Microbial Phytases and Phytate: Exploring Opportunities for Sustainable Phosphorus Management in Agriculture

Myo-inositol phosphates (phytates) are important biological molecules produced largely by plants to store phosphorus. Phytate is very abundant in many different soils making up a large portion of all soil phosphorus. This review assesses current phytase science from the perspective of its substrate, phytate, by examining the intricate relationship between the phytate-hydrolyzing enzymes and phytate as their substrate. Specifically, we examine available data on phytate’s structural features, distribution in nature and functional roles. The role of phytases and their localization in soil and plant tissues are evaluated. We provide a summary of the current biotechnological advances in using industrial or recombinant phytases to improve plant growth and animal nutrition. The prospects of future discovery of novel phytases with improved biochemical properties and bioengineering of existing enzymes are also discussed. Two alternative but complementary directions to increase phosphorus bioavailability through the more efficient utilization of soil phytate are currently being developed. These approaches take advantage of microbial phytases secreted into rhizosphere either by phytase-producing microbes (biofertilizers) or by genetically engineered plants. More research on phytate metabolism in soils and plants is needed to promote environmentally friendly, more productive and sustainable agriculture.

Amino acid and mineral digestibility,bone ash,and plasma inositol is increased by including microbial phytase in diets for growing pigs

Background The effect of microbial phytase on amino acid and energy digestibility is not consistent in pigs,which may be related to the phytase dosage or the adaptation length to the diet.Therefore,an experiment was conducted to test the hypotheses that increasing dietary phytase after an 18-day adaptation period:1)increases nutrient and energy digestibility;2)increases plasma P,plasma inositol,and bone ash of young pigs;and 3)demonstrates that maximum phytate degradation requires more phytase than maximum P digestibility.Results Data indicated that increasing inclusion of phytase[0,250,500,1,000,2,000,and 4,000 phytase units(FTU)/kg feed]in corn-soybean meal-based diets increased apparent ileal digestibility(AID)of Trp(quadratic;P<0.05),and of Lys and Thr(linear;P<0.05),and tended to increase AID of Met(linear;P<0.10).Increasing dietary phytase also increased AID and apparent total tract digestibility(ATTD)of Ca and P(quadratic;P<0.05)and increased ATTD of K and Na(linear;P<0.05),but phytase did not influence the ATTD of Mg or gross energy.Concentrations of plasma P and bone ash increased(quadratic;P<0.05),and plasma inositol also increased(linear;P<0.05)with increasing inclusion of phytase.Reduced concentrations of inositol phosphate(IP)6 and IP5(quadratic;P<0.05),reduced IP4 and IP3(linear;P<0.05),but increased inositol concentrations(linear;P<0.05)were observed in ileal digesta as dietary phytase increased.The ATTD of P was maximized if at least 1,200 FTU/kg were used,whereas more than 4,000 FTU/kg were needed to maximize inositol release.Conclusions Increasing dietary levels of phytase after an 18-day adaptation period increased phytate and IP ester degradation and inositol release in the small intestine.Consequently,increasing dietary phytase resulted in improved digestibility of Ca,P,K,Na,and the first 4 limiting amino acids,and in increased concentrations of bone ash and plasma P and inositol.In a corn-soybean meal diet,maximum inositol release requires approximately 3,200 FTU/kg more phytase than that required for maximum P digestibility.

Expression of Exogenous Gene in Prokaryotes and Eukaryotes

[Objective] This study aimed to investigate the expression of exogenous gene in prokaryotes and eukaryotes. [Method] The expression vector pTYB2-WF harboring target gene was transformed into E. coli ER2566. IPTG was employed to induce the expression of phytase gene. The expression of phytase fusion protein was detected by SDS-PAGE, and the fusion protein was further purified. Phytase gene phyA was expressed in Pichia pastoris expression system. Yeast recombinant vector pPIC9K-phyA was constructed and transformed into P. pastoris GS115 to construct engineering strain GS115-pPIC9K-phyA. [Result] Phytase protein was ex-pressed under methanol induction. Enzyme activity assay indicated that the activity of phytase was 7.3 U/ml. P. pastoris engineering strain GS115-pPIC9K-phyA was successful y constructed. [Conclusion] Methanol yeast expression mechanisms play a certain role in molecular biology and industrial applications.

