wetting/drying cycles and fertilisation will have discrete impacts on the activity of phytases once released in soil and thus the ability to make Po available for uptake by plants and microbes. The optimum acidic pH of A. niger also brings into question its suitability for application in many agricultural soils.

Limestone particle size and Aspergillus niger phytase in the diet of older hens

The aim of this experiment was to evaluate the influence of limestone particle size and phytase (F) on performance, physical egg parameters, mineral digestibility and microbial colonisation in the digestive tract in older hens aged 60–71 weeks. One hundred and sixty Lohmann Brown hens were housed in enriched cages. The 2 × 2 factorial experiment included two levels of 3-phytase NATUPHOS® (0 and 350 FTU/kg), two sizes of limestone grain (coarse limestone (CL) and a mix of CL and fine limestone (FL) (1:1)) in mixed feed. All of the diets contained 4.4 g/kg of total phosphorus and 34 g/kg of calcium. Coarse limestone with F addition significantly increased hen-day egg production (p = .018). The phytase supplement at 350 FTU/kg significantly increased shell quality characteristics. Haugh units (p = .039) and albumen index (p = .008) was higher when using the limestone mix, the same applies to yolk index (p = .013). The phytase addition to the mixed feed non-significantly increased calcium and phosphorus digestibility in the ileum by 3.1 and 3.9%, respectively. Intestinal microbiota from each sample did not cluster according to the treatment; however, bacteria belonging to the genera Turicibacter and Lactobacillus occurred with higher frequency in the ileum of hens that were fed mixed feed enriched with phytase. In conclusion, limestone particle size (1 to 2 mm) with F addition at 350 FTU/kg in a wheat-maize-soybean meal-based diet increased egg production while maintaining egg content and eggshell quality in older hens. Ileal microbiota was influenced by F supplementation in the diet.

Linking the depletion of rhizosphere phosphorus to the heterologous expression of a fungal phytase in Nicotiana tabacum as revealed by enzyme-labile P and solution 31P NMR spectroscopy

Root exudation of phytase could improve the ability of plants to access organic forms of soil phosphorus (P), thereby minimizing fertilizer requirements and improving P use efficiency in agroecosystems. After 75 days growth in a high available P soil, shoot biomass and P accumulation, soil pH, and rhizosphere P depletion were investigated in Nicotiana tabacum wild-type and transgenic plant-lines expressing and exuding Aspergillus niger phytase (ex::phyA), or a null-vector control. Solution 31P NMR analysis revealed a 7% to 11% increase in orthophosphate and a comparable depletion of undefined monoester P compounds (-13 to -18%) in the rhizosphere of tobacco plants relative to the unplanted soil control. Wild-type plants had the greatest impact on the composition of rhizosphere P based on the depletion of other monoester P, polyphosphate, and phosphonate species. The depletion of phytase-labile P by ex::phyA plants was associated with decreased proportions of other monoester P, rather than myo-InsP6 as expected. Rhizosphere pH increased from 6.0 to 6.5-6.7 in transgenic plant soils, beyond the pH optimum for A. niger phyA activity (pH=5), and may explain the limited specificity of ex::phyA plants for phytate in this soil. The efficacy of single exudation traits (e.g., phytase) therefore appear to be limited in P-replete soil conditions and may be improved where soil pH matches the functional requirements of the enzyme or trait of interest.

Phytase-mediated mineral solubilization from cereals under in vitro gastric conditions.

