Coli Phytase Research Articles

Effect of Feeding Different Levels of Escherichia Coli Phytase and Buttiauxella Phytase on the Growth and Digestibility in Broiler

The effect of feeding different levels of bacterial phytase enzymes on the growth performance of broiler was examined using 250 Hubbard broilers, kept at the Poultry Experimental Station, Sindh Agriculture University Tandojam. The products of bacterial phytase enzymes were incorporated in the basal ration at different levels consisted of 0mg/kg (control), 50 mg/kg (group B), 100 mg/kg (group C) E. coli phytase and 50 mg/kg (group D), 100 mg/kg (group E) Buttiauxella spp. phytase. The birds were studied for live body weight, feed and water intake, feed conversion efficiency, carcass weight, dressing percentage, weight of internal edible and non-edible parts, digestibility and economics. The difference in live body weight among treatment and control group was significant (P<0.05). The significantly higher live body weight (2160.00g/bird) was observed in group E (fed 100mg/kg Buttiauxella phytase) and lower (2060g/bird) in group A (control). The effect of treatment on feed and water intake was significant (P<0.05), Feed (3800.00g/bird) and water (3800.00 ml/bird) intake was significantly higher in group A (control) than other treatment groups. Better feed conversion ratio (1.74) was noticed in group E followed by group C, B, D and A respectively. Carcass weight was significantly higher in group E (1350.60) and lower in control group (1219.71), the influence of feeding different sources and levels of bacterial phytase enzymes on dressing percentage was non-significant (P>0.05).Significant (P<0.05) difference was noted for heart weight among the groups and non-significant (P>0.05) difference were examined for gizzard, liver, spleen and proventiculus weight among the groups. The data showed that higher percentage of crude protein digestibility was recorded in group E (75%), then C (72%), B (71%), D (70.8%) and A (70%) respectively. Similarly M.E (kcal) was greater in group E (30.5%), then C (30%), B (29%), D (29%) and A (28.5%) respectively. The comparative net profit among all groups demonstrated that group E generated better net profit (48.70) followed by group C (47.88), B (41.50), D (40.30) and A (35.20) Rs/bird respectively.From the current findings, it was concluded that the supplementation of feed with 100mg/kg Buttiauxella spp phytase enzyme resulted in higher live body weight gain, better FCR and improved digestibility of nutrients. Keywords: phytase enzymes, Buttiauxella spp., E. coli, broiler,

Evolution of E. coli Phytase Toward Improved Hydrolysis of Inositol Tetraphosphate

Protein engineering campaigns are driven by the demand for superior enzyme performance under non-natural process conditions, such as elevated temperature or non-neutral pH, to achieve utmost efficiency and conserve limited resources. Phytases are industrial relevant feed enzymes that contribute to the overall phosphorus (P) management by catalyzing the stepwise phosphate hydrolysis from phytate, which is the main phosphorus storage in plants. Phosphorus is referred to as a critical disappearing nutrient, emphasizing the urgent need to implement strategies for a sustainable circular use and recovery of P from renewable resources. Engineered phytases already contribute today to an efficient phosphorus mobilization in the feeding industry and might pave the way to a circular P-bioeconomy. To date, a bottleneck in its application is the drastically reduced hydrolysis on lower phosphorylated reaction intermediates (lower inositol phosphates, ≤InsP4) and their subsequent accumulation. Here, we report the first KnowVolution campaign of the E. coli phytase toward improved hydrolysis on InsP4 and InsP3. As a prerequisite prior to evolution, a suitable screening setup was established and three isomers Ins(2,4,5)P3, Ins(2,3,4,5)P4 and Ins(1,2,5,6)P4 were generated through enzymatic hydrolysis of InsP6 and subsequent purification by HPLC. Screening of epPCR libraries identified clones with improved hydrolysis on Ins(1,2,5,6)P4 carrying substitutions involved in substrate binding and orientation. Saturation of seven positions and screening of, in total, 10,000 clones generated a dataset of 46 variants on their activity on all three isomers. This dataset was used for training, testing, and inferring models for machine learning guided recombination. The PyPEF method used allowed the prediction of recombinants from the identified substitutions, which were analyzed by reverse engineering to gain molecular understanding. Six variants with improved InsP4 hydrolysis of >2.5 were identified, of which variant T23L/K24S had a 3.7-fold improved relative activity on Ins(2,3,4,5)P4 and concomitantly shows a 2.7-fold improved hydrolysis of Ins(2,4,5)P3. Reported substitutions are the first published Ec phy variants with improved hydrolysis on InsP4 and InsP3.