Phytate:impact on environment and human nutrition.A challenge for molecular breeding

Phytic acid (PA) is the primary storage compound of phosphorus in seeds accounting for up to 80% of the total seed phosphorus and contributing as much as 1.5% to the seed dry weight. The negatively charged phosphate in PA strongly binds to metallic cations of Ca, Fe, K, Mg, Mn and Zn making them insoluble and thus unavailable as nutritional factors. Phytate mainly accumulates in protein storage vacuoles as globoids, predominantly located in the aleurone layer (wheat, barley and rice) or in the embryo (maize). During germination, phytate is hydrolysed by endogenous phytase(s) and other phosphatases to release phosphate, inositol and micronutrients to support the emerging seedling. PA and its derivatives are also implicated in RNA export, DNA repair, signalling, endocytosis and cell vesicular trafficking. Our recent studies on purification of phytate globoids, their mineral composition and dephytinization by wheat phytase will be discussed. Biochemical data for purified and characterized phytases isolated from more than 23 plant species are presented, the dephosphorylation pathways of phytic acid by different classes of phytases are compared, and the application of phytase in food and feed is discussed.

The Effect of Dietary Supplementation with Phytase Transgenic Corn on Growth Performance,Phosphorus Utilization and Excretion in Growing Pigs (Sus scrofa)

Two experiments were conducted to investigate the effect of dietary supplementation with phytase transgenic corn （PTC） on growth performance,phosphorus （P） utilization and excretion in growing pigs.In Exp.1,180 pigs （Large White × Landrace,BW=37.7 kg） were randomly allotted to 4 treatments with 5 replicates of 9 pigs each in order to evaluate the effect of PTC supplementation in low-P diets on growth performance.Four corn soybean meal-based diets consisted of a positive control （PC） diet,a diet containing 500 units （U） of exogenous phytase kg-1 （EP） on the basis of low-P （inorganic P reduced by 0.05% from PC diet） and the low-P＋500 （PTC1） or 750 （PTC2） phytase U of PTC kg-1.In Exp.2,20 barrows （Large White×Landrace,BW=31 kg,4 treatments with 5 replicates of 1 pig each） were randomly selected to evaluate the effect of PTC in low-P diets on serum parameters and nutrient utilization.Diets in Exp.2 were similar to those in Exp.1 except that the EP group was replaced by a low-P diet without exogenous phytase supplementation as a negative control （NC） group.The results from Exp.1 showed that the average daily gain （ADG） in the PTC2 group was significantly higher （P〈0.05） than that in the EP group over all periods.On the other hand,the feed：gain （F：G） ratio of the EP group was significantly higher （P〈0.05） than that of the PTC2 group during 1-21 and 1-42 d,respectively.There were no differences in average daily feed intake （ADFI） among all treatments （P〉0.05）.The results from Exp.2 showed that the concentration of serum Ca in the NC group was the highest （P〈0.05）,while the concentration of serum P in the PTC2 group was the highest （P〈0.05） among all treatments.There was a significant decrease （P〈0.05） in the P apparent digestibility of the NC group compared with the other groups,and that of PTC2 group was the best.Furthermore,fecal P excretion was reduced （P〈0.05） from 1.80 g d-1 in the PC group to 1.28 g d-1 in the PTC2 group.In conclusion,dietary supplementation with PTC could reduce the application of inorganic P,decrease fecal P excretion,and improve the growth performance of growing pigs.