Enzymatic dephosphorylation of phytic acid (inositol hexakisphosphate) in cereals may improve mineral bioavailability in humans. This study quantified enzymatic dephosphorylation of phytic acid by measuring inositol tri- to hexakisphosphate (InsP3-6) degradation and iron and zinc release during microbial phytase action on wheat bran, rice bran and sorghum under simulated gastric conditions. InsP3-6 was depleted within 15-30 min of incubation using an Aspergillus niger phytase or Escherichia coli phytase under simulated gastric conditions, with the two enzymes dephosphorylating cereal phytic acid at similar rates and to similar extents. Microbial phytase-catalyzed phytate dephosphorylation was accompanied by increased iron and zinc release from the cereal substrates. However, for wheat bran at pH 5, the endogenous wheat phytase activity produced mineral release equal to or better than that of the microbial phytases. No increases in soluble cadmium, lead or arsenic were observed with microbial phytase-catalyzed phytate dephosphorylation. Microbial phytase treatment abated phytate chelation hence enhanced the release of iron and zinc from phytate-rich cereals under simulated gastric conditions. The data infer that acid-stable microbial phytases can help improve iron bioavailability from phytate-rich cereal substrates via post-ingestion activity. © 2015 Society of Chemical Industry.

Effect of zinc bacitracin and phytase on growth performance, nutrient digestibility, carcass and meat traits of broilers.

A total of 336 one-day-old Hubbard broiler chickens were randomly distributed among 8 groups, each containing six replicates (7 chickens/replicate). From 1 to 40days of age, the groups fed the same starter, grower and finisher diets. The control group was unsupplemented; zinc bacitracin (ZnB) group received the antibiotic at 0.5g/kg; fungal phytase (FP) groups received 250, 500 and 1000U/kg diet of Aspergillus niger phytase (FP_250, FP_500 and FP_1000 groups), respectively; bacterial phyatse (BP) groups received 250, 500 and 1000U/kg diet of Escherichia coli phytase (BP_250EP, BP_500EP and BP_1000EP groups) respectively. Considering the whole experimental period, body weight gain was unaffected by ZnB and different concentrations of bacterial and fungal phytase; however, the feed conversion ratio of the group fed a diet supplemented with 500U of BP was better (p<0.01) than those fed with a diet supplemented with 500U of FP. BP_250 group had a higher (p<0.05) apparent digestibility of ether extract compared to FP_250 group. In conclusion, bacterial phytase at 500U may enhance performance of broiler chickens fed during days 1-40 of age and yield similar growth performance and economic efficiency to those of eB-supplemented groups.

Computational identification, homology modelling and docking analysis of phytase protein from Fusarium oxysporum

AbstractThe extracellular phytase structural gene was isolated from phytopathogenic fungus Fusarium oxysporum using PCR amplification (GenBank accession number KC708486). The gene possesses an open reading frame of 1,514 bp and two coding regions 1–44 and 156–1458 with one intron (45–155). The phy gene from F. oxysporum (Fophy) encodes a putative phytase protein of F. oxysporum (FoPhy) of 448 amino acids, which includes a putative signal peptide (21 residues). The deduced amino acid sequence of FoPhy exhibits 98% sequence identity with Aspergillus niger and Aspergillus awamori phytases. The deduced protein sequence contains the consensus motifs (RHGXRXP and HD), eight conserved cysteine residues and ten conserved putative N-glycosylation sites, which are conserved among histidine acid phosphatases. Computed structural model of FoPhy was found to consist of mixed α/β motifs and probable loops. The predicted model resembles the structure of Aspergillus niger phytase (root mean square deviation 0.23 Å). Ramachandran plot analysis revealed that 95.0% portion of residues fall into the most favourable regions. The predicted three-dimensional structures of FoPhy on molecular docking with substrates like inositol hexaphosphate, inositol hexasulphate and N-acetyl D-glucosamine showed its interaction with conserved histidine and aspartic acid residues in the active site, as also known for other fungal phytases. This study provides a detailed identification and characterization of the phytase from F. oxysporum, which may be helpful in elucidation of its role in pathogenesis and other transcriptional and expression studies.