Comparative effects of two phytases versus increasing the inorganic phosphorus content of the diet, on nutrient and amino acid digestibility in boilers

Comparative effects of graded doses of two phytases on growth performance, nutrient and amino acid (AA) digestibility were investigated in corn-soybean meal-based broiler diets. A negative control diet (NC) with low phosphorus (P) content (1.8, 4.4 and 2.5 g/kg retainable, total and phytate P respectively) was supplemented with four doses of a Buttiauxella sp phytase (analyzed activity of 303 to 1046 FTU/kg) or an E. coli phytase (analyzed activity of 442 to 1811 FTU/kg), and tested against three positive control diets comprising the NC + 0.6, 1.2 or 1.8 g P from monocalcium phosphate (MCP)/kg feed and increased Ca content (+0.8 g/kg). A total of 1152 male Ross 308 broilers at 5 days of age were assigned to 72 cages with 16 birds/cage and 6 cages/treatment were given free access to pelleted diets until 21 days of age. Feed intake (FI), body weight and mortality were recorded and used to calculate body weight gain (BWG) and feed conversion ratio (FCR) during 5–20 days of age. Excreta was collected on day 18–20 to determine total tract retention of nutrients. Tibias (from 4 birds pooled per cage) and ileal digesta (from all birds and pooled per cage) were sampled at 21 days of age to determine tibia ash, ileal digestibility of nutrients and AA. Compared to NC, increasing phytase dose and MCP levels produced a stepwise increase in FI, BWG and reduction in FCR. Increasing phytase dose produced curvilinear increases in BWG, ileal P digestibility, retention and tibia ash (P < 0.01) but effects were greater for Buttiauxella vs. E. coli phytase. In contrast, only Buttiauxella phytase increased the ileal digestibility of protein and total AA (+3.7% on average vs. NC at 1046 FTU/kg, P < 0.05). Linear increases in ileal digestibility of all individual AA were seen with increasing dose of Buttiauxella (P < 0.05 in all cases) and were greatest for cysteine (+ 7.9% vs. NC at 1046 FTU/kg), whereas only 2 individual AA showed linear increases in digestibility in response to increasing dose of the E. coli phytase (cysteine and proline, P < 0.05). Ileal digestibility of Na (%) was markedly increased (less negative) by phytase, especially for the Buttiauxella phytase which produced a 96% increase in ileal Na digestibility at 1046 FTU/kg vs. NC. Across treatments, ileal digestibility of AA and Na were positively correlated (P < 0.001). These results provide insight into the ‘extra-phosphoric’ effects in broilers fed corn-soybean meal-based diets and suggest that the Buttiauxella and E. coli phytase had markedly different degrees of ‘extra-phosphoric’ effects on AA digestibility per standard FTU. The data showed that the AA digestibility response to increasing phytase dose do not follow the same response curve as for digestible P, and that the response curves are specific for different phytases.

Cloning and High-Level Expression of the Enzymatic Region of Phytase in E. coli

Phytase is an important enzyme poses great nutritional significance in humans and monogastric animals diets. The phytase production yield using wild sources, including micro-organisms, plants, and animals is sorely low. Thus, recombinant expression of phytase has received increasing interest for achieving production rate. Escherichia coli is the most preferred host for expression of heterologous proteins but overexpression of recombinant phytase in E. coli, met with limited success due to the sequestration of the enzyme into inclusion bodies. In the present study, artificial phytases gene with excellent thermostability and activity were designed by detecting the enzymatic region of the E. coli phytase gene by employing bioinformatics tools. Then, the PCR amplified recombinant gene was expressed in E. coli and the active enzyme was recovered from inclusion bodies. Employing cysteine amino acid in the dialysis buffer succeed to the superior activity of the enzyme with a specific activity of 73.8 U/mg. The optimum temperature and pH for enzyme activity were determined at 60 °C and 4, respectively. The novel recombinant enzyme illustrated perfect thermostability up to 70 °C with maintenance 75 of its activity. The enzyme was stable at pH range of 2–10. Moreover, the effects of ions and chemical compounds on enzyme stability and activity were assessed. © 2019, Springer Nature B.V.