Effects of a novel bacterial phytase expressed in Aspergillus Oryzae on digestibility of calcium and phosphorus in diets fed to weanling or growing pigs

In 2 experiments, 48 weanling （initial BW： 13.5 ± 2.4 kg, Exp. 1） were used to determine effects of a novel bacterial 6-phytase and 24 growing pigs （initial BW： 36.2 ± 4.0 kg, Exp. 2） expressed in Aspergillus oryzae on the apparent total tract digestibility （ATTD） of phosphorus and calcium in corn-soybean meal diets fed to weanling and growing pigs. In Exp. 1 and 2, pigs were randomly allotted to 6 dietary treatments using a randomized complete block design and a balanced 2 period changeover design, respectively. In both experiments, 6 diets were formulated. The positive control diet was a corn-soybean meal diet with added inorganic phosphorus （Exp. 1：0.42 and 0.86% standardized total tract digestible phosphorus and total calcium, respectively; Exp. 2：0.32 and 0.79% standardized total tract digestible phosphorus and total calcium, respectively）. A negative control diet and 4 diets with the novel phytase （Ronozyme HiPhos, DSM Nutritional Products Inc., Parsippany, N J） added to the negative control diet at levels of 500, 1,000, 2,000, and 4,000 phytase units （FYT）/kg were also formulated. In Exp. 1, the ATTD of phosphorus was greater （P 〈 0.01） for the positive control diet （60.5%） than for the negative control diet （40.5%）, but increased （linear and quadratic, P 〈 0.01） as phytase was added to the negative control diet （40.5% vs. 61.6%, 65.1%, 68.7%, and 68.0%）. The breakpoint for the ATTD of phosphorus （68.4%） was reached at a phytase inclusion level of 1,016 FYT/kg. In Exp. 2, the ATTD of phosphorus was greater （P〈 0.01） for the positive control diet （59.4%） than for the negative control diet （39.8%） and increased （linear and quadratic, P〈 0.01） as phytase was added to the negative control diet （39.8% vs. 58.1%, 65.4%, 69.1%, and 72.8%）. The breakpoint for the ATTD of phosphorus （69.1%） was reached at a phytase inclusion level of 801 FYT/kg. in conclusion, the novel bacterial 6-phytase improved the ATTD of phosphorus and calcium in both weanling and growing pigs. The optimum level of inclusion for this phytase is 800 to 1,000 FYT/kg of complete feed to maximize ATTD of phosphorus and calcium in weanling and growing pigs

Molecular Characterization and Functional Analysis of OsPHY1,a Purple Acid Phosphatase (PAP)-Type Phytase Gene in Rice (Oryza sativa L.)

As a specific type of acid phosphatses, phytases play diverse roles in plants by catalazing the degradation of phytic acid and its derivatives. In this study, a rice phytase gene referred to OsPHY1 has been functionally characterized. OsPHY1 contains a 1 620 bp of open reading frame, encoding a 539-aa polypeptide. A conserve domain metallophosphatase （MPP） （MPP_PAPs）, generally harbored in phytase and purple acid phosphatases （PAP）, was identified in OsPHY1 （residue 194-398）. Phylogenetic analysis revealed that OsPHY1 shares high similarities with phytase genes and PAP-type genes that derived from diverse plant species. The OsPHY1 transcripts were detected to be abundant in germinating seeds, suggesting that this gene plays potential roles on degradation of seed phytic acid and its derivatives during the germination process. Biochemical analysis confirmed that OsPHY1 possesses strong catalytic activities on phytic acid-Na2, with optimal temperature of 57°C and suitable pH of 3.5. Based on transgene analysis, the putative role of OsPHY1 in plants on utilization of phytate was assessed. Under the condition that phytic acid-Na2 was used as sole P source, the OsPHY1-overexpressing tobacco plants behaved higher phytase activities, higher concentrations of Pi, more accumulative amount of total phosphorus, and much more improved growth traits than those of the control plants. Therefore, OsPHY1 is acted as an important component on degradation of the phytins during the seed germination process in rice. Also, OsPHY1 has a potential use on generation of elite crop germplasms with improved use efficiencies on phytate and its derivatives.