Overexpression and functional characterization of an Aspergillus niger phytase in the fat body of transgenic silkworm, Bombyx mori

In a previous study, we isolated 1,119 bp of upstream promoter sequence from Bmlp3, a gene encoding a member of the silkworm 30 K storage protein family, and demonstrated that it was sufficient to direct fat body-specific expression of a reporter gene in a transgenic silkworm, thus highlighting the potential use of this promoter for both functional genomics research and biotechnology applications. To test whether the Bmlp3 promoter can be used to produce recombinant proteins in the fat body of silkworm pupae, we generated a transgenic line of Bombyx mori which harbors a codon-optimized Aspergillus niger phytase gene (phyA) under the control of the Bmlp3 promoter. Here we show that the Bmlp3 promoter drives high levels of phyA expression in the fat body, and that the recombinant phyA protein is highly active (99.05 and 54.80 U/g in fat body extracts and fresh pupa, respectively). We also show that the recombinant phyA has two optimum pH ranges (1.5-2.0 and 5.5-6.0), and two optimum temperatures (55 and 37 °C). The activity of recombinant phyA was lost after high-temperature drying, but treating with boiling water was less harmful, its residual activity was approximately 84% of the level observed in untreated samples. These results offer an opportunity not only for better utilization of large amounts of silkworm pupae generated during silk production, but also provide a novel method for mass production of low-cost recombinant phytase using transgenic silkworms.

Gene cloning and soluble expression of Aspergillus niger phytase in E. coli cytosol via chaperone co-expression

A phytase gene from Aspergillus niger was isolated and two Escherichia coli expression systems, based on T7 RNA polymerase promoter and tac promoter, were used for its recombinant expression. Co-expression of molecular chaperone, GroES/EL, aided functional cytosolic expression of the phytase in E. coli BL21 (DE3). Untagged and maltose-binding protein-tagged recombinant phytase showed an activity band of ~49 and 92 kDa, respectively, on a zymogram. Heterologously-expressed phytase was fractionated from endogenous E. coli phytase by (NH4)2SO4 precipitation. The enzyme had optimum activity at 50 °C and pH 6.5.

Site-Directed Mutagenesis Improves the Thermostability and Catalytic Efficiency of Aspergillus niger N25 Phytase Mutated by I44E and T252R

Aspergillus niger phytase (PhyA) has been used as a feed supplement to improve the bioavailability of phytate phosphorus to swine and poultry. However, it is unable to maintain its stability due to high temperature during the feed pelleting process. In this study, we performed site-directed mutagenesis in the Aspergillus niger N25 phyA (m) gene at residue 44I and 252 T, and they were replaced by glutamic acid and arginine. Single-site mutants I44E-PhyA and T252R-PhyA, as well as double-site mutant I44E/T252R-PhyA, were constructed to improve the thermostability of PhyA through hydrogen bondings and ionic interactions. The three mutant enzymes all showed more than 20 % improvement in thermostability compared to the wild-type enzyme after being heated at 80 °C for 10 min. Their melting temperatures (T m) were increased by 1, 1, and 1.2 °C, respectively. The k m values of I44E-PhyA, T252R-PhyA, and I44E/T252R-PhyA for sodium phytate were 78, 44, and 79 % lower (P <0.05) than that of the wild-type enzyme. Overall catalytic efficiency (k cat/k m) of I44E-PhyA, T252R-PhyA, and I44E/T252R-PhyA was improved by 310, 155, and 84 % (P <0.05) than that of the wild type, respectively. The catalytic efficiency did not seem to be negatively affected by the improvement in thermostability.

Plant species and organ influence the structure and subcellular localization of recombinant glycoproteins

Many plant-based systems have been developed as bioreactors to produce recombinant proteins. The choice of system for large-scale production depends on its intrinsic expression efficiency and its propensity for scale-up, post-harvest storage and downstream processing. Factors that must be considered include the anticipated production scale, the value and intended use of the product, the geographical production area, the proximity of processing facilities, intellectual property, safety and economics. It is also necessary to consider whether different species and organs affect the subcellular trafficking, structure and qualitative properties of recombinant proteins. In this article we discuss the subcellular localization and N-glycosylation of two commercially-relevant recombinant glycoproteins (Aspergillus niger phytase and anti-HIV antibody 2G12) produced in different plant species and organs. We augment existing data with novel results based on the expression of the same recombinant proteins in Arabidopsis and tobacco seeds, focusing on similarities and subtle differences in N-glycosylation that often reflect the subcellular trafficking route and final destination, as well as differences generated by unique enzyme activities in different species and tissues. We discuss the potential consequences of such modifications on the stability and activity of the recombinant glycoproteins.