A critical review of methods used to determine phosphorus and digestible amino acid matrices when using phytase in poultry and pig diets

SummaryPhytase is applied in animal feed based on its standard activity measured at pH 5.5, however the relative activity at pH 3 (e.g. stomach pH, the main site for the breakdown of phytate) varies among the commercial phytases, ranging from 56% (an E coli phytase) to 235% (Buttiauxella phytase). These diverse sources of phytases have varying capability for degrading phytate and, correspondingly, different P, digestible amino acid and metabolisable energy matrix values. In addition, the matrix values recommended by different phytase suppliers are not comparable, as different methodologies have been used to determine them. Phosphorus (P) and other matrix values can be determined by direct measurement of digestible P (dP) improvements by the addition of phytase above a negative control in large numbers of in vivo studies using increasing phytase doses. Alternatively, matrix values can be assessed by indirect measurement, using inorganic P (usually mono- or dicalcium sources) as a reference, typically based on tibia or metacarpal ash as a response parameter to estimate available P equivalence, either at a single or different phytase doses. When using the indirect measurement, the available P equivalence with increasing phytase doses may be calculated based on a log linear model. Although both methods are acceptable methodologies, direct measurement may under-estimate and indirect measurement may over-estimate matrix values, and a large number of in vivo studies give the best estimates of matrix values. Phytase efficacy can be influenced by phytase source, dose level, dietary composition (Ca level and Ca: P ratio). Phytase end users are encouraged to be aware of the methods used by suppliers to determine matrix values, before applying them in their feed formulations.

Display of Escherichia coli Phytase on the Surface of Bacillus subtilis Spore Using CotG as an Anchor Protein.

Escherichia coli phytase (AppA) has been widely used as an exogenous feed enzyme for monogastric animals; however, the production of this enzyme has been examined primarily in E. coli and yeast expression systems. As an alternative to production of soluble phytase, an enzyme immobilization method using the Bacillus subtilis spore outer-coat protein CotG as an anchoring motif for the display of the AppA was attempted. Using this motif, AppA was successfully produced on the spore surface of B. subtilis as verified by Western blot analysis and phytase activity measurements. Analysis of the pH stability indicated that more than 50% activity was retained after incubation at four different pH values (2.0, 4.0, 7.0, and 8.0) for up to 12h, with maximum activity observed at pH4.5. The highest enzyme activity seen at 55°C and thermal stability measurements demonstrated that more than 30% activity remained after 30min incubation at 60°C. The spore surface-displayed AppA was resistant to pepsin, and more stable than phytase produced previously using a yeast expression system. Furthermore, we present data indicating that the use of peptide linkers may help improve the bioactivity of displayed enzymes on the spore surface of B. subtilis.

Effects of increasing concentrations of an Escherichia coli phytase on the apparent ileal digestibility of amino acids and the apparent total tract digestibility of energy and nutrients in corn-soybean meal diets fed to growing pigs.

An experiment was conducted to test the hypothesis that inclusion of increasing concentrations of an Escherichia coli phytase to a corn-soybean meal (SBM) diet results in improved digestibility of DM, GE, CP, NDF, ADF, macrominerals, microminerals, and AA. Twenty-four growing barrows (initial BW: 37.0 ± 1.4 kg) were equipped with a T-cannula in the distal ileum and placed individually in metabolism crates, and allotted to a 2-period switch-back design with 6 diets and 4 replicate pigs per diet in each period. The positive control diet was a corn-SBM diet that contained limestone and dicalcium phosphate to meet the requirement for standardized total tract digestible (STTD) P and Ca (0.31% STTD P and 0.70% Ca). A negative control diet that was similar to the positive control diet, with the exception that no dicalcium phosphate was used, was also formulated, and this diet contained 0.16% STTD P and 0.43% Ca. Four additional diets were formulated by adding 500, 1,000, 2,000, or 4,000 units of microbial phytase (FTU) to the negative control diet. Each period lasted 14 d. Fecal and urine samples were collected from the feed provided from days 6 to 11 of each period following 5 d of adaptation to the diets. Ileal digesta were collected for 8 h on days 13 and 14. Results indicated that addition of the E. coli phytase to the negative control diet tended to quadratically improve the apparent ileal digestibility of Phe (P = 0.086) and Asp (P = 0.054), and linearly increased (P < 0.05) the apparent total tract digestibility (ATTD) of ADF, K, and Fe. Microbial phytase also quadratically increased (P < 0.05) the ATTD of NDF and Mg, and linearly and quadratically increased (P < 0.05) the ATTD and retention of Ca and P. However, no effects of the phytase on ATTD of GE or the concentration of DE were observed. In conclusion, the increased absorption of several minerals including Ca, P, K, Mg, and Fe that was observed as increasing concentrations of an E. coli phytase was added to a corn-SBM meal diet indicates that the dietary provision of these minerals may be reduced if phytase is fed.