Nonphytate Phosphorus Requirement and Efficacy of a Genetically Engineered Yeast Phytase for Yellow Broilers at 22- to 42-d-Old Age

An experiment was conducted to investigate the requirement of nonphytate phosphorus（nPP） and efficacy of a genetically engineered yeast phytase（PHY A） for Lingnan yellow broilers from 22-to 42-d-old age.A total of 1 320 1-d-old male chicks were randomly divided into 11 dietary treatment groups,which consisted of 4 replicate floor pens with 30 birds per pen.The control group（treatment 1） was fed with basal diet of nPP 0.08% without dicalcium phosphate or phytase supplementation.Dietary levels of nPP were 0.16,0.24,0.32,0.40,0.48,and 0.56%,respectively,for treatments 2 to 7,through addition of dicalcium phosphate（chemistry grade） to the basal diet.Diets of treatments 8 to 11 were supplemented with PHY A at 200,400 and 600 U kg-1,a commercial phytase product（PHY B） at 400 U kg-1 level,respectively.The birds in 0.32-0.56% nPP groups gained more than those of the other groups（P0.05）.The nPP supplementation significantly improved feed intake（P0.05）.The feed gain ratio was significantly decreased by 0.40% nPP diet compared to the control birds（P0.05）.The level of 0.48% nPP was required for optimum tibia development.The additions of PHY A at 400 and 600 U kg-1 level and PHY B all significantly improved ADG（P0.05）,ADFI（P0.05）,and dry defatted tibia weight（P0.05）.Similarly,the percentage of tibia ash was increased by 600 U kg-1 PHY A supplementation（P0.05）.The requirement of nPP for maximal ADG and highest percentage tibia ash both was 0.40%.The phosphorus equivalency value of PHY A was estimated as 685 U kg-1 for male yellow broilers of 22-to 42-d-old age.

Transgenic rice expressing a novel phytase-lactoferricin fusion gene to improve phosphorus availability and antibacterial activity

The developing trends of livestock production are efficiency availability of phytate phosphorus and abuse of antibiotics. safety and sustainability, which face two major challenges： low As a solution phytases and antimicrobial peptides are applied as feed additives. However, phytases and antimicrobial peptides are susceptible to proteases, costly by fermentation and potential toxic to production hosts. We transformed an optimized phytase-lactoferricin fusion gene PhyLfdriven by an en- dosperm-specific promoter Gt13aP and Bar （bialaphos resistance） gene as a selection maker into rice. The Bar and PhyLf genes were integrated into the rice genome, stably inherited and expressed. Their phosphinothricin acetyl transferase （PAT） protein content oftransgenic plants with glufosinate resistance varied between 50.45-93.39 IJg g-l. Fusion protein expressed especially in the seeds of transgenic rice had a summit phytase activity at 32.30 U g-l, which increased by 61.71-fold com- pared to the control/check group （CK） and 7.54-fold compared to un-optimized transgenic plant. The highest inorganic phosphorus （Pi） content of the transgenic seeds reached 13.15 mg g-~, increased by 12.77-fold compared to that of CK. Preliminary antibacterial experiments showed that the enterokinase hydrolysate product of fusion protein could inhibit the growth of Escherichia coil DH5a. These results indicated that the protein PhyLf has the potential to increase availability of feed phytate phosphorus, improve consumer＇s immunity and reduce the use of antibiotics.

Effects of Fermentation Product Containing Phytase on Productive Performance,Egg Quality,and Phosphorous Apparent Metabolism of Laying Hens Fed Different Levels of Phosphorus