Corn seeds as bioreactors for the production of phytase in the feed industry

Corn seed is a major ingredient of animal feed worldwide. However, it contains phytate, a major phosphate storage form that is unavailable to monogastric animals like pigs and poultry. We report a transgenic corn with bioavailable phosphate, achieved by seed-specific overexpression of Aspergillus niger phytase, an enzyme catalyzing the release of phosphate from phytate. We obtained maximal phytase activity of 125 FTU/g kernels, 1000-fold above that of the wild type, with 1000g of kernels containing up to 67 times the feed industry requirement. Enzymatic characterization of Zea mays recombinant phytase (ZmrPhy) showed it to be equivalent to yeast (Pichia pastoris) recombinant phytase (PprPhy), a commercially available phytase product. An animal feeding trial demonstrated that ZmrPhy had similar nutritional effects on broiler chickens to PprPhy in terms of reducing inorganic phosphorus addition to feed and phosphate excretion in animal manure. These results suggest that transgenic phytase corn can be used directly in the feed industry. Experiments were conducted to assess the food safety of the corn; the results demonstrated no difference versus regular corn. This is the first genetically modified corn officially issued with a biosafety certificate in China and has great potential in the animal feed industry.

Efficacy of contrast levels of non-phytate phosphorus and Aspergillus niger phytase in hens fed wheat-maize-based diets

A 2 &amp;times; 2 factorial design experiment examined the effect of dietary non-phytate phosphorus (NPP) (1.3 and 4.0 g/kg) and 3-phytase (F) (0 and 150 FTU/kg) on the performance indicators of hens, physical parameters of eggs, phosphorus (P) content of the eggshells, and the pH of the digestive tract of laying hens. Two hundred and forty hens (ISA Brown) were housed in enriched cages and fed a wheat-maize-based diet. A significant effect of both NPP and F was found for the yolk colour (P = 0.016) and shell thickness (P&amp;nbsp;=&amp;nbsp;0.038). The F supplement or higher level of NPP alone and in combination increased the value of the yolk colour and shell thickness. The supplementation of the basal diet with F significantly increased the laying performance, especially with regard to the egg weight and feed conversion ratio. The higher dose of NPP had a negative effect on the egg production, feed intake, and feed conversion ratio. The lower level of NPP (1.3 g/kg) decreased the internal egg quality and shell quality, except for the shell strength. In contrast, the addition of 3-phytase to the diet increased the shell thickness and shell weight. The P content in the eggshells was not influenced by the dietary treatment. The higher level of NPP or F increased the pH in the gizzard to a value suitable for F activity. The hens fed a diet containing 1.3 g/kg NPP achieved a higher performance; moreover, the F supplement at 150 FTU/kg increased the external quality of the eggs. &amp;nbsp;

Dephytinisation of Sangak and Barbari bread made from different extraction rate flours increases iron and zinc bioaccessibility in Caco‐2 cells

SummaryBread is a staple food in many countries and an important source of iron and zinc. The bioavailability of these minerals is generally low because of the content of phytic acid. Traditional Iranian breads were prepared with flours of different extraction rates, Sangak at 93% and Barbari at 82%. Breads were dephytinised by addition of Aspergillus niger phytase during in vitro digestion. The effect upon iron and zinc bioaccessibility in the Caco‐2 cell model was investigated. The cellular uptake of iron and zinc was lower from Sangak, compared to Barbari, despite higher mineral content in Sangak. Dephytinisation of both breads increased iron uptake in the Caco‐2 cells (0.65 vs. 1.64 in Sangak and 0.77 vs. 1.97 ng mg−1 protein in Barbari). Zinc uptake increased from 0.98 to 2.8 in Sangak and from 1.4 to 2.9 ng mg−1 protein in Barbari. Thus, dephytinisation substantially improves iron and zinc bioaccessibility.