Effects of graded levels of an Escherichia coli phytase on growth performance, apparent total tract digestibility of phosphorus, and on bone parameters of weanling pigs fed phosphorus-deficient corn-soybean meal based diets

An experiment was conducted to evaluate the efficiency of a novel Escherichia (E.) coli phytase on improving growth performance, calcium (Ca) and phosphorus (P) digestibility, and bone ash concentration of weanling pigs fed P-deficient corn-soybean meal based diets. Sixty weanling pigs with an initial body weight of 11.2±1.2kg were randomly allotted to 1 of 6 dietary treatments with 10 replicate pigs per treatment. The dietary treatments were: 1) positive control (PC), 2) negative control (NC), 3) NC+250 phytase units (FTU)/kg diet, 4) NC+500 FTU/kg diet, 5) NC+1000 FTU/kg diet, and 6) NC+2500 FTU/kg diet. Pigs were fed phase I diets during the initial 6days and phase II diets from day 7–27. At the end of the experiment, all pigs were euthanized to collect the 3rd and 4th metacarpals from each front foot. Results indicated that in both phase I and II, pigs fed the NC diet had reduced (P<0.05) apparent total tract digestibility (ATTD) of P compared with pigs fed the PC diet. However, inclusion of graded levels of E. coli phytase to the NC diet increased (linear and quadratic, P<0.01) the ATTD of P. No differences were observed in the ATTD of Ca or the ATTD of dry matter as E. coli phytase inclusion increased in the diets. Pigs fed the PC diet had greater (P<0.05) fat-free dried bone weight (g), bone ash weight (g), bone ash concentration (g/kg), bone Ca (g), and bone P (g and g/kg) and greater (P<0.05) average daily gain (ADG), average daily feed intake (ADFI), and gain: feed ratio (G:F) during phase II and during the overall experimental period than pigs fed the NC diet. Graded levels of E. coli phytase linearly and quadratically increased (P<0.05) fat-free dried bone weight (g), bone ash weight (g), bone ash (g/kg), bone Ca (g), and bone P (g). Inclusion of E. coli phytase also increased (linear, P<0.05) final body weight, ADG, ADFI and G:F during phase II and during the entire experimental period. No differences were observed between diets supplemented with 1000 or 2500 FTU/kg of E. coli phytase for fat-free dried bone (g), bone ash weight (g), bone ash (g/kg), bone Ca (g and g/kg), bone P (g) or final BW, ADG, ADFI, or G:F during phase II or during the overall experimental period. In conclusion, adding up to 1000 FTU/kg of the novel E. coli phytase to P-deficient diets increased growth performance and P utilization in weanling pigs.

Effect of increasing supplemental phytase concentration in diets fed to Hubbard × Cobb 500 male broilers from 1 to 42 days of age