This study investigated the effects of fermentation product containing phytase （FPP） that was fermented using waste vinegar residue （WVR） as substrate from Aspergillusficuum NTG-23 on productive performance, egg quality, and phosphorus apparent metabolism of laying hens. First, 375 22-wk-old Jinghong hens were allocated into 5 treatments （5 replicates of 15 hens each） in an 8-wk experiment for evaluating the parameters of productive performance, egg quality, serum, and tibia. Experimental diets contained 4% FPP and 96% corn-soybean diet. The levels of dicalciurn phosphate （DCP） were 1.34, 1.01, 0.67, 0.34 and 0%. Next, thirty 31-wk-old Jinghong hens were fed 5 types of diets for evaluating phosphorous apparent metabolism rate. Egg productive rate, egg weight, feed conversion ratio, Haugh unit, egg albumen height, serum calcium, tibia ash, tibia ash calcium and tibia breaking strength were not different significantly among 5 treatments. The significant difference of average daily feed intake was not appeared when the DCP content of corn-soybean-FPP diet was reduced to 0.67%; the eggshell hardness, eggshell thickness and serum phosphorus were not reduced significantly until the DCP content of corn-soybean-FPP diet was reduced to 0.34%. The yolk color was improved when the laying hens fed deficient DCP corn-soybean-FPP diet. A 22.14% reduction in excreta phosphorus was observed when the laying hens fed low phosphorus （0.67% DCP） corn-soybean-FPP diet. A 30% elevation of phosphorus apparent metabolism rate was obtained when the DCP content of corn-soybean-FPP diet was decreased from 1.34 to 1.01%. The reducing cost of layer diet was totalized about 120 CNY 1000 kg1 diet when the content of DCP was 0.67% in corn-soybean-FPP diet. These results indicated that FPP could be applied in laying hen as a potential, cost-effective and rational application of WVR.

Effects of a multi-enzyme complex on growth performance,nutrient utilization and bone mineralization of meat duck

Background： Previous studies with broiler have shown dietary supplementation with multi-enzyme complex containing non-starch polysaccharides （NSP） degrading enzymes and phytase is efficient in releasing phosphorus （P）, calcium （Ca）, energy and amino acids from corn-soybean meal diets or corn-sorghum diets, hence compensating considerable levels of nutrients in formulation. Notwithstanding, such potentials have not been well defined in duck nutrition. Giving China being the largest duck producing country, we conducted this study to establish adequate specifications of major nutrients along with multi-enzyme complex to meat duck from day-old to slaughter, focusing on performance, utilization of nutrients and bone mineralization. Five dietary treatments were： Positive control （PC,T1）： the nutrients concentration of diet for 1 to 14 d of age were apparent metabolizable energy（AME） 2,800 kcal/kg, crude protein （CP）1 9.39%, Ca 0.85%, available phosphorus （avP） 0.42%; for 15 to 35 d of age these parameters were AME 2,900 kcal/kg, CP 16.47%,Ca 0.76%,avP 0.38%; Negative control 1（NC1,7-2）, the AME and digestible amino acids （DAA） were reduced by 70 kcal/kg and 2.0%, avP and Ca by 1.0 g/kg from PC diet; Negative control 2（NC2,T4）, the down- spec from PC diet was AME 100 kcal/kg, DAA 2.5%, avP 1.5 g/kg and Ca 1.2 g/kg; The enzyme complex was added at the same dosage （200 mL/1,000 kg） on NC1 （T3） and NC2 （T5） diets. Results： Comparing with the ducks fed on T1, T3 and T5 diets, the birds fed on NC2 diet showed the lowest （P 〈 0.05） body weight （ d 14 and 35）, feed intake （d 35）, tibia ash, Ca and P contents （d 14 and 35）, and the utilization of nutrients （P 〈 0.05）. The supplementation with the enzyme complex to the NC diets restored growth rate, utilization of nutrients and bone mineralization to the level of the PC diet, and increased AME by 60 kcal/kg and 117 kcal/kg, respectively for the NC1 and NC2 diets. Conclusion： These results suggest that down-spec AME by 100 kcal/kg, DAA by 2.5%, avP by 1.5 g/kg and Ca by 1.2 g/kg caused detrimental effects on duck performance compared with those fed on the PC diet, and these performance losses can be compensated by the addition of the multiple-enzyme complex.