Phytase supplementation beyond the standard doses used for phosphorus release has been reported to result in extraphosphoric effects by enhancing nutrient digestibility resulting in improved performance of broilers. A study was conducted to examine the effects of the progressive addition of an enhancedEscherichia Coli phytase (400–1,600 phytase units; FTU) on growth performance and carcass characteristics from 1 to 42 d of age in male broilers. One thousand four hundred Hubbard × Cobb 500 1-d-old chicks were randomly distributed into 56 floor pens (0.08 m2/bird). Seven dietary treatments were provided in a 3-phase feeding program consisting of (1) a positive control (adequate Ca and nonphytate P; PC); (2) 1 negative control (Ca and nonphytate P reduced by 0.14% and 0.13%; NC); (3 to 6) the NC diet with 4 increasing supplemental phytase concentrations (NC + 400 FTU, NC + 800 FTU, NC + 1,200 FTU, and NC + 1,600 FTU, respectively); and (7) a low-energy NC diet without phytase and xylanase (reduced 66 kcal of AMEn/kg). Body weight gain, feed conversion, mortality, weight and yield of whole carcass, abdominal fat, and pectoralis major and minor muscles were evaluated. Progressive supplementation of phytase decreased cumulative FCR linearly. Broilers fed diets containing 1,600 FTU had heavier total breast meat by 49 g compared with birds receiving the PC diets. Broilers consuming the NC + 400 FTU or the low-energy NC diet had similar growth performance and meat yield compared with birds provided PC diet. These data indicated that phytase supplementation beyond the need for phosphorus enhances growth performance and carcass characteristics.

Integration, stability and expression of the E. coli phytase transgene in the Cassie line of Yorkshire Enviropig™

The genomic structure and generational stability of the transgene carried by the Cassie (CA) line of the transgenic Enviropig™, a prospective food animal, are reported here. This transgene is composed of the Escherichia coli phytase coding sequence regulated by the mouse parotid secretory protein promoter to direct secretion of phytase in the saliva. In the CA line the transgene integrated in chromosome 4 is present as a concatemer of three copies, two in a head to tail orientation and the third in a reverse orientation 3' to the other copies with a 6kbp deletion in the 5' promoter region. The overall size of the integrated transgene complex is 46kbp. During integration a 66kbp segment of the chromosome was deleted, but a BLAST search of the segment from a GenBank clone did not reveal any essential genes. The transgene integration site was stable through 9 generations analyzed. Phytase activity in the saliva was similar among 11day old hemizygous boars and gilts and remained relatively constant through nine generations of hemizygous pigs. However, as the pigs grew there generally was a gradual decrease in activity that stabilized when pigs reached the finisher phase of growth (4-6 months old). Homozygous pigs exhibited 1.5 fold higher phytase activity (P<0.0001) than that of hemizygous littermates. Moreover, no differential salivary phytase activity was seen in hemizygotes arising from CA-Yorkshire and CA-Duroc breed outcrosses, suggesting that expression of the transgene is unaffected by genetic background. This data demonstrates that an exogenous phytase gene can be stably transmitted and expressed in the salivary glands of a domestic food animal.

Single-step Purification and Immobilization of MBP–phytase Fusion on Starch Agar Beads: Application in Dephytination of Soy Milk

Periplasmic phytase, appA from E. coli has been noticed as a superior feed and food additive owing to its high specific activity, acidic pH optimum and resistance to gastric proteases. E. coli phytase was expressed as a fusion protein with maltose-binding protein, affinity-purified to homogeneity and, subsequently, immobilized in one step using a cost-effective matrix prepared from starch agar bead. Immobilized enzyme revealed an activity optimum at pH 6, while that of free enzyme was observed at pH 4. Both the immobilized and free enzyme showed a temperature optimum at 60°C. Cleavage of 87kDa fusion protein using factor Xa released 45kDa appA. Hydrolysis of soy milk using immobilized enzyme led to 10% increase in release of inorganic phosphate at 50°C relative to free fusion protein. This study suggests the usability of MBP as an immobilizing linker to other food enzymes for economical use in industry.

Ethanol Tolerance in &amp;lt;i&amp;gt;Aspergillus niger&amp;lt;/i&amp;gt; and &amp;lt;i&amp;gt;Escherichia coli&amp;lt;/i&amp;gt; Phytase

Despite yeast having its own native phytase, the high levels of phytate found in DDGS, a byproduct of ethanol (ETOH) fermentation, suggest that its activity is diminished in the presence of ETOH. Ethanol, a product of grain fermentation, is known to inactivate several hydrolytic enzymes but its effect on phytases is relatively unknown. In this study, two phytases, Aspergillus niger (PhyA) and Escherichia coli (AppA2), were tested for ETOH tolerance. The E. coli phytase displayed greater ethanol tolerance over fungal phytase in the 5% to 10% range. However, ETOH inactivation was found to be reversible for both the enzymes. These differences in ETOH tolerance do suggest that there is a potential to achieve higher ETOH tolerance in phytases by 'structure-function' studies to lower phytic acid levels in DDGS and for other applications.