Optimization of Culture Conditions to Produce Phytase from <i>Aspergillus tubingensis SKA</i>

The effects of nutrients and physical conditions on phytase production were investigated with a recently isolated strain of Aspergillus tubingensis SKA under solid state fermentation on wheat bran. The nutrient factors investigated included carbon source, nitrogen source, phosphate source and concentration, metal ions (salts) and the physical parameters investigated included inoculum size, pH, temperature and fermentation duration. Our investigations revealed that optimal productivity of phytase was achieved using wheat bran supplemented with: 1.5% glucose. 0.5% (NH4)2SO4, 0.1% sodium phytate. Additionally, optimal physical conditions were 1 × 105 spore/g substrate, initial pH of 5.0, temperature of fermentation 30°C and fermentation duration of 96 h. Overall, a 34% improvement in phytase activity was achieved by using the optimal conditions.

Performance and Nutrient Utilization of Layers Fed Diet Supplemented with Microbial Phytase and Cellulase

A 31 week feeding trial was conducted to investigated the effects of dietary supplementation of microbial phytase and cellulase on performance,nutrients utilization and tibia quality of laying hens fed maize and soybean meal diets.192 18 week old Hisex layers were used in the trial A 2×2×2 factorial design was used in the experiment with three factors of two levels each:0 38% and 0 16% of dietary non phytate P(nP).0 and 300 U·kg -1 of phytase (Ph),and 0 and 0 10% of cellulase (Ce).The results showed that supplementation of 300 U·kg -1 phytase significantly improved utilization of dietary crude ash,CP,Ca,total P and copper (P<0 05),and improved tibia breaking strength (P<0 05).No effect of phytase on performance was observed.Addition of 0 10% cellulase decreased feed intake (P<0 05),increased utilization of CF (P<0 05) and Ca(P<0 01),and decreased total tibia ash weight (P<0 05).300 U·kg -1 phytase and 0 10% cellulase exhibited obvious positive interactions to enhance utilization of dietary phytic P and copper (P<0 05).0 16% nP did not reduce performance of the layers,but improved egg shell quality at 20 wks,increased utilization of dietary total P,phytic P and Copper (P<0 01),decreased utilization of dietary CP,increased tibia breaking strength and Ca,Mn contents of tibia(P<0 01)

Transgenic Expression of the Recombinant Phytase in Rice (Oryza sativa)

In most of the cereal crop, phytic acid is the main storage form of phosphorus, which can decrease the bioavailability of phosphate. Transgenic expression of phytase is regarded as an efficient way to release phosphate from phytate in transgenic plants. In this study, a plant expression vector, containing the recombinant phytase gene driven by the maize ubiquitin （Ubi） promoter was constructed and introduced into an elite rice variety via Agrobacterium-mediated transformation. During the experiment, a total of 15 independent transgenic rice lines were regenerated. The results of PCR and Southern blot indicated that the target gene was integrated into the genome of transgenic rice plants. Moreover, the RT-PCR analysis of total RNAs extracted from the immature seeds of several transgenic lines showed that the recombinant phytase gene could be normally expressed. The inorganic phosphorus content, both in the mature seeds and the leaf was significantly higher in the transgenic plants than in the untransformed wild type.

The influence of phytase, pre-pellet cracked maize and dietary crude protein level on broiler performance via response surface methodology