In vitro assessment of the effects of phytate and phytase on nitrogen and phosphorus bioaccessibility within fish digestive tract

The aim of the present work was to assess different possible interactions between phytate (IP6), dietary protein and digestive proteases, as well as the preventive effect of phytase in the formation of protein-IP6 complexes, through the use of a gastrointestinal model simulating conditions existing in the digestive tract of rainbow trout ( Oncorhynchus mykiss). Two commercial phytases, expressed in P. lycii and E. coli, were evaluated. Fungal phytase showed an optimum at pH 5.5 and bacterial phytase showed two peaks at pH 2.5 and 4.5. These phytases evidenced different resistance against gastric proteolytic degradation; E. coli phytase retained 90% of its initial activity after 4 h incubation in the presence of either porcine pepsin or fish stomach proteases, while the dephosphorylation activity of P. lycii phytase was reduced by 90% (P<0.001). The presence of IP6 determined a reduction of 80% in protein solubility (P<0.001) when casein was used as substrate of fish acid proteases. The formation of IP6-protein complexes resulted in lower amino acids liberation from casein (P<0.05). Addition of phytase to soybean meal prevented the formation of such IP6-protein complexes and improved amino acid release by 60% (P<0.01). In contrast, IP6 had not effect on protein solubilisation or amino acid liberation during the intestinal digestion. Under conditions simulating gastric digestion a great amount of IP6 was combined to either porcine pepsin or soluble protein in fish stomach crude extract, precipitating 68 and 100% of such proteins, respectively. A similar, but lower reduction in soluble protein from either pancreatin or fish intestinal extract was observed when simulating intestinal digestion (47 and 33%, respectively). The formation of IP6-protein complexes reduced the activity of all gastric and intestinal proteases assayed (P<0.01) with the exception of chymotrypsin. A positive effect (P<0.05) of low pH on IP6-dephosphorylation was measured when the effect of bacterial phytase (2500 FTU/kg) on the release of soluble P from native IP6 present in soybean meal was tested under different digestion temperatures (6 and 16 °C) and pH (2.0, 3.0 and 4.0). A positive effect of temperature was also observed when the reaction was carried out at pH 2.0 and 3.0 (P<0.001), but not at 4.0, suggesting the existence of a limit in the solubility of IP6 and therefore in its availability for phytase action at this last pH value.

Enzyme mediated silicon–oxygen bond formation; the use of Rhizopus oryzae lipase, lysozyme and phytase under mild conditions

The potential for expanding the variety of enzymic methods for siloxane bond formation is explored. Three enzymes, Rhizopus oryzae lipase (ROL), lysozyme and phytase are reported to catalyse the condensation of the model compound, trimethylsilanol, formed in situ from trimethylethoxysilane, to produce hexamethyldisiloxane in aqueous media at 25 °C and pH 7. Thermal denaturation and reactant inhibition experiments were conducted to better understand the catalytic role of these enzyme candidates. It was found that enzyme activities were significantly reduced following thermal treatment, suggesting a potential key-role of the enzyme active sites in the catalysis. Similarly, residue-specific modification of the key-amino acids believed to participate in the ROL catalysis also had a significant effect on the silicon bio-catalysis, indicating that the catalytic triad of the lipase may be involved during the enzyme-mediated formation of the silicon-oxygen bond. E. coli phytase was found to be particularly effective at catalysing the condensation of trimethylsilanol in a predominantly organic medium consisting of 95% acetonitrile and 5% water. Whereas the use of enzymes in silicon chemistry is still very much a developing and frontier activity, the results presented herein give some grounds for optimism that the variety of enzyme mediated reactions will continue to increase and may one day become a routine element in the portfolio of the synthetic silicon chemist.

Comparative effects of three phytases on phosphorus and calcium digestibility in the growing pig

Abstract Phytase addition to swine diets has generally resulted in a marked increase in phosphorus (P) digestibility and in a reduction of faecal excretion of P. The number of phytases present in the market is growing and the origins of the microbial genes are becoming more diversified. The aim of this study was to compare the effects of 3 different phytases on P and calcium (Ca) digestibility in pigs. The control diet was based on soybean meal, maize and barley. The inclusion levels were 250 (Q1) and 500 U/kg (Q2) for an E. coli phytase (Quantum), 500 U/kg (Nat) for A. niger (Natuphos) and 750 U/kg (Ron) for P. lycii (Ronozyme P) . All phytases significantly reduced faecal concentration of P. For Q2 this reduction was significantly higher than for Q1. P digestibility was improved by 13.8, 18.6, 18.3 and 17.9 percentage units by Q1, Q2, Nat and Ron respectively. The P equivalencies, considered as supplemental P digested compared to the non-supplemented diet of Q1, Q2, Nat and Ron were 0.492, 0.732, 0.678 and 0.678 g of available P/kg of feed respectively. Ca digestibility was also significantly improved by the phytases. It can be concluded that the 3 phytase preparations improved the digestibility and the apparent absorption of P and Ca in the growing pig fed a diet containing P exclusively from plant origin. The effect of including 500 U/kg of E. coli phytase on P digestibility was similar to those induced by the A. niger and P. lycii phytases at their recommended levels of 500 and 750 U/kg, respectively.

Functional Insights Revealed by the Crystal Structures of Escherichia coli Glucose-1-phosphatase

The Escherichia coli periplasmic glucose-1-phosphatase is a member of the histidine acid phosphatase family and acts primarily as a glucose scavenger. Previous substrate profiling studies have demonstrated some of the intriguing properties of the enzyme, including its unique and highly selective inositol phosphatase activity. The enzyme is also potentially involved in pathogenic inositol phosphate signal transduction pathways via type III secretion into the host cell. We have determined the crystal structure of E. coli glucose-1-phosphatase in an effort to unveil the structural mechanism underlying such unique substrate specificity. The structure was determined by the method of multiwavelength anomalous dispersion using a tungstate derivative together with the H18A inactive mutant complex structure with glucose 1-phosphate at 2.4-A resolution. In the active site of glucose-1-phosphatase, there are two unique gating residues, Glu-196 and Leu-24, in addition to the conserved features of histidine acid phosphatases. Together they create steric and electrostatic constraints responsible for the unique selectivity of the enzyme toward phytate and glucose-1-phosphate as well as its unusually high pH optimum for the latter. Based on the structural characterization, we were able to derive simple structural principles that not only precisely explains the substrate specificity of glucose-1-phosphatase and the hydrolysis products of various inositol phosphate substrates but also rationalizes similar general characteristics across the histidine acid phosphatase family.

Efficacy of an E. coli phytase expressed in yeast for releasing phytate-bound phosphorus in young chicks and pigs.

Four chick trials and one pig trial were conducted to investigate the phosphorus-releasing efficacy oftwo commercial phytase enzymes (Natuphos and Ronozyme) and an experimental E. coli phytase enzyme (ECP) when added to corn-soybean meal diets containing no supplemental inorganic P (iP). In the 13- or 14-d chick trials, three or four graded levels of iP (0, 0.05,0.10,0.15%) from KH2PO4 were added to the basal diet to construct standard curves from which bioavailable P release could be calculated for the phytase treatments. In all cases, phytase supplementation levels were based on an assessment of phytase premix activity (i.e., P release from Na phytate at pH 5.5). Linear (P < 0.01) responses in tibia ash and weight gain resulted from iP supplementation in all assays. In the first chick trial, supplementation of 500 phytase units (FTU)/kg of ECP resulted in superior (P < 0.01) weight gain and tibia ash values compared with 500 FTU/kg of Natuphos. Results of the second chick trial revealed P-release values of 0.032 and 0.028% for 500 FTU/kg Natuphos and Ronozyme, respectively, and these were lower (P < 0.01) than the 0.125% P-release value for 500 FTU/kg of ECP. Tibia ash responded quadratically (P < 0.05) in response to graded levels of ECP up to 1,500 FTU/kg in the third chick trial. Combining Natuphos with either Ronozyme or ECP in Chick Trial 4 revealed no synergism between phytases with different initiation sites of P removal. The pig trial involved 10 individually fed weanling pigs per diet, and and phytase enzymes were supplemented to provide 400 FTU/kg in diets containing 0.60% Ca. Based on the linear regression of fibula ash on supplemental iP intake (r2 = 0.87), P-release values were 0.081% for Natuphos, 0.043% for Ronozyme, and 0.108% for ECP. These trials revealed an advantage of the E. coli phytase over the commercial phytases in young chicks.