Background:The reduction of crude protein levels in diets for broiler chickens may generate economic,environmental and flock welfare and health benefits;however,performance is usually compromised.Whole grain feeding and phytase may improve the utilization of reduced crude protein diets.Results:The effects of pre-pellet cracked maize(0,15%and 30%)and phytase(0,750 and 1500 FTU/kg)in isoenergetic maize-soy diets with three levels of crude protein(22%,19.5%and 17%)were evaluated via a BoxBehnken response surface design.Each of 13 dietary treatments were offered to 6 replicate cages(6 birds/cage)of male Ross 308 broiler chicks from 7 to 28 d post-hatch.Model prediction and response surface plots were generated from experimental data via polynomial regression in R and only significant coefficients were included and discussed in the predicted models.Weight gain,feed intake and FCR were all influenced by pre-pellet cracked maize,phytase and crude protein level,where crude protein level had the greatest influence.Consequently,the reduction from 22%to 17%dietary crude protein in non-supplemented diets reduced weight gain,feed intake,relative gizzard weight,relative gizzard content and relative pancreas weight but improved FCR.However,the inclusion of 30%cracked maize to 17%crude protein diets restored gizzard weight and 1500 FTU phytase inclusion to 17%crude protein diets increased relative gizzard contents and pancreas weights.Cracked maize and phytase inclusion in tandem to 17%crude protein diets increased weight gain,feed intake and FCR;however,this FCR was still more efficient than broilers offered the non-supplemented 22%crude protein diet.Broilers offered the prepellet cracked maize and phytase inclusions reduced AME in 22%crude protein diets but improved AME by 2.92 MJ(14.16 versus 11.24 MJ;P<0.001)in diets containing 17%crude protein.Ileal N digestibility was greater in broilers offered diets with 17%crude protein than those offered the 22%crude protein diet;irrespective of phytase and pre-pellet cracked maize.Conclusion:Pre-pellet cracked maize and phytase inclusions will improve the performance of broilers offered reduced crude protein diets.

Cloning of a novel phytase from an anaerobic rumen bacterium, Mitsuokella jalaludinii, and its expression in Escherichia coli

The ful length phytase gene of Mitsuokel a jalaludini was successful y cloned and was found to be 1 047 bp in length, with 348 amino acids, and was designated as PHY7 phytase gene. A comparison of the sequence of PHY7 phytase gene of M. jalaludini with various microbial phytase gene sequences showed that it was not similar to those from other bacteria except Selenomonas ruminatium, thus suggesting that they may both express a new class of phytase. The PHY7 phytase gene was subsequently subcloned into bacterial expression vector, pET32a, for expression in Escherichia coli strain Ro-setta-gami. Expression of the recombinant phytase gene was optimised and characterised. The recombinant phytase was estimated to be approximately 55 kDa by SDS-PAGE analysis. The recombinant phytase exhibited optimum activity at 55°C, pH 4.5 and showed good pH stability from pH 3.5 to 5.5 (>78%relative activity). Metal ions such as Ca2+, Mg2+, and K+were found to exert signiifcant stimulatory effect on the recombinant phytase activity while Cu2+, Fe3+, and Zn2+greatly inhibited the enzyme activity. The recombinant phytase showed moderate resistance to trypsin proteolysis, but susceptible to pepsin proteolysis. The results of the study showed that several characteristics of recombinant phytase were slightly different from the native enzyme. Unfavourable characteristics such as reduced pH stability and metal ion effects should be taken into consideration during feed enzyme formulation.

Treatment of zinc deficiency without zinc fortification

Zinc(Zn) deficiency in animals became of interest until the 1950s.In this paper,progresses in researches on physiology of Zn deficiency in animals,phytate effect on bioavailability of Zn,and role of phytase in healing Zn deficiency of animals were reviewed.Several studies demonstrated that Zn is recycled via the pancreas;the problem of Zn deficiency was controlled by Zn homeostasis.The endogenous secretion of Zn is considered as an important factor influencing Zn deficiency,and the critical molar ratio is 10.Phytate(inositol hexaphosphate) constituted up to 90% of the organically bound phosphorus in seeds.Great improvement has been made in recent years on isolating and measuring phytate,and its structure is clear.Phytate is considered to reduce Zn bioavailability in animal.Phytase is the enzyme that hydrolyzes phytate and is present in yeast,rye bran,wheat bran,barley,triticale,and many bacteria and fungi.Zinc nutrition and bioavailability can be enhanced by addition of phytase to animal feeds.Therefore,using phytase as supplements,the most prevalent Zn deficiency in animals may be effectively corrected without the mining and smelting of several tons of zinc daily needed to correct this deficiency by fortification worldwide.

Effects of phytase supplementation on growth performance, slaughter performance, growth of internal organs and small intestine, and serum biochemical parameters of broilers

The objective of this study was to investigate the effects of phytase supplementation on growth performance, slaughter performance, growth of internal organs and small intestine, and serum biochemical indices of broilers. A total of 360 1-day-old Ross 308 broilers were randomly divided into 4 groups with 3 replicates and 30 broilers per replicate (15 male, 15 female). The treatments were fed with basal diet supplemented with 0%, 0.01%, 0.02% and 0.03% phytase. The results showed that: 1) Phytase supplementation increased the body weight gain and the body weight of Ross 308 broilers (P serum calcium (Ca) consistency was 0.02%. According to the above analysis, the feasible supplementation of phytase (enzyme activity for 5000 u/g) in broiler dietary was 0.02%.

Effect of Phytase on Digestibility and Performance of Growing and Finishing Pigs Fed Diets with Lupins and Rapeseed Meal

Lupin seeds and rapeseed meal(RSM)contain relatively high amounts of poorly digestible phytate.Phytase additive can help in the utilization of nutrients from the diet.The aim of this study was to determine total tract digestibility coefficients of nutrients and performance results of finishing pigs fed diets containing yellow lupin or narrow-leafed lupin seeds and/or RSM with similar or increasing levels of phytase.Three experiments were conducted.In Experiment I the effect of RONOZYME®HiPhos(100 g/t)in diets containing narrow-leafed or yellow lupin seeds and RSM on production parameters of fatteners was analyzed.In Experiment II the effect of phytase RONOZYME®HiPhos(1,000 FTU/t)in similar diets was analyzed but calcium(Ca)and phosphorus(P)levels in diets were reduced.In Experiment III the effect of increasing levels of Quantum Bluephytase(0,5,000,10,000 and 15,000 FTU/t)in diets with yellow lupin seeds with reduced Ca and P level on total tract digestibility coefficients of selected nutrients and performance of pigs was analyzed.In none of the experiments the enzyme additives included in the diet affected pig performance(p>0.05).There were no significant differences(p>0.05)in apparent total tract digestibility coefficients of dry matter(DM)and crude protein(CP).The phytase additives significantly improved P and Ca digestibility coefficients(p<0.05)in comparison with the control diet,but this improvement was not linearly related with phytase dosage.By improving digestibility phytase allows to reduce mineral contents in diets,thus reducing the cost of pig nutrition.

A Single Mutation in the Hepta-Peptide Active Site of <i>Aspergillus niger</i>PhyA Phytase Leads to Myriad Biochemical Changes

The active site motif of proteins belonging to "Histidine Acid Phosphatase" (HAP) contains a hepta-peptide region, RHGXRXP. A close comparison among fungal and yeast HAPs revealed the fourth residue of the hepta-peptide to be E instead of A, which is the case with A. niger PhyA phytase. However, another phytase, PhyB, from the same microorganism has a higher turnover number and it shows E in this position. We mutated A69 residue to E in the fungal PhyA phytase. The mutant phytase shows a myriad of new kinetic properties. The pH profile shifted 0.5 pH unit in both 5.0 and 2.5 bi-hump peaks. The optimum temperature shifted down from 58℃ to 55℃. However, the greatest difference was observed in the mutant protein's reaction to GuCl at a concentration of 0.1 to 0.2 M. The activity of the mutant phytase jumped 100% while the wild type protein showed no activity enhancement in the same concentration range of GuCl. The kinetics performed at higher concentration of GuCl also contrasted the difference between the wild type and mutant phytase. While Km was least affected, the Vmax increased for the mutant and decreased for the wild type. The sensitivity towards myo-inositol hexasulfate, a potent inhibitor, was decreased by the mutation. All in all, A69E mutation has affected a multitude of enzymatic properties of the protein even though the residue was thought to be non-critical for phytase's catalytic function notwithstanding its location in the conserved hepta-peptide region of the biocatalyst.