Heavy metal-mediated crystallization of Escherichia coli phytase and analysis of bridging interactions.

Escherichia coli phytase is a phosphatase that catalyzes the hydrolysis of phytic acid into inorganic phosphate and myo-inositol. Two crystal forms of this enzyme were obtained in the presence of heavy metals. Crystal forms I and II were obtained with the heavy atoms CdCl(2) and HgCl(2) and diffracted to 1.5 A and 2.25 A resolution, respectively. Hg(2+) and Cd(2+) both acted as molecular bridge(s), linking and stabilizing E. coli phytase in the unit cell, and played a crucial role in the crystallization of phytase by bridging neighbouring symmetry related molecules.

The effectiveness of an escherichia coli phytase in improving phosphorus and calcium bioavailabilities in poultry and young pigs

The effectiveness of an Escherichia coli phytase in comparison with a commercially available Aspergillus phytase in improving the bioavailability of phosphorus in broilers, layers and young pigs was studied in three separate experiments. Three basal diets, marginally deficient in dietary P mainly provided as phytate, were formulated. Both phytases were added to the diets at the rate of 500 U/kg diet. The phytases significantly (P ≤ 0.05) improved the availability of phytate P to broilers, layers and young pigs. Aspergillus and E. coli phytases enhanced the pre‐caecal digestibility of P by 11 and 29% for broilers and 18 and 25% for layers, respectively. Total tract digestibility of P (P balance) was also enhanced but with smaller magnitude. In pigs, total tract digestibility of P was improved by 33 and 34% by Aspergillus and E. coli phytases, respectively. Under the conditions of this study, it was observed that E. coli consistently, though with small magnitude in layers and pigs, enhanced the availability of phytate P at the same range or slightly better than Aspergillus phytase. It was only in pigs that the availability of Ca was significantly (P ≤ 0.05) improved by addition of both phytases. It can be concluded that E. coli phytase is highly effective in improving the bioavailability of phytate P to broilers, layers and young pigs. This seems to be based on the high proteolytic stability of the enzyme in the digestive tract, as shown recently.

Comparative studies on the in vitro properties of phytases from various microbial origins

The physical and chemical properties of six crude phytase preparations were compared. Four of these enzymes (Aspergillus A, Aspergillus R, Peniophora and Aspergillus T) were produced at commercial scale for the use as feed additives while the other two (E. coli and Bacillus) were produced at laboratory scale. The encoding genes of the enzymes were from different microbial origins (4 of fungal origin and 2 of bacterial origin, i.e., E. coli and Bacillus phytases). One of the fungal phytases (Aspergillus R) was expressed in transgenic rape. The enzymes were studied for their pH behaviour, temperature optimum and stability and resistance to protease inactivation. The phytases were found to exhibit different properties depending on source of the phytase gene and the production organism. The pH profiles of the enzymes showed that the fungal phytases had their pH optima ranging from 4.5 to 5.5. The bacterial E. coli phytase had also its pH optimum in the acidic range at pH 4.5 while the pH optimum for the Bacillus enzyme was identified at pH 7.0. Temperature optima were at 50 and 60°C for the fungal and bacterial phytases, respectively. The Bacillus phytase was more thermostable in aqueous solutions than all other enzymes. In pelleting experiments performed at 60, 70 and 80°C in the conditioner, Aspergillus A, Peniophora (measurement at pH 5.5) and E. coli phytases were more heat stable compared to other enzymes (Bacillus enzyme was not included). At a temperature of 70°C in the conditioner, these enzymes maintained a residual activity of approximately 70% after pelleting compared to approximately 30% determined for the other enzymes. Incubation of enzyme preparations with porcine proteases revealed that only E. coli phytase was insensitive against pepsin and pancreatin. Incubation of the enzymes in digesta supernatants from various segments of the digestive tract of hens revealed that digesta from stomach inactivated the enzymes most efficiently except E. coli phytase which had a residual activity of 93% after 60 min incubation at 40°C. It can be concluded that phytases of various microbial origins behave differently with respect to their in vitro properties which could be of importance for future developments of phytase preparations. Especially bacterial phytases contain properties like high temperature stability (Bacillus phytase) and high proteolytic stability (E. coli phytase) which make them favourable for future applications as feed additives.